Archive for the ‘Crispr’ Category
The Age of Crispr Medicine Is Here – WIRED
The Age of Crispr Medicine Is Here WIRED
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The Age of Crispr Medicine Is Here - WIRED
The Basics of CRISPR Gene Editing – Cleveland Clinic Health Essentials
In 2013, two biochemists published a paper proclaiming theyd discovered a potentially game-changing method of manipulating genes. CRISPR which sounds like a veggie-forward gastro pub won them each a Nobel Prize.
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In the years since, CRISPR (or Clustered Regularly Interspaced Short Palindromic Repeats) has lived up to the hype. Its altered the global scientific landscape and raised questions about what kinds of revolutionary changes scientists and healthcare providers could and should pursue.
What if we could make foods allergy-free and crops drought-resistant? What if we could eliminate invasive species and protect against infectious diseases like malaria? What if we could revive extinct species? What if we could remove or repair mutations that cause inherited conditions? Or create custom immunotherapies to treat an individuals cancer?
The prospects are that exciting.
If your understanding of genetics starts and ends with high school biology or the (very fictional) Jurassic Park movies youre not alone. This stuff is complicated. Thats why we asked genomics and immunotherapy expert Timothy Chan, MD, PhD, to break CRISPR down for us, so we can better understand why, over a decade later, its still got researchers so excited.
Before we jump into CRISPR, lets start with the concept of gene editing.
Gene editing is the process of altering genetic material (DNA). That could mean changing a few individual genes or an entire sequence. Research has been ongoing for more than a decade thats looking at using gene editing on mutations that cause serious health conditions in people. The goal of this gene editing research is to eliminate or correct the mutation thats causing the health condition, or has the potential to cause one, such as certain cancers. In other research studies, gene editing is being explored so a mutation isnt passed down to children at birth.
For example, the U.S. Food and Drug Administration (FDA) approved a gene therapy in late 2022 that introduces a gene needed for blood clotting into people with hemophilia B. Its one of several cellular and gene therapy products currently in use today.
There are many different techniques and applications for gene editing. CRISPR is one approach to gene editing thats showing promise in ongoing clinical trials.
Now that were clear on what gene editing is, lets focus on a specific approach: CRISPR.
Clustered Regularly Interspaced Short Palindromic Repeats, otherwise known as CRISPR, was originally identified in bacteria, as a bacterial defense system, says Dr. Chan.
Thats right. Bacteria have immune systems, too.
CRISPR contains spacers sequences of DNA left over from unfriendly viruses or other entities as well as repeating sections of genetic material. Those sequences provide acquired immunity, and form the building blocks of the gene editing system or process. It creates a sort of blueprint that allows enzymes in genetic material to make changes to sequences of DNA in living cells. One of the best-known enzymes used for this purpose is called Cas9, which is why youll sometimes hear people talk about CRISPR-Cas9.
Over the years, people have discovered that specific enzymes that allow CRISPR to work Cas9 is one of them.But there are other ones, and they can be tailored to target sequences of interest in the DNA for specific cuts to be made, Dr. Chan explains.
You can think of the underlying mechanism of CRISPR gene editing as being similar to the way magnetic shapes are drawn to each other or the way Lego blocks fit together.
The segments in CRISPR are transcribed into RNA. This RNA includes a guide sequence, which is a match to existing DNA in a persons body.
That guide sequence can be tailored to whatever you want, Dr. Chan says. And as a result, you can make specific alterations or mutations in a part of the genome that you are targeting with a high degree of accuracy.
Along for the ride with this guide sequence is an enzyme like Cas9.
When the guide sequence and enzyme find the desired DNA to edit, the enzyme can then get down to business. It attaches itself to this DNA and makes changes, whether thats a cut or alteration.
CRISPR technology has come a long way, Dr. Chan says. The first generation of CRISPR was a great way to inactivate genes. It only made a break in genes. Then, the DNA would get filled up with natural repair enzymes.
But new versions of CRISPR like CRISPR prime or CRISPR HD are more advanced.
These can allow actual replacements to occur, Dr. Chan continues. You can even very accurately replace one sequence one of the letters in the genome with another letter. And you can make specific mutations.
CRISPRs ability to make very specific, very small cuts has the potential to transform how healthcare providers can address certain genetic diseases.
Dr. Chan is optimistic about the future of CRISPR based on the success of ongoing clinical trials in human subjects. For any type of genetic diseases caused by a single mutated gene, you can use CRISPR to mutate it and make it normal. Thats why its useful. Its a way for us to change errors in the genome.
Right now, CRISPR is geared toward correcting a single change in genes, he adds. While combinations may be possible in the future, were just not there yet.
While gene editing is already in use, CRISPR is still in the clinical trials phase, Dr. Chan says. Its used all the time in research laboratories and industries, he notes. Many clinical trials are testing CRISPR in the setting of genetic diseases and cancer.
Interestingly, CRISPR can be used to detect certain diseases. The best-known example is the Sherlock CRISPR SARS-CoV-2 Kit: A COVID-19 test that received emergency use authorization (EAU) from the FDA in 2020.
But theres no FDA-approved CRISPR therapy right now. The clinical trials are ongoing, he says.
These include trials looking at CRISPR to correct genetic diseases such as cystic fibrosis, Huntingtons disease and muscular dystrophy.
Dr. Chan adds that there are also major clinical trials in process for blood disorders, where CRISPR is being used to correct the gene alteration that causes the condition. As one example, he cites a promising trial looking at CRISPR-Cas9 gene editing for sickle cell disease and -thalassemia, written about in an early 2021 issue of the New England Journal of Medicine. -thalassemia is an inherited blood disorder that impacts the bodys ability to create hemoglobin an iron-dense protein that serves as the primary ingredient in red blood cells.
There are also clinical trials looking to see if CRISPR can be used to treat certain cancers. Dr. Chan notes that chimeric antigen receptor (CAR)T-cell therapy is one of the first gene therapies approved for leukemias. Current research is looking at whether CRISPR technology can make this treatment even more effective.
In CAR T-cell therapy, you take out T-cells from someone and put in a receptor a new way for these cells to target something on cancer cells and then put these cells back in the patient, he explains. Researchers are running trials now where they use CRISPR to alter those T-cells to make them even more active.
CRISPR therapies can take on many different forms. CRISPR has been inserted directly into the body before. It was famously injected into the eyes of seven people with a rare hereditary blindness disorder in 2020, two of whom later told NPR that they regained some ability to see colors. There are human trials in process right now that deliver CRISPR through gels and creams, through food or drink, skin grafts or injections. Ex-vivo delivery is also common: Thats when CRISPR is used to modify a cell outside the body. The cells are then re-inserted into the body using a harmless virus.
The results have been promising so far. I do believe in the next three to five years possibly even sooner were going to see approval to treat some diseases, Dr. Chan states.
With any type of CRISPR therapy, Dr. Chan says theres a risk of getting off-target effects or unexpected side effects.
Whenever youre altering something as fundamental as DNA, you just dont know what might happen he explains. Theres always a chance for the unexpected. You can potentially have effects on your DNA that were not intended.
At the moment, he doesnt have any specific examples of what these effects might be and he notes that existing research suggests the risk is pretty low. Still, data from future research might tell a different story.
Dr. Chan nevertheless sees a lot of potential for CRISPR in the coming years.
The field is moving very quickly, he says. Were seeing continual improvement of the actual CRISPR tools being used.
Its getting more accurate and more flexible in terms of what you can do. There are various engineered modified variants of CRISPR now that are allowing very specific, very accurate changes with fewer off-target effects. So, I think the future is very bright.
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The Basics of CRISPR Gene Editing - Cleveland Clinic Health Essentials
Mechanism and Applications of CRISPR/Cas-9-Mediated Genome Editing
Abstract
Clustered regularly interspaced short palindromic repeat (CRISPR) and their associated protein (Cas-9) is the most effective, efficient, and accurate method of genome editing tool in all living cells and utilized in many applied disciplines. Guide RNA (gRNA) and CRISPR-associated (Cas-9) proteins are the two essential components in CRISPR/Cas-9 system. The mechanism of CRISPR/Cas-9 genome editing contains three steps, recognition, cleavage, and repair. The designed sgRNA recognizes the target sequence in the gene of interest through a complementary base pair. While the Cas-9 nuclease makes double-stranded breaks at a site 3 base pair upstream to protospacer adjacent motif, then the double-stranded break is repaired by either non-homologous end joining or homology-directed repair cellular mechanisms. The CRISPR/Cas-9 genome-editing tool has a wide number of applications in many areas including medicine, agriculture, and biotechnology. In agriculture, it could help in the design of new grains to improve their nutritional value. In medicine, it is being investigated for cancers, HIV, and gene therapy such as sickle cell disease, cystic fibrosis, and Duchenne muscular dystrophy. The technology is also being utilized in the regulation of specific genes through the advanced modification of Cas-9 protein. However, immunogenicity, effective delivery systems, off-target effect, and ethical issues have been the major barriers to extend the technology in clinical applications. Although CRISPR/Cas-9 becomes a new era in molecular biology and has countless roles ranging from basic molecular researches to clinical applications, there are still challenges to rub in the practical applications and various improvements are needed to overcome obstacles.
Keywords: CRISPR, Cas-9, sgRNA, gene-editing, mechanism, applications
Genome editing is a type of genetic engineering in which DNA is deliberately inserted, removed, or modified in living cells.1 The name CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) refers to the unique organization of short, partially repeated DNA sequences found in the genomes of prokaryotes. CRISPR and its associated protein (Cas-9) is a method of adaptive immunity in prokaryotes to defend themselves against viruses or bacteriophages.2 Japanese scientist Ishino and his team accidentally found unusual repetitive palindromic DNA sequences interrupted by spacers in Escherichia coli while analyzing a gene for alkaline phosphatase first discovered CRISPR in 1987. However, they did not ascertain its biological function. In 1990, Francisco Mojica identifies similar sequences in other prokaryotes and he named CRISPR, yet the functions of these sequences were a mystery.3 Later on in 2007, a CRISPR was experimentally conferred as a key element in the adaptive immune system of prokaryotes against viruses. During the adaptation process, bacterial cells become immunized by the insertion of short fragments of viral DNA (spacers) into a genomic region called the CRISPR array. Hence, spacers serve as a genetic memory of previous viral infections.4 The CRISPR defense mechanism protects bacteria from repeated viral attacks via three basic stages: adaptation (spacer acquisition), crRNA synthesis (expression), and target interference. CRISPR loci are an array of short repeated sequences found in chromosomal or plasmid DNA of prokaryotes. Cas gene is usually found adjacent to CRISPR that codes for nuclease protein (Cas protein) responsible to destroy or cleave viral nucleic acid.5
Before the discovery of CRISPR/Cas-9, scientists were relied on two gene-editing techniques using restriction enzymes, zinc finger nucleases (ZFN) and Transcription activator-like effector nucleases (TALENs).6 ZFN has a zinc finger DNA binding domain used to bind a specific target DNA sequence and a restriction endonuclease domain used to cleave the DNA at the target site. TALENs are also composed of DNA binding domain and restriction domain like ZFN but their DNA binding domain has more potential target sequence than the ZFN gene-editing tool. In both cases, the difficulty of protein engineering, being expensive, and time-consuming were the major challenges for researchers and manufacturers.6,7 The development of a reliable and efficient method of a gene-editing tool in living cells has been a long-standing goal for biomedical researchers. After figuring out the CRISPR mechanism in prokaryotes, scientists understood that it could have beneficial use in humans, plants, and other microbes. It was in 2012 that Doudna, J, and Charpentier, E discovered CRISPR/Cas-9 could be used to edit any desired DNA by just providing the right template.8 Since then, CRISPR/Cas-9 becomes the most effective, efficient, and accurate method of genome editing tool in all living cells and utilized in many applied disciplines.9 Thus, this review aims to discuss the mechanisms of genome editing mediated by CRISPR/Cas-9 and to highlight its recent applications as one of the most important scientific discoveries of this century, as well as the current barriers to the transformation of this technology.
Based on the structure and functions of Cas-proteins, CRISPR/Cas system can be divided into Class I (type I, III, and IV) and Class II (type II, V, and VI). The class I systems consist of multi-subunit Cas-protein complexes, while the class II systems utilize a single Cas-protein. Since the structure of type II CRISPR/Cas-9 is relatively simple, it has been well studied and extensively used in genetic engineering.10 Guide RNA (gRNA) and CRISPR-associated (Cas-9) proteins are the two essential components in CRISPR/Cas-9 system. The Cas-9 protein, the first Cas protein used in genome editing was extracted from Streptococcus pyogenes (SpCas-9). It is a large (1368 amino acids) multi-domain DNA endonuclease responsible for cleaving the target DNA to form a double-stranded break and is called a genetic scissor.11 Cas-9 consists of two regions, called the recognition (REC) lobe and the nuclease (NUC) lobe. The REC lobe consists of REC1 and REC2 domains responsible for binding guide RNA, whereas the NUC lobe is composed of RuvC, HNH, and Protospacer Adjacent Motif (PAM) interacting domains. The RuvC and HNH domains are used to cut each single-stranded DNA, while PAM interacting domain confers PAM specificity and is responsible for initiating binding to target DNA.12 Guide RNA is made up of two parts, CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA). The crRNA is an 1820 base pair in length that specifies the target DNA by pairing with the target sequence, whereas tracrRNA is a long stretch of loops that serve as a binding scaffold for Cas-9 nuclease. In prokaryotes, the guide RNA is used to target viral DNA, but in the gene-editing tool, it can be synthetically designed by combining crRNA and tracrRNA to form a single guide RNA (sgRNA) in order to target almost any gene sequence supposed to be edited.11
The mechanism of CRISPR/Cas-9 genome editing can be generally divided into three steps: recognition, cleavage, and repair.13 The designed sgRNA directs Cas-9 and recognizes the target sequence in the gene of interest through its 5crRNA complementary base pair component. The Cas-9 protein remains inactive in the absence of sgRNA. The Cas-9 nuclease makes double-stranded breaks (DSBs) at a site 3 base pair upstream to PAM.14 PAM sequence is a short (25 base-pair length) conserved DNA sequence downstream to the cut site and its size varies depending on the bacterial species. The most commonly used nuclease in the genome-editing tool, Cas-9 protein recognizes the PAM sequence at 5-NGG-3 (N can be any nucleotide base). Once Cas-9 has found a target site with the appropriate PAM, it triggers local DNA melting followed by the formation of RNA-DNA hybrid, but the mechanism of how Cas-9 enzyme melts target DNA sequence was not clearly understood yet. Then, the Cas-9 protein is activated for DNA cleavage. HNH domain cleaves the complementary strand, while the RuvC domain cleaves the non-complementary strand of target DNA to produce predominantly blunt-ended DSBs. Finally, the DSB is repaired by the host cellular machinery.11,15
Non-homologous end joining (NHEJ), and homology-directed repair (HDR) pathways are the two mechanisms to repair DSBs created by Cas-9 protein in CRISPR/Cas-9 mechanism.16 NHEJ facilitates the repair of DSBs by joining DNA fragments through an enzymatic process in the absence of exogenous homologous DNA and is active in all phases of the cell cycle. It is the predominant and efficient cellular repair mechanism that is most active in the cells, but it is an error-prone mechanism that may result in small random insertion or deletion (indels) at the cleavage site leading to the generation of frameshift mutation or premature stop codon.17 HDR is highly precise and requires the use of a homologous DNA template. It is most active in the late S and G2 phases of the cell cycle. In CRISPR-gene editing, HDR requires a large amount of donor (exogenous) DNA templates containing a sequence of interest. HDR executes the precise gene insertion or replacement by adding a donor DNA template with sequence homology at the predicted DSB site.16,17
In just a few years of its discovery, the CRISPR/Cas-9 genome editing tool has already being explored for a wide number of applications and had a massive impact on the world in many areas including medicine, agriculture, and biotechnology. In the future, researchers hope that this technology will continue to advance for treating and curing diseases, develop more nutritious crops, and eradicating infectious diseases.18 Highlights for some of the recent CRISPR/Cas-9 applications and clinical trials being investigated are discussed below.
More than 6000 genetic disorders have been known so far. But the majority of the diseases lack effective treatment strategies.19 Gene therapy is the process of replacing the defective gene with exogenous DNA and editing the mutated gene at its native location. It is the latest development in the revolution of medical biotechnology. From 1998 to August 2019, 22 gene therapies including the novel CRISPR/Cas-9 have been approved for the treatment of human diseases.20
Since its discovery in 2012, CRISPR/Cas-9 gene editing has held the promise of curing most of the known genetic diseases such as sickle cell disease, -thalassemia, cystic fibrosis, and muscular dystrophy.21,22 CRISPR/Cas-9 for targeted sickle cell disease (SCD) therapy and -thalassemia have been also applied in clinical trials.23 SCD is an autosomal recessive genetic disease of red blood cells, which occurs due to point mutation in the -globin chain of hemoglobin leading to sickle hemoglobin (HbS). During the deoxygenation process, HbS polymerization leads to severe clinical complications like hemolytic anemia.24 Either direct repairing the gene of hemoglobin S or boosting fetal -globin are the two main approaches that CRISPR/Cas-9 is being used to treat SCD.25 However, the most common method used in a clinical trial is based on the approach of boosting fetal hemoglobin. First bone marrow cells are removed from patients and the gene that turns off fetal hemoglobin production, called B-cell Lymphoma 11A (BCL11A) is disabled with CRISPR/Cas-9. Then, the gene-edited cells are infused back into the body.26 BCL11A is a 200 base pair gene found on chromosome 2 and its product is responsible to switch -globin into the -globin chain by repressing -globin gene expression.27 Once this gene is disabled using CRISPR/Cas-9, the production of fetal hemoglobin containing -globin in the red blood cells will increase, thereby alleviating the severity and manifestations of SCD.28
Scientists have been also investigating CRISPR/Cas-9 for the treatment of cystic fibrosis. The genetic mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene decreases the structural stability and function of CFTR protein leading to cystic fibrosis.29 CFTR protein is an anion channel protein regulated by protein kinase-A, located at the apical surface of epithelial cells of the lung, intestine, pancreas, and reproductive tract.30 Although there is no cure for cystic fibrosis, symptom-based therapies (such as antibiotics, bronchodilators, and mucus thinning medications) and CFTR modulating drugs have become the first-line treatments to relieve symptoms and reduce the risk of complications.31 Currently, gene manipulation technologies and molecular targets are also being explored. The use of CRISPR/Cas-9 technology for genome editing has great potential, although it is in the early stages of development.32 In 2013, researchers culture intestinal stem cells from two cystic fibrosis patients and corrected the mutation at the CFTR locus resulting in the expression of the correct gene and full function of the protein. Since then, the potential utility of the application of CRISPR/Cas-9 for cystic fibrosis was established.33 Furthermore, Duchenne muscular dystrophy (DMD), which is caused by a mutation in the dystrophin gene and characterized by muscle weakness, has been successfully corrected by CRISPR/Cas-9 in patient-induced pluripotent stem cells.34 Despite considerable efforts, the treatment available for DMD remains supportive rather than curative. Currently, several therapeutic approaches (gene therapy, cell therapy, and exon skipping) have been investigated to restore the expression of dystrophin in DMD muscles.35,36 Deletion/excision of intragenic DNA and removing the duplicated exon by CRISPR/Cas-9 are the new and promising approaches in correcting the DMD gene, which restores the expression of dystrophin protein.37
Moreover, the latest researches show that the CRISPR/Cas-mediated single-base editing and prime editing systems can directly install mutations in cellular DNA without the need for a donor template. The CRISPR/Cas-base editor and prime editor system do not produce DSB, which reduces the possibility of indels that are different from conventional Cas-9.38 So far, two types of base editors have been developed: cytosine base editor (CBE) and adenine base editor (ABE).39 The CBE is a type of base editor composed of cytidine deaminase fused with catalytically deficient or dead Cas-9 (dCas-9). It is one of the novel gene therapy strategies that can produce precise base changes from cytidine (C) to thymidine (T).40 However, the target range of the CBE base editor is still restricted by PAM sequences containing G, T, or A bases. Recently, a more advanced fidelity and efficiency base editor called nNme2-CBE (discovered from Neisseria meningitides) with expanded PAM compatibility for cytidine dinucleotide has been developed in both human cells and rabbits embryos.41 The ABE uses adenosine deaminase fused to dCas-9 to correct the base-pair change from adenosine (A) to guanosine (G).38 Overall, single-base editing through the fusion of dCas-9 to cytidine deaminase or adenosine deaminase is a safe and efficient method to edit point mutations. But both base editors can only fix four-transition mutations (purine to purine or pyrimidine to pyrimidine).42 To overcome this shortcoming, the most recent member of the CRISPR genome editing toolkit called Prime Editor (PE) has been developed to extend the scope of DNA editing beyond the four types of transition mutations.43 PE contains Cas-9 nickase fused with engineered reverse transcriptase and multifunctional primer editing guide RNA (pegRNA). The pegRNA recognizes the target nucleotide sequence; the Cas-9 nickase cuts the non-complementary strand of DNA three bases upstream from the PAM site, exposing a 3-OH nick of genomic DNA. The reverse transcriptase then extends the 3 nick by copying the edit sequence of pegRNA. Hence, PE not only corrects all 12 possible base-to-base transitions, and transversion mutations but also small insertion and deletion mutations in genetic disorders.44
The first CRISPR-based therapy in the human trial was conducted to treat patients with refractory lung cancer. Researchers first extract T-cells from three patients blood and they engineered them in the lab through CRISPR/Cas-9 to delete genes (TRAC, TRBC, and PD-1) that would interfere to fight cancer cells. Then, they infused the modified T-cells back into the patients. The modified T-cells can target specific antigens and kill cancer cells. Finally, no side effects were observed and engineered T-cells can be detected up to 9 months of post-infusion.45 CRISPR/Cas-9 gene-editing technology could also be used to treat infectious diseases caused by microorganisms.46 One focus area for the researchers is treating HIV, the virus that leads to AIDS. In May 2017, a team of researchers from Temple University demonstrated that HIV-1 replication can be completely shut down and the virus eliminated from infected cells through excision of HIV-1 genome using CRISPR/Cas-9 in animal models.47 In addition to the approach of targeting the HIV-genome, CRISPR/Cas-9 technology can also be used to block HIV entry into host cells by editing chemokine co-receptor type-5 (CCR5) genes in the host cells. For instance, an in vitro trial conducted in China reported that genome editing of CCR5 by CRISPR/Cas-9 showed no evidence of toxicity (infection) on cells and they concluded that edited cells could effectively be protected from HIV infection than unmodified cells.48
As the world population continues to grow, the risk of shortage in agricultural resources is real. Hence, there is a need for new technologies for increasing and improving natural food production. CRISPR/Cas-9 is an existing addition to the field since it has been used to genetically modify foods to improve their nutritional value, increase their shelf life, make them drought-tolerant, and enhance disease resistance.18 There are generally three ways that CRISPR is solving the worlds food crisis. It can restore food supplies, help plants to survive in hostile conditions, and could improve the overall health of the plants.49
Beyond genome editing activity, CRISPR/Cas-9 can be used to artificially regulate (activate or repress) a certain target of a gene through advanced modification of Cas-9 protein.15 Researchers had performed an advanced modified Cas-9 endonuclease called dCas-9 nuclease by inactivating its HNH and RuvC domains. The dCas-9 nuclease lacks DNA cleavage activity, but its DNA binding activity is not affected. Then, transcriptional activators or inhibitors can be fused with dCas-9 to form the CRISPR/dCas-9 complex. Therefore, catalytically inactive dCas-9 can be used to activate (CRISPRa) or silence (CRISPRi) the expression of a specific gene of interest.50 Moreover, the CRISPR/dCas-9 can be also used to visualize and pinpoint where specifically the gene of interest is located inside the cell (subcellular localization) by fusing a marker such as Green Fluorescent Proteins (GFP) with dCas-9 enzyme. This enables site-specific labeling and imaging of endogenous loci in living cells for further utilization.51
Despite its great promise as a genome-editing system CRISPR/Cas-9 technology had hampered by several challenges that should be addressed during the process of application. Immunogenicity, lack of a safe and efficient delivery system to the target, off-target effect, and ethical issues have been the major barriers to extend the technology in clinical applications.52 Since the components of the CRISPR/Cas-9 system are derived from bacteria, host immunity can elicit an immune response against these components. Researchers also found that there were both pre-existing humoral (anti-Cas-9 antibody) and cellular (anti-Cas-9 T cells) immune responses to Cas-9 protein in healthy humans. Therefore, how to detect and reduce the immunogenicity of Cas-9 protein is still one of the most important challenges in the clinical trial of the system.53
Safe and effective delivery of the components into the cell is essential in CRISPR/Cas-9 gene editing. Currently, there are three methods of delivering the CRISPR/Cas-9 complex into cells, physical, chemical, and viral vectors. Non-viral (physical and chemical) methods are more suitable for ex vivo CRISPR/Cas-9-based gene editing therapy.54 The physical methods of delivering CRISPR/Cas-9 can include electroporation, microinjection, hydrodynamic injection, and so on. Electroporation applies a strong electric field to the cell membrane to temporarily increase the permeability of the membrane, allowing the CRISPR/Cas-9 complex to enter the cytoplasm of the target cell. However, the main limitation of this method is that it causes significant cell death.55 Microinjection involves injecting the CRISPR/Cas-9 complex directly into cells at the microscopic level for rapid gene editing of a single cell. Nevertheless, this method also has several disadvantages such as cell damage, which is technically challenging and is only suitable for a limited number of cells.56 The hydrodynamic injection is the rapid injection of a large amount of high-pressure liquid into the bloodstream of animals, usually using the tail vein of mice. Although this method is simple, fast, efficient, and versatile, it has not yet been used in clinical applications due to possible complications.57 The chemical methods of CRISPR/Cas-9 delivery involves lipid and polymer-based nanoparticles.58 Lipid nanoparticles/liposomes are spherical structures composed of lipid bilayers membrane and are synthesized in aqueous solutions using Lipofectamine-based reagents. The positively charged liposomes encapsulated with negatively charged nucleic acids thereby facilitate the fusion of the complex across the cell membrane into cells.59 Polymeric nanoparticles, such as polyethyleneimine and poly-L-lysine, are the most widely used carriers of CRISPR/Cas-9 components. Like lipid nanoparticles, polymer-based nanoparticles can also transverse the complex in the membrane through endocytosis.60
Viral vectors are the natural experts for in vivo CRISPR/Cas-9 delivery.61 Vectors, such as adenoviral vectors (AVs), adeno-associated viruses (AAVs), and lentivirus vectors (LVs) are currently being widely used as delivery methods due to their higher delivery efficiency relative to physical and chemical methods. Among them, AAVs are the most commonly used vectors due to their low immunogenicity and non-integration into the host cell genome compared to other viral vectors.62 However, the limited virus cloning capacity and the large size of the Cas-9 protein remain a major problem. One strategy to tackle this hurdle is to package sgRNA and Cas-9 into separate AAVs and then co-transfect them into cells. Recent methods employ a smaller strain of Cas-9 from Staphylococcus aureus (SaCas-9) instead of the more commonly used SpCas-9 to allow packaging of sgRNA and Cas-9 in the same AAVs.54,61 Lately, the development of extracellular vesicles (EVs), for the in vivo delivery of CRISPR/Cas-9 to avoid some of the limitations of viral and non-viral methods has shown a great potential.63
The designed sgRNA will mismatch to the non-target DNA and can result in nonspecific, unexpected genetic modification, which is called the off-target effect.57 The CRISPR/Cas-9 target efficiency is determined by the 20-nucleotide sequences of sgRNA and the PAM sequences adjacent to the target genome. It has been shown that more than three mismatches between the target sequence and the 20-nucleotide sgRNA can result in off-target effects.64 The off-target effect can possibly cause harmful events such as sequence mutation, deletion, rearrangement, immune response, and oncogene activation, which limits the application of the CRISPR/Cas-9 editing system for therapeutic purposes.65 To mitigate the possibility of CRISPR/Cas-9 off-target effect, several strategies have been developed, such as optimization of sgRNA, modification of Cas-9 nuclease, utilization of other Cas-variants, and the use of anti-CRISPR proteins.66 Selecting and designing an appropriate sgRNA for the targeted DNA sequence is an important first step to reduce the off-target effect.67 When designing sgRNA, strategies such as GC content, sgRNA length, and chemical modifications of sgRNA must be considered. Generally speaking, studies revealed that GC content of between 40% and 60%, truncated (short length of sgRNA), and incorporation of 2-O-methyl-3-phosphonoacetate in the sgRNA ribose-phosphate backbone are the preferred methods to increase genome editing efficiency of CRISPR/Cas-9.67,68 Modifying the Cas-9 protein to optimize its nuclease specificity is another way to reduce off-target effects. For instance, mutating either one of the catalytic residues of Cas-9 nuclease (HNH and RuvC) will convert the Cas-9 into nickase that could only generate a single-stranded break instead of a blunt cleavage.69 It has been reported that the use of the inactivated RuvC domain of Cas-9 with sgRNA can reduce the off-target effect by 100 to 1500 times.70 Moreover, the nuclease Cas-12a (previously known as Cpf1) is a type V CRISPR/Cas system that provides high genome editing efficiency.71 Unlike the CRISPR/Cas-9 system, CRISPR/Cas-12a can process pre-crRNA into mature crRNA without tracrRNA, thereby reducing the size of plasmid constructs. The Cas-12a protein recognizes a T-rich (5-TTTN) PAM sequence instead of 5-NGG and provides high accuracy at the target gene loci than Cas-9.69 Recently, the use of multicomponent Class I CRISPR proteins, such as CRISPR/Cas-3 and CRISPR/Cas-10 provides better genome editing efficiency than Cas-9.72 The Cas-3 is an ATP-dependent nuclease/helicase that can delete a large part of DNA from the target site without prominent off-target effect. For instance, the DMD gene were repaired by Cas-3-mediated system in induced pluripotent stem cell.73 The Cas-10 protein does not require the PAM sequence and can identify sequences even in the presence of point mutation.72 Anti-CRISPR (Acr) proteins are phage derived small proteins that inhibit the activity of CRISPR/Cas system. They are a recently discovered method to reduce off-target effects of CRISPR/Cas-9.74 From Acr proteins, AcrIIA4 specifically targets Cas-9 nuclease. AcrIIA4 mimics DNA and binds to the Cas-9 site, making impossible to perform further cleavage in area outside the target region.75 Furthermore, CRISPR/Cas-9 gene editing has been challenged by ethics and safety all over the world. Since the technology is still in its infancy and knowledge about the genome is limited, many scientists restrain that it still needs a lot of work to increase its accuracy and make sure that changes made in one part of the genome do not have unforeseen consequences, especially in the application towards human trials.52
AAVs, adeno-associated viral vectors; ABE, adenine base editor; Acr, anti-CRSPR; AVs, adeno-viral vectors; ATP, adenosine tri-phosphate; BCL11A, B-cell lymphoma 11 A; CAS-9, CRISPR-associated protein-9; CBE, cytidine base editor; CCR5, chemokine receptor type 5; CFTR, cystic fibrosis conductance transmembrane receptor; CRISPR, clustered regularly interspaced short palindromic repeat; CrRNA, CRISPR ribonucleic acid; DMD, Duchenne muscular dystrophy; DNA, deoxyribonucleic acid; DSBs, double-stranded breaks; HDR, homology-directed repair; LVs, lentivirus vectors; NHEJ, non-homologous end Jjining; PAM, protospacer adjacent motif; PD-1, programmed cell death-1; RNA, ribonucleic acid; TALENs, transcriptionactivator like effector nucleases; TRAC, T-cell receptor alpha; TRBC, T-cell receptor beta; TracrRNA, trans-activating CRISPR ribonucleic acid; ZFNs, zinc finger nucleases.
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Mechanism and Applications of CRISPR/Cas-9-Mediated Genome Editing
What is CRISPR gene editing, and how does it work? – The Conversation
Youve probably read stories about new research using the gene editing technique CRISPR, also called CRISPR/Cas9. The scientific world is captivated by this revolutionary technology, since it is easier, cheaper and more efficient than previous strategies for modifying DNA.
The term CRISPR/Cas9 stands for Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9. The names reflect important features identified during its discovery, but dont tell us much about how it works, as they were coined before anyone understood what it was.
CRISPR/Cas9 is a system found in bacteria and involved in immune defence. Bacteria use CRISPR/Cas9 to cut up the DNA of invading bacterial viruses that might otherwise kill them.
Today weve adapted this molecular machinery for an entirely different purpose to change any chosen letter(s) in an organisms DNA code.
We might want to correct a disease-causing error that was inherited or crept into our DNA when it replicated. Or, in some cases, we may want to enhance the genetic code of crops, livestock or perhaps even people.
So do we just snip the unwanted gene out and replace it with a good one?
Read more: Explainer: what is genome editing?
We first have to remember that animals and plants are composed of millions of cells, and each cell contains the same DNA. There is no point editing just one cell: we would have to edit the same gene in every single cell. Wed have to snip out millions of genes and paste in millions of new ones.
And not all cells are easy to get to how could we reach cells buried in our bones or deep within a brain?
A better approach is to start at the beginning and edit the genome while there is only one cell a very early embryo.
So, all we need is a giant microscope and a tiny pair of scissors. And that is basically what we use.
Cas9 is the technical name for the virus-destroying scissors that evolved in bacteria. The CRISPR part of the name comes from repeat DNA sequences that were part of a complex system telling the scissors which part of the DNA to cut.
In order to target our Cas9 scissors, we link them to an artificial guide that directs them to the matching segment of DNA.
Remember, DNA comes in two strands, with one strand fitting alongside the other. We make a guide with a code that will line up with only one part of our 3 billion base pair long genome its like a Google search. Its truly possible for our guide to comb through vast amounts of genetic material to find the one section it matches exactly. Then our scissors can make the cut in exactly the right place.
Once the Cas9 scissors cut the DNA just where we intend, the cell will try to repair the break using any available DNA it can find. So, we also inject the new gene we want to insert.
Read more: Now we can edit life itself, we need to ask how we should use such technology
You can use a microscope and a tiny needle to inject the CRISPR/Cas9 together with the guide and the donor DNA, the new gene. Or, you can punch holes in cells with electric currents and let these things just float in, use guns to shoot them in stuck-on tiny bullets, or introduce them encapsulated in bubbles of fat that fuse with the cell membrane and release their contents inside.
But how does the new gene find the right place to embed itself? Imagine you wanted to put in the last piece of a jigsaw puzzle with 3 billion pieces, and its inside a cell, filled with goop like a passionfruit.
What youd do is fabricate a jigsaw piece of precisely the right shape and inject it into the passionfruit. Then its just a case of jiggling around until eventually the piece finds its way to the correct part of the puzzle and slots into the only place it fits.
You dont need to be able to see the DNA in our genome through the microscope its too small. And you dont really have to jiggle either random diffusion (called Brownian motion) will always deliver the jigsaw piece to the place where it fits in the end.
First, the guide will jiggle along and find the right place for the scissors to cut, and then the new donor DNA will similarly line up where it fits and will be permanently stitched into the DNA strand via natural DNA repair mechanisms.
Recently, though, new CRISPR editing systems have been created that dont even require a cut through the DNA. In this case, the CRIPSR/Cas and guide system can deliver an enzyme to a particular gene and alter it, changing perhaps an A to a G or a C to a T, rather than cutting anything out or putting anything in.
Most experiments use mouse embryos or cells grown in petri dishes in artificial liquid designed to be like blood. Other researchers are modifying stem cells that may then be re-injected into patients to repopulate damaged organs.
Only a few labs around the world are actually working with early human embryos. This research is highly regulated and carefully watched. Others work on plant cells, as whole plants can be grown from a few cells.
As we learn more, the scope of what we can do with CRISPR/Cas9 will improve. We can do a lot, but every organism and every cell is different. Whats more, everything in the body is connected, so we must think about unexpected side effects and consider the ethics of changing genes. Most of all we, as a society, should discuss and agree what we wish to achieve.
Read more: Why we can trust scientists with the power of new gene-editing technology
Read the other articles in our precision medicine series here.
Read more here:
What is CRISPR gene editing, and how does it work? - The Conversation
What is CRISPR/Cas9? – PMC – National Center for Biotechnology Information
Arch Dis Child Educ Pract Ed. 2016 Aug; 101(4): 213215.
1HYMS Centre for Education Development (CED), Hull, York Medical School, University of York, York, UK
2Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK
3Department of Haematology, Oxford University NHS Foundation Trust, Churchill Hospital, Oxford, UK
4Academic Unit of Child Health, Sheffield Children's Hospital, Sheffield, UK
1HYMS Centre for Education Development (CED), Hull, York Medical School, University of York, York, UK
2Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK
3Department of Haematology, Oxford University NHS Foundation Trust, Churchill Hospital, Oxford, UK
4Academic Unit of Child Health, Sheffield Children's Hospital, Sheffield, UK
Received 2016 Jan 5; Revised 2016 Feb 18; Accepted 2016 Feb 19.
Keywords: CRISPR/cas9, gene editing, children, genome engineering
Clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 is a gene-editing technology causing a major upheaval in biomedical research. It makes it possible to correct errors in the genome and turn on or off genes in cells and organisms quickly, cheaply and with relative ease. It has a number of laboratory applications including rapid generation of cellular and animal models, functional genomic screens and live imaging of the cellular genome.1 It has already been demonstrated that it can be used to repair defective DNA in mice curing them of genetic disorders,2 and it has been reported that human embryos can be similarly modified.3 Other potential clinical applications include gene therapy, treating infectious diseases such as HIV and engineering autologous patient material to treat cancer and other diseases. In this review we will give an overview of CRISPR/Cas9 with an emphasis on how it may impact on the specialty of paediatrics. Although it is likely to have a significant effect on paediatrics through its impact in the laboratory, here we will concentrate on its potential clinical applications. We will also describe some of the difficulties and ethical controversies associated with this novel technology.
CRISPR/Cas9 is a gene-editing technology which involves two essential components: a guide RNA to match a desired target gene, and Cas9 (CRISPR-associated protein 9)an endonuclease which causes a double-stranded DNA break, allowing modifications to the genome (see ).
The CRISPR/Cas9 system.1 Clustered regularly interspaced palindromic repeats (CRISPR) refers to sequences in the bacterial genome. They afford protection against invading viruses, when combined with a series of CRISPR-associated (Cas) proteins. Cas9, one of the associated proteins, is an endonuclease that cuts both strands of DNA. Cas9 is directed to its target by a section of RNA. This can be synthesised as a single strand called a synthetic single guide RNA (sgRNA); the section of RNA which binds to the genomic DNA is 1820 nucleotides. In order to cut, a specific sequence of DNA of between 2 and 5 nucleotides (the exact sequence depends upon the bacteria which produces the Cas9) must lie at the 3 end of the guide RNA: this is called the protospacer adjacent motif (PAM). Repair after the DNA cut may occur via two pathways: non-homologous end joining, typically leading to a random insertion/deletion of DNA, or homology directed repair where a homologous piece of DNA is used as a repair template. It is the latter which allows precise genome editing: the homologous section of DNA with the required sequence change may be delivered with the Cas9 nuclease and sgRNA, theoretically allowing changes as precise as a single base-pair.
One of the most exciting applications of CRISPR/Cas9 is its potential use to treat genetic disorders caused by single gene mutations. Examples of such diseases include cystic fibrosis (CF), Duchenne's muscular dystrophy (DMD) and haemoglobinopathies. The approach so far has currently only been validated in preclinical models, but there is hope it can soon be translated to clinical practice.
Schwank et al used CRISPR/Cas9 to investigate the treatment of CF. Using adult intestinal stem cells obtained from two patients with CF, they successfully corrected the most common mutation causing CF in intestinal organoids. They demonstrated that once the mutation had been corrected, the function of the CF transmembrane conductor receptor (CFTR) was restored.4
Another disease in which CRISPR/Cas9 has been investigated is DMD. Tabebordbar et al recently used adeno-associated virus (AAV) delivery of CRISPR/Cas9 endonucleases to recover dystrophin expression in a mouse model of DMD, by deletion of the exon containing the original mutation. This produces a truncated, but still functional protein. Treated mice were shown to partially recover muscle functional deficiencies.5 Significantly, it was demonstrated that the dystrophin gene was edited in muscle stem cells which replenish mature muscle tissue. This is important to ensure any therapeutic effects of CRISPR/Cas9 do not fade over time. Two similar studies have described using the CRISPR/Cas9 system in vivo to increase expression of the dystrophin gene and improve muscle function in mouse models of DMD.67 Other studies have used CRISPR/Cas9 to target duplication of exons in the human dystrophin gene in vitro and have shown that this approach can lead to production of full-length dystrophin in the myotubules of an individual with DMD.8
CRISPR/Cas9 could also be used to treat haemoglobinopathies. Canver et al9 recently showed BCL11A enhancer disruption by CRISPR/Cas9 could induce fetal haemoglobin in both mice and primary human erythroblast cells. In the future such an approach could allow fetal haemoglobin to be expressed in patients with abnormal adult haemoglobin. This would represent a novel therapeutic strategy in patients with diseases such as sickle cell disease or thalassaemias. Knock-in of a fully functional -globin gene is much more challenging, which is the reason for this somewhat unusual approach.
Another potential clinical application of CRISPR/Cas9 is to treat infectious diseases, such as HIV. Although antiretroviral therapy provides an effective treatment for HIV, no cure currently exists due to permanent integration of the virus into the host genome. Hu et al showed the CRISPR/Cas9 system could be used to target HIV-1 genome activity. This inactivated HIV gene expression and replication in a variety of cells which can be latently infected with HIV, without any toxic effects. Furthermore, cells could also be immunised against HIV-1 infection. This is a potential therapeutic advance in overcoming the current problem of how to eliminate HIV from infected individuals. After further refinement, the authors suggest their findings may enable gene therapies or transplantation of genetically altered bone marrow stem cells or inducible pluripotent stem cells to eradicate HIV infection.10
There has been increasing interest in the possibility of using CRISPR/Cas9 to modify patient-derived T-cells and stem/progenitor cells which can then be reintroduced into patients to treat disease. This approach may overcome some of the issues associated with how to efficiently deliver gene editing to the right cells.
T-cell genome engineering has already shown success in treating haematological malignancies and has the potential to treat solid cancers, primary immune deficiencies and autoimmune diseases. Genetic manipulation of T-cells has previously been inefficient. However, Schumann et al recently reported a more effective approach in human CD4+ T-cells based on the CRISPR/Cas9 system. Their technique allowed experimental and therapeutic knock-out and knock-in editing of the genome in primary human T-cells. They demonstrated T-cells could be manipulated to prevent expression of the protein PD-1, which other work has shown may allow T-cells to target solid cancers.11
There is also interest in using CRISPR/Cas9-mediated genome editing in pluripotent stem cells or primary somatic stem cells to treat disease. For example Xie et al12 showed the mutation causing -thalassaemia could be corrected in human induced pluripotent stem cells ex vivo. They suggest that in the future such an approach could provide a source of cells for bone marrow transplantation to treat -thalassaemia and other similar monogenic diseases.
A number of challenges remain before the potential of CRISPR/Cas9 can be translated to effective treatments at the bedside. A particular issue is how to deliver gene editing to the right cells, especially if the treatment is to be delivered in vivo. To safely deliver Cas9-nuclease encoding genes and guide RNAs in vivo without any associated toxicity, a suitable vector is needed. AAV has previously been a favoured option for gene delivery.1 However, this delivery system may be too small to allow efficient transduction of the Cas9 gene.1 A smaller Cas9 gene could be used, but this has additional implications on efficacy.1 A number of other non-viral delivery systems are under investigation and this process requires further optimisation.
Another significant concern is the possibility of off-target effects on parts of the genome separate from the region being targeted. Unintentional edits of the genome could have profound long-term complications for patients, including malignancy. The concentration of the Cas9 nuclease enzyme and the length of time Cas9 is expressed are both important when limiting off-target activity.1 Although recent modifications in the nuclease have increased specificity, further work is required to minimise off-target effects and to establish the long-term safety of any treatment.
The therapeutic applications of CRISPR/Cas9 considered in this article have predominantly been directed at somatic cells. A particularly controversial issue surrounding CRISPR/Cas9 is that of gene editing in embryos. It has already been shown that CRISPR/Cas9 technology can alter the genome of human embryos3 which theoretically could prove useful in the preimplantation treatment of genetic diseases. However, any genetic modification of the germline would be permanent and the long-term consequences are unclear. Many oppose germline modification under any circumstances, reasoning that an eventual consequence could be non-therapeutic genetic enhancement.13 It is clear that the ethical boundaries, within which CRISPR/Cas9 can be used, remain to be fully determined.
Clinical bottom line
CRISPR/Cas9 technology has the potential to revolutionise the treatment of many paediatric conditions.
A number of practical and ethical challenges must be overcome before this potential can be realised at the bedside.
Contributors: DK conceived the idea for this article. All authors were involved in writing and reviewing the final manuscript.
Funding: AK is supported by a Wellcome Trust Fellowship (108785/Z/15/Z).
Competing interests: None declared.
Provenance and peer review: Not commissioned; externally peer reviewed.
See the article here:
What is CRISPR/Cas9? - PMC - National Center for Biotechnology Information
CRISPR, 10 Years On: Learning to Rewrite the Code of Life
Ten years ago this week, Jennifer Doudna and her colleagues published the results of a test-tube experiment on bacterial genes. When the study came out in the journal Science on June 28, 2012, it did not make headline news. In fact, over the next few weeks, it did not make any news at all.
Looking back, Dr. Doudna wondered if the oversight had something to do with the wonky title she and her colleagues had chosen for the study: A Programmable Dual RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity.
I suppose if I were writing the paper today, I would have chosen a different title, Dr. Doudna, a biochemist at the University of California, Berkeley, said in an interview.
Far from an esoteric finding, the discovery pointed to a new method for editing DNA, one that might even make it possible to change human genes.
I remember thinking very clearly, when we publish this paper, its like firing the starting gun at a race, she said.
In just a decade, CRISPR has become one of the most celebrated inventions in modern biology. It is swiftly changing how medical researchers study diseases: Cancer biologists are using the method to discover hidden vulnerabilities of tumor cells. Doctors are using CRISPR to edit genes that cause hereditary diseases.
The era of human gene editing isnt coming, said David Liu, a biologist at Harvard University. Its here.
But CRISPRs influence extends far beyond medicine. Evolutionary biologists are using the technology to study Neanderthal brains and to investigate how our ape ancestors lost their tails. Plant biologists have edited seeds to produce crops with new vitamins or with the ability to withstand diseases. Some of them may reach supermarket shelves in the next few years.
CRISPR has had such a quick impact that Dr. Doudna and her collaborator, Emmanuelle Charpentier of the Max Planck Unit for the Science of Pathogens in Berlin, won the 2020 Nobel Prize for chemistry. The award committee hailed their 2012 study as an epoch-making experiment.
Dr. Doudna recognized early on that CRISPR would pose a number of thorny ethical questions, and after a decade of its development, those questions are more urgent than ever.
Will the coming wave of CRISPR-altered crops feed the world and help poor farmers or only enrich agribusiness giants that invest in the technology? Will CRISPR-based medicine improve health for vulnerable people across the world, or come with a million-dollar price tag?
The most profound ethical question about CRISPR is how future generations might use the technology to alter human embryos. This notion was simply a thought experiment until 2018, when He Jiankui, a biophysicist in China, edited a gene in human embryos to confer resistance to H.I.V. Three of the modified embryos were implanted in women in the Chinese city of Shenzhen.
In 2019, a court sentenced Dr. He to prison for illegal medical practices. MIT Technology Review reported in April that he had recently been released. Little is known about the health of the three children, who are now toddlers.
Scientists dont know of anyone else who has followed Dr. Hes example yet. But as CRISPR continues to improve, editing human embryos may eventually become a safe and effective treatment for a variety of diseases.
Will it then become acceptable, or even routine, to repair disease-causing genes in an embryo in the lab? What if parents wanted to insert traits that they found more desirable like those related to height, eye color or intelligence?
Franoise Baylis, a bioethicist at Dalhousie University in Nova Scotia, worries that the public is still not ready to grapple with such questions.
Im skeptical about the depth of understanding about whats at issue there, she said. Theres a difference between making people better and making better people.
Dr. Doudna and Dr. Charpentier did not invent their gene-editing method from scratch. They borrowed their molecular tools from bacteria.
In the 1980s, microbiologists discovered puzzling stretches of DNA in bacteria, later called Clustered Regularly Interspaced Short Palindromic Repeats. Further research revealed that bacteria used these CRISPR sequences as weapons against invading viruses.
The bacteria turned these sequences into genetic material, called RNA, that could stick precisely to a short stretch of an invading viruss genes. These RNA molecules carry proteins with them that act like molecular scissors, slicing the viral genes and halting the infection.
As Dr. Doudna and Dr. Charpentier investigated CRISPR, they realized that the system might allow them to cut a sequence of DNA of their own choosing. All they needed to do was make a matching piece of RNA.
To test this revolutionary idea, they created a batch of identical pieces of DNA. They then crafted another batch of RNA molecules, programming all of them to home in on the same spot on the DNA. Finally, they mixed the DNA, the RNA and molecular scissors together in test tubes. They discovered that many of the DNA molecules had been cut at precisely the right spot.
For months Dr. Doudna oversaw a series of round-the-clock experiments to see if CRISPR might work not only in a test tube, but also in living cells. She pushed her team hard, suspecting that many other scientists were also on the chase. That hunch soon proved correct.
In January 2013, five teams of scientists published studies in which they successfully used CRISPR in living animal or human cells. Dr. Doudna did not win that race; the first two published papers came from two labs in Cambridge, Mass. one at the Broad Institute of M.I.T. and Harvard, and the other at Harvard.
Lukas Dow, a cancer biologist at Weill Cornell Medicine, vividly remembers learning about CRISPRs potential. Reading the papers, it looked amazing, he recalled.
Dr. Dow and his colleagues soon found that the method reliably snipped out pieces of DNA in human cancer cells.
It became a verb to drop, Dr. Dow said. A lot of people would say, Did you CRISPR that?
Cancer biologists began systematically altering every gene in cancer cells to see which ones mattered to the disease. Researchers at KSQ Therapeutics, also in Cambridge, used CRISPR to discover a gene that is essential for the growth of certain tumors, for example, and last year, they began a clinical trial of a drug that blocks the gene.
Caribou Biosciences, co-founded by Dr. Doudna, and CRISPR Therapeutics, co-founded by Dr. Charpentier, are both running clinical trials for CRISPR treatments that fight cancer in another way: by editing immune cells to more aggressively attack tumors.
Those companies and several others are also using CRISPR to try to reverse hereditary diseases. On June 12, researchers from CRISPR Therapeutics and Vertex, a Boston-based biotech firm, presented at a scientific meeting new results from their clinical trial involving 75 volunteers who had sickle-cell anemia or beta thalassemia. These diseases impair hemoglobin, a protein in red blood cells that carries oxygen.
The researchers took advantage of the fact that humans have more than one hemoglobin gene. One copy, called fetal hemoglobin, is typically active only in fetuses, shutting down within a few months after birth.
The researchers extracted immature blood cells from the bone marrow of the volunteers. They then used CRISPR to snip out the switch that would typically turn off the fetal hemoglobin gene. When the edited cells were returned to patients, they could develop into red blood cells rife with hemoglobin.
Speaking at a hematology conference, the researchers reported that out of 44 treated patients with beta thalassemia, 42 no longer needed regular blood transfusions. None of the 31 sickle cell patients experienced painful drops in oxygen that would have normally sent them to the hospital.
CRISPR Therapeutics and Vertex expect to ask government regulators by the end of year to approve the treatment.
Other companies are injecting CRISPR molecules directly into the body. Intellia Therapeutics, based in Cambridge and also co-founded by Dr. Doudna, has teamed up with Regeneron, based in Westchester County, N.Y., to begin a clinical trial to treat transthyretin amyloidosis, a rare disease in which a damaged liver protein becomes lethal as it builds up in the blood.
Doctors injected CRISPR molecules into the volunteers livers to shut down the defective gene. Speaking at a scientific conference last Friday, Intellia researchers reported that a single dose of the treatment produced a significant drop in the protein level in volunteers blood for as long as a year thus far.
The same technology that allows medical researchers to tinker with human cells is letting agricultural scientists alter crop genes. When the first wave of CRISPR studies came out, Catherine Feuillet, an expert on wheat, who was then at the French National Institute for Agricultural Research, immediately saw its potential for her own work.
I said, Oh my God, we have a tool, she said. We can put breeding on steroids.
At Inari Agriculture, a company in Cambridge, Dr. Feuillet is overseeing efforts to use CRISPR to make breeds of soybeans and other crops that use less water and fertilizer. Outside of the United States, British researchers have used CRISPR to breed a tomato that can produce vitamin D.
Kevin Pixley, a plant scientist at the International Maize and Wheat Improvement Center in Mexico City, said that CRISPR is important to plant breeding not only because its powerful, but because its relatively cheap. Even small labs can create disease-resistant cassavas or drought-resistant bananas, which could benefit poor nations but would not interest companies looking for hefty financial returns.
Because of CRISPRs use for so many different industries, its patent has been the subject of a long-running dispute. Groups led by the Broad Institute and the University of California both filed patents for the original version of gene editing based on CRISPR-Cas9 in living cells. The Broad Institute won a patent in 2014, and the University of California responded with a court challenge.
In February of this year, the U.S. Patent Trial and Appeal Board issued what is most likely the final word on this dispute. They ruled in favor of the Broad Institute.
Jacob Sherkow, an expert on biotech patents at the University of Illinois College of Law, predicted that companies that have licensed the CRISPR technology from the University of California will need to honor the Broad Institute patent.
The big-ticket CRISPR companies, the ones that are farthest along in clinical trials, are almost certainly going to need to write the Broad Institute a really big check, he said.
The original CRISPR system, known as CRISPR-Cas9, leaves plenty of room for improvement. The molecules are good at snipping out DNA, but theyre not as good at inserting new pieces in their place. Sometimes CRISPR-Cas9 misses its target, cutting DNA in the wrong place. And even when the molecules do their jobs correctly, cells can make mistakes as they repair the loose ends of DNA left behind.
A number of scientists have invented new versions of CRISPR that overcome some of these shortcomings. At Harvard, for example, Dr. Liu and his colleagues have used CRISPR to make a nick in one of DNAs two strands, rather than breaking them entirely. This process, known as base editing, lets them precisely change a single genetic letter of DNA with much less risk of genetic damage.
Dr. Liu has co-founded a company called Beam Therapeutics to create base-editing drugs. Later this year, the company will test its first drug on people with sickle cell anemia.
Dr. Liu and his colleagues have also attached CRISPR molecules to a protein that viruses use to insert their genes into their hosts DNA. This new method, called prime editing, could enable CRISPR to alter longer stretches of genetic material.
Prime editors are kind of like DNA word processors, Dr. Liu said. They actually perform a search and replace function on DNA.
Rodolphe Barrangou, a CRISPR expert at North Carolina State University and a founder of Intellia Therapeutics, predicted that prime editing would eventually become a part of the standard CRISPR toolbox. But for now, he said, the technique was still too complex to become widely used. Its not quite ready for prime time, pun intended, he said.
Advances like prime editing didnt yet exist in 2018, when Dr. He set out to edit human embryos in Shenzen. He used the standard CRISPR-Cas9 system that Dr. Doudna and others had developed years before.
Dr. He hoped to endow babies with resistance to H.I.V. by snipping a piece of a gene called CCR5 from the DNA of embryos. People who naturally carry the same mutation rarely get infected by H.I.V.
In November 2018, Dr. He announced that a pair of twin girlshad been born with his gene edits. The announcement took many scientists like Dr. Doudna by surprise, and they roundly condemned him for putting the health of the babies in jeopardy with untested procedures.
Dr. Baylis of Dalhousie University criticized Dr. He for the way he reportedly presented the procedure to the parents, downplaying the radical experiment they were about to undertake. You could not get an informed consent, unless you were saying, This is pie in the sky. Nobodys ever done it, she said.
In the nearly four years since Dr. Hes announcement, scientists have continued to use CRISPR on human embryos. But they have studied embryos only when theyre tiny clumps of cells to find clues about the earliest stages of development. These studies could potentially lead to new treatments for infertility.
Bieke Bekaert, a graduate student in reproductive biology at Ghent University in Belgium, said that CRISPR remains challenging to use in human embryos. Breaking DNA in these cells can lead to drastic rearrangements in the chromosomes. Its more difficult than we thought, said Ms. Bekaert, the lead author of a recent review of the subject. We dont really know what is happening.
Still, Ms. Bekaert held out hope that prime editing and other improvements on CRISPR could allow scientists to make reliably precise changes to human embryos. Five years is way too early, but I think in my lifetime it may happen, she said.
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CRISPR, 10 Years On: Learning to Rewrite the Code of Life
What Is CRISPR, and Why Is It So Important? – Scientific American
CRISPR is the basis of a revolutionary gene editing system. One day, it could make it possible to do everything from resurrect extinct species to develop cures for chronic disease.
Its built on a natural adaptation found in the DNA of bacteria and single-celled organisms.
CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats.
Theyre really just bits of genetic code with a specific, recognizable format. They contain a sequence that shows up over and over again, though its often reversed each time.
Thats what makes it "Palindromic: palindromes are words that can be read the same backwards as forwards. Palindromes are common in DNA. Some serve as backups for damage to our genetic code, while others are common in cancer mutations.
With CRISPR, a group of enzymes recognize certain repeats, and break the DNA there to insert important information in the middle. These insertions are called spacers, and they contain the genetic code of different viruses that have invaded in the past.
Such previous invasions served a very important evolutionary purpose: immunizing against foreign threats.
Researchers first discovered CRISPR in E. Coli in the 1980s. When E. coli survives viral attacks, it incorporates some of the virus DNA into its own genetic code.
E. Coli isnt unique in using this strategy. Between the 1980s and 2000, scientists found that lots of bacteria and single-celled organisms incorporate viral DNA this way.
Cells use these sequences as templates for transcribing complementary strands of RNA.
When viruses matching the template sequence enter the cell, the complementary RNA binds to them, and directs a series of CRISPR-associated enzymes, or Cas enzymes, to attackcutting invader DNA at the binding site. That neutralizes the viral threat.
The CRISPR-Cas system is incredibly effective. Its also easy to manipulate, letting us alter a cells genetic code however we want.
In 2012, French microbiologist Emmanuelle Charpentier and American biochemist Jennifer Doudna discovered that Cas enzymesspecifically Cas9can be reprogrammed to cut nearly any part of the genome, using RNA sequences made in a lab. Those guide RNA molecules tell Cas9 where to cut DNA in a cell.
For their discovery, Charpentier and Doudna won the Nobel Prize in Chemistry in 2020.
And the use of CRISPR has taken off in science since their breakthrough.
But scientists are still far from realizing CRISPRs potential.
Cas9 is great at suppressing or knocking out unwanted genes. But for most medical purposes, its not enough to cut unwanted DNA out. Scientists need to control how the DNA repairs itself.
Left to their own devices, cells tend to repair broken DNA using a method that introduces lots of random errors. Researchers can provide cells with templates to guide the repair process, but theyre still working on making that more reliable.
Researchers have found lots of applications for CRISPR in animals, like making disease-resistant chickens and pigs, and mosquitos that cant bite or lay eggs. But theyve got many projects underway, like making disease-resistant cropsincluding wine grapes. More ambitiously, theyre working to genetically alter pigs so their organs could be transplanted into humans. And bring extinct species such as the passenger pigeon back to life, by tweaking the genomes of similar birds.
When it comes to the human genome, though, scientists have been more hesitant. Editing our own DNA could easily end up causing more problems than it solves.
While Cas9 reliably cuts DNA where we want it to, recent experiments have shown it can also affect genes far off-target. And even if we could get it to work reliably, many experts have flagged ethical concerns about using the technology for eugenics and designer babies. If parents can one day pay scientists to edit their babies DNA, making them stronger and smarter, CRISPR could make the world even more unequal and prejudiced.
In 2018, Chinese researcher HEH JEE'-an-qway claimed hed used CRISPR to make HIV-resistant children. Whether or not he succeeded, his work violated Chinas National Health Commission rules, and he was sentenced to three years in prison.
Using CRISPR on babies is widely illegal. But there are some cases where using CRISPR on humans may be worth the risk.
In 2020, American researchers began the first clinical trials injecting CRISPR directly into living humans, aiming to repair a genetic mutation that causes blindness.
Many researchers hope CRISPR-based therapies could eventually cure hereditary diseases; theyve already seen promising results in various animal studies. Though given the risks of editing the human genome, were still a ways off from widespread use of CRISPR in medicine.
CRISPR has given science a tool to reliably tinker with the code of life. But the question remains: can we do so safely and ethically, while avoiding the unintended consequences of such power?
Read more from the original source:
What Is CRISPR, and Why Is It So Important? - Scientific American
What is CRISPR and why is it controversial? | CNN
CNN
Two women have won the Nobel prize in chemistry for the development of a revolutionary gene editing tool thats been described as rewriting the code of life.
The technique discovered by Emmanuelle Charpentier, the director at the Max Planck Institute for Infection Biology, and Jennifer A. Doudna, a biochemist at the University of California Berkeley, is known as CRISPR/Cas9.
It hit the headlines in 2018 when a Chinese scientist used the technology to create the first gene-edited babies, shocking the world and sparking a highly charged ethical debate about its use.
What is CRISPR (pronounced crisper) and why has it been controversial?
DNA is like the instruction manual for life on our planet, and CRISPR/Cas9 can target sites in genetic material.
This allows scientists to change it by knocking out a particular gene or inserting new genetic material at a predetermined site in our DNA.
Cas9, a type of modified protein, acts like a pair of scissors that can snip parts of DNA strands. CRISPR stands for clustered regularly interspaced short palindromic repeats a repeated DNA sequence in genomes.
Doudna and Charpentier showed that CRISPR works like a pair of scissors that can be targeted to cut specific DNA sequences, said Andrew Holland, an assistant professor in the Department of Molecular Biology and Genetics at Johns Hopkins School of Medicine. After cutting, the repair of the DNA code enables it to be altered. This has allowed scientists to change the DNA code in a targeted way to help understand and treat genetic disease, he told CNN via email.
The technology has worked in pretty much every organism that it has been used on, including plants, microbes and humans.
What the system does is that it can recognize (a) certain specific gene in the genome of ourselves and correct mutations, do some copy pasting, do some editing like we edit a text. The system can edit the genome and change the properties of the genes, Charpentier said in 2016 when she was interviewed by CNN.
It is already having a major impact on biomedical research, clinical medicine and agriculture. For example, its been used to grow rice that accumulates lower levels of potentially toxic heavy metals and create livestock with more desirable traits.
It was used for the first time in humans in 2016 and a trial is underway in the United States to use the experimental technology to treat a dozen patients with sickle cell disease, a group of inherited blood disorders.
Related technologies may be able to potentially correct up to 89% of genetic defects, scientists have said.
Its not an exaggeration to say that the technology that arose from Doudna and Charpentiers discoveries has revolutionized the field, Jessica Downs, the deputy head of the Division of Cancer Biology at the Institute of Cancer Research in the UK, told the Science Media Centre in London.
We adopted the technology in our lab to investigate molecular changes that lead to cancer development. Its been transformative in terms of what we can achieve, but there is also great potential for using this technology in the clinic. And on a more personal level, its inspiring and uplifting to see two women honored for their work in this way.
While it has immense potential to transform our lives, the technology has raised many ethical questions.
Chinese scientist He Jiankui was jailed for three years in 2019 after announcing that twin girls had been born with modified DNA to make them resistant to HIV, which he had managed using the gene-editing tool CRISPR/Cas9 before birth.
An associate professor at the Southern University of Science and Technology in Shenzhen at the time, he said that he was proud of the achievement. But he was condemned by many of his peers, with the experiment labeled monstrous, unethical and a huge blow to the reputation of Chinese biomedical research.
Claes Gustafsson, secretary of the Nobel committee in chemistry and a professor of biochemistry and biophysics at Stockholm University, said that with every really powerful technology, in life sciences or elsewhere, theres a possibility of misuse.
Clearly this Chinese researcher was way out of line in applying it in this particular way, he told CNN.
Everyone has agreed that it cannot be used for germline engineering. You cant make heritable changes to human DNA. That is far too uncertain at this point, added Gustafsson. There are specific genetic diseases you can think of curing for the individual but not in a heritable way.
Scientists have called for a moratorium on human germline editing, while efforts are being made to better regulate use of the technology. An international commission said in September it was too early for gene-edited human embryos to be used to create a pregnancy.
Doudna has expressed deep concern about Hes work, telling CNN it was not medically necessary and there was no way to defend using an experimental technology when there were established ways of avoiding HIV transmission.
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What is CRISPR and why is it controversial? | CNN
CRISPR | Description, Technology, Uses, & Ethical Concerns
CRISPR, in full clustered regularly interspaced short palindromic repeats, short palindromic repeating sequences of DNA, found in most bacterial genomes, that are interrupted by so-called spacer elements, or spacerssequences of genetic code derived from the genomes of previously encountered bacterial pathogens. CRISPR elements are found naturally in many bacteria and archaea, where they provide a sort of genetic memory, enabling the cells to efficiently detect and destroy pathogens, particularly viruses known as bacteriophages.
As a naturally occurring adaptive defense system, CRISPR functions by destroying nucleic acids from pathogens that invade the cell. The effectiveness and efficiency of CRISPR immunity is directly linked to the presence of spacer elements. Spacer elements essentially are recognition sequences that match sequences inpathogen genomes; as spacers from newly encountered pathogens are added to the bacterial genome, the cell gains the ability to recognize those pathogens on repeat encounters. Most new spacer elements are inserted only at one end of the CRISPR region; hence, across the length of the CRISPR region exists a record of pathogens that have been encountered by the cell and its ancestors over time. Less often, spacers are added in other places in a process called ectopic integration.
The CRISPR system works by producing small guide RNA sequences that correspond to specific DNA targets. Guide RNAs, generated via transcription of the CRISPR region,include hairpin formations, derived from the palindromic repeats, that are linked to sequencesderivedfrom the spacer elements. When guide RNAs bind to their DNA targets, an RNA-DNA heteroduplex is formed. The heteroduplex binds to a nuclease called CRISPR-associated (Cas), which catalyzes the cleavage of double-stranded DNA at a position near the junction of the target-specific sequence and the palindromic repeat in the guide RNA. In this way, the nuclease destroys invading pathogenic genomes.
CRISPR interacts with multiple Cas proteins as part of the defense response, and thus there are distinct CRISPR-Cas defense systems. The three major systems are type I, type II, and type III. The type I system is defined by the presence of Cas3 protein. Cas3 forms part of the so-called CRISPR-associated complex for antiviral defense (or Cascade) -like complex, which binds a guide RNA and identifies the target sequence for destruction. The type II system is based on the presence of several proteins, namely Cas1, Cas2, Cas9, and, in some cases, Cas4. The Cas9 protein is considered a signature element of the type II system, owing to its essential role in facilitating cellular adaptation to new pathogens and to its participation in RNA processing and cleaving of target DNA. The signature protein of the type III system is Cas10. The type III system differs from its two counterparts in that, in addition to targeting DNA, it identifies RNA targets.
The high sequence specificity of the CRISPR system has drawn significant interest in the field of gene editing. The functional precision of CRISPR allows researchers to remove and insert DNA in desired locations within a genome, making it possible to correct genetic defects in animals and to modify DNA sequences in embryonicstem cells. These types of sequence corrections and alterations are possible because RNA-DNA heteroduplexes are stable and because designing an RNA sequence that binds specifically to a unique target DNA sequence is based simply on the Watson-Crickbase-pairingrules (adenine binds to thymine [or uracil in RNA], and cytosine binds to guanine).
The possibility of using CRISPR as a gene-editing technology was recognized in 2012 by American scientistJennifer Doudna, French scientistEmmanuelle Charpentier, and colleagues. These researchers discovered that guide RNAs produced by CRISPR bind to nucleases, which then target particular DNA sequences, and that such RNAs could be modified to bind to a desired sequence. The researchers found that the type II CRISPR-Cas9 system was especially versatile for correcting or altering desired target sequences. Doudna and Charpentier shared the 2020 Nobel Prize in Chemistry for their work.
In 2015 American scientistFeng Zhang and colleagues developed a new version of CRISPR technology using the microbial nuclease CRISPR from Prevotella and Francisella 1 (Cpf1) in place of Cas9. Unlike Cas9, Cpf1 requires only a single CRISPR guide RNA for specificity and introduces staggered (rather than blunt) cuts in double-stranded DNA, which in certain instances can give greater control over the modification of target DNA sequences. Zhang and colleagues subsequently developed multiple other CRISPR gene-editing tools, including CRISPR-Cas13 systems, which target RNA.
CRISPR gene-editing technology has a wide array of research and medical applications. For example, in the laboratory, CRISPR systems can be used to modify genes in bacteria and in animal and plant models, enabling researchers to gain new understanding of the effects of genetic modification. Although preexisting genetic engineering technologies have allowed researchers to investigate various types of genetic modifications and alterations for decades, CRISPR is less costly, more efficient, and more reliable.
In addition, different CRISPR-based therapies are being explored in clinical trials for the treatment of certain human diseases. Some examples include novel treatments for diabetes; for sickle cell disease; for cancers of blood-forming tissues, such as multiple myeloma, leukemia,and lymphoma; for chronic infectious diseases, such as AIDS; and for a form of inherited impairment in vision known as Leber congenital amaurosis. Investigations of CRISPR-based therapies in humans are helping to shed light on how DNA alterations induced by CRISPR enzymes affect cells, on how the human immune system responds to CRISPR-derived interventions, and on risks associated with unwanted off-target alterations in DNA.
The ability to easily and accurately edit genes using CRISPR technology has raised significant ethical issues. In particular, CRISPR can be used to modify DNA sequences in embryonicstem cells, such as in germ-line (spermandegg) genome modification in humans. Critics point out that this ability, applied to embryos in the womb, may be used to improve traits such as intelligence, appearance, and athletic ability, potentially introducing permanent changes in human DNA. The generation of such designer babies sparked debates about the morality of tampering with human development and the ethics of who would have access to the technology. The worlds first gene-edited human babies were born in late 2018 in China; the infants, twin girls, carried an edited gene that reduced the risk of HIV infection.
Following the birth of gene-edited babies, some medical and bioethics researchers, including Charpentier, called for a moratorium on editing human genes in eggs, sperm, or embryos. They contend that because there remain many unknowns about the technology, scientists may unintentionally introduce as many genetic errors as they attempt to fix. Nonetheless, critics argue that CRISPR technology is a remarkable achievement with significant potential to improve human health, although under rigorously controlled conditions.
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CRISPR | Description, Technology, Uses, & Ethical Concerns
In vivo CRISPR screening reveals nutrient signaling processes … – PubMed
Figure 4.. Terminal differentiation of T EFF cells is dependent upon Pofut1 and associated with
(A) Differentially expressed genes in sgPofut1- compared to sgNTC-transduced P14 cells at day 7.5 post-infection (p.i.). Upregulated (orange) or downregulated (blue) transcripts [false discovery rate (FDR) < 0.05] are highlighted. Selective MP- and TE-associated genes are labelled. (B) Enrichment plots of cell cycle-related signatures. NES, normalized enrichment score. (C) Flow cytometry (left) and quantification (right) of BrdU incorporation. (D) UMAP plot of published scRNA-seq dataset of P14 cells at day 8 p.i. (Chen et al., 2019). Each dot corresponds to an individual cell. The number and frequency of cells in each of the color-coded clusters (clusters 13) are indi cated. (E) Violin plots of Klrg1, Cxcr3 or Il7r expression in clusters 13 from (D). (F) Gating str ategy (left) and quantification (right) of the proportions of TE (KLRG1hiCXCR3loCD127lo), MP (KLRG1loCXCR3hiCD127hi) and TINT (CXCR3hiCD127lo) cells among WT P14 cells. (G) PCA plot of TE, MP and TINT cells [gating strategy in (F)] at day 7.5 p.i., with the percentage of variance shown. (H) Quantification of the relative frequency of BrdU+ cells in MP and TINT cells compared to TE cells. (I) Diagram of the in vivo differentiation assay (left), flow cytometry of KLRG1 versus CXCR3 expression (middle), and quantification of TINT, TE and CXCR3hiCD127hi cells (right). Only representative plots of KLRG1 versus CXCR3 are shown (TE population is largely defined by KLRG1hiCXCR3lo cells, which constitute ~ 95% of TE cells). (J) Quantification of TE, MP and TINT cells in the indicated P14 cells. (K) UMAP plot of Pofut1-dependent signature [downregulated genes as identified in (A)] in published scRNA-seq dataset from (D) (Chen et al., 2019). (L) UMAP plot of scRNA-seq data from sgNTC- (in black, left) and sgPofut1- (in red, right) transduced P14 cells (from dual-color transfer system) at day 7 p.i. Gray shadow indicates location of all cells; the number of analyzed cells in each group is indicated. (M) UMAP plot of the expression of Klrg1 (left), Cxcr3 (middle) and Il7r (right) in scRNA-seq data described in (L). (N) Flow cytometry of KLRG1 versus CXCR3 expression (left) and quantification (right) of TE cells in the in vivo differentiation assay similar as (I), except for the use of both wild-type and Pofut1-null TINT groups as the pretransfer cells. Data are from one (A, B, D, E, G, and KM), representative of two (C, H, and N), or compiled from at least two (I, J, and N) independent experiments, with 4 (A, C, G, H, and I), 17 (F), 11 (J), or 3 (L and N) biological replicates per group. *P < 0.05, **P < 0.01, and ***P < 0.001; NS, not significant; two-tailed paired Students t-test (C), two-tailed unpaired Students t-test (I, J, and N), or one-way analysis of variance (ANOVA) (F and H). Data are presented as mean s.e.m. See also Figures S4S6 and Tables S3S6.
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In vivo CRISPR screening reveals nutrient signaling processes ... - PubMed
What is CRISPR? | New Scientist
CRISPR is a technology that can be used to edit genes and, as such, will likely change the world.
The essence of CRISPR is simple: its a way of finding a specific bit of DNA inside a cell. After that,the next step in CRISPR gene editing is usually to alter that piece of DNA. However, CRISPR has also been adapted to do other things too, such as turning genes on or off without altering their sequence.
There were ways to edit the genomes of some plants and animals before the CRISPR method was unveiled in 2012 but it took years and cost hundreds of thousands of dollars. CRISPR has made it cheap and easy.
CRISPR is already widely used for scientific research, and in the not too distant future many ofthe plantsandanimalsinour farms, gardens or homes may have been altered with CRISPR. In fact, some people already are eating CRISPRed food.
CRISPR technology also has the potential to transform medicine, enabling us to not onlytreatbut alsopreventmany diseases. We may even decide to use it tochange the genomesofour children. An attempt to do this in Chinahasbeen condemned as premature and unethical, but some think it could benefit children in the future.
CRISPR is being used for all kinds of other purposes too, from fingerprinting cells andlogging what happensinside them todirecting evolutionand creatinggene drives.
The key to CRISPR is the many flavours of Cas proteins found in bacteria, where they help defend against viruses. The Cas9 protein is the most widely used by scientists. This protein can easily be programmed to find and bind to almost any desired target sequence, simply by giving it a piece of RNA to guide it in its search.
When the CRISPR Cas9 protein is added to a cell along with a piece of guide RNA, the Cas9 protein hooks up with the guide RNA and then moves along the strands of DNA until it finds and binds to a 20-DNA-letter long sequence that matches part of the guide RNA sequence. Thats impressive, given thatthe DNA packed into each of our cellshas six billion letters and is two metres long.
What happens next can vary. The standard Cas9 protein cuts the DNA at the target. When the cut is repaired, mutations are introduced that usually disable a gene. This is by far the most common use of CRISPR. Its called genome editing or gene editing but usually the results arenot as preciseas that term implies.
CRISPR can also be used tomake precise changessuch as replacing faulty genes true genome editing but this is far more difficult.
Customised Cas proteins have been created that do not cut DNA or alter it in any way,but merely turn genes on or off: CRISPRa and CRISPRi respectively. Yet others, called base editors,change one letter of the DNA code to another.
So why do we call it CRISPR? Cas proteins are used by bacteria to destroy viral DNA. They add bits of viral DNA to their own genome to guide the Cas proteins, and the odd patterns of these bits of DNA are what gave CRISPR its name: clustered regularly interspaced short palindromic repeats. Michael Le Page
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What is CRISPR? | New Scientist
CRISPR-Cas9 Structures and Mechanisms – PubMed
Many bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems employ the dual RNA-guided DNA endonuclease Cas9 to defend against invading phages and conjugative plasmids by introducing site-specific double-stranded breaks in target DNA. Target recognition strictly requires the presence of a short protospacer adjacent motif (PAM) flanking the target site, and subsequent R-loop formation and strand scission are driven by complementary base pairing between the guide RNA and target DNA, Cas9-DNA interactions, and associated conformational changes. The use of CRISPR-Cas9 as an RNA-programmable DNA targeting and editing platform is simplified by a synthetic single-guide RNA (sgRNA) mimicking the natural dual trans-activating CRISPR RNA (tracrRNA)-CRISPR RNA (crRNA) structure. This review aims to provide an in-depth mechanistic and structural understanding of Cas9-mediated RNA-guided DNA targeting and cleavage. Molecular insights from biochemical and structural studies provide a framework for rational engineering aimed at altering catalytic function, guide RNA specificity, and PAM requirements and reducing off-target activity for the development of Cas9-based therapies against genetic diseases.
Keywords: CRISPR; Cas9; genome engineering; mechanism; off-target; structure.
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CRISPR-Cas9 Structures and Mechanisms - PubMed
A CRISPR cure for HIV? Gene-editing technology may be able stop viral replication in its tracks and wipe out infections – Genetic Literacy Project
In July, an HIV-positive man became the first volunteer in a clinical trial aimed at using Crispr gene editing to snip the AIDS-causing virus out of his cells. For an hour, he was hooked up to an IV bag that pumped the experimental treatment directly into his bloodstream. The one-time infusion is designed to carry the gene-editing tools to the mans infected cells to clear the virus.
Later this month, the volunteer will stop taking the antiretroviral drugs hes been on to keep the virus at undetectable levels. Then, investigators will wait 12 weeks to see if the virus rebounds. If not, theyll consider the experiment a success. What were trying to do is return the cell to a near-normal state, says Daniel Dornbusch, CEO of Excision BioTherapeutics, the San Francisco-based biotech company thats running the trial.
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Crispr isbeing used in several other studiesto treat a handful of conditions that arise from genetic mutations. In those cases, scientists are using Crispr to edit peoples own cells. But for the HIV trial, Excision researchers are turning the gene-editing tool against thevirus. The Crispr infusion contains gene-editing molecules that target two regions in the HIV genome important for viral replication. The virus can only reproduce if its fully intact, so Crispr disrupts that process by cutting out chunks of the genome.
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Editas Medicine Presents Preclinical Data on EDIT-103 for Rhodopsin-associated Autosomal Dominant Retinitis Pigmentosa at the European Society of Gene…
Studies in non-human primates demonstrated nearly 100% gene editing and knockout of endogenous RHO gene and more than 30% replacement protein levels using a dual vector AAV approach
Treated eyes showed morphological and functional photoreceptor preservation
EDIT-103 advancing towards IND-enabling studies
CAMBRIDGE, Mass., Oct. 13, 2022 (GLOBE NEWSWIRE) -- Editas Medicine, Inc. (Nasdaq: EDIT), a leading genome editing company, today announced ex vivo and in vivo preclinical data supporting its experimental medicine EDIT-103 for the treatment of rhodopsin-associated autosomal dominant retinitis pigmentosa (RHO-adRP). The Company reported these data in an oral presentation today at the European Society of Gene and Cell Therapy 29th Annual Meeting in Edinburgh, Scotland, UK.
EDIT-103 is a mutation-independent CRISPR/Cas9-based, dual AAV5 vectors knockout and replace (KO&R) therapy to treat RHO-adRP. This approach has the potential to treat any of over 150 dominant gain-of-function rhodopsin mutations that cause RHO-adRP with a one-time subretinal administration.
These promising preclinical data demonstrate the potential of EDIT-103 to efficiently remove the defective RHO gene responsible for RHO-adRP while replacing it with an RHO gene capable of producing sufficient levels of RHO to preserve photoreceptor structure and functions. The program is progressing towards the clinic, said Mark S. Shearman, Ph.D., Executive Vice President and Chief Scientific Officer, Editas Medicine. EDIT-103 uses a dual AAV gene editing approach, and also provides initial proof of concept for the treatment of other autosomal dominant disease indications where a gain of negative function needs to be corrected.
Key findings include:
Full details of the Editas Medicine presentations can be accessed in the Posters & Presentations section on the Companys website.
About EDIT-103EDIT-103 is a CRISPR/Cas9-based experimental medicine in preclinical development for the treatment of rhodopsin-associated autosomal dominant retinitis pigmentosa (RHO-adRP), a progressive form of retinal degeneration. EDIT-103 is administered via subretinal injection and uses two adeno-associated virus (AAV) vectors to knockout and replace mutations in the rhodopsin gene to preserve photoreceptor function. This approach can potentially address more than 150 gene mutations that cause RHO-adRP.
AboutEditas MedicineAs a leading genome editing company, Editas Medicine is focused on translating the power and potential of the CRISPR/Cas9 and CRISPR/Cas12a genome editing systems into a robust pipeline of treatments for people living with serious diseases around the world. Editas Medicine aims to discover, develop, manufacture, and commercialize transformative, durable, precision genomic medicines for a broad class of diseases. Editas Medicine is the exclusive licensee of Harvard and Broad Institutes Cas9 patent estates and Broad Institutes Cas12a patent estate for human medicines. For the latest information and scientific presentations, please visit http://www.editasmedicine.com.
Forward-Looking StatementsThis press release contains forward-looking statements and information within the meaning of The Private Securities Litigation Reform Act of 1995. The words "anticipate," "believe," "continue," "could," "estimate," "expect," "intend," "may," "plan," "potential," "predict," "project," "target," "should," "would," and similar expressions are intended to identify forward-looking statements, although not all forward-looking statements contain these identifying words. The Company may not actually achieve the plans, intentions, or expectations disclosed in these forward-looking statements, and you should not place undue reliance on these forward-looking statements. Actual results or events could differ materially from the plans, intentions and expectations disclosed in these forward-looking statements as a result of various factors, including: uncertainties inherent in the initiation and completion of preclinical studies and clinical trials and clinical development of the Companys product candidates; availability and timing of results from preclinical studies and clinical trials; whether interim results from a clinical trial will be predictive of the final results of the trial or the results of future trials; expectations for regulatory approvals to conduct trials or to market products and availability of funding sufficient for the Companys foreseeable and unforeseeable operating expenses and capital expenditure requirements. These and other risks are described in greater detail under the caption Risk Factors included in the Companys most recent Annual Report on Form 10-K, which is on file with theSecurities and Exchange Commission, as updated by the Companys subsequent filings with theSecurities and Exchange Commission, and in other filings that the Company may make with theSecurities and Exchange Commissionin the future. Any forward-looking statements contained in this press release speak only as of the date hereof, and the Company expressly disclaims any obligation to update any forward-looking statements, whether because of new information, future events or otherwise.
More Foods Will Be Gene-Edited Than You Think – The Epoch Times
Gene editing has long been primarily used for research, treatment, and disease prevention. Currently, this technology is increasingly being applied to modify agricultural products to create more perfect species. More and more genetically edited foods are appearing on the market, including high-nutrient tomatoes and zero-trans-fat soybean oil.
Some argue that gene-edited foods are safer than genetically modified (GM) foods (pdf). The U.S. Department of Agriculture (USDA) specified in 2018 that most genetically edited foods do not need to be regulated. However, are these foods, which will increasingly appear on the table, really risk-free?
In September 2021, the first gene-edited foodSicilian Rouge tomatoesmade with CRISPR-Cas9 technology were officially on sale.
This gene-edited tomato contains high levels of gamma-aminobutyric acid (GABA), which helps lower blood pressure and aids relaxation.
Japanese researchers remove a gene from the genome of the common tomato. After the gene is removed, the activity of an enzyme in tomatoes increases, promoting the production of GABA. The GABA content in this tomato is four to five times higher than that of a regular tomato.
Warren H. J. Kuo, an emeritus professor of the Department of Agronomy at National Taiwan University, explains that both gene editing and transgenic organisms are genetic modification, also known as genetic engineering.
The earliest technique was genetic modification, that is, transgenicin which a plant or animal is being inserted a gene from another species, such as a specific bacterial gene. The purpose of artificially modifying plants and animals is to improve their resistance against diseases and droughts, promote growth rates, increase yields, or improve nutrient content. However, the finished product will exhibit the foreign species genes.
Kuo says that transgenic modification is genetic modification 1.0, while gene editing is genetic modification 2.0. Gene editing is directly modifies the genes of the organism itself, so most of them do not exhibit foreign genes. However, the most common gene editing technique, CRISPR-Cas9, introduces foreign genes as the editing tool, and then removes the transplanted foreign genes.
While gene-edited tomatoes were on the market, Japan also approved two types of fish genetically edited with CRISPRtiger pufferfish and red seabream. These fish are genetically edited to accelerate muscle growth. Among them, the gene-edited tiger pufferfish weighs nearly twice that of the ordinary species.
Back in 2019, the United States had used another earlier gene-editing technique to create soybean oil with zero trans fat and introduced it into the market.
Gene-edited foods which have also been approved for sale worldwide by now include soybeans, corn, mushrooms, canola, and rice.
The number of genetically edited foods on the market is likely to increase. Patent applications relating to CRISPR-edited commercial agricultural products have skyrocketed since the 2014/2015 period.
Proponents ofgenetic modification believe this is a method to perfect agricultural produce and solve problems such as pests, droughts, and nutritional deficiencies. But the technology is still a double-edged sword.
Genetic engineering indeed has its benefits in the short term, but it may bring long-term pitfalls, said Joe Wang, molecular biologist. Wang is currently a columnist with The Epoch Times.
Hornless cattle were once the celebrity of the animal kingdom, appearing in news stories one after another.
Many breeds of dairy cattle have horns, but they are dehorned to prevent them from harming humans and other animals, and to save more feeding trough space. To solve the problem of horns, the gene editing company Recombinetics successfully produced hornless cattle with gene-editing techniques many years ago.
The company simply added a few letters of DNA to the genome of ordinary cattle and their offspring didnt grow horns, either.
However, a few years later, an accident happened.
The FDA found that a modified genetic sequence of a bull contained a stretch of bacterial DNA including a gene conferring antibiotic resistance, which has been one of the global health crises in recent years. Scientists arent clear whether this gene in gene-edited cattle will pose a greater risk than expected or not, and the FDA has stressed that its hazard-free. However, John Heritage, a retired microbiologist from Leeds University, told MIT Technology Review that the antibiotic resistance gene could be absorbed by gut bacteria in cattle and could create unpredictable opportunities for its spread.
In fact, this is one of the currently perceived risks of genetically edited foods.
The problem with unexpected accidents in the genetic modification process occurs in GM foods because transgenic techniques cannot control where the foreign gene is embedded in the chromosome.
Kuo used the example of a study that compared the protein of transgenic soybeans and non-transgenic soybeans. These transgenic soybeans were initially embedded with one foreign gene, and should have had only one protein that didnt exist before. However, the comparison showed that there was a difference of about 40 proteins between the two: Half of the proteins were originally present, but disappeared after transgenic modification; the other half were not present but were added after the transgenic modification.
In contrast, emerging gene editing techniques allow for more precise modification of specific genes (pdf). Its like a tailor modifying a section of a zipper by cutting off a specific segment and replacing it with a new one. However, there may be mistakes and unexpected changes in the process of cutting and repairing, and another similar section of the zipper may also be cut off.
Kuo says that this process may have unforeseen side effects; for example, if during this, new allergy-causing proteins or new toxins are produced.
The genetic engineering procedure, and this includes gene editing, has the potential to damage DNA, said molecular geneticist Dr. Michael Antoniou, head of the Gene Expression and Therapy Group at Kings College London, in an interview in April 2022. If you alter gene function, you automatically alter the biochemistry of the plant included within that altered biochemistry can be the production of novel toxins and allergens that is my main concern.
Another major concern with GM foods is herbicide residue.
Most crops, whether genetically edited or genetically modified, have herbicide-resistant genes incorporated into them. This is done so that when herbicides are applied to crops for weed control, the crops themselves wont be harmed.
When planting herbicide-resistant crops, farmers can use herbicides rather liberally. But, long term, the weeds the farmers are targeting become increasingly herbicide-resistant as well, resulting in a cycle of increased herbicide use and resistance.
Since the introduction of herbicide-resistant GM crops in 1996, herbicides have experienced a significant growth in application every year. The herbicides residue in the crops grown are increasing as well.
One of the most widely used herbicides is glyphosate under the trade name Roundup. The International Agency for Research on Cancer (IARC) classifies glyphosate as a Group 2A carcinogen that is probably carcinogenic to humans.
Massachusetts Institute of Technology (MIT) researcher Stephanie Sene and scientific consultant Anthony Samsel said in their study that 80 percent of GM crops, especially corn, soybeans, canola, cotton, sugar beets, and alfalfa, are specifically introduced with glyphosate resistance genes.
In addition to carcinogenic concerns, glyphosate may have more harmful effects. They have collected and reviewed 286 studies and indicated that glyphosate inhibits the activity of an enzyme in the mitochondria of liver cellscytochrome P450which has the ability to detoxify and decompose foreign toxic substances. Moreover, glyphosate also has adverse effects on the gut microbiota.
These effects are not immediately apparent, but in the long run may contribute to inflammatory bowel disease, obesity, depression, attention deficit hyperactivity disorder (ADHD), autism, Alzheimers, Parkinsons, amyotrophic lateral sclerosis (ALS), multiple sclerosis, cancer, infertility, and developmental abnormalities.
An animal study published in Environmental Health shows that long-term exposure to ultra-low doses of glyphosate still causes liver and kidney diseases in rats.
The debate over whether GM food is safe or not has not yet settled. Many advocates of transgenic modification and gene editing believe that people have been eating GM crops for 20-plus years and still there is no evidence that they have caused problems to human health. Other argue they contribute to long term harm that is still being measured.
Kuo said that GM food is not a highly toxic drug causing immediate problems. Health problems can be the result of something cumulative, and hard to relate back to a single food cause. Whether GM foods are the culprit of such health problems has not been proven, nor ruled out.
At present, various countries have adopted an early warning principle for GM foods, stipulating that merchants label their products. It is the consumers decision to purchase them or not.
Will gene-edited food require specific labeling? Some argue that because these foods do not exhibit foreign genes, there should not be such regulation. Kuo believes this is a misleading argument, given that the tool used to edit the original genes were in fact foreign genes, and the method carries the risk that these foreign genes may not be completely removed.
Currently, the regulations for gene-edited foods in various countries are much looser than those for GM foods.
The USDA has consistently stated that gene-edited agricultural products are not regulated. Plant technologists are usually given the green light within months after submitting inquiries to the agency, allowing them to grow gene-edited foods without oversight.
In addition to the United States, Brazil and Australia and other countries have also adopted similar regulatory approaches. European regulations are still more stringent.
Antoniou argues that since these GM agricultural products are not monitored, the unexpected genes that they carry are released into the environment and will cause harm to it. They may also cause harm to the public due to the scientific communitys insufficient understanding of their risks.
Wang said that scientists who support gene editing believe that what they are doing now will also happen in nature, albeit at a slower pace. They simply speed it up. However, humans are not gods and cannot control everything. When humans do such things, the odds of mistakes and danger are definitely higher than what happens naturally, Wang said.
We humans have violated the laws of nature for a long time, Kuo said.
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Camille Su is a health reporter covering disease, nutrition, and investigative topics. Have a tip? kuanmi.su@epochtimes.com
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More Foods Will Be Gene-Edited Than You Think - The Epoch Times