Posts Tagged ‘article’

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Testosterone deficiency in men with end stage renal disease and kidney transplantation: a narrative review ... - Nature.com

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Excerpt from:
Adeno-associated virus as a delivery vector for gene therapy of human diseases | Signal Transduction and Targeted ... - Nature.com

Advances in the treatment of ophthalmic conditions with gene therapies have led to a growing market with huge potential for the future, according to research from DelveInsight.

The industry analyst has prepared a reportindicating that the market size for gene therapies in ophthalmology reached roughly $132 million across mature markets last year.

Taking into account new therapies expected to come online in future years, this market could grow at

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Research shows stellar growth for gene therapies in ophthalmology - The Pharma Letter

CRISPR technology offers the promise to cure human genetic diseases with gene editing. This promise became a reality when the worlds first CRISPR therapy was approved by regulators to treat patients with sickle cell disease and beta-thalassemia last year.

American biopharma Vertex Pharmaceuticals CASGEVY works by turning on the BCL11A gene, which codes for fetal hemoglobin. While this form of hemoglobin is produced before a baby is born, the body begins to deactivate the gene after birth. As both sickle cell disease and beta-thalassemia are blood disorders that affect hemoglobin, by switching on the gene responsible for fetal hemoglobin production, CASGEVY presents a curative, one-time treatment for patients.

As CASGEVYs clearance is a significant milestone, the technology has come a long way. CRISPR/Cas9 was first used as a gene-editing tool in 2012. Over the years, the technology exploded in popularity thanks to its potential for making gene editing faster, cheaper, and easier than ever before.

CRISPR is short for clustered regularly interspaced short palindromic repeats. The term makes reference to a series of repetitive patterns found in the DNA of bacteria that form the basis of a primitive immune system, defending them from viral invaders by cutting their DNA.

Using this natural process as a basis, scientists developed a gene-editing tool called CRISPR/Cas that can cut a specific DNA sequence by simply providing it with an RNA template of the target sequence. This allows scientists to add, delete, or replace elements within the target DNA sequence. Slicing a specific part of a genes DNA sequence with the help of the Cas9 enzyme, aids in DNA repair.

This system represented a big leap from previous gene-editing technologies, which required designing and making a custom DNA-cutting enzyme for each target sequence rather than simply providing an RNA guide, which is much simpler to synthesize.

CRISPR gene editing has already changed the way scientists do research, allowing a wide range of applications across multiple fields. Here are some of the diseases that scientists aim to tackle using CRISPR/Cas technology, testing its possibilities and limits as a medical tool.

Cancer is a complex, multifactorial disease, and a cure remains elusive. There are hundreds of different types of cancer, each with a unique mutation signature. CRISPR technology is a game-changer for cancer research and treatment as it can be used for many things, including screening for cancer drivers, identifying genes and proteins that can be targeted by cancer drugs, cancer diagnostics, and as a treatment.

China spearheaded the first in-human clinical trials using CRISPR/Cas9 as a cancer treatment. The study tested the use of CRISPR to modify immune T cells extracted from a patient with late-stage lung cancer. The gene-editing technology was used to remove the gene that encodes for a protein called PD-1 that some tumor cells can bind to to block the immune response against cancer. This protein found on the surface of immune cells is the target of some cancer drugs termed checkpoint inhibitors.

CRISPR technology has also been applied to improve the efficacy and safety profiles of cancer immunotherapy, such as CAR-T cell and natural killer cell therapies. In the U.S., CRISPR Therapeutics is one of the leading companies in this space, developing off-the-shelf, gene-edited T cell therapies using CRISPR, with two candidates targeting CD19 and CD70 proteins in clinical trials.

In 2022, the FDA granted Orphan Drug designation to Intellia Therapeutics CRISPR/Cas9-gene-edited T cell therapy for acute myeloid leukemia (AML). Currently, Vor BioPharmas VOR33 is undergoing phase 2 trials to treat AML, and the CRISPR trial is one to watch, according to a report published by Clinical Trials Arena earlier this year.

However, CRISPR technology still has limitations, including variable efficiency in the genome-editing process and off-target effects. Some experts have recommended that the long-term safety of the approach remain under review. Others have suggested using more precise gene-editing approaches such as base editing, an offshoot of CRISPR that hit the clinic in the U.S. last year.

There are several ways CRISPR could help us in the fight against AIDS. One is using CRISPR to cut the viral DNA that the HIV virus inserts within the DNA of immune cells. This approach could be used to attack the virus in its hidden, inactive form, which is what makes it impossible for most therapies to completely get rid of the virus.

The first ever patient with HIV was dosed with a CRISPR-based gene-editing therapy in a phase 1/2 trial led by Excision Biotherapeutics and researchers at the Lewis Katz School of Medicine at Temple University in Philadelphia back in 2022.

The decision to move the therapy to the clinic was bolstered by the success of an analog of the drug EBT-101 called EBT-001 in rhesus macaques infected with simian immunodeficiency virus (SIV). In a phase 1/2 study, EBT-101 was found to be safe.

Another approach could make us resistant to HIV infections. A small percentage of the worlds population is born with a natural resistance to HIV, thanks to a mutation in a gene known as CCR5, which encodes for a protein on the surface of immune cells that HIV uses as an entry point to infect the cells. The mutation changes the structure of the protein so that the virus is no longer able to bind to it.

This approach was used in a highly controversial case in China in 2018, where human embryos were genetically edited to make them resistant to HIV infections. The experiment caused outrage among the scientific community, with some studies pointing out that the CRISPR babies might be at a higher risk of dying younger.

The general consensus seems to be that more research is needed before this approach can be used in humans, especially as recent studies have pointed out this practice can have a high risk of unintended genetic edits in embryos.

Cystic fibrosis is a genetic disease that causes severe respiratory problems. Cystic fibrosis can be caused by multiple different mutations in the target gene CFTR more than 700 of which have been identified making it difficult to develop a drug for each mutation. With CRISPR technology, mutations that cause cystic fibrosis can be individually edited.

In 2020, researchers in the Netherlands used base editing to repair CFTR mutations in vitro in the cells of people with cystic fibrosis without creating damage elsewhere in their genetic code. Moreover, aiming to strike again with yet another win is the duo Vertex Pharmaceuticals and CRISPR Therapeutics, which have collaborated to develop a CRISPR-based medicine for cystic fibrosis. However, it might be a while until it enters the clinic as it is currently in the research phase.

Duchenne muscular dystrophy is caused by mutations in the DMD gene, which encodes for a protein necessary for the contraction of muscles. Children born with this disease experience progressive muscle degeneration, and existing treatments are limited to a fraction of patients with the condition.

Research in mice has shown CRISPR technology could be used to fix the multiple genetic mutations behind the disease. In 2018, a group of researchers in the U.S. used CRISPR to cut at 12 strategic mutation hotspots covering the majority of the estimated 3,000 different mutations that cause this muscular disease. Following this study, Exonics Therapeutics was spun out to further develop this approach, which was then acquired by Vertex Pharmaceuticals for approximately $1 billion to accelerate drug development for the disorder. Currently, Vertex is in the research stage, and is on a mission to restore dystrophin protein expression by targeting mutations in the dystrophin gene.

However, a CRISPR trial run by the Boston non-profit Cure Rare Disease targeting a rare DMD mutation resulted in the death of a patient owing to toxicity back in November 2022. Further research is needed to ensure the safety of the drug to treat the disease.

Huntingtons disease is a neurodegenerative condition with a strong genetic component. The disease is caused by an abnormal repetition of a certain DNA sequence within the huntingtin gene. The higher the number of copies, the earlier the disease will manifest itself.

Treating Huntingtons can be tricky, as any off-target effects of CRISPR in the brain could have very dangerous consequences. To reduce the risk, scientists are looking at ways to tweak the genome-editing tool to make it safer.

In 2018, researchers at the Childrens Hospital of Philadelphia revealed a version of CRISPR/Cas9 that includes a self-destruct button. A group of Polish researchers opted instead for pairing CRISPR/Cas9 with an enzyme called nickase to make the gene editing more precise.

More recently, researchers at the University of Illinois Urbana-Champaign used CRISPR/Cas13, instead of Cas9, to target and cut mRNA that codes for the mutant proteins responsible for Huntingtons disease. This technique silences mutant genes while avoiding changes to the cells DNA, thereby minimizing permanent off-target mutations because RNA molecules are transient and degrade after a few hours.

In addition, a 2023 study published in Nature went on to prove that treatment of Huntingtons disease in mice delayed disease progression and that it protected certain neurons from cell death in the mice.

With CASGEVYs go-ahead to treat transfusion-dependent beta-thalassemia and sickle cell disease in patients aged 12 and older, this hints that CRISPR-based medicines could even be a curative therapy to treat other blood disorders like hemophilia.

Hemophilia is caused by mutations that impair the activity of proteins that are required for blood clotting. Although Intellia severed its partnership with multinational biopharma Regeneron to advance its CRISPR candidate for hemophilia B a drug that was recently cleared by the FDA to enter the clinic the latter will take the drug ahead on its own.

As hemophilia B is caused by mutations in the F9 gene, which encodes a clotting protein called factor IX (FIX), Regenerons drug candidate uses CRISPR/Cas9 gene editing to place a copy of the F9 gene in cells in order to get the taps running for FIX production.

The two biopharmas will continue their collaboration in developing their CRISPR candidate to treat hemophilia A, which manifests as excessive bleeding because of a deficit of factor VIII. The therapy is currently in the research phase.

While healthcare companies were creating polymerase chain reaction (PCR) tests to screen for COVID-19 in the wake of the pandemic, CRISPR was also being put to use for speedy screening. A study conducted by researchers in China in 2023, found that the CRISPR-SARS-CoV-2 test had a comparable performance with RT-PCR, but it did have several advantages like short assay time, low cost, and no requirement for expensive equipment, over RT-PCRs.

To add to that, the gene editing tool could fight COVID-19 and other viral infections.

For instance, scientists at Stanford University developed a method to program a version of the gene editing technology known as CRISPR/Cas13a to cut and destroy the genetic material of the virus behind COVID-19 to stop it from infecting lung cells. This approach, termed PAC-MAN, helped reduce the amount of virus in solution by more than 90 percent.

Another research group at the Georgia Institute of Technology used a similar approach to destroy the virus before it enters the cell. The method was tested in live animals, improving the symptoms of hamsters infected with COVID-19. The treatment also worked on mice infected with influenza, and the researchers believe it could be effective against 99 percent of all existing influenza strains.

As European, U.S., and U.K. regulators have given their stamp of approval for the first-ever CRISPR-based drug to treat patients, who is to say we wont see another CRISPR-drug hitting this milestone in the near future.

And apart from the diseases mentioned, CRISPR is also being studied to treat other conditions like vision and hearing loss. In blindness caused by mutations, CRISPR gene editing could eliminate mutated genes in the DNA and replace them with normal versions of the genes. Researchers have also demonstrated how getting rid of the mutations in the Atp2b2 and Tmc1 genes helped partially restore hearing.

However, one of the biggest challenges to turn CRISPR research into real cures is the many unknowns regarding the potential risks of CRISPR therapy. Some scientists are concerned about possible off-target effects as well as immune reactions to the gene-editing tool. But as research progresses, scientists are proposing and testing a wide range of approaches to tweak and improve CRISPR in order to increase its efficacy and safety.

Hopes are high that CRISPR technology will soon provide a way to address complex diseases such as cancer and AIDS, and even target genes associated with mental health disorders.

New technologies related to CRISPR research:

This article was originally published in June 2018, and has since been updated by Roohi Mariam Peter.

Read more here:
Seven diseases that CRISPR technology could cure - Labiotech.eu

image:

Surgery team ICREC-IGTP

Credit: IGTP

The promising results obtained in a clinical trial with a pioneering advanced therapy drug named PeriCord, which aims to repair the heart of patients who have suffered a heart attack, confirm the feasibility of new therapies based on the application of stem cells and tissue engineering to promote the regeneration of damaged tissues.

This new medicine, derived from umbilical cord and pericardium stem cells from tissue donors, is a world-first tissue engineering product (a type of advanced therapy combining cells and tissues optimised in the laboratory). The drug is applied in patients undergoing coronary bypass, utilising the procedure to repair the scar in the heart area affected by the infarction, which has lost the ability to beat when blood flow stopped.

Thefirst interventionof this new therapy was almost 4 years ago, resulting from a collaboration between the ICREC Group (Heart Failure and Cardiac Regeneration) at Germans Trias i Pujol Research Institute (IGTP) and Banc de Sang i Teixits (BST). Following its success, a study was initiated to demonstrate its clinical safety. The study included 12 coronary bypass candidates, 7 treated with bioimplants and 5 without, to compare the outcomes.

Dr Antoni Bays, ICREC researcher and first author of the article:"This pioneering human clinical trial comes after many years of research in tissue engineering, representing a very innovative and hopeful treatment for patients with a heart scar resulting from a heart attack", referring to PeriCord.

While the current study aimed to demonstrate the safety of this new drug in the context of myocardial infarction, its positive outcomes have shown that PeriCord possesses other exceptional properties. It has proven to be a medicine with excellent biocompatibility, drastically minimising the risk of rejection and ensuring perfect tolerance by the body. Additionally, it has anti-inflammatory properties, paving the way for broader applications in pathologies involving inflammation."Its potential could be much wider; we believe it can be a valuable tool for modulating inflammatory processes", explains Dr Sergi Querol, head of the Cellular and Advanced Therapies Service at BST.

Severe but stable patients

The patients included in the therapy are individuals who have suffered a heart attack and have reduced quality and life expectancy. The bypass ensures blood circulation in the area, and the bioimplant goes a step further to stimulate the scar, initiating cellular mechanisms involved in tissue repair.

"Voluntarily provided substances of human origin are used, both in terms of multi-tissue donor pericardial tissue and mesenchymal stem cells from umbilical cord donors at the birth of a baby", explains Querol. It is very gratifying to think that"thanks to this and the donors, we provide a new therapeutic tool that can improve a patient's quality of life", he adds.

PeriCord consists of a membrane that comes from the pericardium of a tissue donor, which BST has decellularised and lyophilised. It has then been recellularised with these umbilical cord stem cells.

Once in the operating theatre, surgeons attach the laboratory-generated bioimplant to the affected area of the patient's heart. After a year, the implanted tissue adheres and adapts perfectly to the structure of the heart, covering the scar left by the heart attack.

Randomized controlled/clinical trial

People

Implantation of a double allogeneic human engineered tissue graft on damaged heart: insights from the PERISCOPE phase I clinical trial

14-Mar-2024

Disclaimer: AAAS and EurekAlert! are not responsible for the accuracy of news releases posted to EurekAlert! by contributing institutions or for the use of any information through the EurekAlert system.

Original post:
First cardiac bioimplants for the treatment of patients with myocardial infarction using umbilical cord stem cells - EurekAlert

Abstract

Genome editing is the modification of genomic DNA at a specific target site in a wide variety of cell types and organisms, including insertion, deletion and replacement of DNA, resulting in inactivation of target genes, acquisition of novel genetic traits and correction of pathogenic gene mutations. Due to the advantages of simple design, low cost, high efficiency, good repeatability and short-cycle, CRISPR-Cas systems have become the most widely used genome editing technology in molecular biology laboratories all around the world. In this review, an overview of the CRISPR-Cas systems will be introduced, including the innovations, the applications in human disease research and gene therapy, as well as the challenges and opportunities that will be faced in the practical application of CRISPR-Cas systems.

Keywords: CRISPR, Cas9, Genome editing, Human disease models, Rabbit, Gene therapy, Off target effects

Genome editing is the modification of genomic DNA at a specific target site in a wide variety of cell types and organisms, including insertion, deletion and replacement of DNA, resulting in inactivation of target genes, acquisition of novel genetic traits and correction of pathogenic gene mutations [1], [2], [3]. In recent years, with the rapid development of life sciences, genome editing technology has become the most efficient method to study gene function, explore the pathogenesis of hereditary diseases, develop novel targets for gene therapy, breed crop varieties, and so on [4], [5], [6], [7].

At present, there are three mainstream genome editing tools in the world, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the RNA-guided CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) nucleases systems [8], [9], [10]. Due to the advantages of simple design, low cost, high efficiency, good repeatability and short-cycle, CRISPR-Cas systems have become the most widely used genome editing technology in molecular biology laboratories all around the world [11], [12]. In this review, an overview of the CRISPR-Cas systems will be introduced, including the innovations and applications in human disease research and gene therapy, as well as the challenges and opportunities that will be faced in the practical application of CRISPR-Cas systems.

CRISPR-Cas is an adaptive immune system existing in most bacteria and archaea, preventing them from being infected by phages, viruses and other foreign genetic elements [13], [14]. It is composed of CRISPR repeat-spacer arrays, which can be further transcribed into CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA), and a set of CRISPR-associated (cas) genes which encode Cas proteins with endonuclease activity [15]. When the prokaryotes are invaded by foreign genetic elements, the foreign DNA can be cut into short fragments by Cas proteins, then the DNA fragments will be integrated into the CRISPR array as new spacers [16]. Once the same invader invades again, crRNA will quickly recognize and pair with the foreign DNA, which guides Cas protein to cleave target sequences of foreign DNA, thereby protecting the host [16].

CRISPR-Cas systems can be classified into 2 classes (Class 1 and Class 2), 6 types (I to VI) and several subtypes, with multi-Cas protein effector complexes in Class 1 systems (Type I, III, and IV) and a single effector protein in Class 2 systems (Type II, V, and VI) [17], [18]. The classification, representative members, and typical characteristics of each CRISPR-Cas system are summarized in [10], [12], [15], [16], [17], [18].

Summary of CRISPR-Cas systems.

Type II CRISPR-Cas9 system derived from Streptococcus pyogenes (SpCas9) is one of the best characterized and most commonly used category in numerous CRISPR-Cas systems [18], [19]. The main components of CRISPR-Cas9 system are RNA-guided Cas9 endonuclease and a single-guide RNA (sgRNA) [20]. The Cas9 protein possesses two nuclease domains, named HNH and RuvC, and each cleaves one strand of the target double-stranded DNA [21]. A single-guide RNA (sgRNA) is a simplified combination of crRNA and tracrRNA [22]. The Cas9 nuclease and sgRNA form a Cas9 ribonucleoprotein (RNP), which can bind and cleave the specific DNA target [23]. Furthermore, a protospacer adjacent motif (PAM) sequence is required for Cas9 proteins binding to the target DNA [20].

During genome editing process, sgRNA recruits Cas9 endonuclease to a specific site in the genome to generate a double-stranded break (DSB), which can be repaired by two endogenous self-repair mechanisms, the error-prone non-homologous end joining (NHEJ) pathway or the homology-directed repair (HDR) pathway [24]. Under most conditions, NHEJ is more efficient than HDR, for it is active in about 90% of the cell cycle and not dependent on nearby homology donor [25]. NHEJ can introduce random insertions or deletions (indels) into the cleavage sites, leading to the generation of frameshift mutations or premature stop codons within the open reading frame (ORF) of the target genes, finally inactivating the target genes [26], [27]. Alternatively, HDR can introduce precise genomic modifications at the target site by using a homologous DNA repair template [28], [29] (). Furthermore, large fragment deletions and simultaneous knockout of multiple genes could be achieved by using multiple sgRNAs targeting one single gene or more [30], [31].

Mechanism of genome editing. Double-strand break (DSB) induced by nucleases can be repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathways. NHEJ can introduce random insertions or deletions (indels) of varying length at the site of the DSB. Alternatively, HDR can introduce precise genomic modifications at the target site by using a homologous DNA donor template.

CRISPR-Cas systems have become the most favorite genome editing tool in the molecular biology laboratory since they were confirmed to have genome editing capabilities in 2012 [23]. They have made numerous achievements in the field of correcting pathogenic mutations, searching for essential genes for cancer immunotherapy, and solving key problems in organ xenotransplantation [5], [32], [33]. Unfortunately, there are still some limitations which need to solve in CRISPR-Cas systems, such as potential off-target effects, limited genome-targeting scope restricted by PAM sequences, and low efficiency and specificity [34], [35]. Therefore, many research teams have been trying to improve this tool.

By introducing two point mutations, H840A and D10A, into HNH and RuvC nuclease domain, researchers have obtained a nuclease dead Cas9 (dCas9) [36]. The dCas9 lacks DNA cleavage activity, but DNA binding activity is not affected. Then, by fusing transcriptional activators or repressors to dCas9, the CRISPR-dCas9 system can be used to activate (CRISPRa) or inhibit (CRISPRi) transcription of target genes [37], [38]. Additionally, dCas9 can be fused to various effector domains, which enables sequence-specific recruitment of fluorescent proteins for genome imaging and epigenetic modifiers for epigenetic modification [39], [40]. Furthermore, this system is easy to operate and allows simultaneous manipulation of multiple genes within a cell [38].

In order to improve the efficiency of site-directed mutagenesis, base editing systems containing dCas9 coupled with cytosine deaminase (cytidine base editor, CBE) or adenosine deaminase (adenine base editor, ABE) have been developed [41], [42]. It can introduce CG to TA or AT to GC point mutations into the editing window of the sgRNA target sites without double-stranded DNA cleavage [41], [42]. Since base editing systems avoid the generation of random insertions or deletions to a great extent, the results of gene mutation are more predictive. However, owing to the restriction of base editing window, base editing systems are not suitable for any target sequence in the genome. Accordingly, C-rich sequences, for example, would produce a lot of off-target mutations [43]. Therefore, researchers have always been trying to develop and optimize novel base editing systems to overcome this drawback [44]. At present, base editing systems have been widely used in various cell lines, human embryos, bacteria, plants and animals for efficient site-directed mutagenesis, which may have broad application prospects in basic research, biotechnology and gene therapy [45], [46], [47]. In theory, 3956 gene variants existing in Clin var database could be repaired by base substitution of C-T or G-A [42], [48].

An NGG PAM at the 3 end of the target DNA site is essential for the recognization and cleavage of the target gene by Cas9 protein [20]. Besides classical NGG PAM sites, other PAM sites such as NGA and NAG also exist, but their efficiency of genome editing is not high [49]. However, such PAM sites only exist in about one-sixteenth of the human genome, thereby largely restricting the targetable genomic loci. For this purpose, several Cas9 variants have been developed to expand PAM compatibility.

In 2018, David Liu et al.[50] developed xCas9 by phage-assisted continuous evolution (PACE), which can recognize multiple PAMs (NG, GAA, GAT, etc.). In the latter half of the same year, Nishimasu et al. developed SpCas9-NG, which can recognize relaxed NG PAMs [51]. In 2020, Miller et al. developed three new SpCas9 variants recognizing non-G PAMs, such as NRRH, NRCH and NRTH PAMs [52]. Later in the same year, Walton et al. developed a SpCas9 variant named SpG, which is capable of targeting an expanded set of NGN PAMs [53]. Subsequently, they optimized the SpG system and developed a near-PAMless variant named SpRY, which is capable of editing nearly all PAMs (NRN and NYN PAMs) [53].

By using these Cas9 variants, researchers have repaired some previously inaccessible disease-relevant genetic variants [51], [52], [53]. However, there are still some drawbacks in these variants, such as low efficiency and cleavage activity [50], [51]. Therefore, they should be further improved by molecular engineering in order to expand the applications of SpCas9 in disease-relevant genome editing.

In addition to editing DNA, CRISPR-Cas systems can also edit RNA. Class 2 Type VI CRISPR-Cas13 systems contain a single RNA-guided Cas13 protein with ribonuclease activity, which can bind to target single-stranded RNA (ssRNA) and specifically cleave the target [54]. To date, four Cas13 proteins have been identified: Cas13a (also known as C2c2), Cas13b, Cas13c and Cas13d [55]. They have successfully been applied in RNA knockdown, transcript labeling, splicing regulation and virus detection [56], [57], [58]. Later, Feng Zhang et al. developed two RNA base edting systems (REPAIR system, enables A-to-I (G) replacement; RESCUE system, enables C-to-U replacement) by fusing catalytically inactivated Cas13 (dCas13) with the adenine/cytidine deaminase domain of ADAR2 (adenosine deaminase acting on RNA type 2) [59], [60].

Compared with DNA editing, RNA editing has the advantages of high efficiency and high specificity. Furthermore, it can make temporary, reversible genetic edits to the genome, avoiding the potential risks and ethical issues caused by permanent genome editing [61], [62]. At present, RNA editing has been widely used for pre-clinical studies of various diseases, which opens a new era for RNA level research, diagnosis and treatment.

Recently, Anzalone et al. developed a novel genome editing technology, named prime editing, which can mediate targeted insertions, deletions and all 12 types of base substitutions without double-strand breaks or donor DNA templates [63]. This system contains a catalytically impaired Cas9 fused to a reverse transcriptase and a prime editing guide RNA (pegRNA) with functions of specifying the target site and encoding the desired edit [63]. After Cas9 cleaves the target site, the reverse transcriptase uses pegRNA as a template for reverse transcription, and then, new genetic information can be written into the target site [63]. Prime editing can effectively improve the efficiency and accuracy of genome editing, and significantly expand the scope of genome editing in biological and therapeutic research. In theory, it is possible to correct up to 89% known disease-causing gene mutations [63]. Nevertheless, as a novel genome editing technique, more research is still needed to further understand and improve prime editing system.

So far, as a rapid and efficient genome editing tool, CRISPR-Cas systems have been extensively used in a variety of species, including bacteria, yeast, tobacco, Arabidopsis, sorghum, rice, Caenorhabditis elegans, Drosophila, zebrafish, Xenopus laevis, mouse, rat, rabbit, dog, sheep, pig and monkey [64], [65], [66], [67], [68], [69], [70], [71], [72], [73], [74], [75], [76], [77], [78], as well as various human cell lines, such as tumor cells, adult cells and stem cells [79], [80]. In medical field, the most important application of CRISPR-Cas systems is to establish genetically modified animal and cell models of many human diseases, including gene knockout models, exogenous gene knock-in models, and site directed mutagenesis models [80], [81].

(1)

Establishing animal models of human diseases

Animal models are crucial tools for understanding gene function, exploring pathogenesis of human diseases and developing new drugs. However, traditional methods for generating animal models are complex, costly and time-consuming, which severely limit the application of animal models in basic medical research and preclinical studies [82]. Since the discovery of CRISPR-Cas systems, a series of genetically modified animal models have successfully been generated in a highly efficient manner [72], [73], [74], [75], [76], [77], [78].

Among numerous model animals, mice are widely used for scientific studies and recognized as the most important model animals in human disease research [83]. So far, researchers have successfully generated many genetically modified mouse models, such as cancer, cardiovascular disease, cardiomyopathy, Huntington's disease, albino, deafness, hemophilia B, obesity, urea cycle disorder and muscular dystrophy [84], [85], [86], [87], [88], [89], [90], [91], [92], [93]. Nevertheless, owing to the great species differences between humans and rodents, they cant provide effective assessment and long-term follow-up for research and treatment of human diseases [94]. Therefore, the application of larger model animals, such as rabbits, pigs and non-human primates, is becoming more and more widespread [74], [77], [78]. With the development of CRISPR-Cas systems, generating larger animal models for human diseases has become a reality, which greatly enriches the disease model resource bank.

Our research focuses on the generation of genetically modified rabbit models using CRISPR-Cas systems. Compared with mice, rabbits are closer to humans in physiology, anatomy and evolution [95]. In addition, rabbits have a short gestation period and less breeding cost. All these make them suitable for studies of the cardiovascular, pulmonary and metabolism diseases [95], [96]. Nowadays, we have generated a series of rabbit models for simulating human diseases, including congenital cataracts, duchenne muscular dystrophy (DMD), X-linked hypophosphatemia (XLH), etc (summarized in ) [97], [98], [99], [100], [101], [102], [103], [104], [105], [106], [107], [108], [109], [110], [111], [112], [113], [114]. Take the generation of PAX4 gene knockout rabbits as an example, the procedure we used to establish genetically modified rabbit models is summarized in and .

CRISPR-Cas system mediated rabbit models of human diseases.

Generation of PAX4 gene knockout (KO) rabbits using CRISPR-Cas9 system. (A) Schematic diagram of the sgRNA target sites located in the rabbit PAX4 locus. PAX4 exons are indicated by yellow rectangles; target sites of the two sgRNA sequences, sgRNA1 and sgRNA2, are highlighted in green; protospacer-adjacent motif (PAM) sequence is highlighted in red. Primers F and R are used for mutation detection in pups. (B) Microinjection and embryo transfer. First a mixture of Cas9 mRNA and sgRNA is microinjected into the cytoplasm of the zygote at the pronuclear stage. Then the injected embryos are transferred into the oviduct of recipient rabbits. After 30days gestation, PAX4 KO rabbits are born. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Summary of the PAX4 KO rabbits generated by CRISPR-Cas9 system.

In addition, the pig is an important model animal extensively used in biomedical research. Compared with mice, their body/organ size, lifespan, anatomy, physiology, metabolic profile and immune characteristics are more similar to those of humans, which makes the pig an ideal model for studying human cardiovascular diseases and xenotransplantation [115]. At present, several genetically modified pig models have been successfully generated, including neurodegenerative diseases, cardiovascular diseases, cancer, immunodeficiency and xenotransplantation model [116], [117], [118], [119], [120], [121], [122].

To date, non-human primates are recognized as the best human disease models. Their advantage is that their genome has 98% homology with the human genome; also, they are highly similar to humans in tissue structure, immunity, physiology and metabolism [123]. Whats more, they can be infected by human specific viruses, which makes them very important models in infectious disease research [124]. Nowadays, researchers have generated many genetically modified monkey models, such as cancer, muscular dystrophy, developmental retardation, adrenal hypoplasia congenita and Oct4-hrGFP knockin monkeys [125], [126], [127], [128], [129].

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Establishing cell models of human diseases

It was found that the efficiency of CRISPR-Cas mediated genome editing is higher in vitro than in vivo, thus the use of genetically modified cell models can greatly shorten the research time in medical research [130]. Until now, researchers have used CRISPR-Cas systems to perform genetic manipulations on various cell lines, such as tumor cells, adult cells and stem cells, in order to simulate a variety of human diseases [79], [80].

Fuchs et al. generated the RPS25-deficient Hela cell line by knocking out ribosomal protein eS25 (RPS25) gene using CRISPR-Cas9 system [131]. Drost et al. edited four common colorectal cancer-related genes (APC, P53, KRAS and SMAD4) in human intestinal stem cells (hISCs) by CRISPR-Cas9 technology [132]. The genetically modified hISCs with 4 gene mutations possessed the biological characteristics of intestinal tumors and could simulate the occurrence of human colorectal cancer [132]. Jiang et al. induced site-specific chromosome translocation in mouse embryonic stem cells by CRISPR-Cas9, in order to establish a cell and animal model for subsequent research on congenital genetic diseases, infertility, and cancer related to chromosomal translocation [133].

In addition, induced pluripotent stem cells (iPSCs) have shown great application prospect in disease model establishment, drug discovery and patient-specific cellular therapy development [134]. iPSCs have the ability of self-renewal and multiple differentiation potential, which are of great significance in disease model establishment and regenerative medicine research [135]. In recent years, by combining CRISPR-Cas systems with iPSC technology, researchers have generated numerous novel and reliable disease models with isogenic backgrounds and provided new solutions for cell replacement therapy and precise therapy in a variety of human diseases, including neurodegenerative diseases, acquired immunodeficiency syndrome (AIDS), -thalassemia, etc [134], [135], [136].

With the development of CRISPR-Cas systems and the discovery of novel Cas enzymes (Cas12, Cas13, etc.), CRISPR-based molecular diagnostic technology is rapidly developing and has been selected as one of the world's top ten science and technology advancements in 2018 [137].

Unlike Cas9, Cas13 enzymes possess a collateral cleavage activity, which can induce cleavage of nearby non-target RNAs after cleavage of target sequence [54]. Based on the collateral cleavage activity of Cas13, Feng Zhang et al.[138] developed a Cas13a-based in vitro nucleic acid detection platform, named SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing). It is composed of Cas13a, sgRNA targeting specific RNA sequences and fluorescent RNA reporters. After Cas13a protein recognizes and cleaves the target RNA, it will cut the report RNA and release the detectable fluorescence signal, so as to achieve the purpose of diagnosis [138]. Researchers have used this method to detect viruses, distinguish pathogenic bacteria, genotype human DNA and identify tumor DNA mutations [137], [138]. Later, Feng Zhang et al. improved SHERLOCK system and renamed it as SHERLOCKv2, which can detect four virus at the same time [139].

In addition to Cas13, Cas12 enzymes are also found to possess collateral cleavage activity [140]. Doudna et al.[141] developed a nucleic acid detection system based on Cas12a (also known as Cpf1), named DETECTR (DNA endonuclease-targeted CRISPR trans reporter). DETECTR has been used to detect cervical cancer associated HPV subtypes (HPV16 and HPV18) in either virus-infected human cell lines or clinical patient samples [141]. Furthermore, Doudna et al. are trying to use the newly discovered Cas14 and CasX proteins in molecular diagnosis, which may further enrich the relevant techniques of CRISPR-based molecular diagnosis [142], [143].

CRISPR-based molecular diagnostic technology has incomparable advantages over traditional molecular diagnostic methods, such as high sensitivity and single-base specificity, which is suitable for early screening of cancer, detection of cancer susceptibility genes and pathogenic genes [137], [144]. Meanwhile, CRISPR diagnostics is inexpensive, simple, fast, without special instrument, and is suitable for field quick detection and detection in less-developed areas [137], [144]. At present, many companies are trying to develop CRISPR diagnostic kits for family use, to detect HIV, rabies, Toxoplasma gondi, etc.

CRISPR-Cas9 system enables genome-wide high-throughput screening, making it a powerful tool for functional genomic screening [145]. The high efficiency of genome editing with CRISPR-Cas9 system makes it possible to edit multiple targets in parallel, thus a mixed cell population with gene mutation can be produced, and the relationship between genotypes and phenotypes could be confirmed by these mutant cells [146]. CRISPR-Cas9 library screening can be divided into two categories: positive selection and negative selection [147]. It has been utilized to identify genes associated with cancer cell survival, drug resistance and virus infection in various models [148], [149], [150]. Compared with RNAi-based screening, high-throughput CRISPR-Cas9 library screening has the advantages of higher transfection efficiency, minimal off-target effects and higher data reproducibility [151]. At present, scientists have constructed human and mouse genome-wide sgRNA libraries, and they have been increasingly improved according to different requirements [152], [153]. In the future, CRISPR-Cas9-based high-throughput screening technology will definitely get unprecedented development and application.

Gene therapy refers to the introduction of foreign genes into target cells to treat specific diseases caused by mutated or defective genes [154]. Target cells of gene therapy are mainly divided into two categories: somatic cells and germ line cells. However, since germ line gene therapy is complicated in technique as well as involves ethical and security issues, today gene therapy is limited to somatic cell gene therapy [155]. Traditional gene therapy is usually carried out by homologous recombination or lentiviral delivery. Nevertheless, the efficiency of homologous recombination is low, and lentiviral vectors are randomly inserted into the recipient genome, which may bring potential security risks to clinical applications [156]. Currently, with the rapid development of CRISPR-Cas systems, they have been widely applied in gene therapy for treating various of human diseases, monogenic diseases, infectious diseases, cancer, etc [155], [156], [157]. Furthermore, some CRISPR-mediated genome-editing therapies have already reached the stage of clinical testing. briefly summarizes the ongoing clinical trials of gene therapy using genome-editing technology, including ZFN, TALEN and CRISPR-Cas systems.

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Monogenic diseases

Monogenic diseases refer to the genetic diseases caused by mutations of a single allele or a pair of alleles on a pair of homologous chromosomes [158]. There are more than 6600 known monogenic diseases around the world, -thalassaemia, sickle cell disease (SCD), hemophilia B (HB), retinitis pigmentosa (RP), leber congenital amaurosis type 10 (LCA10), duchenne muscular dystrophy (DMD), hutchinson-gilford progeria syndrome (HGPS), hereditary tyrosinemia (HT), cystic fibrosis (CF), etc [159]. Most of the monogenic diseases are rare diseases lacking of effective treatment, which will greatly affect the life quality of patients. Nowadays, many animal models of monogenic diseases have been treated with CRISPR-mediated gene therapy. Furthermore, even some CRISPR clinical trials for monogenic diseases are going on [160].

Summary of clinical trials of gene therapy using genome-editing technology.

-Thalassaemia, a hereditary hemolytic anemia disease, is one of the most common and health-threatening monogenic diseases in the world. It is characterized by mutations in the -globin (HBB) gene, leading to severe anemia caused by decreased hemoglobin (Hb) level [161]. For the moment, the only way to cure -thalassemia is hematopoietic stem cell transplantation (HSCT). Yet, high cost of treatment and shortage of donors limit its clinical application [162]. Other therapy, for example, blood transfusion, can only sustain the life of patients but cant cure the disease [161]. To better treat -thalassemia, researchers have turned their attention to gene therapy. A major technical idea is to repair the defective -globin gene of iPSCs from patients with -thalassemia by CRISPR-Cas9 technology, then red blood cells can be produced normally and the disease could be cured [163], [164]. Besides, reactivating fetal hemoglobin (HbF) expression has also been proposed to be an effective method to treat -thalassemia through knockout of BCL11A gene, which suppresses the expression of fetal hemoglobin [165], [166].

Additionally, CRISPR-Cas systems have also been used for the treatment of other hematologic diseases, such as sickle cell disease (SCD) and hemophilia B (HB). SCD is a monogenic disease caused by a single-nucleotide mutation in human -globin gene, leading to a substitution of glutamic acid by valine and the production of an abnormal version of -globin, which is known as hemoglobin S (HbS) [167]. CRISPR-Cas9 system has been used to treat SCD by repairing the -globin gene mutation or reactivating HbF expression [168], [169]. HB is an X-linked hereditary bleeding disorder caused by deficiency of coagulation factor IX, and the most common treatment for hemophilia B is supplement blood coagulation factor [170], [171]. Huai et al. injected naked Cas9-sgRNA plasmid and donor DNA into the adult mice of F9 mutation HB mouse model for gene correction [172]. Meanwhile, Cas9/sgRNA were also microinjected into germline cells of this HB mouse model for gene correction. Both in vivo and ex vivo experiment were sufficient to remit the coagulation deficiency [172]. Guan et al. corrected the F9 Y371D mutation in HB mice using CRISPR-Cas9 mediated in situ genome editing, which greatly improved the hemostatic efficiency and increased the survival of HB mice [173].

Duchenne muscular dystrophy (DMD) is an X-chromosome recessive hereditary disease, with clinical manifestations of muscle weakness or muscle atrophy due to a progressive deterioration of skeletal muscle function [174]. It is usually caused by mutations in the DMD gene, a gene encoding dystrophin protein [174]. Deletions of one or more exons of the DMD gene will result in frameshift mutations or premature termination of translation, thereby normal dystrophin protein can not be synthesized [175]. Currently, there is no effective treatment for DMD. Conventional drug treatment can only control the disease to a certain extent, but can not cure it. It was found that a functional truncated dystrophin protein can be obtained by removing the mutated transcripts with CRISPR-Cas9 system [176], [177], [178]. In addition, base editing systems can also be applied in DMD treatment by repairing single base mutation or inducing exon skipping by introducing premature termination codons (PTCs) [179].

Retinitis pigmentosa (RP) is a group of hereditary retinal degenerative diseases characterized by progressive loss of photoreceptor cells and retinal pigment epithelium (RPE) function [180]. RP has obvious genetic heterogeneity, and the inheritance patterns include autosomal dominant, autosomal recessive, and X-linked recessive inheritance [180]. To date, there is still no cure for RP. In recent years, with the rapid development of gene editing technology, there has been some progress in the treatment of RP. Several gene mutations causing RP have been corrected by CRISPR-Cas9 in mouse models to prevent retinal degeneration and improve visual function, for example, RHO gene, PRPF31 gene and RP1 gene [181], [182].

Leber Congenital Amaurosis type 10 (LCA10) is an autosomal retinal dystrophy with severe vision loss at an early age. The most common gene mutation found in patients with LCA10 is IVS26 mutation in the CEP290 gene, which disrupts the coding sequence by generating an aberrant splice site [183]. Ruan et al. used CRISPR-Cas9 system to knock out the intronic region of the CEP290 gene and restored normal CEP290 expression [184]. In addition, subretinal injection of EDIT-101 in humanized CEP290 mice showed rapid and sustained CEP290 gene editing [185], [186].

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare lethal genetic disorder with the characteristic of accelerated aging [187]. A point mutation within exon 11 of lamin A gene activates a cryptic splice site, leading to the production of a truncated lamin A called progerin [188]. However, CRISPR-Cas based gene therapy has opened up a broad prospect in HGPS treatment. Administration of AAV-delivered CRISPR-Cas9 components into HGPS mice can reduce the expression of progerin, thereby improved the health condition and prolonged the lifespan of HGPS mice [189], [190]. In addition, Suzuki et al. repaired G609G mutation in a HGPS mouse model via single homology arm donor mediated intron-targeting gene integration (SATI), which ameliorated aging-associated phenotypes and extended the lifespan of HGPS mice [191].

CRISPR-Cas systems have also showed their advantages in gene therapy of hereditary tyrosinemia (HT) and cystic fibrosis (CF). HT is a disorder of tyrosine metabolism caused by deficiency of fuarylacetoacetate hydrolase (Fah) [192]. Yin et al. corrected a Fah mutation in a HT mouse model by injecting CRISPR-Cas9 components into the liver of the mice [193]. Then, the wild-type Fah protein in the liver cells began to express and the body weight loss phenotype was rescued [193]. CF, an autosomal recessive inherited disease with severe respiratory problems and infections, has a high mortality rate at an early age [194]. It is caused by mutations in the CFTR gene, which encodes an epithelial chloride anion channel, the cystic fibrosis transmembrane conductance regulator (CFTR) [194]. Until now, genome editing strategies have been carried out in cell models to correct CFTR mutations. In cultured intestinal stem cells and induced pluripotent stem cells from cystic fbrosis patients, the CFTR homozygous 508 mutation has been corrected by CRISPR-Cas9 technology, leading to recovery of normal CFTR expression and function in differentiated mature airway epithelial cells and intestinal organoids [195], [196].

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Infectious diseases

In recent years, gene therapy has gradually been applied to the treatment of viral infectious diseases. Transforming host cells to avoid viral infection or preventing viral proliferation and transmission are two main strategies for gene therapy of viral infectious diseases [197].

Human immunodeficiency virus (HIV), a kind of retrovirus, mainly attacks the human immune system, especially the CD4 T lymphocytes. When human cells are invaded by HIV, the viral sequences can be integrated into the host genome, blocking cellular and humoral immunity while causing acquired immunodeficiency syndrome (AIDS) [198]. There is still no known cure for AIDS but it could be treated. Although antiretroviral therapy can inhibit HIV-1 replication, the viral sequences still exist in the host genome, and they could be reactivated at any time [199]. CRISPR-Cas9 system can target long terminal repeat (LTR) and destruct HIV-1 proviruses, thus it is possible to completely eliminate HIV-1 from genome of infected host cells [200], [201]. In addition, resistance to HIV-1 infection could be induced by knockout of the HIV co-receptor CCR5 gene in CD4 T cells [202], [203].

Cervical cancer is the second most common gynecologic malignant tumor. The incidence is increasing year by year and young people are especially prone to this disease. It was found that the occurrence of cervical cancer is closely related to HPV (human papillomavirus) infection [204]. HPV is a double-stranded cyclic DNA virus, E6 and E7 genes located in HPV16 early regions are carcinogenic genes [205]. Researchers designed sgRNAs targeting E6 and E7 genes to block the expression of E6 and E7 protein, subsequently the expression of p53 and pRb was restored to normal, finally increasing tumor cells apoptosis and suppressing subcutaneous tumor growth in in vivo experiments [206], [207], [208]. Moreover, HPV virus proliferation was blocked through cutting off E6/E7 genes, and the virus in the bodies could be eliminated [206], [207], [208].

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Cancer

Cancer is the second leading cause of death worldwide after cardiovascular diseases, and it is also a medical problem that needs to be solved urgently. A variety of genetic or epigenetic mutations have been accumulated in the cancer genome, which can activate proto-oncogenes, inactivate tumor suppressors and produce drug resistance [209], [210]. So far, CRISPR-Cas systems have been used to correct the oncogenic genome/epigenome mutations in tumor cells and animal models, resulting in inhibition of tumor cell growth and promotion of cell apoptosis, thereby inhibiting tumor growth [211], [212], [213].

In addition, immunotherapy is considered to be a major breakthrough in cancer treatment, especially chimeric antigen receptor-T (CAR-T) cell therapy, which has a significantly therapeutic effect on leukemia, lymphoma and certain types of solid tumors [214], [215], [216]. CAR-T cells are genetically manipulated, patient-specific T cells, which express receptors targeting antigens specially expressed on tumor cells, for example, CD19 CAR-T cells for B cell malignancies. Then these cells will be transfused back to patients to fight against cancer [217]. However, CAR-T cell therapy is complex, time-consuming and expensive, and it is greatly limited by the quality and quantity of autologous T cells. Therefore, researchers have used CRISPR-Cas9 system to develop universal CAR-T cells, such as simultaneously removing endogenous T cell receptor gene and HLA class I encoding gene on T cells of healthy donors and introducing CAR sequence [218], [219], [220]. Thereby, it could be used in multiple patients without causing graft versus host reaction (GVHR). In addition, CRISPR-Cas mediated genome editing has also been used to enhance the function of CAR-T cells by knocking out genes encoding signaling molecules or T cell inhibitory receptors, such as programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte antigen 4 (CTLA-4) [221], [222].

Though CRISPR-Cas mediated efficient genome editing technologies have been broadly applied in a variety of species and different types of cells, there are still some important issues needed to be addressed during the process of application, such as off-target effects, delivery methods, immunogenicity and potential risk of cancer.

It was found that designed sgRNAs will mismatch with non-target DNA sequences and introduce unexpected gene mutations, called off-target effects [223]. Off-target effects seriously restrict the widespread application of CRISPR-Cas mediated genome editing in gene therapy, for it might lead to genomic instability and increase the risk of certain diseases by introducing unwanted mutations at off-target sites [224]. At present, several strategies have been used to predict and detect off-target effects, online prediction software, whole genome sequencing (WGS), genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-seq), discovery of in situ cas off-targets and verification by sequencing (DISCOVER-Seq), etc [225]. Furthermore, to minimize off-target effects, researchers have systematically studied the factors affecting off-target effects and developed a number of effective approaches.

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Rational design and modification of sgRNAs

The specific binding of sgRNA with the target sequence is the key factor in CRISPR-Cas mediated genome editing. Rational design of highly specific sgRNAs might minimize off-target effects [224]. The length and GC content of sgRNAs, and mismatches between sgRNA and its off-target site will all affect the frequency of off-target effects [226]. In addition, on the basis of rational design of sgRNAs, the specificity of CRISPR-Cas systems can be further improved by modifying sgRNAs, such as engineered hairpin sgRNAs and chemical modifications of sgRNAs [227], [228].

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Modification of Cas9 protein

As we know, the interaction between Cas9 and DNA affects the stability of DNA-Cas9/sgRNA complex as well as tolerance to mismatch [229]. Therefore, high-fidelity SpCas9 variants have been developed by introducing amino substitution(s) into Cas9 protein in order to destabilize the function structure of the CRISPR complex [230]. Researchers have developed several highly effective Cas9 mutants, high-fidelity Cas9 (SpCas9-HF1), enhanced specificity Cas9 (eSpCas9), hyper-accurate Cas9 (HypaCas9), etc [231], [232], [233]. All of them can significantly reduce off-target effects while retain robust target cleavage activity.

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Adoption of double nicking strategy

Recently, a double-nicking strategy has been developed to minimize off-target effects, which employs two catalytic mutant Cas9-D10A nickases and a pair of sgRNAs to produce a cleavage on each strand of the target DNA, thus forming a functional double strand break [234]. Additionally, it was proven that the fusion protein generated by combining dCas9 with Fok nuclease can also reduce off-target effects [235]. Only when the two fusion protein monomers are close to each other to form dimers, can they perform the cleavage function [235]. This strategy could greatly reduce DNA cleavage at non-target sites.

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Anti-CRISPRs

Off switches for CRISPR-Cas9 system was first discovered by Pawluk et al. in 2016. They identified three naturally existing protein families, named as anti-CRISPRs, which can specifically inhibit the CRISPR-Cas9 system of Neisseria meningitidis[236]. Later, Rauch et al. discovered four unique type IIA CRISPR-Cas9 inhibitor proteins encoded by Listeria monocytogenes prophages, and two of them (AcrllA2 and AcrllA4) can block SpCas9 when assayed in Escherichia coli and human cells [237]. Recently, Doudna et al. discovered two broad-spectrum inhibitors of CRISPR-Cas9 system (AcrllC1 and AcrllC3) [238]. Therefore, in order to reduce off-target effects, the anti-CRISPRs could be used to prevent the continuous expression of Cas9 protein in cells to be edited.

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Others

The concentration of Cas9/sgRNA can also affect the frequency of off-target mutations [239]. Thus, the optimal concentration of Cas9 and sgRNA needs to be determined by pre-experiment. Besides, the formulation of CRISPR-Cas9 can affect the frequency of off-target mutations as well. Cas9 nucleases can be delivered into target cells in 3 different forms: DNA expression plasmid, mRNA or recombination protein [240]. Currently, the use of Cas9/sgRNA ribonucleoprotein complexes (Cas9-RNPs), which are composed of purified Cas9 proteins in combination with sgRNA, is becoming more and more widespread. It was found that delivery as plasmid usually produces more off-targets than delivery as RNPs, since the CRISPR-Cas system is active for a shorter time without Cas9 transcription and translation stages [241], [242].

Nowadays, how to effectively deliver CRISPR-Cas components to specific cells, tissues and organs for precisely directed genome editing is still a major problem in gene therapy. Ideal delivery vectors should have the advantages of non-toxicity, well targeting property, high efficiency, low cost, and biodegradability [35], [156]. At present, three main delivery methods have been employed in delivering CRISPR-Cas components, including physical, viral and non-viral methods [243]. Physical methods are the simplest way to deliver CRISPR-Cas components, including electroporation, microinjection and mechanical cell deformation. They are simple and efficient, which can also improve the expression of genes, and being widely applied in in vitro experiments [243], [244]. In addition, viral vectors, such as adenovirus, adeno-associated virus (AAV) and lentivirus viral vectors, are being widely used for both in vitro/ex vivo and in vivo delivery due to their high delivery efficiency. They are commonly used for gene delivery in gene therapy, and some of them have been approved for clinical use [245], [246]. However, safety issue of viral vectors is still a major problem needed to be solved in pre-clinical trials. Therefore, researchers have turned their attention to non-viral vectors, for instance, liposomes, polymers and nanoparticles [247]. Based on the advantages of safety, availability and cost-effectiveness, they are becoming a hotspot for the delivery of CRISPR-Cas components [248].

Since all these delivery methods have both advantages and disadvantages, its necessary to design a complex of viral vectors and non-viral vectors, which combines the advantages of both vectors. Along with the deepening of research, various carriers could be modified by different methods to increase the delivery efficiency and reduce the toxicity [249]. In addition, more novel vectors, such as graphene and carbon nanomaterials (CNMs), could also be applied in the delivery of CRISPR-Cas components [250], [251].

Since the components of CRISPR-Cas systems are derived from bacteria, host immune response to Cas gene and Cas protein is regarded as one of the most important challenges in the clinical trials of CRISPR-Cas system [156], [252]. It was found that in vivo delivery of CRISPR-Cas components can elicit immune responses against the Cas protein [252], [253]. Furthermore, researchers also found that there were anti-Cas9 antibodies and anti-Cas9 T cells existing in healthy humans, suggesting the pre-existing of humoral and celluar immune responses to Cas9 protein in humans [254]. Therefore, how to detect and reduce the immunogenicity of Cas proteins is a major challenge will be faced in clinical application of CRISPR-Cas systems. Researchers are trying to handle this problem by modifying Cas9 protein or using Cas9 homologues [255].

Recently, two independent research groups found that CRISPR-Cas mediated double-stranded breaks (DSBs) can activate the p53 signaling pathway [256], [257]. This means that genetically edited cells are likely to become potential cancer initiating cells, and clinical treatment with CRISPR-Cas systems might inadvertently increase the risk of cancer [256], [257], [258]. Although there is still no direct evidence to confirm the relationship between CRISPR-Cas mediated genome editing and carcinogenesis, these studies once again give a warning on the application of CRISPR-Cas systems in gene therapy. It reminds us that there is still a long way to go before CRISPR-Cas systems could be successfully applied to humans.

CRISPR-Cas mediated genome editing has attracted much attention since its advent in 2012. In theory, each gene can be edited by CRISPR-Cas systems, even genes in human germ cells [259]. However, germline gene editing is forbidden in many countries including China, for it could have unintended consequences and bring ethical and safety concerns [260].

However, in March 2015, a Chinese scientist, Junjiu Huang, published a paper about gene editing in human tripronuclear zygotes in the journal Protein & Cell, which brings the ethical controversy of human embryo gene editing to a climax [261]. Since then, genome editing has been challenged by ethics and morality, and legal regulation of genome editing has triggered a heated discussion all around the world.

Then, on Nov. 28, 2018, the day before the opening of the second international human genome editing summit, Jiankui He, a Chinese scientist from the Southern University of Science and Technology, announced that a pair of gene-edited babies, named Lulu and Nana, were born healthy in China this month. They are the worlds first gene-edited babies, whose CCR5 gene has been modified, making them naturally resistant to HIV infection after birth [262]. The announcement has provoked shock, even outrage among scientists around the world, causing widespread controversy in the application of genome editing.

The society was shocked by this breaking news, for it involves genome editing in human embryos and propagating into future generations, triggering a chorus of criticism from the scientific community and bringing concerns about ethics and security in the use of genome editing. Therefore, scientists call on Chinese government to investigate the matter fully and establish strict regulations on human genome editing. Global supervisory system is also needed to ensure genome editing of human embryos moving ahead safely and ethically [263].

Since CRISPR-Cas mediated genome editing technologies have provided an accessible and adaptable means to alter, regulate, and visualize genomes, they are thought to be a major milestone for molecular biology in the 21st century. So far, CRISPR-Cas systems have been broadly applied in gene function analysis, human gene therapy, targeted drug development, animal model construction and livestock breeding, which fully prove their great potential for further development. However, there are still some limitations to overcome in the practical applications of CRISPR-Cas systems, and great efforts still need to be made to evaluate their long-term safety and effectiveness.

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