Addgene: CRISPR Guide
Posted: October 7, 2018 at 4:43 am
Generating a Knockout Using CRISPR
You can use CRISPR to generate knockout cells or animals by co-expressing an endonuclease like Cas9 or Cpf1 and a gRNA specific to the gene to be targeted. The genomic target can be any 20 nucleotide DNA sequence, provided it meets two conditions:
The PAM sequence is essential for target binding, but the exact sequence depends on which Cas protein you use. We'll use the popular S. pyogenes Cas9 (SpCas9) as an example, but check out our list of additional Cas proteins and PAM sequences. Once expressed, the Cas9 protein and the gRNA form a ribonucleoprotein complex through interactions between the gRNA scaffold and surface-exposed positively-charged grooves on Cas9. Cas9 undergoes a conformational change upon gRNA binding that shifts the molecule from an inactive, non-DNA binding conformation into an active DNA-binding conformation. Importantly, the spacer region of the gRNA remains free to interact with target DNA.
Cas9 will only cleave a given locus if the gRNA spacer sequence shares sufficient homology with the target DNA. Once the Cas9-gRNA complex binds a putative DNA target, the seed sequence (8-10 bases at the 3 end of the gRNA targeting sequence) will begin to anneal to the target DNA. If the seed and target DNA sequences match, the gRNA will continue to anneal to the target DNA in a 3 to 5 direction. The zipper-like annealing mechanics of Cas9 may explain why mismatches between the target sequence in the 3 seed sequence completely abolish target cleavage, whereas mismatches toward the 5 end distal to the PAM often still permit target cleavage.
The Cas9 nuclease has two functional endonuclease domains: RuvC and HNH. Cas9 undergoes a second conformational change upon target binding that positions the nuclease domains to cleave opposite strands of the target DNA. The end result of Cas9-mediated DNA cleavage is a double-strand break (DSB) within the target DNA (3-4 nucleotides upstream of the PAM sequence).
The resulting DSB is then repaired by one of two general repair pathways:
The NHEJ repair pathway is the most active repair mechanism, and it frequently causes small nucleotide insertions or deletions (indels) at the DSB site. The randomness of NHEJ-mediated DSB repair has important practical implications, because a population of cells expressing Cas9 and a gRNA will result in a diverse array of mutations (for more information, jump to Plan Your Experiment.) In most cases, NHEJ gives rise to small indels in the target DNA that result in amino acid deletions, insertions, or frameshift mutations leading to premature stop codons within the open reading frame (ORF) of the targeted gene. The ideal end result is a loss-of-function mutation within the targeted gene. However, the strength of the knockout phenotype for a given mutant cell must be validated experimentally. Learn more about non-homologous end joining (NHEJ).
Browse Plasmids: Double-Strand Break (Cut)
CRISPR specificity is partially determined by how specific the gRNA targeting sequence is for the genomic target compared to the rest of the genome. Ideally, a gRNA targeting sequence will have perfect homology to the target DNA with no homology elsewhere in the genome. Realistically, a given gRNA targeting sequence will have additional sites throughout the genome where partial homology exists. These sites are called off-targets and need to be considered when designing a gRNA for your experiment (see the Plan Your Experiment section below).
In addition to optimizing gRNA design, CRISPR specificity can also be increased through modifications to Cas9. As discussed previously, Cas9 generates double-strand breaks (DSBs) through the combined activity of two nuclease domains, RuvC and HNH. Cas9 nickase, a D10A mutant of SpCas9, retains one nuclease domain and generates a DNA nick rather than a DSB.
Thus, two nickases targeting opposite DNA strands are required to generate a DSB within the target DNA (often referred to as a double nick or dual nickase CRISPR system). This requirement dramatically increases target specificity, since it is unlikely that two off-target nicks will be generated within close enough proximity to cause a DSB. Therefore, if high specificity is crucial to your experiment, you might consider using the dual nickase approach to create a double nick-induced DSB. The nickase system can also be combined with HDR-mediated gene editing for specific gene edits.
In 2015, researchers used rational mutagenesis to develop two high fidelity Cas9s: eSpCas9(1.1) and SpCas9-HF1. eSpCas9(1.1) contains alanine substitutions that weaken the interactions between the HNH/RuvC groove and the non-target DNA strand, preventing strand separation and cutting at off-target sites. Similarly, SpCas9-HF1 lowers off-target editing through alanine substitutions that disrupt Cas9's interactions with the DNA phosphate backbone. HypaCas9, developed in 2017, contains mutations in the REC3 domain that increase Cas9 proofreading and target discrimination. All three high fidelity enzymes generate less off-target editing than wildtype Cas9.
Browse Plasmids: Single-Strand Break (Nick)
While NHEJ-mediated DSB repair often disrupts the open reading frame of the gene, homology directed repair (HDR) can be used to generate specific nucleotide changes ranging from a single nucleotide change to large insertions like the addition of a fluorophore or tag.
In order to utilize HDR for gene editing, a DNA repair template containing the desired sequence must be delivered into the cell type of interest with the gRNA(s) and Cas9 or Cas9 nickase. The repair template must contain the desired edit as well as additional homologous sequence immediately upstream and downstream of the target (termed left & right homology arms.) The length of each homology arm is dependent on the size of the change being introduced, with larger insertions requiring longer homology arms.
Depending on the application, the repair template may be a single-stranded oligonucleotide, double-stranded oligonucleotide, or a double-stranded DNA plasmid. When designing the repair template, do not include the PAM sequence present in the genomic DNA. This step prevents the repair template from being a suitable target for Cas9 cleavage. For example, you could alter the DNA sequence of the PAM with a silent mutation that does not change the amino acid sequence.
The efficiency of HDR is generally low (<10% of modified alleles) even in cells that express Cas9, gRNA and an exogenous repair template. For this reason, many laboratories try to enhance HDR by synchronizing the cells, since HDR takes place during the S and G2 phases of the cell cycle. Chemically or genetically inhibiting genes involved in NHEJ may also increase HDR frequency.
Since the efficiency of Cas9 cleavage is relatively high and the efficiency of HDR is relatively low, a large portion of the Cas9-induced DSBs will be repaired via NHEJ. In other words, the resulting population of cells will contain some combination of wild-type alleles, NHEJ-repaired alleles, and/or the desired HDR-edited allele. Therefore, it is important to confirm the presence of the desired edit experimentally and to isolate clones containing the desired edit (see the validation section in Plan Your Experiment). Learn more about homology directed repair (HDR).
Browse Plasmids: Endogenous Tagging
As discussed above, the efficiency of HDR is very low due to the number of DSBs repaired by NHEJ. To make point mutations without using HDR, researchers have developed CRISPR base editors that fuse Cas9 nickase or dCas9 to a cytidine deaminase like APOBEC1. Base editors are targeted to a specific locus by a gRNA, and they can convert cytidine to uridine within a small editing window near the PAM site. Uridine is subsequently converted to thymidine through base excision repair, creating a C->T change (or G->A on the opposite strand.) This class of base editors is available with multiple Cas9 variants and using high fidelity Cas9s. In addition, new base editors have been engineered to convert adenosine to inosine, which is treated like guanosine by the cell, creating an A->G (or T->C) change. Learn more about CRISPR DNA base editors.
Browse Plasmids: Base Edit
Type VI CRISPR systems, including the enzymes Cas13a/C2c2 and Cas13b, target RNA rather than DNA. Fusing an ADAR2(E488Q) adenosine deaminase to catalytically dead Cas13b creates a programmable RNA base editor that converts adenosine to inosine in RNA (termed REPAIR.) Since inosine is functionally equivalent to guanosine, the result is an A->G change in RNA. dPspCas13b does not appear to require a specific sequence adjacent to the RNA target, making this a very flexible editing system. Editors based on ADAR2(E488Q/T375G) display improved specificity, and editors carrying the delta-984-1090 ADAR truncation retain RNA editing capabilities and are small enough to be packaged in AAV particles.
Browse Plasmids: RNA Editing
CRISPR is a remarkably flexible tool for genome manipulation, as Cas enzymes bind target DNA independently of their ability to cleave target DNA. Specifically, both RuvC and HNH nuclease domains can be rendered inactive by point mutations (D10A and H840A in SpCas9), resulting in a nuclease dead Cas9 (dCas9) molecule that cannot cleave target DNA. The dCas9 molecule retains the ability to bind to target DNA based on the gRNA targeting sequence.
Early experiments demonstrated that targeting dCas9 to transcription start sites was sufficient to repress transcription by blocking initiation. dCas9 can also be tagged with transcriptional repressors or activators, and targeting these dCas9 fusion proteins to the promoter region results in robust transcriptional repression or activation of downstream target genes. The simplest dCas9-based activators and repressors consist of dCas9 fused directly to a single transcriptional activator, A (e.g. VP64) or transcriptional repressor, R (e.g. KRAB; see panel A to the right).
Additionally, more elaborate activation strategies have been developed for more potent activation of target genes in mammalian cells. These include: co-expression of epitope-tagged dCas9 and antibody-activator effector proteins (e.g. SunTag system, panel B), dCas9 fused to several different activation domains in series (e.g. dCas9-VPR, panel C) or co-expression of dCas9-VP64 with a modified scaffold gRNA and additional RNA-binding helper activators (e.g. SAM activators, panel D). Importantly, unlike the genome modifications induced by Cas9 or Cas9 nickase, dCas9-mediated gene activation or repression is reversible, since it does not permanently modify the genomic DNA.
Browse Plasmids: Activate, Repress/Interfere
Cas enzymes can be fused to epigenetic modifiers like p300 and TET1 to create programmable epigenome-engineering tools. Like CRISPR activators and repressors, these tools alter gene expression without inducing double-strand breaks. However, they are much more specific for particular chromatin and DNA modifications, allowing researchers to isolate the effects of a single epigenetic mark.
Another potential advantage of CRISPR epigenetic tools is their persistence and inheritance. CRISPR activators and repressors are thought to be reversible once the effector is inactivated/removed from the system. In contrast, epigenetic marks left by targeted epigenetic modifiers may be more frequently inherited by daughter cells. In certain cases, epigenetic modifiers may work better than activators/repressors in modulating transcription. However, since the effects of these tools are likely cell type- and context-dependent, it might be beneficial to try multiple CRISPR strategies when setting up your experimental system.
Browse Plasmids: Epigenetics
Expressing several gRNAs from the same plasmid ensures that each cell containing the plasmid will express all of the desired gRNAs, thus increasing the likelihood that all desired genomic edits will be carried out by Cas9. Such multiplex CRISPR applications include:
Current multiplex CRISPR systems enable researchers to target anywhere from 2 to 7 genetic loci by cloning multiple gRNAs into a single plasmid. These multiplex gRNA vectors can conceivably be combined with any of the aforementioned CRISPR derivatives to not only knock out target genes, but activate or repress target genes as well. Read more about Cas9 multiplexing and Cpf1 multiplexing.
Browse Plasmids: Multiplex gRNA vectors
The ease of gRNA design and synthesis, as well as the ability to target almost any genomic locus, make CRISPR the ideal genome engineering system for large-scale forward genetic screening. Forward genetic screens are particularly useful for studying diseases or phenotypes for which the underlying genetic cause is not known. In general, the goal of a genetic screen is to generate a large population of cells with mutations in a wide variety of genes and then use these mutant cells to identify the genetic perturbations that result in a desired phenotype.
Before CRISPR, genetic screens relied heavily on shRNA technology, which is prone to off-target effects and false negatives due to incomplete knockdown of target genes. In contrast, CRISPR is capable of making highly specific, permanent genetic modifications that are more likely to ablate target gene function. CRISPR has already been used extensively to screen for novel genes that regulate known phenotypes, including resistance to chemotherapy drugs, resistance to toxins, cell viability, and tumor metastasis. Currently, the most popular method for conducting genome-wide screens using CRISPR involves the use of pooled lentiviral CRISPR libraries.
Pooled lentiviral CRISPR libraries (often referred to as CRISPR libraries) are a heterogenous population of lentiviral transfer vectors, each containing an individual gRNA targeting a single gene in a given genome.
Guide RNAs are designed in silico and synthesized (see panel A below), then cloned in a pooled format into lentiviral transfer vectors (panel B). CRISPR libraries have been designed for common CRISPR applications including genetic knockout, activation, and repression for human and mouse genes.
Each CRISPR library is different, as libraries can target anywhere from a single class of genes up to every gene in the genome. However, there are several features that are common across most CRISPR libraries. First, each library typically contains 3-6 gRNAs per gene to ensure modification of every target gene, so CRISPR libraries contain thousands of unique gRNAs targeting a wide variety of genes. Guide RNA design for CRISPR libraries is usually optimized to select for guide RNAs with high on-target activity and low off-target activity.
Keep in mind that the exact region of the gene to be targeted varies depending on the specific application. For example, knockout libraries often target 5 constitutively expressed exons, but activation and repression libraries will target promoter or enhancer regions. Be sure to check the library page/original publication to see if a library is suitable for your experiment. Libraries may be available in a 1-plasmid system, in which Cas9 is included on the gRNA-containing plasmid, or a 2-plasmid system in which Cas9 must be delivered separately.
CRISPR libraries from Addgene are available in two formats: as DNA, or in select cases, as pre-made lentivirus.
In the case of DNA libraries, the CRISPR library will be shipped at a concentration that is too low to be used in experiments. Thus, the first step in using your library is to amplify the library (panel C) to increase the total amount of DNA. When amplifying the library, it is important to maintain good representation of gRNAs so that the composition of your amplified library matches that of the original library. You'll use next-generation sequencing (NGS) to verify that this is the case. Learn more about library verification.
Once the library has been amplified/verified, the next step is to generate lentivirus containing the entire CRISPR library (panel D). Then, you will transduce cells with the lentiviral library (panel E). Remember - if you are using a 2-vector system, you will transduce cells that are already expressing Cas9. After applying your screening conditions, you will look for relevant genes (hits) using NGS technology. For more detail on using CRISPR for both positive and negative screens, see our pooled library guide.
As noted above, forward genetic screens are most useful for situations in which the physiology or cell biology behind a particular phenotype or disease is well understood, but the underlying genetic causes are unknown. Therefore, genome-wide screens using CRISPR libraries are a great way to gather unbiased information regarding which genes play a causal role in a given phenotype. With any experiment, it is important to verify that the hits you identify are actually important for your phenotype of interest. You can individually test the gRNAs identified in your screen to ensure that they reproduce the phenotype of interest.
Find more information on factors to consider before starting your pooled library experiment in Practical Considerations for Using Pooled Lentiviral CRISPR Libraries (McDade et al., 2016).
Browse Libraries: CRISPR Pooled Libraries
Using catalytically inactive Cas9 (dCas9) fused to a fluorescent marker like GFP, researchers have turned dCas9 into a customizable DNA labeler compatible with fluorescence microscopy in living cells. Alternatively, gRNAs can be fused to protein-interacting RNA aptamers, which recruit specific RNA-binding proteins (RBPs) tagged with fluorescent proteins to visualize targeted genomic loci.
CRISPR imaging has numerous advantages over other imaging techniques, including that it is easy to implement due to the simplicity of gRNA design, programmable for different genomic loci, capable of detecting multiple genomic loci, and compatible with live cell imaging. Compared to techniques like fluorescence in situ hybridization (FISH), CRISPR imaging offers a unique method for detecting the chromatin dynamics in living cells.
Multicolor CRISPR imaging allows for simultaneous tracking of multiple genomic loci in living cells. One method uses orthogonal dCas9s (e.g., S. pyogenes dCas9 and S. aureus dCas9) tagged with different fluorescent proteins. Alternatively, one can fuse gRNAs to orthogonal protein-interacting RNA aptamers, which recruit specific orthogonal RNA-binding proteins (RBPs) tagged with different fluorescent proteins, as seen in the popular CRISPRainbow kit.
The fluorescent CRISPR system has been used for dynamic tracking of repetitive and non-repetitive genomic loci, as well as chromosome painting in living cells. Visualizing a specific genomic locus requires recruitment of many copies of labeled proteins to the given region. For example, chromosome-specific repetitive loci can be efficiently visualized in living cells using a single gRNA that has multiple targeted sequences in close proximity. A non-repetitive genomic locus can also be labeled by co-delivering multiple gRNAs that tile the locus. Chromosome painting requires delivery of hundreds of gRNAs with target sites throughout the chromosome.
Browse Plasmids: Label
Identifying molecules associated with a genomic region of interest in vivo is essential to understanding locus function. Using CRISPR, researchers have expanded chromatin immunoprecipitation (ChIP) to allow purification of any genomic sequence specified by a particular gRNA.
In the enChIP (engineered DNA-binding molecule-mediated ChIP) system, catalytically inactive dCas9 is used to purify genomic DNA bound by the gRNA. An epitope tag(s) can be fused to dCas9 or gRNA for efficient purification. Various epitope tags including 3xFLAG-tag, PA, and biotin tags, can be used for enChIP, as well as an anti-Cas9 antibody. Biotin tagging of dCas9 can be achieved by fusing a biotin acceptor site to dCas9 and co-expressing BirA biotin ligase, as seen in the CAPTURE system. The locus is subsequently isolated by affinity purification against the epitope tag.
After purification of the locus, molecules associated with the locus can be identified by mass spectrometry (proteins), RNA-sequencing (RNAs), and next-generation sequencing (NGS) (other genomic regions). Compared to conventional methods for genomic purification, CRISPR-based purification methods are more straightforward and enable direct identification of molecules associated with a genomic region of interest in vivo.
Browse Plasmids: Purify
In bacteria, type VI CRISPR systems recognize ssRNA rather than dsDNA. Many type VI enzymes also have the ability to process crRNA precursors to mature crRNAs. Upon ssRNA recognition by the crRNA, the target RNA is degraded. In bacteria, Cas13 enzymes can also cleave RNAs nonspecifically after the initial crRNA-guided cleavage. This promiscuous cleavage activity slows bacterial cell growth and may further protect bacteria from viral pathogens. Non-specific cleavage does not occur in mammalian cells. Similar to Cas9 and Cpf1, Cas13 can be converted to an RNA-binding protein through mutation of its catalytic domain. Learn more about Cas13a.
Browse Plasmids: RNA Targeting
While S. pyogenes Cas9 (SpCas9) is certainly the most commonly used CRISPR endonuclease for genome engineering, it may not be the best endonuclease for every application. For example, the PAM sequence for SpCas9 (5 NGG 3) is abundant throughout the human genome, but an NGG sequence may not be positioned correctly to target your desired genes for modification. This limitation is of particular concern when trying to edit a gene using homology directed repair (HDR), which requires PAM sequences in very close proximity to the region to be edited. Kleinstiver et al. generated synthetic SpCas9-derived variants with non-NGG PAM sequences. Gao et al. subsequently engineered Cpf1 PAM variants. The inclusion of these variants into the CRISPR arsenal effectively doubles the targeting range of CRISPR in the human genome. Read more about Cas9 variants.
Additional Cas9 orthologs from various species bind a variety of PAM sequences. These enzymes may have other characteristics that make them more useful than SpCas9 for specific applications. For example, the relatively large size of SpCas9 (4kb coding sequence) means that plasmids carrying the SpCas9 cDNA cannot be efficiently packaged into adeno-associated virus (AAV). Since the coding sequence for Staphylococcus aureus Cas9 (SaCas9) is 1 kilobase shorter than SpCas9, SaCas9 can be efficiently packaged into AAV. Similar to SpCas9, the SaCas9 endonuclease is capable of modifying target genes in mammalian cells in vitro and in mice in vivo.
Another limitation of SpCas9 is the low efficiency of making specific genetic edits via HDR. For specific point edits, CRISPR base editing is a useful alternative to HDR. For larger edits, Cpf1, first described by Zetsche et al., may be a better option. Unlike Cas9 nucleases, which create blunt DSBs, Cpf1-mediated DNA cleavage creates DSBs with a short 3 overhang. Cpf1s staggered cleavage pattern opens up the possibility of directional gene transfer, analogous to traditional restriction enzyme cloning, which may increase the efficiency of gene editing. Like the Cas9 variants and orthologs described above, Cpf1 also expands the range of sites that can be targeted by CRISPR to AT-rich regions or AT-rich genomes that lack the NGG PAM sites favored by SpCas9.
CRISPR is a powerful system that enables researchers to manipulate the genome like never before. This section will provide a general framework to get you started using CRISPR in your research. Although we will use the example of CRISPR/Cas9 in mammalian cells, many of these principles apply to using CRISPR in other organisms. First, consider the genetic manipulation that is necessary to model your specific disease or process of interest. Do you want to:
Once you have a clear understanding of your experimental goal, you are ready to start navigating the different reagents that are available for your particular experiment.
Different genetic manipulations require different CRISPR components. Selecting a specific genetic manipulation can be a good way to narrow down which reagents are appropriate for a given experiment. Make sure to check whether reagents are available to carry out your experiment in your particular model organism. There may not be a perfect plasmid for your specific application, and in such cases, it may be necessary to customize an existing reagent to suit your needs.
To use CRISPR, you will need both Cas9 and a gRNA expressed in your target cells. For easy-to-transfect cell types (e.g. HEK293 cells), transfection with standard transfection reagents may be sufficient to express the CRISPR machinery. For more difficult cells (e.g. primary cells), viral delivery of CRISPR reagents may be more appropriate. In cases where off-target editing is a major concern, Cas9-gRNA ribonucleoprotein (RNP) complexes are advantageous due to the transient Cas9 expression.
The table below summarizes the major expression systems and variables for using CRISPR in mammalian cells. Some of the variables include:
Once you have selected your CRISPR components and method of delivery, you are ready to select a target sequence and design your gRNA.
When possible, you should sequence the region you are planning to modify prior to designing your gRNA, as sequence variation between your gRNA targeting sequence and target DNA may result in reduced cleavage. The number of alleles for each gene may vary depending on the specific cell line or organism, which may affect the observed efficiency of CRISPR knockout or knockin.
In order to manipulate a given gene using CRISPR, you will have to identify the genomic sequence for the gene you are trying to target. However, the exact region of the gene you target will depend on your specific application. For example:
A PAM sequence is absolutely necessary for Cas9 to bind target DNA. As such, one can start by identifying all PAM sequences within the genetic region to be targeted. If there are no PAM sequences for your chosen enzyme within your desired sequence, you may want to consider alternative Cas enzymes (see Cas9 variants and PAM sequences). Once possible PAM sequences and putative target sites have been identified, it is time to choose which site is likely to result in the most efficient on-target cleavage.
The gRNA target sequence needs to match the target locus, but it is also critical to ensure that the gRNA target sequence does NOT match additional sites within the genome. In a perfect world, your gRNA target sequence would have perfect homology to your target with no homology elsewhere in the genome. Realistically, a given gRNA target sequence will have partial homology to additional sites throughout the genome. These sites are called off-targets and should be examined during gRNA design. In general, off-target sites are not cleaved as efficiently when mismatches occur near the PAM sequence, so gRNAs with no homology or those with mismatches close to the PAM sequence will have the highest specificity. To increase specificity, you can also consider using a high-fidelity Cas enzyme.
In addition to off-target activity, it is also important to consider factors that maximize cleavage of the desired target sequence or on-target activity. Two gRNA targeting sequences with 100% homology to their DNA targets may not result in equivalent cleavage efficiency. In fact, cleavage efficiency may increase or decrease depending upon the specific nucleotides within the selected target sequence. For example, gRNA targeting sequences containing a G nucleotide at position 20 (1 bp upstream of the PAM) may be more efficacious than gRNAs containing a C nucleotide at the same position in spite of being a perfect match for the target sequence.
Many gRNA design programs can locate potential PAM and target sequences and rank the associated gRNAs based on their predicted on-target and off-target activity (see gRNA design software). Additionally, many plasmids containing validated gRNAs are now available through Addgene. These plasmids contain gRNAs that have been used successfully in genome engineering experiments. Using validated gRNAs can save your lab valuable time and resources when carrying out CRISPR experiments. Read more about how to design your gRNA.
Browse Plasmids: Validated gRNAs
Once you have selected your target sequences it is time to design your gRNA oligos and clone these oligos into your desired vector. In many cases, targeting oligos are synthesized, annealed, and inserted into plasmids containing the gRNA scaffold using standard restriction-ligation cloning. However, the exact cloning strategy will depend on the gRNA vector you have chosen, so it is best to review the protocol associated with the specific plasmid in question (see CRISPR protocols from Addgene depositors).
Choose a delivery method that is compatible with your experimental system. CRISPR efficiency will vary based on the method of delivery and the cell type. Before proceeding with your experiment, it may be necessary to optimize your delivery conditions. Learn more about CRISPR delivery in mammalian systems.
Once you have successfully delivered the gRNA and Cas enzyme to your target cells, it is time to validate your genome edit. CRISPR editing produces several possible genotypes within the resulting cell population. Some cells may be wild-type due to either (1) a lack of gRNA and/or Cas9 expression or (2) a lack of efficient target cleavage in cells expressing both Cas9 and gRNA.
Edited cells may be homozygous or heterozygous for edits at your target locus. Furthermore, in cells containing two mutated alleles, each mutated allele may be different owing to the error-prone nature of NHEJ. In HDR gene editing experiments, most mutated alleles will not contain the desired edit, as a large percentage of DSBs are still repaired by NHEJ.
How do you determine that your desired edit has occurred? The exact method necessary to validate your edit will depend upon your specific application. However, there are several common ways to verify that your cells contain your desired edit, including but not limited to:
More information on each of these techniques can be found in our blog post CRISPR 101: Validating your Genome Edit.
The majority of the CRISPR plasmids in Addgenes collection are from S. pyogenes unless otherwise noted.
Engineered Cpf1 variants with altered PAM specificities. 2017. Gao L, Cox DBT, Yan WX, Manteiga JC, Schneider MW, Yamano T, Nishimasu H, Nureki O, Crosetto N, Zhang F. Nat Biotechnol. 35(8):789-792. PMID: 28581492
Enhanced proofreading governs CRISPR-Cas9 targeting accuracy. 2017. Chen JS, Dagdas YS, Kleinstiver BP, Welch MM, Sousa AA, Harrington LB, Sternberg SH, Joung JK, Yildiz A, Doudna JA. Nature. 550(7676):407-410. PMID: 28931002
Enhanced proofreading governs CRISPR-Cas9 targeting accuracy. 2017. Chen JS, Dagdas YS, Kleinstiver BP, Welch MM, Sousa AA, Harrington LB, Sternberg SH, Joung JK, Yildiz A, Doudna JA. Nature. 550(7676):407-410. PMID: 28931002
Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity. 2017. Komor AC, Zhao KT, Packer MS, Gaudelli NM, Waterbury AL, Koblan LW, Kim YB, Badran AH, Liu DR. Sci Adv. 3(8):eaao4774. PMID: 28875174
Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery. 2017. Rees HA, Komor AC, Yeh WH, Caetano-Lopes J, Warman M, Edge ASB, Liu DR. Nat Commun. 8:15790. PMID: 28585549
Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions. 2017. Kim YB, Komor AC, Levy JM, Packer MS, Zhao KT, Liu DR. Nat Biotechnol. 35(4):371-376. PMID: 28191901
Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array. 2017. Zetsche B, Heidenreich M, Mohanraju P, Fedorova I, Kneppers J, DeGennaro EM, Winblad N, Choudhury SR, Abudayyeh OO, Gootenberg JS, Wu WY, Scott DA, Severinov K, van der Oost J, Zhang F. Nat Biotechnol. 35(1):31-34. PMID: 27918548
Nucleic acid detection with CRISPR-Cas13a/C2c2. 2017. Gootenberg JS, Abudayyeh OO, Lee JW, Essletzbichler P, Dy AJ, Joung J, Verdine V, Donghia N, Daringer NM, Freije CA, Myhrvold C, Bhattacharyya RP, Livny J, Regev A, Koonin EV, Hung DT, Sabeti PC, Collins JJ, Zhang F. Science. 356(6336):438-442. PMID: 28408723
Programmable base editing of AT to GC in genomic DNA without DNA cleavage. 2017. Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, Liu DR. Nature. 551(7681):464-471. PMID: 29160308
RNA editing with CRISPR-Cas13. 2017. Cox DBT, Gootenberg JS, Abudayyeh OO, Franklin B, Kellner MJ, Joung J, Zhang F. Science. pii: eaaq0180. PMID: 29070703
RNA targeting with CRISPR-Cas13. 2017. Abudayyeh OO, Gootenberg JS, Essletzbichler P, Han S, Joung J, Belanto JJ, Verdine V, Cox DBT, Kellner MJ, Regev A, Lander ES, Voytas DF, Ting AY, Zhang F. . 550(7675):280-284. PMID: 28976959
High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. 2016. Kleinstiver BP, Pattanayak V, Prew MS, Tsai SQ, Nguyen NT, Zheng Z, Joung JK. Nature. 529(7587):490-5. PMID: 26735016
Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow. 2016. Ma H, Tu LC, Naseri A, Huisman M, Zhang S, Grunwald D, Pederson T. Nat Biotechnol. . PMID: 27088723
Naturally occurring off-switches for CRISPR-Cas9. 2016. Pawluk A, Amrani N, Zhang Y, Garcia B, Hidalgo-Reyes Y, Lee J, Edraki A, Shah M, Sontheimer EJ, Maxwell KL, Davidson AR. Cell. 167(7):1829-1838. PMID: 27984730
Practical Considerations for Using Pooled Lentiviral CRISPR Libraries. 2016. McDade JR, Waxmonsky NC, Swanson LE, Fan M. Curr Protoc Mol Biol. 115:31.5.1-31.5.13. PMID: 27366891
Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. 2016. Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR. Nature. 533(7603):420-4. PMID: 27096365
Rationally engineered Cas9 nucleases with improved specificity. 2016. Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F. Science. 351(6268):84-8. PMID: 26628643
A Scalable Genome-Editing-Based Approach for Mapping Multiprotein Complexes in Human Cells. 2015. Dalvai M, Loehr J, Jacquet K, Huard CC, Roques C, Herst P, Ct J, Doyon Y. Cell Rep. 13(3):621-33. PMID: 26456817
An updated evolutionary classification of CRISPR-Cas systems. 2015. Makarova KS, Wolf YI, Alkhnbashi OS, Costa F, Shah SA, Saunders SJ, Barrangou R, Brouns SJ, Charpentier E, Haft DH, Horvath P, Moineau S, Mojica FJ, Terns RM, Terns MP, White MF, Yakunin AF, Garrett RA, van der Oost J, Backofen R, Koonin EV. Nat Rev Microbiol. 13(11):722-36. PMID: 26411297
Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. 2015. Zuris JA, Thompson DB, Shu Y, Guilinger JP, Bessen JL, Hu JH, Maeder ML, Joung JK, Chen ZY, Liu DR. Nat Biotechnol. 33(1):73-80. PMID: 25357182
CETCh-seq: CRISPR epitope tagging ChIP-seq of DNA-binding proteins. 2015. Savic D, Partridge EC, Newberry KM, Smith SB, Meadows SK, Roberts BS, Mackiewicz M, Mendenhall EM, Myers RM. Genome Res. 25(10):1581-9. PMID: 26355004
Read this article:
Addgene: CRISPR Guide
- What's the Latest in CRISPR Gene-Editing Technology? - Technology Networks - March 12th, 2024
- In vivo genome-wide CRISPR screening identifies CITED2 as a driver of prostate cancer bone metastasis | Oncogene - Nature.com - March 12th, 2024
- Investigating the mechanisms underlying resistance to chemoterapy and to CRISPR-Cas9 in cancer cell lines ... - Nature.com - March 12th, 2024
- Here's Why CRISPR Therapeutics Stock Climbed 34% in February - The Motley Fool - March 12th, 2024
- SXSW Panel Recap: The First CRISPR Foods Have Arrived - Austin Chronicle - March 12th, 2024
- CRISPR-Cas systems: Overview, innovations and applications in human ... - March 4th, 2024
- CRISPR Therapeutics Stock Has 32% Upside, According to 1 Wall Street Analyst - The Motley Fool - March 4th, 2024
- Missed Out on CRISPR Therapeutics? My Best Gene-Editing Stock to Buy and Hold - The Motley Fool - March 4th, 2024
- MEGA-CRISPR tool gives a power boost to cancer-fighting cells - Nature.com - February 23rd, 2024
- 3 Biotech Stocks to Buy That Have CRISPR-Like Breakthrough Potential - InvestorPlace - February 23rd, 2024
- CRISPR 'will provide cures for genetic diseases that were incurable before,' says renowned biochemist Virginijus iknys - Livescience.com - February 23rd, 2024
- Opinion: The Promise and Challenges of CRISPR-Based Treatments - BioSpace - February 23rd, 2024
- There's Reason For Concern Over CRISPR Therapeutics AG's (NASDAQ:CRSP) Massive 26% Price Jump - Simply Wall St - February 23rd, 2024
- Move over, CRISPR: RNA-editing therapies pick up steam - Nature.com - February 23rd, 2024
- If You Invested $10000 in CRISPR Therapeutics in 2019, This Is How Much You Would Have Today - The Motley Fool - February 23rd, 2024
- CRISPR Therapeutics Joins Rank Of Stocks With 95-Plus Composite Rating - Investor's Business Daily - February 23rd, 2024
- CRISPR Therapeutics (NASDAQ:CRSP) Hits New 12-Month High on Better-Than-Expected Earnings - AmericanBankingNEWS - February 23rd, 2024
- CRISPR Therapeutics Provides Business Update and Reports Fourth Quarter and Full Year 2023 Financial Results - GlobeNewswire - February 23rd, 2024
- CRISPR Therapeutics AG (CRSP) Moves 6.9% Higher: Will This Strength Last? - Yahoo Finance - February 23rd, 2024
- Advancements in RNA for HIV Treatment: CRISPR Cas9, mRNA Therapeutics, and Next-Generation Sequencing ... - Medriva - February 23rd, 2024
- Intellia Therapeutics Charges Ahead: A Glimpse into the Future of CRISPR-Based Therapies - BNN Breaking - February 23rd, 2024
- The FDA Approved The First CRISPR-Based Therapy. What's Next? - Science Friday - February 5th, 2024
- Using CRISPR technology, researchers succeed in growing tomatoes that consume less water without compromising yield - Phys.org - February 5th, 2024
- CRISPR-Cas9 gene-editing tool repairs defective T cells to treat rare hereditary disease - News-Medical.Net - February 5th, 2024
- New CRISPR Technology Increases Recognition of Cancer Cells by the Immune System - Inside Precision Medicine - February 5th, 2024
- Is CRISPR Therapeutics a Buy in the New Bull Market? - The Motley Fool - February 5th, 2024
- Stocks Flashing Renewed Technical Strength: CRISPR Therapeutics - Investor's Business Daily - February 5th, 2024
- AI at Davos, new CRISPR therapies and health tech's bad marketing - Marketplace - January 20th, 2024
- FDA expands use of newly approved CRISPR therapy - Axios - January 20th, 2024
- CRISPR-based therapy receives expanded approval for beta thalassemia - STAT - January 20th, 2024
- CRISPR Therapeutics And Vertex's CRISPR Breakthrough: How And Why They Got There First - Scrip - January 20th, 2024
- Pharmalittle: We're reading about a CRISPR approval, selling meds directly to patients, and more - STAT - January 20th, 2024
- Here's Why CRISPR Therapeutics Stock Rose 54% Last Year - The Motley Fool - January 20th, 2024
- Vertex's CRISPR Gene Therapy Lands Another FDA Nod in a Rare Blood Disease - MedCity News - January 20th, 2024
- Groundbreaking CRISPR/Cas9-based Genome Editing Therapy Secured the Second FDA Approval - geneonline - January 20th, 2024
- What Does It Mean for Investors if CRISPR Therapeutics Gets Bought Out in 2024? - The Motley Fool - January 20th, 2024
- First FDA-approved CRISPR-based gene therapy cleared for 2nd indication - LabPulse - January 20th, 2024
- CRISPR Therapeutics Announces U.S. Food and Drug Administration (FDA) Approval of CASGEVY ... - GlobeNewswire - January 20th, 2024
- CRISPR Gene Editing And Its Role In Hematology | TheHealthSite.com - TheHealthSite - January 20th, 2024
- Doudna institute hatches plan to 'cure hundreds of diseases' left behind by CRISPR revolution - STAT - January 11th, 2024
- Discover the recent progress of nonviral delivery carriers for CRISPR/Cas9 systems - News-Medical.Net - January 11th, 2024
- How CRISPR could yield the next blockbuster crop - Nature.com - January 11th, 2024
- Weight-loss drugs, malaria vaccines and more: CRISPR innovations headline the science breakthroughs of 2023 - Genetic Literacy Project - January 11th, 2024
- CRISPR Therapeutics Highlights Strategic Priorities and 2024 Outlook - GlobeNewswire - January 11th, 2024
- CRISPR Therapeutics AG (CRSP) 42nd Annual J.P. Morgan Healthcare Conference (Transcript) - Seeking Alpha - January 11th, 2024
- CRISPR to be used to genetically modify crops - FoodNavigator.com - January 11th, 2024
- Casgevy approval unlikely to be followed up by another CRISPR drug in near future - BioPharma-Reporter.com - January 11th, 2024
- Vertex Announces Approval of First CRISPR/Cas9 Gene-Edited Therapy, CASGEVY, for the Treatment of Sickle Cell ... - Business Wire - January 11th, 2024
- The Science Behind CRISPR: Germline Genome Editing and Its Applications - Medriva - January 11th, 2024
- Here's Why 2024 Could Be a Big Year for CRISPR Therapeutics - The Motley Fool - January 11th, 2024
- Revolutionizing acne treatment with CRISPR technology - Labiotech.eu - January 11th, 2024
- What Is CRISPR Gene Editing and How Does It Work? - December 25th, 2023
- This first CRISPR treatment is just the beginning. Heres what's next - Fast Company - December 25th, 2023
- The Age of Crispr Medicine Is Here - WIRED - December 25th, 2023
- 6 Words That Explain Why CRISPR Stock Isn't Soaring Despite the Recent FDA Approval for Its Gene-Editing Therapy - Yahoo Finance - December 25th, 2023
- Crispr Therapeutics Medical Chief Morrow to Resign - The Wall Street Journal - December 25th, 2023
- Crispr Therapeutics chief medical officer is resigning - MarketWatch - December 25th, 2023
- 3 Reasons to Buy CRISPR Therapeutics Stock Like There's No Tomorrow - Yahoo Finance - December 25th, 2023
- CAR T Therapy May Cause Rare Cancer & How CRISPR Could Be The Solution - Forbes - December 25th, 2023
- CRSP Stock Alert: CRISPR Therapeutics Is Losing Its Medical Chief - InvestorPlace - December 25th, 2023
- With the promise of saving millions of lives, CRISPR medicine is born - EL PAS USA - December 25th, 2023
- Casgevy: the world's first CRISPR therapy - Epigram - December 25th, 2023
- The Basics of CRISPR Gene Editing - Cleveland Clinic Health Essentials - November 27th, 2023
- Mechanism and Applications of CRISPR/Cas-9-Mediated Genome Editing - November 27th, 2023
- What is CRISPR gene editing, and how does it work? - The Conversation - October 16th, 2023
- What is CRISPR/Cas9? - PMC - National Center for Biotechnology Information - October 16th, 2023
- CRISPR, 10 Years On: Learning to Rewrite the Code of Life - April 26th, 2023
- What Is CRISPR, and Why Is It So Important? - Scientific American - March 23rd, 2023
- Global CRISPR Technology Market Is Projected To Grow At A 22% Rate Through The Forecast Period - EIN News - March 14th, 2023
- What is CRISPR and why is it controversial? | CNN - February 2nd, 2023
- CRISPR | Description, Technology, Uses, & Ethical Concerns - February 2nd, 2023
- In vivo CRISPR screening reveals nutrient signaling processes ... - PubMed - December 12th, 2022
- What is CRISPR? | New Scientist - October 16th, 2022
- CRISPR-Cas9 Structures and Mechanisms - PubMed - October 16th, 2022
- A CRISPR cure for HIV? Gene-editing technology may be able stop viral replication in its tracks and wipe out infections - Genetic Literacy Project - October 16th, 2022
- Editas Medicine Presents Preclinical Data on EDIT-103 for Rhodopsin-associated Autosomal Dominant Retinitis Pigmentosa at the European Society of Gene... - October 16th, 2022
- More Foods Will Be Gene-Edited Than You Think - The Epoch Times - October 16th, 2022
- What is CRISPR? - MD Anderson Cancer Center - September 21st, 2022
- CRISPR infusion eliminates swelling in those with rare genetic disease - Science - September 21st, 2022
- Crispr Therapeutics becomes the latest biotech to open in the Seaport - The Boston Globe - September 21st, 2022