CRISPR Genome Editing Solutions

The CRISPR system can be used for a growing variety of applications, and each CRISPR approach requires at least two basic components: a Cas protein and guide RNA (gRNA). The most commonly used Cas endonuclease is engineered from Streptococcus pyogenes (a.k.a. SpCas9 or, with codon optimization for expression in human cells, hCas9). It is an RNA-guided DNA nuclease which can generate double-strand breaks (DSBs) at target sites (Figure 1). Cas9 is localized to a target site in the host genome by the complementary gRNA, and the DSB is introduced if the gRNA was designed to target a region immediately adjacent to a Protospacer Adjacent Motif (PAM). The PAM sequence is dependent on the particular Cas enzyme being used: for SpCas9, the PAM sequence is NGG or NAG (orange DNA in Figure 1).

Figure 1. Mechanisms of CRISPR-induced DNA repair to produce gene knockout or precise sequence changes.

Once DSBs are generated by the CRISPR system, cells most commonly activate the non-homologous end joining (NHEJ) pathway to repair the DNA breaks, which usually results in small random deletions, or more rarely insertions and base substitutions. When these mutations disrupt a protein-coding region (e.g. a deletion causing a frameshift), they may lead to functional gene knockout. Dual gRNAs can be introduced with Cas9 to create two DSBs, thereby deleting a DNA fragment located between the two target sites (Figure 4), or dual gRNAs can be used to target two different genes simultaneously.

Alternatively, and less efficiently, DSBs can be repaired via the homology-directed repair (HDR) pathway when a DNA template is present. When donor DNA is introduced with the CRISPR components, HDR can result in replacement of the target genomic DNA sequence with the donor sequence, generating precise changes such as point mutations or knockin. The donor DNA template can be a single-stranded oligo nucleotide (ssODN) or a dsDNA fragment (usually linearized DNA derived from either regular plasmid or AAV). ssODNs are suitable for introducing point mutations or small tag insertions, while dsDNA fragments are widely used to introduce large fragment knockins like targeted stable integration of transgenes.

Another widely used Cas9 variant, Cas9 “nickase” (e.g. Cas9(D10A)), generates single-stranded cuts in DNA. If Cas9 nickase is used in conjunction with two gRNAs targeting the two opposite strands flanking a single target region, the nickase will generate two single-stranded cuts, one on each strand, resulting in wide-spread DSBs around the target region as shown in Figure 2. Because single nicks are generally repaired with high fidelity, efficient cleavage occurs only when both offset gRNAs bind correctly, creating a staggered DSB. This double-nicking strategy, therefore, dramatically reduces off-target activity while maintaining high on-target efficiency at many loci. 

Figure 2. Nickase activity with two gRNAs.

A catalytically inactive form of Cas9 (dCas9) can be combined with different transcriptional complexes to achieve CRISPR-mediated transcriptional regulation of endogenous genomic loci. CRISPR-mediated gene regulation can be used for a wide variety of applications including testing regulatory elements on chromatin, reprogramming of target cells, or for whole genome screening to identify genes that drive proliferation and differentiation.

To activate gene transcription (CRISPRa), the Synergistic Activation Mediator (SAM) system can be used. This system utilizes three components: a dCas9/VP64 fusion protein, an MS2/P65/HSF1 helper complex, and a modified gRNA (msgRNA). The dCas9 protein is engineered with VP64, a synthetic transcriptional activator, and the gRNA is modified to include MS2 aptamers which recruit the MS2 complex consisting of the transactivation domains of NF-kB (p65) and HSF1. Together, the dCas9/VP64 and MS2/P65/HSF1 complexes work synergistically to recruit endogenous transcriptional machinery and cofactors to activate target gene expression, as shown in Figure 5. Beyond the SAM system, VectorBuilder offers the dCas9/VP64-p65-Rta (VPR) system which replaces the transactivation domain of HSF1 with the Epstein-Barr virus R transactivator. The dCas9/VPR system has been reported to exhibit higher levels of endogenous activation, although due to it's smaller size, dCas9/VP64 is preferable for AAV.

In contrast, to repress target gene transcription, dCas9 can be engineered with a transcriptional repressor, such as a Kruppel-associated box domain (KRAB) and MeCP2. Two versions of the dCas9/KRAB helper vector are currently available: the original dCas9/KRAB vector and an improved dCas9/KRAB/MeCP2 version which drives the expression of dCas9 fused to a bipartite repressor domain KRAB/MeCP2 for achieving more potent transcriptional repression of DNA target sites. The KRAB domain is commonly found in human transcription factors and is one of the most potent transcriptional repressors currently identified in the human genome. MeCP2 further enhances KRAB-mediated repression through recruitment of histone deacetylases, resulting in chromatin condensation and epigenetic-mediated repression. When the gRNA is designed to recruit dCas9/KRAB/MeCP2 to an endogenous promoter, as shown in Figure 6, transcription is heavily repressed.

CRISPR-mediated genome editing or regulation requires target cells to co-express Cas9 and a target site-specific gRNA. Regardless of the application, CRISPR components must be introduced into target cells. Specific components can be transfected or transduced via different approaches including:

• gRNA and Cas9 plasmid
• gRNA and Cas9 virus (i.e. lentivirus, AAV, adenovirus, etc.)
• Mixture of gRNA and Cas9 mRNA
• Preformed RNP consisting of gRNA and Cas9 protein

Figure 3 summarizes the various methods for CRISPR delivery and the case studies in Figures 7, 8, 9 and 10 illustrate the effectiveness of each delivery method.

Figure 3. Popular methods for delivering CRISPR components into target cells.

The table below lists key advantages and disadvantages for each delivery approach and can be used as a reference to help you decide the most suitable approach for your experiments:

VectorBuilder’s online CRISPR vector design tool features optimized, whole-genome gRNA databases for human, mouse, and rat enabling you to design CRISPR vectors with high targeting efficiency. We are also expanding our gRNA database to include more Cas enzymes and more host species. We follow the algorithm utilized in CRISPR library design (CLD) to calculate specificity scores for gRNAs. Briefly, for a given gRNA intended to target a N(20)NGG sequence in a species, we search for all potential off-target sites in the genome of that species that have ≤3 mismatches with the target sequence. For each potential off-target site identified this way, a single off-target score is calculated. Scores for all the off-target sites are then used in aggregate to calculate the final specificity score of the gRNA, which is between 0 and 100, with higher values indicating greater targeting specificity. Please note that specificity scores are a theoretical guide. Actual targeting efficiency and specificity could depart from what the scores predict. gRNAs with low scores may still work well.

When you design CRISPR vectors on VectorBuilder’s online platform, you will have the option to search for your target genes in our database. Upon entering your gene name, you will see detailed information on all available gRNA designs against your target gene available in our database.

Figure 4. Generating homozygous knockout (KO) mutants using the gRNA/Cas9 ribonucleoprotein (RNP) approach with dual gRNAs for deletion of a DNA fragment. (A) The editing RNP is electroporated into target cells with two sites on the targeted gene to delete a 13 kb region, and single clones are isolated and screened. The genotypes of the candidates are validated using PCR and Sanger sequencing. (B) Four primers, P1 to P4, were used in three PCRs to differentiate KO and WT clones. Based on the (C) PCR results, clone 1 is validated to be homozygous KO mutant, which is also confirmed by (D) sequencing results.

Figure 5. Up-regulation of gene expression achieved by lentivirus-based CRISPRa. NIH3T3 cells stably expressing SAM complex dCas9/VP64 and MS2/P65/HSF1 were transduced with msgRNA-expressing lentivirus followed by antibiotic selection. (A) Illustration of transcriptional activation activity. (B) Diagram of msgRNA design targeting the promoter region of the mouse Brn2 gene. (C) Relative gene expression of Brn2 in NIH3T3 cells transduced with scramble or targeting msgRNA or no treatment control (NC), measured by qRT-PCR. Mean±SD, *P<0.05, ANOVA with Tukey’s post hoc test.

Figure 6. Down-regulation of gene expression achieved by lentivirus-based CRISPRi. Jurkat cells stably expressing the dCas9/KRAB/MeCP2 transcriptional repressor complex were transduced with gRNA expression lentivirus followed by antibiotic selection. (A) Illustration of repressor complex activity. (B) Diagram of gRNA design targeting the promoter region of the human CXCR4 gene. (C) CXCR4 protein levels in Jurkat cells transduced with scramble or targeting gRNA or no treatment control (NC), measured by Western blot. (D) Relative CXCR4 gene expression measured by qRT-PCR. Mean±SD, ***P<0.001, ****P<0.0001, ANOVA with Tukey’s post hoc test. (E) The surface-expressed CXCR4 was quantified by flow cytometry. CXCR4 was labeled with monoclonal primary antibodies (Ab) and fluorophore-conjugated secondary Ab. Unlabeled and secondary Ab only Jurkat cells were used as negative controls. (F) The amount of CXCR4 on the surface of cells transduced with the CXCR4 targeting gRNA was reduced by about 50% compared to the cells transduced with the scramble gRNA. Mean±SD.

Several methods are available for delivering CRISPR components into target cells, each with its own unique advantages and limitations.

1. gRNA and Cas9 plasmid
2. gRNA and Cas9 virus
3. Mixture of gRNA and Cas9 mRNA
4. RNP containing gRNA and Cas9 protein