CRISPR Genome Editing Solutions

CRISPR-mediated genome editing

The conventional use of CRISPR system contains two components: Cas9 protein and guide RNA (gRNA). The most commonly used Cas9 is engineered from Streptococcus pyogenes (a.k.a. SpCas9 or hCas9). It is an RNA-guided DNA nuclease which can generate double-stranded breaks (DSBs) at target sites (Figure 1). 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 DSBs at the target region. Once DSBs are generated by the CRISPR system, cells 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.

Figure 1. Mechanisms of CRISPR-induced DNA repair.

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

Figure 2. Generating homozygous CD274 knockout (KO) mutants using the gRNA-Cas9 ribonucleoprotein (RNP) approach. (A) The editing RNP is electroporated into target cells, and single clones are isolated and screened. The genotypes of the candidates are validated using PCR and Sanger sequencing. (B) In this case study of editing a murine colon adenocarcinoma cell line, cells were electroporated with RNP binding to two sites on the targeted gene to KO a 13-kbp region. Four primers, P1 to P4, were used in three PCR to differentiate KO and WT clones. Based on the (C) PCR results, clone 1 are validated to be homozygous KO mutants, which is also confirmed by (D) sequencing results.

CRISPR-mediated gene regulation

Catalytically inactive form of the Cas9 (a.k.a. dCas9) can be combined with different transcriptional complexes to achieve CRISPR-mediated transcriptional regulation of endogenous genomic loci. To activate gene transcription, the Synergistic Activation Mediator (SAM) system can be used. This system utilizes three components: dCas9/VP64 fusion protein, MS2/P65/HSF1 helper, and a modified gRNA. 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. VP64 consists of four tandem repeats of herpes simplex virus protein 16 and is fused to the C-terminus of the Cas9 protein which results in the recruitment of transcriptional machinery that activates gene expression. The transactivation domains of p65 and HSF1 are potent transcriptional activators that act to further recruit cofactors involved in transcription to promoters or enhancers. Together, the dCas9/VP64 and MS2/P65/HSF1 complexes work synergistically to activate target gene expression, as shown in Figure 2. Beyond the SAM system, VectorBuilder offers the dCas9-VP64-p65-Rta (VPR) transactivation domain system which replaces the transactivation domain of HSF1 with the Epstein-Barr virus R transactivator.

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 helper vector version and an improved dCas9/KRAB/MeCP2 helper vector 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. When gRNA recruits dCas9/KRAB/MeCP2 to an endogenous promoter, as in Figure 3, transcription is repressed. 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. CRISPR-mediated gene regulation can be used for a wide variety of applications including testing regulatory elements on chromatin, regulating target genes of interest, or for whole genome screening.

Figure 3. Up-regulation of gene expression achieved by the lentivirus-based CRISPRa. NIH3T3 cells stably expressing SAM complex, dCas9/VP64, and MS2/P65/HSF1 were transduced with msgRNA expression lentivirus followed by antibiotic selection. (A) Illustration of SAM system regulated transcriptional activation. (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 4. Down-regulation of gene expression achieved by the 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 dCas9/KRAB/MeCP2 regulated gene transcriptional inhibition. (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 in Jurkat cells transduced with scramble or targeting gRNA or no treatment control (NC), measured by qRT-PCR. Mean±SD, ***P<0.001, ****P<0.0001, ANOVA with Tukey’s post hoc test. (E) The surface expressed CXCR4 in the Jurkat cells transduced with scramble or targeting gRNA were quantified by flowcytometry. 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 averagely reduced by about 50% compared to the cells transduced with the scramble gRNA. Mean±SD.

CRISPR delivery approaches

CRISPR components can be introduced into mammalian cells via different approaches (Figure 4), including:

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

Figure 5. Methods for delivering CRISPR components into 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:

gRNA databases

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 follow the algorithm developed in Feng Zhang’s lab 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 only a rough 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 guide RNA designs against your target gene available in our database. Our whole-genome gRNA database allows you to easily pick appropriate guide sequence for your target genes without looking for and analyzing the target sites on your own, thereby providing you with advantages offered by popular gRNA design tools or software.

VectorBuilder's online “Learning Center” under "Resources" contains rich educational resources to help you to successfully plan, execute and troubleshoot your CRISPR experiments.

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• CRISPR genome editing solutions