In CRISPR/Cas9 genome editing, targeting the Cas9 nuclease to a specific genomic locus is solely mediated by a user-defined sgRNA. Currently available web-based tools for sgRNA design will return a variety of candidate sgRNAs for a single gene target. Despite these in silico predictions, not every sgRNA will exhibit equivalent cleavage efficiency. Given this inconsistency, it is necessary to screen multiple sgRNAs to identify the most effective one.

An in vitro assay to test sgRNA cleavage efficiency

The Guide-it sgRNA Screening Kit is a complete system for predicting the cleavage efficacy of sgRNAs in vitro, prior to use for genome editing in cells (Figure 1). With this kit, a template containing a sgRNA-target site is created by PCR; then the test sgRNA and recombinant Cas9 nuclease are added. The efficiency of Cas9-mediated cleavage can be measured by agarose gel electrophoresis.

sgRNAs exhibit different cleavage efficiencies

CRISPR/Cas9 genome editing was used to disrupt the CXCR4 locus in HeLa cells. CXCR4 encodes a cell surface chemokine receptor that interacts with the CXCL12 chemokine and plays an important role in the immune system. In this experiment, four different sgRNAs targeting the CXCR4 locus were tested using the Guide-it sgRNA Screening Kit. Briefly, sgRNAs targeting the CXCR4 gene were synthesized using the Guide-it sgRNA In Vitro Transcription Kit. A PCR fragment containing the sgRNA target sequence was mixed with recombinant Cas9 protein and each sgRNA. The cleavage reaction was analyzed by agarose gel electrophoresis. Densitometry (Cong et al., 2013) showed that sgRNA3 had the lowest cleavage efficiency (Figure 2).

In vitro cleavage efficiency predicts in vivo cleavage

HeLa cells were cotransfected with plasmids encoding Cas9 and each of the four different sgRNAs tested above. The presence of mutations in the CXCR4 locus as was assayed using the Guide-it Mutation Detection Kit. This assay uses a mismatch-specific nuclease, Guide-it Resolvase, to identify insertions or deletions in specific loci in cells treated with engineered nucleases. Mismatches were detected with high efficiency in cells treated with sgRNAs 1, 2, and 4 (Figure 3). However, cells treated with sgRNA3 exhibited a very low efficiency of mismatches, consistent with the efficiency predicted by the Guide-it sgRNA Screening Kit (Figure 2).

CXCR4 gene disruption was also assessed by flow cytometry; since CXCR4 is a cell surface receptor, it can be detected by flow cytometry using a FITC-labeled CXCR4 antibody. Disruption in CXCR4 expression could be detected in cells transfected with Cas9 and sgRNAs 1, 2, and 4 (Figure 4). In contrast, for cells transfected with Cas9 and sgRNA3, a much smaller proportion of the cells had disruption of CXCR4 expression. These functional data confirm the results obtained by both the Guide-it sgRNA Screening Kit and the Guide-it Mutation Detection Kit.

There is a clear correlation between in vitro sgRNA cleavage efficiency as predicted by the Guide-it sgRNA Screening Kit and in vivo sgRNA-mediated cleavage as assessed by the presence of indels and functional gene knockout (Figure 5). These results indicate that the Guide-it sgRNA Screening Kit is an ideal method for screening for ineffective sgRNAs during CRISPR/Cas9 genome editing projects.

Cong, L. et al. (2013) Multiplex genome engineering using CRISPR/Cas9 systems. Science 339(6121):819–23.