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TECH NOTE

In Vitro Cleavage Efficiency of sgRNAs Correlates with Functional Genome Editing in Target Cells

Overview

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.

Guide-it sgRNA Screening Kit workflow

Figure 1. Overview of the Guide-it sgRNA Screening Kit protocol. A PCR amplicon containing a sgRNA target site is synthesized from genomic DNA (Step 1). The PCR fragment is then combined with a candidate sgRNA and recombinant Cas9 (Step 2). The entire reaction is separated by agarose gel electrophoresis (Step 3). Since the sgRNA-target sequence is located asymmetrically within the amplicon, cleavage by the Cas9-sgRNA complex results in two bands of unequal length that can be easily distinguished on an agarose gel.

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 sgRNA cleavage efficiency

Figure 2. Differences in in vitro cleavage efficiency as determined by the Guide-it sgRNA Screening Kit. The cleavage efficiency of four different sgRNAs targeting the CXCR4 locus were tested. A PCR fragment containing the CXCR4 target sequence was synthesized and mixed with Cas9 and each sgRNA. A negative control that lacked sgRNA was included for comparison (NC). Cleavage efficiency was assessed by agarose gel electrophoresis and measured using densitometry (%).

In Vitro Cleavage Efficiency Predicts In Vivo Cleavage

HeLa cells were co-transfected with plasmids encoding Cas9 and one 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 sgRNA1, 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).

Mutation detection

Figure 3. sgRNA-mediated cleavage in HeLa cells as determined by the Mutation Detection Kit. HeLa cells were co-transfected with plasmids encoding Cas9 and one of the four different sgRNAs using Xfect Transfection Reagent. Six days after transfection, cells were assayed for the presence of mutations using Guide-it Resolvase, a mismatch-specific nuclease. Cleavage fragments were present for all sgRNAs except sgRNA3, indicating low Cas9 guiding efficiency for this particular sgRNA.

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 sgRNA1, 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 Mutation Detection Kit.

Functional knockout of CXCR4

Figure 4. Flow cytometric analysis detects sgRNA-mediated loss of CXCR4 function. HeLa cells cotransfected with plasmids encoding Cas9 and one of four sgRNAs, were stained with a FITC-labeled antibody against CXCR4. Knockout of CXCR4 gene by CRISPR/Cas9 editing will result in in reduced protein expression. Therefore, FITC staining is inversely correlated with efficient genome editing. The percentage (%) of the cell population that were not labeled with FITC is shown in blue. Cells treated with Cas9 and sgRNA3 exhibit the greatest percentage of FITC+ cells and the least efficient genome editing.

Conclusions: Accurate Prediction of sgRNA Cleavage Efficiency

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 ideal method for screening for ineffective sgRNAs during CRISPR/Cas9 genome editing projects.

Correlation between in vitro cleavage efficiency and gene knockout

Figure 5. Guide-it sgRNA Screening Kit accurately predicts in vivo sgRNA efficacy. Cleavage efficiency was assessed by in vitro cleavage (Figure 2) and the Guide-it Mutation Detection Kit (Figure 3); functional knockout was assessed by flow cytometry (Figure 4, % of CXCR4- cells). There is a clear correlation between the efficiency predicted by the Guide-it sgRNA Screening Kit, and estimation of in vivo cleavage (Mutation Detection Kit) and functional knockout (Flow Cytometry).

References

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

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