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.
Tech Note
In vitro cleavage efficiency of sgRNAs correlates with functional genome editing in target cells
- A novel in vitro assay to test sgRNA cleavage efficiency
Screen various sgRNAs to determine the most effective sgRNAs prior to delivering to your cells - Accurate prediction of sgRNA cleavage efficiency
sgRNA cleavage efficiency predicted in vitro correlates with in vivo cleavage as assessed by both a nuclease assay and functional analysis
Introduction
Results
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.
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).
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 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).
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 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.
Figure 4. Flow cytometric analysis detects sgRNA-mediated loss of CXCR4 function. Knockout of the CXCR4 gene by CRISPR/Cas9 editing results in in reduced protein expression; therefore, FITC staining is inversely correlated with efficient genome editing. In this experiment, HeLa cells were cotransfected with plasmids encoding Cas9 and each of four sgRNAs, and then stained with a FITC-labeled antibody against CXCR4. The percentage (%) of the cell population that was not labeled with FITC is shown in blue. Cells treated with Cas9 and sgRNA3 exhibited the greatest percentage of FITC+ cells and the least efficient genome editing.
Conclusions
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.
Figure 5. The 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, the estimation of in vivo cleavage provided by the Mutation Detection Kit, and the level of functional knockout (via flow cytometry).
References
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