Abstract
The CRISPR/Cas system uses guide RNAs (gRNAs) to direct sequence-specific DNA cleavage. Not every gRNA elicits cleavage and the mechanisms that govern gRNA activity have not been resolved. Low activity could result from either failure to form a functional Cas9–gRNA complex or inability to recognize targets in vivo. Here we show that both phenomena influence Cas9 activity by comparing mutagenesis rates in zebrafish embryos with in vitro cleavage assays. In vivo, our results suggest that genomic factors such as CTCF inhibit mutagenesis. Comparing near-identical gRNA sequences with different in vitro activities reveals that internal gRNA interactions reduce cleavage. Even though gRNAs containing these structures do not yield cleavage-competent complexes, they can compete with active gRNAs for binding to Cas9. These results reveal that both genomic context and internal gRNA interactions can interfere with Cas9-mediated cleavage and illuminate previously uncharacterized features of Cas9–gRNA complex formation.
Highlights
The CRISPR/Cas system uses guide RNAs to direct sequence-specific DNA cleavage
Comparing the results of this assay with cleavage in zebrafish embryos[8], we identified guide RNAs (gRNAs) with high in vitro cleavage activity but low in vivo mutagenesis rates (Fig. 1a)
We found that four of the five gRNAs were located within short, sometimes fewer than 50-nucleotide, regions that were refractory to cleavage (Fig. 1b,c)
Summary
The CRISPR/Cas system uses guide RNAs (gRNAs) to direct sequence-specific DNA cleavage. There are two potential mechanisms that might contribute to poor gRNA performance: first, some gRNAs could be inherently poor at forming active Cas9–gRNA complexes, or second, they do form active complexes but their target sites could be refractory in vivo By measuring both the in vitro cleavage and in vivo activity in zebrafish embryos, we find that there are gRNAs that fail for both reasons. By further dissection of the gRNAs that fail to form a functional Cas[9] complex, we found that they contain internal gRNA interactions, providing experimental support for suggestions that gRNA secondary structure modulates Cas[9] cleavage efficiency[9,10,12] Their fold prevents recognition activity, we found that these gRNAs can still bind to the Cas[9] protein, competing effectively with active gRNAs. Strikingly, some inactive complexes can regain high levels of activity when internal gRNA interactions are disrupted. These results contribute to the groundwork for future structural studies on inactive Cas9–gRNA complexes and the mechanism of gRNA loading, and provide new rules for improving gRNA design methods
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