Abstract
Cas12a (Cpf1) is an RNA-guided endonuclease in the bacterial type V-A CRISPR-Cas anti-phage immune system that can be repurposed for genome editing. Cas12a can bind and cut dsDNA targets with high specificity in vivo, making it an ideal candidate for expanding the arsenal of enzymes used in precise genome editing. However, this reported high specificity contradicts Cas12a's natural role as an immune effector against rapidly evolving phages. Here, we employed high-throughput in vitro cleavage assays to determine and compare the native cleavage specificities and activities of three different natural Cas12a orthologs (FnCas12a, LbCas12a, and AsCas12a). Surprisingly, we observed pervasive sequence-specific nicking of randomized target libraries, with strong nicking of DNA sequences containing up to four mismatches in the Cas12a-targeted DNA-RNA hybrid sequences. We also found that these nicking and cleavage activities depend on mismatch type and position and vary with Cas12a ortholog and CRISPR RNA sequence. Our analysis further revealed robust nonspecific nicking of dsDNA when Cas12a is activated by binding to a target DNA. Together, our findings reveal that Cas12a has multiple nicking activities against dsDNA substrates and that these activities vary among different Cas12a orthologs.
Highlights
Cas12a (Cpf1) is an RNA-guided endonuclease in the bacterial type V-A CRISPR-Cas anti-phage immune system that can be repurposed for genome editing
Our findings reveal that Cas12a has multiple nicking activities against dsDNA substrates and that these activities vary among different Cas12a orthologs
The CRISPR locus is transcribed into a long preCRISPR RNA, which is processed by Cas12a into small mature CRISPR RNA [3]
Summary
Genome editing activity and specificity of Cas12a have previously been characterized in vivo (10 –12). To directly observe the cleavage activity and specificity of Cas12a, we performed in vitro cleavage assays using a plasmid library followed by high-throughput sequencing analysis (Fig. 1A and Fig. S1). Only a fraction of pLibrary was linearized, and some of the plasmid remained supercoiled, indicating the presence of sequences within the pool that could not be cleaved by Cas12a in the time span tested (Fig. 1 (B and C) and Figs. We observed variable cleavage rates and patterns for the three pLibraries and Cas12a orthologs (Fig. 1C and Fig. S3 (A and B)) Both FnCas12a and LbCas12a cleaved most pLibraries with a relatively high efficiency, resulting in substantial depletion of supercoiled DNA to ϳ10% of the total DNA at the longest time point (3 h). We could not PCRamplify and analyze target sequences present in the linearized DNA pool due to the double-strand break generated in the tar-
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