CRISPR-Cas12a has widespread off-target and dsDNA-nicking effects

Date
2020-03-11
Authors
Seetharam, Arun
Murugan, Karthik
Seetharam, Arun
Severin, Andrew
Severin, Andrew
Sashital, Dipali
Major Professor
Advisor
Committee Member
Journal Title
Journal ISSN
Volume Title
Publisher
Altmetrics
Authors
Research Projects
Organizational Units
Journal Issue
Series
Department
Office of BiotechnologyBiochemistry, Biophysics and Molecular BiologyGenome Informatics FacilityMolecular, Cellular and Developmental Biology
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 (crRNA) sequence. Our analysis further revealed robust non-specific 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.

Comments

This research was originally published in the Journal of Biological Chemistry. Murugan, Karthik, Arun S. Seetharam, Andrew J. Severin, and Dipali G. Sashital. "CRISPR-Cas12a has widespread off-target and dsDNA-nicking effects." Journal of Biological Chemistry (2020): jbc-RA120. © the Author(s). doi: 10.1074/jbc.RA120.012933.

Description
Keywords
Citation
DOI
Collections