“CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity” (2018), by Janice S. Chen et al. Genetics and Biochemistry research paper. Original abstract below, unchanged. A free PDF is available.

CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity

Janice S. Chen, Enbo Ma, Lucas B. Harrington et al. · February 15, 2018 · Genetics and Biochemistry

Abstract

CRISPR-Cas12a (Cpf1) proteins are RNA-guided DNA targeting enzymes that bind and cut DNA as components of bacterial adaptive immune systems. Like CRISPR-Cas9, Cas12a can be used as a powerful genome editing tool based on its ability to induce genetic changes in cells at sites of double-stranded DNA (dsDNA) cuts. Here we show that RNA-guided DNA binding unleashes robust, non-specific single-stranded DNA (ssDNA) cleavage activity in Cas12a sufficient to completely degrade both linear and circular ssDNA molecules within minutes. This activity, catalyzed by the same active site responsible for site-specific dsDNA cutting, indiscriminately shreds ssDNA with rapid multiple-turnover cleavage kinetics. Activation of ssDNA cutting requires faithful recognition of a DNA target sequence matching the 20-nucleotide guide RNA sequence with specificity sufficient to distinguish between closely related viral serotypes. We find that target-dependent ssDNA degradation, not observed for CRISPR-Cas9 enzymes, is a fundamental property of type V CRISPR-Cas12 proteins, revealing a fascinating parallel with the RNA-triggered general RNase activity of the type VI CRISPR-Cas13 enzymes. One Sentence Summary Cas12a (Cpf1) and related type V CRISPR interference proteins possess non-specific, single-stranded DNase activity upon activation by guide RNA-dependent DNA binding.

Free PDF DOI: 10.1126/science.aar6245