@article {Chen226993, author = {Janice S. Chen and Enbo Ma and Lucas B. Harrington and Xinran Tian and Jennifer A. Doudna}, title = {CRISPR-Cas12a target binding unleashes single-stranded DNase activity}, elocation-id = {226993}, year = {2017}, doi = {10.1101/226993}, publisher = {Cold Spring Harbor Laboratory}, 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.}, URL = {https://www.biorxiv.org/content/early/2017/11/29/226993}, eprint = {https://www.biorxiv.org/content/early/2017/11/29/226993.full.pdf}, journal = {bioRxiv} }