CRISPR-Cas12a Possesses Unconventional DNase Activity that Can Be Inactivated by Synthetic Oligonucleotides

Bin Li; Jingyue Yan; Youxi Zhang; Wenqing Li; Chunxi Zeng; Weiyu Zhao; Xucheng Hou; Chengxiang Zhang; Yizhou Dong

doi:10.1016/j.omtn.2019.12.038

CRISPR-Cas12a Possesses Unconventional DNase Activity that Can Be Inactivated by Synthetic Oligonucleotides

Mol Ther Nucleic Acids. 2020 Mar 6:19:1043-1052. doi: 10.1016/j.omtn.2019.12.038. Epub 2020 Jan 14.

Authors

Bin Li¹, Jingyue Yan², Youxi Zhang², Wenqing Li², Chunxi Zeng², Weiyu Zhao², Xucheng Hou², Chengxiang Zhang², Yizhou Dong³

Affiliations

¹ Division of Pharmaceutics and Pharmacology, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA; Department of Infectious Disease, Shenzhen People's Hospital, The Second Clinical Medical College of Jinan University, The First Affiliated Hospital of Southern University of Science and Technology, Shenzhen 518020, China.
² Division of Pharmaceutics and Pharmacology, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA.
³ Division of Pharmaceutics and Pharmacology, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA; Department of Biomedical Engineering, The Ohio State University, Columbus, OH 43210, USA; The Center for Clinical and Translational Science, The Ohio State University, Columbus, OH 43210, USA; James Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA; Dorothy M. Davis Heart & Lung Research Institute, The Ohio State University, Columbus, OH 43210, USA; Department of Radiation Oncology, The Ohio State University, Columbus, OH 43210, USA. Electronic address: dong.525@osu.edu.

Abstract

CRISPR-Cas12a (CRISPR-Cpf1) was reported to have multiple types of cleavage activities. Without the assistance of CRISPR RNA (crRNA), we investigated DNase activity and substrate specificity of Cas12a orthologs in the presence of diverse divalent metal ions. Cas12a from different species are capable of degrading single-stranded DNA (ssDNA) and/or double-stranded DNA (dsDNA), depending on the metal ions used. In spite of sharing high sequence similarity and functional domains among diverse Cas12a orthologs, only Acidaminococcus sp. Cas12a (AsCas12a) showed a predominant preference for cleaving ssDNA, but no detectable activity toward dsDNA substrate in the presence of magnesium (II) ions. In addition, we found that both AsCas12a and Francisella novicida Cas12a (FnCas12a) caused substantial dsDNA cleavage in the presence of manganese (II) ion. More importantly, the DNase activities can be inhibited by synthetic DNA oligonucleotides with phosphorothioate linkage modifications. Overall, ssDNase activity of the Cas12a orthologs uncovered a distinct approach for DNA cleavage compared with crRNA-guided dsDNA breaks, and provided insights into potential biological and therapeutic applications.

Keywords: CRISPR-Cas12a; DNase activity; inhibition; metal ions; synthetic oligonucleotide.

Grants and funding

R01 HL136652/HL/NHLBI NIH HHS/United States