Structural Basis for the Inhibition of CRISPR-Cas12a by Anti-CRISPR Proteins

Heng Zhang; Zhuang Li; Courtney M Daczkowski; Clinton Gabel; Andrew D Mesecar; Leifu Chang

doi:10.1016/j.chom.2019.05.004

Structural Basis for the Inhibition of CRISPR-Cas12a by Anti-CRISPR Proteins

Cell Host Microbe. 2019 Jun 12;25(6):815-826.e4. doi: 10.1016/j.chom.2019.05.004. Epub 2019 May 30.

Authors

Heng Zhang¹, Zhuang Li¹, Courtney M Daczkowski², Clinton Gabel¹, Andrew D Mesecar³, Leifu Chang⁴

Affiliations

¹ Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA.
² Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA.
³ Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA; Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA; Purdue University Center for Cancer Research, Purdue University, West Lafayette, IN 47907, USA.
⁴ Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA; Purdue University Center for Cancer Research, Purdue University, West Lafayette, IN 47907, USA. Electronic address: lchang18@purdue.edu.

PMID: 31155345
DOI: 10.1016/j.chom.2019.05.004

Abstract

CRISPR-Cas12a (Cpf1), a type V CRISPR-associated nuclease, provides bacterial immunity against bacteriophages and plasmids but also serves as a tool for genome editing. Foreign nucleic acids are integrated into the CRISPR locus, prompting transcription of CRISPR RNAs (crRNAs) that guide Cas12a cleavage of foreign complementary DNA. However, mobile genetic elements counteract Cas12a with inhibitors, notably type V-A anti-CRISPRs (AcrVAs). We present cryoelectron microscopy structures of Cas12a-crRNA bound to AcrVA1 and AcrVA4 at 3.5 and 3.3 Å resolutions, respectively. AcrVA1 is sandwiched between the recognition (REC) and nuclease (NUC) lobes of Cas12a and inserts into the binding pocket for the protospacer-adjacent motif (PAM), a short DNA sequence guiding Cas12a targeting. AcrVA1 cleaves crRNA in a Cas12a-dependent manner, inactivating Cas12a-crRNA complexes. The AcrVA4 dimer is anchored around the crRNA pseudoknot of Cas12a-crRNA, preventing required conformational changes for crRNA-DNA heteroduplex formation. These results uncover molecular mechanisms for CRISPR-Cas12a inhibition, providing insights into bacteria-phage dynamics.

Keywords: CRISPR; Cas12a; Cpf1; anti-CRISPR proteins; cryoelectron microscopy; genome editing.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

CRISPR-Cas Systems*
Cryoelectron Microscopy
Endodeoxyribonucleases / antagonists & inhibitors*
Endodeoxyribonucleases / metabolism*
Endodeoxyribonucleases / ultrastructure
Enzyme Inhibitors / metabolism*
Protein Binding
Protein Conformation
RNA, Guide, CRISPR-Cas Systems / metabolism*
RNA, Guide, CRISPR-Cas Systems / ultrastructure
Ribonucleases / metabolism*
Ribonucleases / ultrastructure

Substances

Enzyme Inhibitors
RNA, Guide, CRISPR-Cas Systems
Endodeoxyribonucleases
Ribonucleases