Synthetic Oligonucleotides Inhibit CRISPR-Cpf1-Mediated Genome Editing

Abstract

Summary: Previously, researchers discovered a series of anti-CRISPR proteins that inhibit CRISPR-Cas activity, such as Cas9 and Cpf1 (Cas12a). Herein, we constructed crRNA variants consisting of chemically modified DNA-crRNA and RNA-crRNA duplexes and identified that phosphorothioate (PS)-modified DNA-crRNA duplex completely blocked the function of Cpf1. More important, without prehybridization, these PS-modified DNA oligonucleotides showed the ability to suppress DNA double-strand breaks induced by two Cpf1 orthologs, AsCpf1 and LbCpf1. Time-dependent inhibitory effects were validated in multiple loci of different human cells. Further studies demonstrated that PS-modified DNA oligonucleotides were able to serve as Cpf1 inhibitors in a sequence-independent manner. Mechanistic studies indicate that PS-modified DNA oligonucleotides hinder target DNA binding and recognition by Cpf1. Consequently, these synthetic DNA molecules expand the sources of CRISPR inhibitors, providing a platform to inactivate Cpf1-mediated genome editing. : Li et al. show that phosphorothioate-modified DNA (psDNA) oligonucleotides inhibit Cpf1-mediated genome-editing activity in a sequence-independent manner in human cells. These psDNA oligonucleotides interact with Cpf1 protein and block the formation of Cpf1-crRNA-target DNA complex. They also display inhibitory effects on the CRISPR-Cas9 system. Keywords: synthetic DNA oligonucleotides, phosphorothioate oligonucleotides, CRISPR-Cpf1, Cas12a, Cas9, genome editing