In vivo high-throughput profiling of CRISPR–Cpf1 activity

Abstract

Programmable nuclease is useful tools for genome editing at site-specific locus. For high efficiency of genome editing, it is important that programmable nuclease has high activity. Because Cpf1 is a newly reported nuclease protein of the class 2 CRISPR-Cas system (Zetsche, Gootenberg et al. 2015), it has limited information about Cpf1 activity profiles in mammalian cells. For this reason, Cpf1 preclude its wide use for genome editing. Here, we developed a new high-throughput method for evaluating Cpf1 activity, based on target sequence composition in mammalian cells. A library of >11,000 guide RNA and target sequence pairs was delivered into human cells by lentiviral vectors. Subsequent delivery of Cpf1 in various methods into this cell library induced indels at the integrated synthetic target sequences, which allowed en masse evaluation of guide RNA and Cpf1 activity using deep sequencing. Regardless of delivery method of Cpf1, our hight-throughput method showed similar results and the indel frequencies measured at the integrated target sequences correlated with those at the corresponding endogenous sites. In this high-throughput approach, we determined target sequence-dependent activity profiles and newly protospacer adjacent motif sequences of Cpf1 in mammalian cells. And we found that sequence features of high activity AsCpf1 guide RNAs are distinct from those of SpCas9. Evaluation of activity at mismatched target sequences showed that Cpf1 target sequences can be divided into seed, trunk, and promiscuous regions depending on mismatch tolerability. Both the Cpf1 characterization profile and the in vivo high-throughput evaluation method will greatly facilitate Cpf1-based genome editing.