CRISPR/Cpf1-mediated DNA-free plant genome editing

Abstract

Cpf1, a type V CRISPR effector, recognizes a thymidine-rich protospacer-adjacent motif and induces cohesive double-stranded breaks at the target site guided by a single CRISPR RNA (crRNA). Here we show that Cpf1 can be used as a tool for DNA-free editing of plant genomes. We describe the delivery of recombinant Cpf1 proteins with in vitro transcribed or chemically synthesized target-specific crRNAs into protoplasts isolated from soybean and wild tobacco. Designed crRNAs are unique and do not have similar sequences (<= 3 mismatches) in the entire soybean reference genome. Targeted deep sequencing analyses show that mutations are successfully induced in FAD2 paralogues in soybean and AOC in wild tobacco. Unlike SpCas9, Cpf1 mainly induces various nucleotide deletions at target sites. No significant mutations are detected at potential off-target sites in the soybean genome. These results demonstrate that Cpf1-crRNA complex is an effective DNA-free genome-editing tool for plant genome editing.