CRISPR/Cpf1-mediated DNA-free plant genome editing

Hyeran Kim,Sang-Gyu Kim,Jin-Soo Kim,Sang-Tae Kim,Jahee Ryu,Beum-Chang Kang

doi:10.1038/ncomms14406

Hyeran Kim, Sang-Gyu Kim + Show 4 more

Open Access

https://doi.org/10.1038/ncomms14406

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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.

Highlights

Cpf[1], a type V Clustered regularly interspaced short palindromic repeats (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
The results show that Cpf1–CRISPR RNA (crRNA) complexes can introduce targeted mutations in plant genomes
In our previous Cpf[1] study[12], we showed that Cpf1–crRNA complexes could induce mutations at one- or two-base mismatches sites

Summary

Introduction

Cpf[1], 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). 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. CRISPR-Cpf[1] (CRISPR from Prevoltella and Francisella1) has recently been reported as a new type of genome-editing tool[9]; similar to the type II CRISPR-Cas system, a single Cpf[1] protein functions in crRNA processing[10], target-site recognition and DNA cleavage[9]. To test whether the Cpf[1] protein can be used as an alternative DNA-free genomeediting tool in plants, we delivered the recombinant LbCpf[1] and AsCpf[1] proteins mixed with crRNAs into protoplasts isolated from soybean and wild tobacco plants and analysed insertion and deletion (indel) frequencies and patterns at the targeted loci (Fig. 1). The results show that Cpf1–crRNA complexes can introduce targeted mutations in plant genomes

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Conclusion