Synthesis-dependent repair of Cpf1-induced double strand DNA breaks enables targeted gene replacement in rice.

Shaoya Li,Jiahui Zhang,Lanqin Xia,Suhas Sutar,Wenming Du,Jindong Fu,Jingying Li,Yunde Zhao

doi:10.1093/jxb/ery245

Abstract

The recently developed CRISPR (clustered regularly interspaced short palindromic repeats)/Cpf1 system expands the range of genome editing and is emerging as an alternative powerful tool for both plant functional genomics and crop improvement. Cpf1-CRISPR RNA (crRNA) produces double strand DNA breaks (DSBs) with long 5'-protruding ends, which may facilitate the pairing and insertion of repair templates through homology-directed repair (HDR) for targeted gene replacement and introduction of the desired DNA elements at specific gene loci for crop improvement. However, the potential mechanism underlying HDR of DSBs generated by Cpf1-crRNA remains to be investigated, and the inherent low efficiency of HDR and poor availability of exogenous donor DNA as repair templates strongly impede the use of HDR for precise genome editing in crop plants. Here, we provide evidence of synthesis-dependent repair of Cpf1-induced DSBs, which enables us precisely to replace the wild-type ALS gene with the intended mutant version that carries two discrete point mutations conferring herbicide resistance to rice plants. Our observation that the donor repair template (DRT) with only the left homologous arm is sufficient for precise targeted allele replacement offers a better understanding of the mechanism underlying HDR in plants, and greatly simplifies the design of DRTs for precision genome editing in crop improvement.

Highlights

Double strand DNA breaks (DSBs) in target genes generated by Cas endonucleases are repaired by the error-prone non-homologous end joining (NHEJ) pathway or the precise homology-directed repair (HDR), or both NHEJ and HDR (Jinek et al, 2012; Cong et al, 2013; Zetsche et al, 2015)
When dsDNAs are used as repair templates, different DSBs generated by Cas9 variants engage in different repair pathways and the polarity of the overhang structure is a critical determinant of DSB repair pathway choice in human cells (Bothmer et al, 2017)
We demonstrated that donor repair template (DRT) with either only the left homologous arm or two homologous arms function efficiently in achieving precise targeted replacement of the Acetolactate synthase (ALS) gene, which encodes a key enzyme for the biosynthesis of the branched chain amino acids leucine, isoleucine, and valine, and is a major target for ALS-inhibiting herbicides such as chlorsulfuron and bispyribac sodium (BS) in rice (Mazur et al, 1987)

Summary

Introduction

Double strand DNA breaks (DSBs) in target genes generated by Cas endonucleases are repaired by the error-prone non-homologous end joining (NHEJ) pathway or the precise homology-directed repair (HDR), or both NHEJ and HDR (Jinek et al, 2012; Cong et al, 2013; Zetsche et al, 2015). Three potential mechanisms have been proposed for HDR of DSBs: single-strand annealing (SSA), synthesis-dependent strand annealing (SDSA), and the so-called double strand break repair (DSBR) model (Puchta, 1998). In DSBR, DNA synthesis occurs at both broken ends so that genetic information is copied from both strands of the homologous sequences, which may lead to a crossover event (Puchta and Fauser, 2013). Analyses of the sequence requirements for efficient repair of DSBs generated by Cas in human cells indicated that the repair process is more consistent with SDSA when either ssODNs or dsDNAs are used as repair templates (Paix et al, 2017). The mechanism underlying HDR of DSBs when a dsDNA is used as the DRT still remains inconclusive, especially in plants

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Results

Conclusion