A Universal System of CRISPR/Cas9-Mediated Gene Targeting Using All-in-One Vector in Plants.

Ayako Nishizawa-Yokoi,Masafumi Mikami,Seiichi Toki

doi:10.3389/fgeed.2020.604289

Abstract

Homologous recombination-mediated genome editing, also called gene targeting (GT), is an essential technique that allows precise modification of a target sequence, including introduction of point mutations, knock-in of a reporter gene, and/or swapping of a functional domain. However, due to its low frequency, it has been difficult to establish GT approaches that can be applied widely to a large number of plant species. We have developed a simple and universal clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated DNA double-strand break (DSB)-induced GT system using an all-in-one vector comprising a CRISPR/Cas9 expression construct, selectable marker, and GT donor template. This system enabled introduction of targeted point mutations with non-selectable traits into several target genes in both rice and tobacco. Since it was possible to evaluate the GT frequency on endogenous target genes precisely using this system, we investigated the effect of treatment with Rad51-stimulatory compound 1 (RS-1) on the frequency of DSB-induced GT. GT frequency was slightly, but consistently, improved by RS-1 treatment in both target plants.

Highlights

Genome editing techniques have come to be required for both the development of basic research and plant molecular breeding in recent years
The desired point mutations were located on the protospacer adjacent motif (PAM) sequences following the DNA region targeted by SpCas9, indicating that the gene targeting (GT) donor template on the vectors was not cleaved by the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system
cleaved amplified polymorphic sequence (CAPS) analysis, i.e., PCR analysis coupled with MfeI digestion, revealed that there were no GT positive callus lines from among 960 independent transgenic calli harboring all-in-one GT vector treated with dimethyl sulfoxide (DMSO) (Table 1), while MfeI-digested PCR fragments were detected in two independent callus lines, designated as OsALS-GT RS-1_A and B, among the 960 allin-one GT vector transgenic calli treated with 25 μM Rad51-stimulatory compound 1 (RS-1), suggesting that, in these callus lines, W548L/S627I mutations were introduced into the OsALS locus by homologous recombination (HR) between the GT vector that could be integrated into the genome and the target locus

Summary

Introduction

Genome editing techniques have come to be required for both the development of basic research and plant molecular breeding in recent years. It is difficult to deliberately introduce a desired mutation into target locus using this method because the mutations occur randomly in the process of repair via the non-homologous end joining (NHEJ) pathway of DNA double-strand breaks (DSBs) induced by SSNs. On the other hand, homologous recombination (HR)-mediated gene targeting (GT) allows precise genome engineering (the introduction of nucleotide substitutions, swapping of functional domains, and knock-in of reporter genes, etc.) of endogenous target genes via “copy and paste” of sequences from a repair template. Homologous recombination (HR)-mediated gene targeting (GT) allows precise genome engineering (the introduction of nucleotide substitutions, swapping of functional domains, and knock-in of reporter genes, etc.) of endogenous target genes via “copy and paste” of sequences from a repair template Establishing a universal GT method that can be applied to a large number of plant species remains a challenge

Methods

Results

Conclusion