High‐efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR/Cas9

Abstract

SUMMARYThe ability to edit plant genomes through gene targeting (GT) requires efficient methods to deliver both sequence-specific nucleases (SSNs) and repair templates to plant cells. This is typically achieved using Agrobacterium T-DNA, biolistics or by stably integrating nuclease-encoding cassettes and repair templates into the plant genome. In dicotyledonous plants, such as Nicotinana tabacum (tobacco) and Solanum lycopersicum (tomato), greater than 10-fold enhancements in GT frequencies have been achieved using DNA virus-based replicons. These replicons transiently amplify to high copy numbers in plant cells to deliver abundant SSNs and repair templates to achieve targeted gene modification. In the present work, we developed a replicon-based system for genome engineering of cereal crops using a deconstructed version of the wheat dwarf virus (WDV). In wheat cells, the replicons achieve a 110-fold increase in expression of a reporter gene relative to non-replicating controls. Furthermore, replicons carrying CRISPR/Cas9 nucleases and repair templates achieved GT at an endogenous ubiquitin locus at frequencies 12-fold greater than non-viral delivery methods. The use of a strong promoter to express Cas9 was critical to attain these high GT frequencies. We also demonstrate gene-targeted integration by homologous recombination (HR) in all three of the homoeoalleles (A, B and D) of the hexaploid wheat genome, and we show that with the WDV replicons, multiplexed GT within the same wheat cell can be achieved at frequencies of~1%. In conclusion, high frequencies of GT using WDV-based DNA replicons will make it possible to edit complex cereal genomes without the need to integrate GT reagents into the genome.

Gene Targeting by Homology-Directed Repair in Rice Using a Geminivirus-Based CRISPR/Cas9 System

“…The wheat dwarf virus (WDV) infects a variety of grasses including most cereals and has been previously used to express foreign proteins in wheat and maize cells (Suarez-Lopez and Gutierrez, 1997). Recently, WDV was used to achieve high efficiency gene targeting in hexaploid wheat calli, and it was also shown to express in rice protoplast (Gil-Humanes et al, 2017).…”

Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens

“…This result was obtained by putting the Cas9 nuclease under the regulation of a strong promoter reinforcing the action of the replicon LIR. Simultaneous HR-mediated knock-in at two endogenous genes and in all three homeoalleles of the bread wheat genome is also reported [94].…”

“…For monocots, other viruses from the same mastrevirus genius could be used, such as the wheat dwarf virus (WDV) or maize streak virus (MSV) [93]. Recently, a modified replicon from WDV carrying the CRISPR-Cas9 system and a repair template allowed to increase by 12-fold the efficiency of CRISPRinduced gene knock-in (5.85%) at an endogenous ubiquitin locus in wheat cells [94] (Table 1). This result was obtained by putting the Cas9 nuclease under the regulation of a strong promoter reinforcing the action of the replicon LIR.…”

“…Transient transformation can then be performed either by direct transformation using non linearized cassettes, by Agrobacterium transformation of viral replicons that are released from the T-DNA and not integrated [94], or direct transfection of in vitro transcribed sgRNAs associated to RNA or protein versions of the Cas9 nuclease. The efficiency of the first two types of methods was demonstrated for targeted mutagenesis in different plant models and crops (as described above).…”

Genomic innovation for crop improvement

“…Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 technologies are being applied to crops for genome editing. The delivery of Cas9 and DNA templates to crop genomes can be carried out transiently 72 or using a viral vector 73 , without an intermediate transgenic step. This challenges the present definitions of genetic engineering according to European Union regulations.…”