Genome editing in rice and wheat using the CRISPR/Cas system

Abstract

Targeted genome editing nucleases, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), are powerful tools for understanding gene function and for developing valuable new traits in plants. The clustered regularly interspersed short palindromic repeats (CRISPR)/Cas system has recently emerged as an alternative nuclease-based method for efficient and versatile genome engineering. In this system, only the 20-nt targeting sequence within the single-guide RNA (sgRNA) needs to be changed to target different genes. The simplicity of the cloning strategy and the few limitations on potential target sites make the CRISPR/Cas system very appealing. Here we describe a stepwise protocol for the selection of target sites, as well as the design, construction, verification and use of sgRNAs for sequence-specific CRISPR/Cas-mediated mutagenesis and gene targeting in rice and wheat. The CRISPR/Cas system provides a straightforward method for rapid gene targeting within 1-2 weeks in protoplasts, and mutated rice plants can be generated within 13-17 weeks.

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

“…The presence of these sites is a parameter that can be taken into account when selecting the sgRNAs. Knock-in events presenting disrupted restriction sites will resist to the digestion and be available for a much more efficient and detectable amplification [11]. Furthermore, positive and/or negative selection markers, as well as a high selection pressure for gene modifications during the regeneration process (when the desired phenotype is amenable to it), can be used to increase the probability to see knock-in events [27][28][29].…”

“…As for free DNA fragments, they varied from 72 to 476 bp including homology arms, of even length or not, of 46 to 146 bp [6,11,67,69]. Rare are the situations (involving the same protocol, target and plant species) allowing to compare the impact of the nature and length of the homologies on knock-in efficiency.…”

“…Shorter templates (with short homology arms or small replacement sequences) can be introduced into the cells as ssDNA oligonucleotides (sense or antisense) [6,11,67,75].…”

“…However, some of these techniques are still in their early developments in plants, and gene knock-in, in particular, needs to be optimized for many species. Indeed, whereas the knock-out of endogenous plant loci is generally easy to achieve (with mutagenesis efficiencies varying from 2% [6] to 100% [7] of mutated individuals), CRISPR-induced gene knock-in of a donor DNA by homology-driven repair (HDR) is much more difficult to perform in higher plants, with efficiencies rarely reaching a few percent [2,6,[8][9][10][11]. It explains why among the CRISPR-induced traits already reported in the literature, such as diseases tolerances [12][13][14], adaptation to drought [2], modified ripening profiles [15], male sterility and factors impacting yield [9,16,17], most of them are derived from gene knock-out, and still very few from gene knock-in.…”

Advances in detection of stress tolerance in plants through metabolomics approaches

“…In this system, only 20-nucleotide target sequences (or even one nucleotide) form the target gene with a desirable sequence of gene needs to be altered (Fig 3). Shan et al (2014) suggested the (CRISPR)/Cas9 as an appealing system due to its simplicity. They described a stepwise protocol for the selection of target sites, design, construction, verification and use of single guide RNA (sgRNAs) for sequence-specific mutagenesis in target genes of rice and wheat.…”