The CRISPR/Cas system can be used as nuclease for in planta gene targeting and as paired nickases for directed mutagenesis in Arabidopsis resulting in heritable progeny

Abstract

These authors contributed equally to this work. SUMMARYThe CRISPR/Cas nuclease is becoming a major tool for targeted mutagenesis in eukaryotes by inducing double-strand breaks (DSBs) at pre-selected genomic sites that are repaired by non-homologous end joining (NHEJ) in an error-prone way. In plants, it could be demonstrated that the Cas9 nuclease is able to induce heritable mutations in Arabidopsis thaliana and rice. Gene targeting (GT) by homologous recombination (HR) can also be induced by DSBs. Using a natural nuclease and marker genes, we previously developed an in planta GT strategy in which both a targeting vector and targeting locus are activated simultaneously via DSB induction during plant development. Here, we demonstrate that this strategy can be used for natural genes by CRISPR/Cas-mediated DSB induction. We were able to integrate a resistance cassette into the ADH1 locus of A. thaliana via HR. Heritable events were identified using a PCR-based genotyping approach, characterised by Southern blotting and confirmed on the sequence level. A major concern is the specificity of the CRISPR/Cas nucleases. Off-target effects might be avoided using two adjacent sgRNA target sequences to guide the Cas9 nickase to each of the two DNA strands, resulting in the formation of a DSB. By amplicon deep sequencing, we demonstrate that this Cas9 paired nickase strategy has a mutagenic potential comparable with that of the nuclease, while the resulting mutations are mostly deletions. We also demonstrate the stable inheritance of such mutations in A. thaliana.

Coselected genes determine adaptive variation in herbivore resistance throughout the native range of Arabidopsis thaliana

“…Using the same method, we found that, among accessions included in field experiments, the three loci and their interactions explained nearly 36% of the variation in overall GSL profile among accessions. Recent genome-editing techniques may allow formal testing of fitness effects of combinations of GSL alleles in the future (37).…”

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

“…So far, reports about CRISPR-assisted knock-in experiments in plants describe circular donor templates ranging from 0.47 to 5.2 kb in total length, including homology sequences of 46 bp to 1.6 kb on each side of the sequence of interest [2,6,8,10,43,67,68] ( Table 1). 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].…”

“…Regarding the donor DNA, long donor templates (with long sequences of interest or long homology arms) can be delivered to the cells on the same or on another plasmid as the CRISPR-Cas system [2,6,8,27,41,69], or as linear dsDNA fragments, provided as free molecules [67] or released in planta from a plasmid by inserting them between CRISPR target sites [3,10,67]. 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].…”

“…1A) [10]. This strategy was efficient for gene knockin in rice by Agrobacterium transformation of embryonic cells whether the donor template contained homology sequences (25% of lines regenerated on selective media) [67] or not (2% of lines regenerated on selective media) [3].…”

“…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.…”