CRISPR‐Cas9‐mediated efficient directed mutagenesis and RAD51‐dependent and RAD51‐independent gene targeting in the moss Physcomitrella patens

Abstract

SummaryThe ability to address the CRISPR‐Cas9 nuclease complex to any target DNA using customizable single‐guide RNAs has now permitted genome engineering in many species. Here, we report its first successful use in a nonvascular plant, the moss Physcomitrella patens. Single‐guide RNAs (sgRNAs) were designed to target an endogenous reporter gene, PpAPT, whose inactivation confers resistance to 2‐fluoroadenine. Transformation of moss protoplasts with these sgRNAs and the Cas9 coding sequence from Streptococcus pyogenes triggered mutagenesis at the PpAPT target in about 2% of the regenerated plants. Mainly, deletions were observed, most of them resulting from alternative end‐joining (alt‐EJ)‐driven repair. We further demonstrate that, in the presence of a donor DNA sharing sequence homology with the PpAPT gene, most transgene integration events occur by homology‐driven repair (HDR) at the target locus but also that Cas9‐induced double‐strand breaks are repaired with almost equal frequencies by mutagenic illegitimate recombination. Finally, we establish that a significant fraction of HDR‐mediated gene targeting events (30%) is still possible in the absence of PpRAD51 protein, indicating that CRISPR‐induced HDR is only partially mediated by the classical homologous recombination pathway.

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

“…Finally, it was demonstrated in Physcomitrella patens that a strong bias towards MMEJ (microhomology end-joining)-driven targeted mutagenesis was observed when inducing DSBs in the vicinity of sequences presenting microhomologies [41]. To avoid this phenomenon that could compete with the insertion of the donor template, selecting sgRNAs inducing DSBs at sites that do not present obvious microhomologies of more than 2 bp in the vicinity of the cleavage site (usually occuring 3 bp upstream the PAM) is preferable.…”

“…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].…”

“…The CRISPR-Cas9 system was successfully applied to P. patens for targeted mutagenesis using up to 5 sgRNAs simultaneously [41,110]. The mutations observed included a diversity of deletions, insertions and/or substitutions mainly resulting from NHEJ, but also deletions resulting from microhomology-mediated end joining (MMEJ) when micro-homologies were located on both sides of the cleavage sites [41].…”

“…The mutations observed included a diversity of deletions, insertions and/or substitutions mainly resulting from NHEJ, but also deletions resulting from microhomology-mediated end joining (MMEJ) when micro-homologies were located on both sides of the cleavage sites [41].…”

“…Three separate plasmids carrying the Cas9, the sgRNA and a donor template bearing an antibiotic resistance gene framed by homologies to the reporter gene PpAPT were co-transformed by PEG fusion of protoplasts [41]. As previously described in P. patens [111][112][113][114] and as frequently observed in other plants [115], the donor DNA template can be inserted in two different ways: either by homologous recombination on both sides leading to targeted gene replacement (TGR) or by HDR on one side and NHEJ on the other, upstream or downstream of the targeted locus, leading to targeted gene insertions (TGI).…”