Transient cotransformation of CRISPR/Cas9 and oligonucleotide templates enables efficient editing of target loci in Physcomitrella patens

A blueprint for gene function analysis through Base Editing in the model plant Physcomitrium (Physcomitrella) patens

“…We provide information on the nature of the edited products, windows of editing, simplex versus multiplex systems and selection strategies, which should facilitate their use in this model plant. BE editors extend the already imposing tool box for precise genome editing in P. patens, such as gene replacement through homologous recombination (Schaefer, 2001), that could be made more efficient and precise using a CRISPR-Cas9 strategy (Collonnier et al, 2017b), or via an elegant recent strategy also based on CRISPR-Cas9 but using oligonucleotide templates (Yi & Goshima, 2020). In theory, all these strategies could benefit from the SMART co-editing selection system described here.…”

“…These studies have shown that CBE or ABE activities can vary between plants, but also between target sites in term of editing efficiency of an effective deamination window and of occurrence of byproducts (insertions, deletions or unpredicted substitutions). In the model plant bryophyte Physcomitrium ( Physcomitrella ) patens , the use of CRISPR‐Cas9 or CRISPR‐Cas12a strategies has permitted efficient gene knock‐out (Nomura et al ., 2016; Lopez‐Obando et al ., 2016; Pu et al ., 2019; Mallett et al ., 2019) or gene knock‐in (Collonnier et al ., 2017a; Yi & Goshima, 2020), but no base‐editing strategies have been reported so far.…”

Co-targeting strategy for precise, scarless gene editing with CRISPR/Cas9 and donor ssODNs inChlamydomonas

“…In higher plants, ssODNs have been used for precise gene editing, as templates for the repair of DSBs generated by CRISPR/Cas9, but very few edited plants/calli have been analyzed by sequencing precluding any inference on the repair mechanism (Shan et al, 2013; Svitashev et al, 2015; Sauer et al, 2016). In the moss Physcomitrella patens , it has been suggested that a template (ssODN)-target pairing mechanism is involved in the repair of DSBs generated by CRISPR/Cas9 (Yi and Goshima, 2020). In our work with Chlamydomonas, several lines of evidence, taken together, support the interpretation that SSTR is responsible for HDR.…”

“…To overcome the outlined problems, we explored whether selection for precise editing of an endogenous gene in Chlamydomonas, which would allow exclusive selection of cells taking up the editing components and capable of carrying out HDR, may facilitate the recovery of precise, scarless edits in another gene of interest, when both genes were simultaneously targeted by co-electroporation of CRISPR/Cas9 RNPs and template donor DNAs. We used single-stranded oligodeoxynucleotides (ssODNs) as donor DNA because prior work demonstrated their usefulness as templates in the repair of CRISPR/Cas-induced DSBs in Chlamydomonas (Ferenczi et al, 2017; Greiner et al, 2017; Jiang and Weeks, 2017) and land plants (Shan et al, 2013; Svitashev et al, 2015; Sauer et al, 2016; Yi and Goshima, 2020).…”

A Fully Functional ROP Fluorescent Fusion Protein Reveals Roles for This GTPase in Subcellular and Tissue-Level Patterning

“…To generate mutations in the ROP genes, we 545 transformed 10-15 µg of pMH-∆ROP123 plasmid and also incorporated 5 µL of each of 546 the three 50-60 bp double-stranded homology oligos (Supplemental Table 1) at 50 mM 547 concentration. These oligos were homologous to the region adjacent to the protospacer, 548 with a few mismatches deleting the PAM sequence and inserting an in-frame stop 549 codon, adapting the methods described in (Yi and Goshima, 2020b). To quantify the size and polarity of ROP null mutant plants, 5 to 7-day-old ground 583 tissue was used to generated protoplasts.…”