Efficient CRISPR/Cas9-Mediated Genome Editing Using a Chimeric Single-Guide RNA Molecule

Abstract

The CRISPR/Cas9 system has been applied in diverse eukaryotic organisms for targeted mutagenesis. However, targeted gene editing is inefficient and requires the simultaneous delivery of a DNA template for homology-directed repair (HDR). Here, we used CRISPR/Cas9 to generate targeted double-strand breaks and to deliver an RNA repair template for HDR in rice (Oryza sativa). We used chimeric single-guide RNA (cgRNA) molecules carrying both sequences for target site specificity (to generate the double-strand breaks) and repair template sequences (to direct HDR), flanked by regions of homology to the target. Gene editing was more efficient in rice protoplasts using repair templates complementary to the non-target DNA strand, rather than the target strand. We applied this cgRNA repair method to generate herbicide resistance in rice, which showed that this cgRNA repair method can be used for targeted gene editing in plants. Our findings will facilitate applications in functional genomics and targeted improvement of crop traits.

Efficient in planta gene targeting in Arabidopsis using egg cell‐specific expression of the Cas9 nuclease of Staphylococcus aureus

“…Interestingly, a superiority of 5 0 over 3 0 overhangs on GT efficiencies has been reported for mammalian cells before (Mali et al, 2013;Bothmer et al, 2017). A similar result was also recently found in transient assays in tobacco cells ( Cerm ak et al, 2017), although no GT events were regenerated and characterized on the molecular level. Our study demonstrates the importance of molecular characterization for the evaluation of GT events, as for most of the seedlings the IM resistance was due to ectopic and not due precise GT in the case of the 5 0 overhang approach (30 out of 34 tested).…”

“…Using Cas9-mediated induction of DSBs, several strategies have been developed to enhance HR-mediated GT frequencies in plants (for a recent review see Steinert et al, 2016). The group of Dan Voytas, for instance, developed an innovative geminivirus replicon-based strategy which enabled them to improve GT frequencies in some plant species (Baltes et al, 2014;Cerm ak et al, 2015). However, at least for monocots, no fertile plants have been reported to be obtained with this technology, as yet Wang et al, 2017).…”

“…The use of DNA oligonucleotides (Svitashev et al, 2015;Sauer et al, 2016) has also been applied successfully for gene correction in plants. In one attempt, a chimeric sgRNA was used as a template for DSB repair (Butt et al, 2017). Additionally, inhibition of NHEJ (Endo et al, 2016;Nishizawa-Yokoi et al, 2016) can help to improve GT frequencies.…”

Application of protoplast technology to CRISPR/Cas9 mutagenesis: from single‐cell mutation detection to mutant plant regeneration

“…Numerous published studies have described various vectors to improve CRISPR-mediated target mutagenesis. These studies describe parameters such as promoters, different versions of Cas9, and the use of multiple sgRNAs (Ali et al, 2015(Ali et al, , 2016Belhaj et al, 2013;Bortesi and Fischer, 2015;Butt et al, 2017;Cermak et al, 2017;Eid et al, 2016;Kaya et al, 2016;Ma et al, 2015;Murovec et al, 2017;Shimatani et al, 2017;Wang et al, 2015b;Yan et al, 2015). Not only Cas9, but other endonucleases such as Cpf1 can induce mutations Kim et al, 2017;Mahfouz, 2017;Xu et al, 2017).…”

Engineering Plant Architecture via CRISPR/Cas9-mediated Alteration of Strigolactone Biosynthesis

“…Genome engineering requires molecular scissors capable of making precise double strand breaks (DSBs) in the genome (Sander and Joung 2014; Voytas and Gao 2014). Such DSBs are repaired by either the imprecise non-homologous end joining (NHEJ) repair or the precise homology directed repair (HDR) pathways (Symington and Gautier 2011; Voytas and Gao 2014; Butt et al 2017). Harnessing the cellular repair pathways of the DSBs, a variety of user-desired genetic outcomes can be generated.…”