Repositorium
Journal Article / 2014
Johnson, Ross A.; Gurevich, Vyacheslav; Filler, Shdema; Samach, Aviva; Levy, Avraham A.
Custom-designed nucleases can enable precise plant genome editing by catalyzing DNA-breakage at specific targets to stimulate targeted mutagenesis or gene replacement. The CRISPR-Cas system, with its target-specifying RNA molecule to direct the Cas9 nuclease, is a recent addition to existing nucleases that bind and cleave the target through linked protein domains (e.g. TALENs and zinc-finger nucleases). We have conducted a comparative study of these different types of custom-designed nucleases and we have assessed various components of the CRISPR-Cas system. For this purpose, we have adapted our previously reported assay for cleavage-dependent luciferase gene correction in Nicotiana benthamiana leaves (Johnson et al. in Plant Mol Biol 82(3):207-221, 2013). We found that cleavage by CRISPR-Cas was more efficient than cleavage of the same target by TALENs. We also compared the cleavage efficiency of the Streptococcus pyogenes Cas9 protein based on expression using three different Cas9 gene variants. We found significant differences in cleavage efficiency between these variants, with human and Arabidopsis thaliana codon-optimized genes having the highest cleavage efficiencies. We compared the activity of 12 de novo-designed single synthetic guide RNA (sgRNA) constructs, and found their cleavage efficiency varied drastically when using the same Cas9 nuclease. Finally, we show that, for one of the targets tested with our assay, we could induce a germinally-transmitted deletion in a repeat array in A. thaliana. This work emphasizes the efficiency of the CRISPR-Cas system in plants. It also shows that further work is needed to be able to predict the optimal design of sgRNAs or Cas9 variants.
Techniques
ID | Corresponding Author Country |
Plant Species | GE Technique Sequence Identifier |
Trait Type of Alteration |
Progress in Research Key Topic |
---|---|---|---|---|---|
187 |
Johnson, Ross A. Israel, USA |
Arabidopsis thaliana |
CRISPR/Cas9 CRU3 |
No information SDN1 |
Basic research Basic research |
188 |
Johnson, Ross A. Israel, USA |
Nicotiana benthamiana |
CRISPR/Cas9 LUC |
No information SDN1 |
Basic research Basic research |
189 |
Johnson, Ross A. Israel, USA |
Solanum lycopersicum |
CRISPR/Cas9 CrtISO |
No information SDN1 |
Basic research Basic research |
190 |
Johnson, Ross A. Israel, USA |
Solanum lycopersicum |
CRISPR/Cas9 PSY1 |
Albino phenotype SDN1 |
Basic research Basic research |