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Comparative assessments of CRISPR-Cas nucleases’ cleavage efficiency in planta

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Abstract

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.

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Acknowledgments

We thank all members of Avraham A. Levy’s laboratory at the Weizmann Institute of Science, including Dr. Cathy Melamed-Bessudo for technical advice, and Tal Dahan for her help maintaining N. benthamiana plants. We thank Prof. Holger Puchta from Karlsruhe Institute of Technology and Prof. Daniel Voytas at the University of Minnesota for providing Cas9 gene constructs. Further thanks to Prof. Sophien Kamoun from the Sainsbury Laboratory for providing another Cas9 gene construct. We also thank Dario Breitel and Prof. Asaph Aharoni who developed the A. thaliana seed genomic DNA extraction protocol that was used here. This work was funded by an EU-FP7 TRACTAR grant from the European Research Council.

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot, Israel

    Ross A. Johnson, Vyacheslav Gurevich, Shdema Filler, Aviva Samach & Avraham A. Levy

  2. Department of Plant Biology, University of California, Davis, Davis, CA, 95616, USA

    Ross A. Johnson

  3. Faculty of Medicine in the Galilee, Bar-Ilan University, Safed, Israel

    Vyacheslav Gurevich

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  1. Ross A. Johnson
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  2. Vyacheslav Gurevich
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Correspondence to Ross A. Johnson.

Electronic supplementary material

Below is the link to the electronic supplementary material.

The sequences and applications of synthesized molecules that were used in the study

The sequences and intended applications of synthesized molecules that we could not clone during the study

11103_2014_266_MOESM3_ESM.docx

Testing the CRISPR-Cas cleavage efficiency using a single sgRNA, delivered in one of two vectors, as well as using double sgRNAs with juxtaposed target sites

An additional sgRNA expression cassette was not found to increase the CRISPR-Cas cleavage efficiency in planta

11103_2014_266_MOESM5_ESM.docx

Target sites for de novo-developed CRISPR-Cas sgRNAs in the tomato (Solanum lycopersicum) phytoene synthase-encoding gene 1 (SlPSY1) and the carotene cis–trans-isomerase-encoding gene (SlCrtISO)

Features of CRISPR-Cas sgRNAs relative to their cleavage performance, for our work and reports of others

11103_2014_266_MOESM7_ESM.docx

The cleavage efficiency of the CRISPR-Cas sgRNA designed to cleave the ‘3m’ target sequences naturally repeated in the Arabidopsis thaliana CRU3 gene

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Johnson, R.A., Gurevich, V., Filler, S. et al. Comparative assessments of CRISPR-Cas nucleases’ cleavage efficiency in planta . Plant Mol Biol 87, 143–156 (2015). https://doi.org/10.1007/s11103-014-0266-x

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