Genome editing in plants using the compact editor CasΦ

Author:

Li Zheng1,Zhong Zhenhui1ORCID,Wu Zhongshou1,Pausch Patrick2,Al-Shayeb Basem34,Amerasekera Jasmine5,Doudna Jennifer A.36789ORCID,Jacobsen Steven E.110

Affiliation:

1. Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, CA 90095

2. Life Sciences Center - European Molecular Biology Laboratory (LSC-EMBL) Partnership for Genome Editing Technologies, Vilnius University – Life Sciences Center, Vilnius 10257, Lithuania

3. Innovative Genomics Institute, University of California, Berkeley, CA 94720

4. Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720

5. Department of Human Genetics, University of California, Los Angeles, CA 90095

6. HHMI, University of California, Berkeley, CA 94720

7. Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720

8. California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, CA 94720

9. Department of Chemistry, University of California, Berkeley, CA 94720

10. HHMI, University of California, Los Angeles, CA 90095

Abstract

Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) systems have been developed as important tools for plant genome engineering. Here, we demonstrate that the hypercompact CasΦ nuclease is able to generate stably inherited gene edits in Arabidopsis , and that CasΦ guide RNAs can be expressed with either the Pol-III U6 promoter or a Pol-II promoter together with ribozyme mediated RNA processing. Using the Arabidopsis fwa epiallele, we show that CasΦ displays higher editing efficiency when the target locus is not DNA methylated, suggesting that CasΦ is sensitive to chromatin environment. Importantly, two CasΦ protein variants, vCasΦ and nCasΦ, both showed much higher editing efficiency relative to the wild-type CasΦ enzyme. Consistently, vCasΦ and nCasΦ yielded offspring plants with inherited edits at much higher rates compared to WTCasΦ. Extensive genomic analysis of gene edited plants showed no off-target editing, suggesting that CasΦ is highly specific. The hypercompact size, T-rich minimal protospacer adjacent motif (PAM), and wide range of working temperatures make CasΦ an excellent supplement to existing plant genome editing systems.

Publisher

Proceedings of the National Academy of Sciences

Subject

Multidisciplinary

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