PAM-Less CRISPR-SpRY Genome Editing in Plants

Simon Sretenovic; Xu Tang; Qiurong Ren; Yong Zhang; Yiping Qi

doi:10.1007/978-1-0716-3131-7_1

PAM-Less CRISPR-SpRY Genome Editing in Plants

Methods Mol Biol. 2023:2653:3-19. doi: 10.1007/978-1-0716-3131-7_1.

Authors

Simon Sretenovic¹, Xu Tang², Qiurong Ren², Yong Zhang³, Yiping Qi^{4

5}

Affiliations

¹ Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD, USA.
² Department of Biotechnology, School of Life Sciences and Technology, Center for Informational Biology, University of Electronic Science and Technology of China, Chengdu, China.
³ Department of Biotechnology, School of Life Sciences and Technology, Center for Informational Biology, University of Electronic Science and Technology of China, Chengdu, China. zhangyong916@uestc.edu.cn.
⁴ Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD, USA. Yiping@umd.edu.
⁵ Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD, USA. Yiping@umd.edu.

PMID: 36995616
DOI: 10.1007/978-1-0716-3131-7_1

Abstract

Engineered SpCas9 variant, SpRY, has been demonstrated to facilitate protospacer adjacent motif (PAM) unrestricted targeting of genomic DNA in various biological systems. Here we describe fast, efficient, and robust preparation of SpRY-derived genome and base editors that can be easily adapted to target various DNA sequences in plants due to modular Gateway assembly. Presented are detailed protocols for preparing T-DNA vectors for genome and base editors and for assessing genome editing efficiency through transient expression of these reagents in rice protoplasts.

Keywords: Base editing; CRISPR-Cas9; Gateway assembly; Genome editing; Rice protoplast assay; SpRY.

Publication types

Research Support, U.S. Gov't, Non-P.H.S.
Research Support, Non-U.S. Gov't

MeSH terms

CRISPR-Associated Protein 9 / genetics
CRISPR-Cas Systems / genetics
Gene Editing* / methods
Genome, Plant
Oryza* / genetics
Oryza* / metabolism

Substances

CRISPR-Associated Protein 9