Expanding plant genome-editing scope by an engineered iSpyMacCas9 system that targets A-rich PAM sequences

Simon Sretenovic; Desuo Yin; Adam Levav; Jeremy D Selengut; Stephen M Mount; Yiping Qi

doi:10.1016/j.xplc.2020.100101

Expanding plant genome-editing scope by an engineered iSpyMacCas9 system that targets A-rich PAM sequences

Plant Commun. 2020 Jul 22;2(2):100101. doi: 10.1016/j.xplc.2020.100101. eCollection 2021 Mar 8.

Authors

Simon Sretenovic¹, Desuo Yin^{1

2}, Adam Levav^{1

3}, Jeremy D Selengut⁴, Stephen M Mount⁵, Yiping Qi^{1

6}

Affiliations

¹ Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD 20742, USA.
² Hubei Academy of Agricultural Sciences, Wuhan 430064, China.
³ Montgomery Blair High School, Silver Spring, MD 20901, USA.
⁴ Center for Bioinformatics and Computational Biology, University of Maryland, College Park, MD 20742, USA.
⁵ Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA.
⁶ Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD 20850, USA.

Abstract

The most popular CRISPR-SpCas9 system recognizes canonical NGG protospacer adjacent motifs (PAMs). Previously engineered SpCas9 variants, such as Cas9-NG, favor G-rich PAMs in genome editing. In this manuscript, we describe a new plant genome-editing system based on a hybrid iSpyMacCas9 platform that allows for targeted mutagenesis, C to T base editing, and A to G base editing at A-rich PAMs. This study fills a major technology gap in the CRISPR-Cas9 system for editing NAAR PAMs in plants, which greatly expands the targeting scope of CRISPR-Cas9. Finally, our vector systems are fully compatible with Gateway cloning and will work with all existing single-guide RNA expression systems, facilitating easy adoption of the systems by others. We anticipate that more tools, such as prime editing, homology-directed repair, CRISPR interference, and CRISPR activation, will be further developed based on our promising iSpyMacCas9 platform.

Keywords: PAM; adenine base editing; cytosine base editing; iSpyMacCas9; plant genome editing.

Publication types

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

MeSH terms

CRISPR-Cas Systems*
Gene Editing / methods*
Genome, Plant*
Oryza / genetics*
Triticum / genetics*
Zea mays / genetics*