SpRY greatly expands the genome editing scope in rice with highly flexible PAM recognition

Background Plant genome engineering mediated by various CRISPR-based tools requires specific protospacer adjacent motifs (PAMs), such as the well-performed NGG, NG, and NNG, to initiate target recognition, which notably restricts the editable range of the plant genome. Results In this study, we thor...

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Published in:Genome Biology Vol. 22; no. 1; p. 6
Main Authors: Xu, Ziyan, Kuang, Yongjie, Ren, Bin, Yan, Daqi, Yan, Fang, Spetz, Carl, Sun, Wenxian, Wang, Guirong, Zhou, Xueping, Zhou, Huanbin
Format: Journal Article
Language:English
Published: London BioMed Central 04.01.2021
Springer Nature B.V
BMC
Subjects:
Adenosine
Adenosine deaminase
Animal Genetics and Genomics
Base editing
Bioinformatics
Biomedical and Life Sciences
CRISPR
CRISPR-Associated Protein 9
CRISPR-Cas Systems
Cytidine deaminase
Deoxyribonucleic acid
DNA
DNA, Plant
Efficiency
Evolutionary Biology
Gene Editing
Gene loci
Genes, Plant - genetics
Genetic engineering
genetically modified organisms
genome
Genome editing
Genome, Plant
Genomes
genomics
Genotype
Human Genetics
Life Sciences
Microbial Genetics and Genomics
Mutation
Nuclease
Oryza - genetics
Plant Genetics and Genomics
Plants, Genetically Modified
Rice
Self-targeting
SpG
SpRY
T-DNA
CRISPR
SpRY
Base editing
Self-targeting
Genome editing
SpG
L
Oryza sativa L
ISSN:1474-760X, 1474-7596, 1474-760X
Online Access:Get full text
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Summary:Background Plant genome engineering mediated by various CRISPR-based tools requires specific protospacer adjacent motifs (PAMs), such as the well-performed NGG, NG, and NNG, to initiate target recognition, which notably restricts the editable range of the plant genome. Results In this study, we thoroughly investigate the nuclease activity and the PAM preference of two structurally engineered SpCas9 variants, SpG and SpRY, in transgenic rice. Our study shows that SpG nuclease favors NGD PAMs, albeit less efficiently than the previously described SpCas9-NG, and that SpRY nuclease achieves efficient editing across a wide range of genomic loci, exhibiting a preference of NGD as well as NAN PAMs. Furthermore, SpRY-fused cytidine deaminase hAID*Δ and adenosine deaminase TadA8e are generated, respectively. These constructs efficiently induce C-to-T and A-to-G conversions in the target genes toward various non-canonical PAMs, including non-G PAMs. Remarkably, high-frequency self-editing events (indels and DNA fragments deletion) in the integrated T-DNA fragments as a result of the nuclease activity of SpRY are observed, whereas the self-editing of SpRY nickase-mediated base editor is quite low in transgenic rice lines. Conclusions The broad PAM compatibility of SpRY greatly expands the targeting scope of CRISPR-based tools in plant genome engineering.
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ISSN:1474-760X
1474-7596
1474-760X
DOI:10.1186/s13059-020-02231-9