Streptococcus pyogenes Cas9 (SpCas9) is the most popular system for plant genome editing. With its robust efficiency, it is considered as the default platform to develop multiple tools for plant genome engineering.
However, the system strictly requires a canonical NGG protospacer-adjacent motif (PAM) immediately downstream of targets thus limiting the scope of application especially for precise genome editing, including base editing, prime editing,and targeting of small regulatory elements.
Dr. Pengcheng Wei's team recently published an article in Molecular Plant titled "Genome editing mediated by SpCas9 variants with broad non-canonical protospacer-adjacent motif compatibility in plants", reporting five SpCas9 variants engineered to recognize non-canonical PAMs for plant genome editing and developing base editors from those variants (Molecular Plant, 2020). In this study, SpCas9 variants including SpCas9-NRRH, SpCas9-NRCH, SpCas9-NRTH, SpG, and SpRY, were engineered and comprehensively evaluated for their efficacy to conduct targeted mutagenesis and base editing in rice genome.
The cassettes of Cas9 and sgRNA in binary vectors for targeted mutagenesis (Molecular Plant, 2020). OsU3, rice U3 promoter; ZmUBI, maize Ubiquitin 1 promoter; Poly T, poly T terminator; 35S Ter, CaMV 35S terminator. The amino acid mutations to develop the variants from the SpCas9-WT are listed.
As showed in the above figure, the plant SpCas9-NRRH, SpCas9-NRCH, and SpCas9-NRTH variants were developed by introducing 13-16 amino acid mutations in a rice codon-optimized SpCas9-WT. The results obtained from this study reveled that those three variants can enable gene editing with expanded PAM compatibility in rice. To introduce precise base editing atnon-canonical PAMs in plants, the researchers further adopted those variants for constructing base editors and achieved precise nucleotide substitutions mediated by SpG-derivated base editors in the rice genome.
As concluded by the authors of this article, a complete solution for nearly PAM-unlimited genome editing in plant may have been established. SpCas9-WT and SpG are applied to mediate efficient targeting at NGG and NGH PAM, respectively; SpCas9-NRRH, SpCas9-NRCH and SpCas9-NRTH collectively target NRNH PAMs, particularly NANH PAMs; and practicable editing at NYN and some NA sites could be attempted by SpRY.
The five novel tools would largely break the restriction on PAM requirement of CRISPR-SpCas9 system and tremendously accelerate the usage of genome editing in plant functional genomics research as well as crop breeding.
Plasmids from this article have been deposited on MolecularCloud
|pHUC-SpCas9-NRRH||SpCas9-NRRH binary vector|
SpCas9-NRTH binary vector
|MC_0101195||pHUC-SpCas9-NRCH||SpCas9-NRCH binary vector|
|MC_0101196||pHUC-SpG||SpG binary vector|
|MC_0101197||pHUC-SpRY||SpRY binary vector|
|MC_0101198||pHUC-SpRY-eBE3||SpRY CBE binary vector with extra UGI copies|
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