MolecularCloud February Newsletter: Plant Genome Editing by SpCas9 Variants

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

Cat. No.

Plasmid Name


SpCas9-NRRH binary vector



SpCas9-NRTH binary vector

SpCas9-NRCH binary vector
SpG binary vector
SpRY binary vector
SpRY CBE binary vector with extra UGI copies

If you want to get those plasmids, please leave a massage on the comment area below or send your request to

*This material is available to academic and nonprofit organizations for research use only. Please contact MolecularCloud at and the Depositor directly if you are from industrial institute or attempt for profit application. 

Time-limited Campaign

With the help of Dr. Pengcheng Wei's team, MolecularCloud has initiated a reward-winning campaign for this specific topic "plant genome editing". As long as you get the highest score or the first perfect score from the quiz below, you'll win yourself a $20 gift card or a free plasmid.

*MolecularCloud reserves the right of final explanation for details of this campaign. If there's any further question, please feel free to contact us at

Previous Newsletters

MolecularCloud January Newsletter: Detergent-free Extraction of Membrane Protein
MolecularCloud December Newsletter: Methods and Protocols for Cereal Genomics
MolecularCloud November Newsletter: Predicting the effect of mutations on protein–RNA binding

Q1: Which of the following is the earliest tool for precise genome editing of plants? (1 mark)

  • ZFN
  • Base editor

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Q2: Which of the following is the most widely used genome editing system in plants? (2 marks)

  • Cas13
  • Cas12a
  • SaCas9
  • SpCas9

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Q3: Is protospacer adjacent motif a component of the bacterial CRISPR locus? (3 marks)

  • Yes
  • No

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Q4: How long is a typical protospacer adjacent motif of Type II-A? (4 marks)

  • 1-2 base pairs
  • 2-6 base pairs
  • 6-15 base pairs
  • 15-20 base pairs

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Q5: Which of the following was not a variant developed from SpCas9? (5 marks)

  • SpCas9-NRRH
  • SpCas9-NRCH
  • SpCas9-NRAH
  • SpCas9-NRTH
  • SpG

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6 Reply

Participated. Again wonderful quiz and read a lot to finish this. Thanks again Molecular Cloud and team for awesome effort. Please vote to Neha Mittal and her team members for molecular cloud award which is based on this area.

Participating first time in this type of quiz on the scientific platform. Well worked by the molecular cloud communication. #Neha Mittal and peer's team members

Well that was fun. I can remember some of this from coaching Science Olympiad Protein Modeling...

Awesome quiz. #Neha Mittal and team members for Molecular Cloud Award.

Excellent quiz. Enjoyed a lot. I am also supporting the team of Neha Mittal who has a good work in the genome editing filed for molecular distinguished award. #Neha Mittal Team for Molecular Distinguished Research Award.

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