Plant biotechnology journalMay 7, 2024

Expanding plant genome editing using CRISPR-FrCas9 systems that target palindrome TA DNA sites

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗OA ↗

Updated Mar 30, 2026

Abstract

FrCas9 recognizes a concise 5'-NNTA-3' , enabling targeted mutagenesis in rice.

FrCas9 targets more abundant palindromic TA sites in plant genomes compared to SpCas9's PAM requirements.
It demonstrates cleavage activities at all tested 5'-NNTA-3' PAM sites, producing editing outcomes typical of conventional CRISPR systems.
FrCas9 enables high-efficiency targeted mutagenesis and generates biallelic mutants in stable rice lines.
The fusion of FrCas9 with TREX2 enhances its capacity to produce larger deletions while maintaining editing efficiency.
FrCas9-based cytosine and adenine base editors can create targeted C-to-T and A-to-G edits in rice plants.
Whole-genome sequencing indicates that while FrCas9 is highly specific, TREX2-FrCas9 may lead to off-target mutations, primarily single nucleotide variants.

Simplified

CRISPR-Cas9 is widely used for genome editing, but its sequence requirements limit its efficiency. In this study, we explore Faecalibaculum rodentium Cas9 (FrCas9) for plant genome editing, especially in rice. FrCas9 recognizes a concise 5'-NNTA-3' PAM, targeting more abundant palindromic TA sites in plant genomes than the 5'-NGG-3' PAM sites of the most popular SpCas9. FrCas9 shows cleavage activities at all tested 5'-NNTA-3' PAM sites with editing outcomes sharing the same characteristics of a typical CRISPR-Cas9 system. FrCas9 induces high-efficiency targeted mutagenesis in stable rice lines, readily generating biallelic mutants with expected phenotypes. We augment FrCas9's ability to generate larger deletions through fusion with the exonuclease, TREX2. TREX2-FrCas9 generates much larger deletions than FrCas9 without compromise in editing efficiency. We demonstrate TREX2-FrCas9 as an efficient tool for genetic knockout of a microRNA gene. Furthermore, FrCas9-derived cytosine base editors (CBEs) and adenine base editors (ABE) are developed to produce targeted C-to-T and A-to-G base edits in rice plants. Whole-genome sequencing-based off-target analysis suggests that FrCas9 is a highly specific nuclease. Expression of TREX2-FrCas9 in plants, however, causes detectable guide RNA-independent off-target mutations, mostly as single nucleotide variants (SNVs). Together, we have established an efficient CRISPR-FrCas9 system for targeted mutagenesis, large deletions, C-to-T base editing, and A-to-G base editing in plants. The simple palindromic TA motif in the PAM makes the CRISPR-FrCas9 system a promising tool for genome editing in plants with an expanded targeting scope.

Key numbers

2.7% to 53.9%

Editing Efficiency Range

Measured at 16 endogenous sites in rice protoplasts.

6–20 bp

Larger Deletions Achieved

Deletion sizes generated by TREX2-FrCas9 compared to FrCas9.

59.5%

C-to-T Conversion Rate

Achieved by CBE V2.1 at four target sites.

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Funding

Competing interests

The authors declare no competing interests.

PubMed

Expanding plant genome editing using CRISPR-FrCas9 systems that target palindrome TA DNA sites

Abstract

Key numbers

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Abstract

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Full Text

Funding

Competing interests

Related papers

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What Lands in Your Inbox Each Week:

Recent issues from the crispr gene editing brief