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Summary Class 2 Type V‐A CRISPR‐Cas (Cas12a) nucleases are powerful genome editing tools, particularly effective in A/T‐rich genomic regions, complementing the widely used CRISPR‐Cas9 in plants. To enhance the utility of Cas12a, we investigate three Cas12a orthologs—Mb3Cas12a, PrCas12a, and HkCas12a—in plants. Protospacer adjacent motif (PAM) requirements, editing efficiencies, and editing profiles are compared in rice. Among these orthologs, Mb3Cas12a exhibits high editing efficiency at target sites with a simpler, relaxed TTV PAM which is less restrictive than the canonical TTTV PAM of LbCas12a and AsCas12a. To optimize Mb3Cas12a, we develop an efficient single transcription unit (STU) system by refining the linker between Mb3Cas12a and CRISPR RNA (crRNA), nuclear localization signal (NLS), and direct repeat (DR). This optimized system enables precise genome editing in rice, particularly for fine‐tuning target gene expression by editing promoter regions. Further, we introduced Arginine (R) substitutions at Aspartic acid (D) 172, Asparagine (N) 573, and Lysine (K) 579 of Mb3Cas12a, creating two temperature‐tolerant variants: Mb3Cas12a‐R (D172R) and Mb3Cas12a‐RRR (D172R/N573R/K579R). These variants demonstrate significantly improved editing efficiency at lower temperatures (22 °C and 28 °C) in rice cells, with Mb3Cas12a‐RRR showing the best performance. We extend this approach by developing efficient Mb3Cas12a‐RRR STU systems in maize and tomato, achieving biallelic mutants targeting single or multiple genes in T₀lines cultivated at 28 °C and 25 °C, respectively. This study significantly expands Cas12a's targeting capabilities in plant genome editing, providing valuable tools for future research and practical applications. 

Abstract Cytosine base editors (CBEs) and adenine base editors (ABEs) enable precise C-to-T and A-to-G edits. Recently, ABE8e, derived from TadA-8e, enhances A-to-G edits in mammalian cells and plants. Interestingly, TadA-8e can also be evolved to confer C-to-T editing. This study compares engineered CBEs derived from TadA-8e in rice and tomato cells, identifying TadCBEa, TadCBEd, and TadCBEd_V106W as efficient CBEs with high purity and a narrow editing window. A dual base editor, TadDE, promotes simultaneous C-to-T and A-to-G editing. Multiplexed base editing with TadCBEa and TadDE is demonstrated in transgenic rice, with no off-target effects detected by whole genome and transcriptome sequencing, indicating high specificity. Finally, two crop engineering applications using TadDE are shown: introducing herbicide resistance alleles inOsALSand creating synonymous mutations inOsSPL14to resistOsMIR156-mediated degradation. Together, this study presents TadA-8e derived CBEs and a dual base editor as valuable additions to the plant editing toolbox. 

Summary CRISPR‐Cas9 is widely used for genome editing, but its PAM sequence requirements limit its efficiency. In this study, we exploreFaecalibaculum rodentiumCas9 (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. 

Abstract Among CRISPR-Cas genome editing systems,Streptococcus pyogenesCas9 (SpCas9), sourced from a human pathogen, is the most widely used. Here, through in silico data mining, we have established an efficient plant genome engineering system using CRISPR-Cas9 from probioticLactobacillus rhamnosus. We have confirmed the predicted 5’-NGAAA-3’ PAM via a bacterial PAM depletion assay and showcased its exceptional editing efficiency in rice, wheat, tomato, and Larix cells, surpassing LbCas12a, SpCas9-NG, and SpRY when targeting the identical sequences. In stable rice lines, LrCas9 facilitates multiplexed gene knockout through coding sequence editing and achieves gene knockdown via targeted promoter deletion, demonstrating high specificity. We have also developed LrCas9-derived cytosine and adenine base editors, expanding base editing capabilities. Finally, by harnessing LrCas9’s A/T-rich PAM targeting preference, we have created efficient CRISPR interference and activation systems in plants. Together, our work establishes CRISPR-LrCas9 as an efficient and user-friendly genome engineering tool for diverse applications in crops and beyond. 

Abstract CRISPR-Cas12a is a promising genome editing system for targeting AT-rich genomic regions. Comprehensive genome engineering requires simultaneous targeting of multiple genes at defined locations. Here, to expand the targeting scope of Cas12a, we screen nine Cas12a orthologs that have not been demonstrated in plants, and identify six, ErCas12a, Lb5Cas12a, BsCas12a, Mb2Cas12a, TsCas12a and MbCas12a, that possess high editing activity in rice. Among them, Mb2Cas12a stands out with high editing efficiency and tolerance to low temperature. An engineered Mb2Cas12a-RVRR variant enables editing with more relaxed PAM requirements in rice, yielding two times higher genome coverage than the wild type SpCas9. To enable large-scale genome engineering, we compare 12 multiplexed Cas12a systems and identify a potent system that exhibits nearly 100% biallelic editing efficiency with the ability to target as many as 16 sites in rice. This is the highest level of multiplex edits in plants to date using Cas12a. Two compact single transcript unit CRISPR-Cas12a interference systems are also developed for multi-gene repression in rice and Arabidopsis . This study greatly expands the targeting scope of Cas12a for crop genome engineering.