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1. Expanding plant genome editing scope and profiles with CRISPR‐FrCas9 systems targeting palindromic TA sites

   https://doi.org/10.1111/pbi.14363  

   He, Yao; Han, Yangshuo; Ma, Yanqin; Liu, Shishi; Fan, Tingting; Liang, Yanling; Tang, Xu; Zheng, Xuelian; Wu, Yuechao; Zhang, Tao; et al (May 2024, Plant Biotechnology Journal)

   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. 

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2. Efficient plant genome engineering using a probiotic sourced CRISPR-Cas9 system

   https://doi.org/10.1038/s41467-023-41802-9  

   Zhong, Zhaohui; Liu, Guanqing; Tang, Zhongjie; Xiang, Shuyue; Yang, Liang; Huang, Lan; He, Yao; Fan, Tingting; Liu, Shishi; Zheng, Xuelian; et al (December 2023, Nature Communications)

   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. 

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3. CRISPR/Cas9-mediated targeted T-DNA integration in rice

   https://doi.org/10.1007/s11103-018-00819-1  

   Lee, L (January 2019, Plant molecular biology)

   Agrobacterium-mediated T-DNA integration into the plant genomes is random, which often causes variable transgene expression and insertional mutagenesis. Because T-DNA preferentially integrates into double-strand DNA breaks, we adapted a CRISPR/Cas9 system to demonstrate that targeted T-DNA integration can be achieved in the rice genome. Using a standard Agrobacterium binary vector, we constructed a T-DNA that contains a CRISPR/Cas9 system using SpCas9 and a gRNA targeting the exon of the rice AP2 domain-containing protein gene Os01g04020. The T-DNA also carried a red fluorescent protein and a hygromycin resistance (hptII) gene. One version of the vector had hptII expression driven by an OsAct2 promoter. In an effort to detect targeted T-DNA insertion events, we built another T-DNA with a promoterless hptII gene adjacent to the T-DNA right border such that integration of T-DNA into the targeted exon sequence in-frame with the hptII gene would allow hptII expression. Our results showed that these constructs could produce targeted T-DNA insertions with frequencies ranging between 4 and 5.3% of transgenic callus events, in addition to generating a high frequency (50−80%) of targeted indel mutations. Sequencing analyses showed that four out of five sequenced T-DNA/gDNA junctions carry a single copy of full-length T-DNA at the target site. Our results indicate that Agrobacterium-mediated transformation combined with a CRISPR/Cas9 system can efficiently generate targeted T-DNA insertions. 

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4. Predictable NHEJ insertion and assessment of HDR editing strategies in plants

   https://doi.org/10.3389/fgeed  

   Molla, Kutubuddin A; Shih, J; Wheatley, Matthew S; Yang, Yinong (March 2022, Frontiers in genome editing)

   Canonical CRISPR-Cas9 genome editing technique has profoundly impacted the fields of plant biology, biotechnology, and crop improvement. Since non-homologous end joining (NHEJ) is usually considered to generate random indels, its high efficiency mutation is generally not pertinent to precise editing. Homology-directed repair (HDR) can mediate precise editing with supplied donor DNA, but it suffers from extreme low efficiency in higher plants. Therefore, precision editing in plants will be facilitated by the ability to predict NHEJ repair outcome and to improve HDR efficiency. Here, we report that NHEJ-mediated single nucleotide insertion at different rice genes is predictable based on DNA sequences at the target loci. Three mutation prediction tools (inDelphi, FORECasT, and SPROUT) have been validated in the rice plant system. We also evaluated the chimeric guide RNA (cgRNA) and Cas9-Retron precISe Parallel Editing via homologY (CRISPEY) strategies to facilitate donor template supply for improving HDR efficiency in Nicotiana benthamiana and rice. However, neither cgRNA nor CRISPEY improved plant HDR editing efficiency in this study. Interestingly, our data indicate that tethering of 200–250 nucleotides long sequence to either 5′ or 3′ ends of guide RNA did not significantly affect Cas9 cleavage activity. 

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5. Status on Genetic Resistance to Rice Blast Disease in the Post-Genomic Era

   https://doi.org/10.3390/plants14050807  

   Pedrozo, Rodrigo; Osakina, Aron; Huang, Yixiao; Nicolli, Camila Primieri; Wang, Li; Jia, Yulin (March 2025, Plants)

   Rice blast, caused by Magnaporthe oryzae, is a major threat to global rice production, necessitating the development of resistant cultivars through genetic improvement. Breakthroughs in rice genomics, including the complete genome sequencing of japonica and indica subspecies and the availability of various sequence-based molecular markers, have greatly advanced the genetic analysis of blast resistance. To date, approximately 122 blast-resistance genes have been identified, with 39 of these genes cloned and molecularly characterized. The application of these findings in marker-assisted selection (MAS) has significantly improved rice breeding, allowing for the efficient integration of multiple resistance genes into elite cultivars, enhancing both the durability and spectrum of resistance. Pangenomic studies, along with AI-driven tools like AlphaFold2, RoseTTAFold, and AlphaFold3, have further accelerated the identification and functional characterization of resistance genes, expediting the breeding process. Future rice blast disease management will depend on leveraging these advanced genomic and computational technologies. Emphasis should be placed on enhancing computational tools for the large-scale screening of resistance genes and utilizing gene editing technologies such as CRISPR-Cas9 for functional validation and targeted resistance enhancement and deployment. These approaches will be crucial for advancing rice blast resistance, ensuring food security, and promoting agricultural sustainability. 

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