Aug 2026· New Phytologist· 1 citation· 60 references
Medicine
TL;DR
This study expands Cas12a targeting scope in plants and provides the first evidence for Cas12-mediated MMEJ-based large-fragment deletion in plants.
Abstract
Cas12a is widely used in plant genome editing, but its targeting scope is constrained by stringent protospacer adjacent motif (PAM) requirements and variable activity across species, limiting its application at diverse genomic loci. LbCas12a-RRV-based editing system was established in nonheading Chinese cabbage, and T5exo-PF-LbCas12a was generated by introducing a triple mutation (D535G/S551F/D665N) and fusing with T5 exonuclease. This engineered system recognizes an expanded PAM sequence from 5'-VTTV-3' to 5'-NYHV-3'. The system exhibited efficient editing at noncanonical PAM sites in cabbage, tomato, and rice. Additionally, it successfully mediated large-fragment deletions via microhomology-mediated end joining (MMEJ) in plants. This study expands Cas12a targeting scope in plants and provides the first evidence for Cas12-mediated MMEJ-based large-fragment deletion. The toolkit facilitates functional genomics and crop improvement, and the methodology is readily adaptable to other plant species.
The results demonstrate that the AI-designed OpenCRISPR-1 system is a highly active and versatile nuclease for dicot genome engineering, offering a powerful, commercially unencumbered tool to accelerate global crop trait improvement.
A substantial decrease in menthofuran content in the essential oil of the edited line #10 compared to the wild-type control is revealed, thereby demonstrating a viable strategy for improving mint essential oil quality through genome-editing.
Natural variation in PAM recognition among SaCas9 orthologs is analyzed and StaCas9 is identified as a compact and efficient nuclease recognizing an NNG PAM, establishing StaCas9 as a high-performance genome-editing tool for therapeutic applications.
The CRISPR-Cas12f system is an ultracompact genome-editing platform, yet only a few orthologs exhibit robust activity in mammalian cells. Here, we systematically screened 23 Cas12f orthologs and identified two active nucleases, PspCas12f1 and TcCas12f1, capable of genome editing in human cells. sgRNA scaffold optimization enhanced the basal activity of PspCas12f1. To further improve its performance, we combined structure-guided rational design with protein language model-assisted filtering. Candidate mutations predicted by SaProt were further screened based on structural proximity to the DNA-binding interface and electrostatic compatibility. This integrative strategy identified Q100R and E293R, whose combination yielded the optimized variant enPspCas12f1. enPspCas12f1 achieved genome-editing efficiencies comparable to SpCas9 across multiple endogenous loci while maintaining high specificity. Collectively, our results demonstrate that integrating protein language model-assisted filtering with structure-guided rational design provides an effective strategy for engineering PspCas12f1 and may facilitate the optimization of additional compact CRISPR nucleases.
Jingtong Liu, Sheng-Zhou Wang, Bei Wang et al.· Molecular Therapy· 0 citations
iPB-REG is established as a practical strategy for producing uniform genome-edited fruit trees and provide a valuable platform for DNA-free genetic improvement and functional genomics in clonally propagated perennial crops.
C. Nishitani, Nozomi Tsujino, Misa Kuroki et al.· bioRxiv· 0 citations
A novel highly efficient CRISPR/Cas9-based dual-sgRNA expression editing system for F. oxysporum suitable not only for single-gene editing but also for large-fragment deletion and multiplex gene editing, although the editing efficiency is somewhat lower.
Wangjie Zhu, Jiao Liao, Yuanyuan Liu et al.· ACS Synthetic Biology· 0 citations
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