ArticleNucleic acids research2025
Directed evolution expands CRISPR-Cas12a genome-editing capacity.
Article in Nucleic acids research, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 8 papers.
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The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.
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Who cites it
8 citing papers in PubMed.
- Lachnospiraceae in the Food-Gut Axis: A Critical Review of Dietary Modulation, Immune Regulation, and Translational Barriers.Comprehensive reviews in food science and food safety · 2026Review
- Establishment of an efficient and PAM-relaxed LbCas12a genome editing tool in plants.The New phytologist · 2026Article
- Molecular mechanisms and biotechnology applications of CRISPR-Cas12a.Nature reviews. Molecular cell biology · 2026Review
- Structure and evolution-guided design of minimal RNA-guided nucleases.Science (New York, N.Y.) · 2026Article
- Structure-Guided Engineering of a Cas12i Nuclease Unlocks Near-PAMless Genome Editing.Advanced science (Weinheim, Baden-Wurttemberg, Germany) · 2026Article
- crRNA scaffold remodeling controls CRISPR-Cas12a activity for enhanced performance.Nucleic acids research · 2026Article
- Functional decoupling of crRNA enables customizable CRISPR diagnostics.Nucleic acids research · 2026Article
- Structure and evolution-guided design of minimal RNA-guided nucleases.bioRxiv : the preprint server for biology · 2025Article
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Authors and funding
9 authors.
Funding
Abstract
CRISPR-Cas12a enzymes are versatile RNA-guided genome-editing tools with applications encompassing viral diagnosis, agriculture, and human therapeutics. However, their dependence on a 5'-TTTV-3' protospacer adjacent motif (PAM) next to DNA target sequences restricts Cas12a's gene targeting capability to only ∼1% of a typical genome. To mitigate this constraint, we used a bacterial-based directed evolution assay combined with rational engineering to identify variants of Lachnospiraceae bacterium Cas12a with expanded PAM recognition. The resulting Cas12a variants use a range of noncanonical PAMs while retaining recognition of the canonical 5'-TTTV-3' PAM. In particular, biochemical and cell-based assays show that the variant Flex-Cas12a utilizes 5'-NYHV-3' PAMs that expand DNA recognition sites to ∼25% of the human genome. With enhanced targeting versatility, Flex-Cas12a unlocks access to previously inaccessible genomic loci, providing new opportunities for both therapeutic and agricultural genome engineering.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.