Small RNA Toxin‐Assisted Evolution of GC‐Preferred ErCas12a for Enhanced Genome Targeting Range

Small RNA Toxin‐Assisted Evolution of GC‐Preferred ErCas12a for Enhanced Genome Targeting Range

Abstract CRISPR/Cas12a, a promising gene editing technology, faces limitations due to its requirement for a thymine (T)‐rich protospacer adjacent motif (PAM). Despite the development of Cas12a variants with expanded PAM profiles, many genomic loci, especially those with guanine‐cytosine (GC)‐rich PA...

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Bibliographic Details
Main Authors: Zehua Chen, Junyuan Xue, Ziying Wang, Jinyuan Sun, Yinglu Cui, Tong Zhu, Huaiyi Yang, Ming Li, Bian Wu
Format: Article
Language:English
Published: Wiley 2025-08-01
Series:Advanced Science
Subjects:
CRISPR/Cas12a
expanded PAM profiles
gene editing
RNA toxin‐assisted evolution
Online Access:https://doi.org/10.1002/advs.202417105
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Summary:Abstract CRISPR/Cas12a, a promising gene editing technology, faces limitations due to its requirement for a thymine (T)‐rich protospacer adjacent motif (PAM). Despite the development of Cas12a variants with expanded PAM profiles, many genomic loci, especially those with guanine‐cytosine (GC)‐rich PAMs, have remained inaccessible. This study develops a small RNA toxin‐aided strategy to evolve ErCas12a for targeting GC‐rich PAMs, resulting in the creation of enhanced ErCas12a (enErCas12a). EnErCas12a demonstrates the ability to recognize GC‐rich PAMs and target five times more PAM sequences than the wild‐type ErCas12a. Furthermore, enErCas12a achieves efficient gene editing in both bacterial and mammalian cells at various sites with non‐canonical PAMs, including GC‐rich PAMs such as GCCC, CGCC, and GGCC, which are inaccessible to previous Cas12a variants. Moreover, enErCas12a effectively targets PAM sequences with a GC content exceeding 75% in mammalian cells, providing a valuable alternative to the existing Cas12a toolkit. Importantly, enErCas12a maintains high specificity at targets with canonical PAMs, while also demonstrating enhanced specificity at targets with non‐canonical PAMs. Collectively, this work establishes enErCas12a as a promising tool for gene editing in both eukaryotes and prokaryotes.
ISSN:2198-3844

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