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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Wiley
2025-08-01
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| Series: | Advanced Science |
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| Online Access: | https://doi.org/10.1002/advs.202417105 |
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| author | Zehua Chen Junyuan Xue Ziying Wang Jinyuan Sun Yinglu Cui Tong Zhu Huaiyi Yang Ming Li Bian Wu |
| author_facet | Zehua Chen Junyuan Xue Ziying Wang Jinyuan Sun Yinglu Cui Tong Zhu Huaiyi Yang Ming Li Bian Wu |
| author_sort | Zehua Chen |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-eda50ec03a4b4b5cbd58008292a5043f |
| institution | Kabale University |
| issn | 2198-3844 |
| language | English |
| publishDate | 2025-08-01 |
| publisher | Wiley |
| record_format | Article |
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| spelling | doaj-art-eda50ec03a4b4b5cbd58008292a5043f2025-08-20T03:41:08ZengWileyAdvanced Science2198-38442025-08-011229n/an/a10.1002/advs.202417105Small RNA Toxin‐Assisted Evolution of GC‐Preferred ErCas12a for Enhanced Genome Targeting RangeZehua Chen0Junyuan Xue1Ziying Wang2Jinyuan Sun3Yinglu Cui4Tong Zhu5Huaiyi Yang6Ming Li7Bian Wu8AIM center College of Life Sciences and Technology Beijing University of Chemical Technology Institute of Microbiology Chinese Academy of Sciences Beijing 100101 ChinaState Key Laboratory of Microbial Diversity and Innovative Utilization Institute of Microbiology Chinese Academy of Sciences Beijing 100101 ChinaSenior Department of Orthopedics the Fourth Medical Center of PLA General Hospital Beijing 100000 ChinaUniversity of Chinese Academy of Sciences Beijing 100049 ChinaAIM center College of Life Sciences and Technology Beijing University of Chemical Technology Institute of Microbiology Chinese Academy of Sciences Beijing 100101 ChinaAIM center College of Life Sciences and Technology Beijing University of Chemical Technology Institute of Microbiology Chinese Academy of Sciences Beijing 100101 ChinaState Key Laboratory of Microbial Diversity and Innovative Utilization Institute of Microbiology Chinese Academy of Sciences Beijing 100101 ChinaState Key Laboratory of Microbial Diversity and Innovative Utilization Institute of Microbiology Chinese Academy of Sciences Beijing 100101 ChinaAIM center College of Life Sciences and Technology Beijing University of Chemical Technology Institute of Microbiology Chinese Academy of Sciences Beijing 100101 ChinaAbstract 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.https://doi.org/10.1002/advs.202417105CRISPR/Cas12aexpanded PAM profilesgene editingRNA toxin‐assisted evolution |
| spellingShingle | Zehua Chen Junyuan Xue Ziying Wang Jinyuan Sun Yinglu Cui Tong Zhu Huaiyi Yang Ming Li Bian Wu Small RNA Toxin‐Assisted Evolution of GC‐Preferred ErCas12a for Enhanced Genome Targeting Range Advanced Science CRISPR/Cas12a expanded PAM profiles gene editing RNA toxin‐assisted evolution |
| title | Small RNA Toxin‐Assisted Evolution of GC‐Preferred ErCas12a for Enhanced Genome Targeting Range |
| title_full | Small RNA Toxin‐Assisted Evolution of GC‐Preferred ErCas12a for Enhanced Genome Targeting Range |
| title_fullStr | Small RNA Toxin‐Assisted Evolution of GC‐Preferred ErCas12a for Enhanced Genome Targeting Range |
| title_full_unstemmed | Small RNA Toxin‐Assisted Evolution of GC‐Preferred ErCas12a for Enhanced Genome Targeting Range |
| title_short | Small RNA Toxin‐Assisted Evolution of GC‐Preferred ErCas12a for Enhanced Genome Targeting Range |
| title_sort | small rna toxin assisted evolution of gc preferred ercas12a for enhanced genome targeting range |
| topic | CRISPR/Cas12a expanded PAM profiles gene editing RNA toxin‐assisted evolution |
| url | https://doi.org/10.1002/advs.202417105 |
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