Engineered CRISPR-OsCas12f1 and RhCas12f1 with robust activities and expanded target range for genome editing

Xiangfeng Kong, Hainan Zhang, Guoling Li, Zikang Wang, Xuqiang Kong, Lecong Wang, Mingxing Xue, Weihong Zhang, Yao Wang, Jiajia Lin, Jingxing Zhou, Xiaowen Shen, Yinghui Wei, Na Zhong, Weiya Bai, Yuan Yuan, Linyu Shi, Yingsi Zhou () and Hui Yang ()
Additional contact information
Xiangfeng Kong: HUIEDIT Therapeutics Co., Ltd.
Hainan Zhang: HUIEDIT Therapeutics Co., Ltd.
Guoling Li: HUIDAGENE Therapeutics Co., Ltd.
Zikang Wang: HUIEDIT Therapeutics Co., Ltd.
Xuqiang Kong: HUIEDIT Therapeutics Co., Ltd.
Lecong Wang: HUIEDIT Therapeutics Co., Ltd.
Mingxing Xue: HUIEDIT Therapeutics Co., Ltd.
Weihong Zhang: HUIEDIT Therapeutics Co., Ltd.
Yao Wang: HUIEDIT Therapeutics Co., Ltd.
Jiajia Lin: Fujian Medical University
Jingxing Zhou: HUIEDIT Therapeutics Co., Ltd.
Xiaowen Shen: HUIEDIT Therapeutics Co., Ltd.
Yinghui Wei: HUIEDIT Therapeutics Co., Ltd.
Na Zhong: HUIEDIT Therapeutics Co., Ltd.
Weiya Bai: HUIDAGENE Therapeutics Co., Ltd.
Yuan Yuan: HUIDAGENE Therapeutics Co., Ltd.
Linyu Shi: HUIDAGENE Therapeutics Co., Ltd.
Yingsi Zhou: HUIEDIT Therapeutics Co., Ltd.
Hui Yang: HUIEDIT Therapeutics Co., Ltd.

Nature Communications, 2023, vol. 14, issue 1, 1-13

Abstract: Abstract The type V-F CRISPR-Cas12f system is a strong candidate for therapeutic applications due to the compact size of the Cas12f proteins. In this work, we identify six uncharacterized Cas12f1 proteins with nuclease activity in mammalian cells from assembled bacterial genomes. Among them, OsCas12f1 (433 aa) from Oscillibacter sp. and RhCas12f1 (415 aa) from Ruminiclostridium herbifermentans, which respectively target 5’ T-rich Protospacer Adjacent Motifs (PAMs) and 5’ C-rich PAMs, show the highest editing activity. Through protein and sgRNA engineering, we generate enhanced OsCas12f1 (enOsCas12f1) and enRhCas12f1 variants, with 5’-TTN and 5’-CCD (D = not C) PAMs respectively, exhibiting much higher editing efficiency and broader PAMs, compared with the engineered variant Un1Cas12f1 (Un1Cas12f1_ge4.1). Furthermore, by fusing the destabilized domain with enOsCas12f1, we generate inducible-enOsCas12f1 and demonstate its activity in vivo by single adeno-associated virus delivery. Finally, dead enOsCas12f1-based epigenetic editing and gene activation can also be achieved in mammalian cells. This study thus provides compact gene editing tools for basic research with remarkable promise for therapeutic applications.

Date: 2023
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DOI: 10.1038/s41467-023-37829-7

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