Fan Peng, Wang Hejun, Zhao Feiyu, Zhang Tao, Li Jinze, Sun Xiaodi, Yu Yongduo, Xiong Haoyang, Lai Liangxue, Sui Tingting
State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, 130062, China.
Key Laboratory of Regenerative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, Guangdong, China.
Cell Mol Life Sci. 2024 Jan 28;81(1):63. doi: 10.1007/s00018-023-05100-3.
SpCas9 and AsCas12a are widely utilized as genome editing tools in human cells, but their applications are largely limited by their bulky size. Recently, AsCas12f1 protein, with a small size (422 amino acids), has been demonstrated to be capable of cleaving double-stranded DNA protospacer adjacent motif (PAM). However, low editing efficiency and large differences in activity against different genomic loci have been a limitation in its application. Here, we show that engineered AsCas12f1 sgRNA has significantly improved the editing efficiency in human cells and mouse embryos. Moreover, we successfully generated three stable mouse mutant disease models using the engineered CRISPR-AsCas12f1 system in this study. Collectively, our work uncovers the engineered AsCas12f1 system expands mini CRISPR toolbox, providing a remarkable promise for therapeutic applications.
SpCas9和AsCas12a作为基因组编辑工具在人类细胞中被广泛应用，但其应用在很大程度上受到其庞大体积的限制。最近，体积较小（422个氨基酸）的AsCas12f1蛋白已被证明能够切割双链DNA原间隔相邻基序（PAM）。然而，编辑效率低以及对不同基因组位点的活性存在巨大差异一直是其应用的一个限制因素。在此，我们表明经过工程改造的AsCas12f1 sgRNA显著提高了在人类细胞和小鼠胚胎中的编辑效率。此外，在本研究中，我们使用经过工程改造的CRISPR-AsCas12f1系统成功构建了三种稳定的小鼠突变疾病模型。总的来说，我们的工作揭示了经过工程改造的AsCas12f1系统扩展了微型CRISPR工具箱，为治疗应用带来了巨大希望。
