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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.
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http://dx.doi.org/10.1007/s00018-023-05100-3 | DOI Listing |
ACS Synth Biol
August 2025
Department of Chemical Engineering, Guangdong Technion-Israel Institute of Technology, 515063 Shantou, China.
, a nonconventional yeast, has become an industrial workhorse to synthesize valuable compounds, including lipids, oleochemicals, and nutraceuticals. While the synthetic biology toolkits to engineer the genome and endogenous metabolic pathways are not as developed as Baker's yeast, it has emerged as a promising microbial host for industrial applications. In this study, we examined the multiplexed editing capability of the CRISPR-AsCpf1 coupled with gRNAs generated from either a yeast native promoter or an orthogonal T7 promoter, which yielded 73.
View Article and Find Full Text PDFBiotechnol Lett
July 2025
School of Life Sciences, University of Science and Technology of China, Hefei, 230027, Anhui, China.
Kluyveromyces marxianus is an attractive chassis for microbial cell factories due to its rapid growth, thermotolerance, and wide substrate spectrum. However, gene disruption in this organism is challenging primarily due the prevalence of dominant nonhomologous recombination. AsCas12f1, a hypercompact CRISPR-associated protein consisting of 422 amino acids-approximately one-third the size of Cas9 or Cas12a-enables more efficient packaging into delivery vehicles than its larger counterparts.
View Article and Find Full Text PDFNat Commun
May 2025
Cancer Research Institute, School of Basic Medical Sciences, State Key Laboratory of Multi-organ Injury Prevention and Treatment, Guangdong Province Key Laboratory of Immune Regulation and Immunotherapy, Southern Medical University, Guangzhou, China.
Compact CRISPR-Cas systems have demonstrated potential for effective packaging into adeno-associated viruses (AAVs) for use in gene therapy. However, their applications are currently limited due to modest gene-editing activity. Here we introduce an engineered compact CRISPR-Cas12f (hpCasMINI, 554 aa), with hyper editing efficiency in mammalian cells via adding an α-helix structure to the N-terminus of an Un1Cas12f1 variant CasMINI (529 aa).
View Article and Find Full Text PDFBiotechnol Bioeng
June 2025
Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, Zhejiang, China.
Miniature CRISPR/Cas systems possess delivery advantages for gene therapy. The type V-F Cas12f1 from Acidibacillus sulfuroxidans is exceptionally compact (422 amino acids) and has been engineered by several studies as compact genome editing tools through protein and single guide RNA (sgRNA) engineering. However, a comparative evaluation of gene editing and activation efficiencies mediated by different AsCas12f1 variants and sgRNA scaffolds is lacking.
View Article and Find Full Text PDFNucleic Acids Res
December 2024
School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China.
Type V-F CRISPR-Cas12f is a group of hypercompact RNA-guided nucleases that present a versatile in vivo delivery platform for gene therapy. Upon target recognition, Acidibacillus sulfuroxidans Cas12f (AsCas12f1) distinctively engenders three DNA break sites, two of which are located outside the protospacer. Combining ensemble and single-molecule approaches, we elucidate the molecular details underlying AsCas12f1-mediated DNA cleavages.
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