The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system is a valuable genome editing tool for microorganisms. However, the commonly used Cas9 nuclease derived from Streptococcus pyogenes (SpCas9) is not applicable to many industrially relevant bacteria, due to its cytotoxicity and large size (1368 amino acids [aa]). We developed an alternative genome editing system using a miniature Cas12f1 nuclease (529 aa) derived from an uncultured archaeon, Un1Cas12f1. When editing four dispensable genes in Escherichia coli MG1655 and BW25113, the CRISPR/Un1Cas12f1 system showed higher efficiency (63%–100%) than the CRISPR/SpCas9 system (50%–79%). The CRISPR/Un1Cas12f1 genome editing system is expected to be applied to the genome editing of a wide variety of bacteria.

成簇的规则间隔短回文重复序列 (CRISPR)/CRISPR 相关 (Cas) 系统是微生物的一种有价值的基因组编辑工具。然而，由于其细胞毒性和大尺寸（1368个氨基酸[aa]），常用的源自化脓性链球菌（SpCas9）的Cas9核酸酶不适用于许多工业相关细菌。我们使用源自未培养古菌 Un1Cas12f1 的微型 Cas12f1 核酸酶 (529 aa) 开发了一种替代基因组编辑系统。在大肠杆菌中编辑四个可有可无的基因时MG1655 和 BW25113，CRISPR/Un1Cas12f1 系统显示出比 CRISPR/SpCas9 系统（50%–79%）更高的效率（63%–100%）。CRISPR/Un1Cas12f1基因组编辑系统有望应用于多种细菌的基因组编辑。