Gene therapy would benefit from a miniature CRISPR system that fits into the small adeno-associated virus (AAV) genome and has high cleavage activity and specificity in eukaryotic cells. One of the most compact CRISPR-associated nucleases yet discovered is the archaeal Un1Cas12f1. However, Un1Cas12f1 and its variants have very low activity in eukaryotic cells. In the present study, we redesigned the natural guide RNA of Un1Cas12f1 at five sites: the 5′ terminus of the trans-activating CRISPR RNA (tracrRNA), the tracrRNA–crRNA complementary region, a penta(uridinylate) sequence, the 3′ terminus of the crRNA and a disordered stem 2 region in the tracrRNA. These optimizations synergistically increased the average indel frequency by 867-fold. The optimized Un1Cas12f1 system enabled efficient, specific genome editing in human cells when delivered by plasmid vectors, PCR amplicons and AAV. As Un1Cas12f1 cleaves outside the protospacer, it can be used to create large deletions efficiently. The engineered Un1Cas12f1 system showed efficiency comparable to that of SpCas9 and specificity similar to that of AsCas12a.

基因治疗将受益于适合小型腺相关病毒 (AAV) 基因组并在真核细胞中具有高切割活性和特异性的微型 CRISPR 系统。迄今为止发现的最紧凑的 CRISPR 相关核酸酶之一是古细菌 Un1Cas12f1。然而，Un1Cas12f1 及其变体在真核细胞中的活性非常低。在本研究中，我们在五个位点重新设计了 Un1Cas12f1 的天然向导 RNA：反式的 5' 末端-激活 CRISPR RNA (tracrRNA)、tracrRNA-crRNA 互补区、五（尿苷酸）序列、crRNA 的 3' 末端和 tracrRNA 中的无序茎 2 区。这些优化协同地将平均插入缺失频率提高了 867 倍。当通过质粒载体、PCR 扩增子和 AAV 传递时，优化的 Un1Cas12f1 系统能够在人类细胞中进行高效、特异性的基因组编辑。由于 Un1Cas12f1 在 protospacer 外切割，它可用于有效地创建大的缺失。工程化的 Un1Cas12f1 系统显示出与 SpCas9 相当的效率和与 AsCas12a 相似的特异性。