Abstract
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.
Highlights
Gene therapy would benefit from a miniature clustered regularly interspaced short palindromic repeats (CRISPR) system that fits into the small adeno-associated virus (AAV) genome and has high cleavage activity and specificity in eukaryotic cells
Efforts to engineer gRNAs have contributed to improving CRISPR tools with respect to efficiency of indel generation[27], simplicity[4], multiplexing[28], imaging[29] and specificity[4,30]
Considering the sizes of genes encoding validated regulators, we propose that the Cas12f system could provide a scaffold for various applications including CRISPR interference (CRISPRi)[40], CRISPR activation (CRISPRa), base editing[8,9], prime editing[10] and site-specific epigenetic regulations[5,6] (Fig. 3h)
Summary
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. Precise genetic modifications have been achieved by homology-driven repair[7], base editing systems[8,9] and prime editing technology[10] These diverse genome-editing tools have facilitated cell engineering, generation of model animals, development of new plant varieties[11] and genetic screening[12]. These methods hold promise for gene therapy in the treatment of cancer[13], genetic disorders[14] and infectious diseases[15,16]. In neither case was the feasibility of AAV delivery demonstrated
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