Efficient CRISPR editing with a hypercompact Cas12f1 and engineered guide RNAs delivered by adeno-associated virus

Taeyoung Koo, Daesik Kim, Jeong Mi Lee, Yong-Sam Kim, Do Yon Kim, Su Bin Moon, Hyun Jung Chin, Seyeon Park, Youjung Lim, Jeong‐Heon Ko, Do Yon Kim
2021-09-02

SCID:  54.1/7fy48npk
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.
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2021-09-02
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Taeyoung Koo
Daesik Kim
Jeong Mi Lee
Yong-Sam Kim
Do Yon Kim
Su Bin Moon
Hyun Jung Chin
Seyeon Park
Youjung Lim
Jeong‐Heon Ko
Do Yon Kim
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