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

Cited 41 time in scopus
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Title
Efficient CRISPR editing with a hypercompact Cas12f1 and engineered guide RNAs delivered by adeno-associated virus
Author(s)
Doyon Kim; Jeongmi Lee; Subin Moon; Hyun Jung Chin; S Park; Y Lim; Daesik Kim; T Koo; Jeong Heon KoYong-Sam Kim
Bibliographic Citation
Nature Biotechnology, vol. 40, no. 1, pp. 94-102
Publication Year
2022
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.
ISSN
1087-0156
Publisher
Springer-Nature Pub Group
DOI
http://dx.doi.org/10.1038/s41587-021-01009-z
Type
Article
Appears in Collections:
Critical Diseases Diagnostics Convergence Research Center > 1. Journal Articles
Synthetic Biology and Bioengineering Research Institute > Genome Editing Research Center > 1. Journal Articles
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