Citation
- Authors: Kim DY, Lee JM. et al.
- Year: 2021
- Journal: Nat Biotechnol
- Applications: in vitro / DNA / PEIpro
- Cell type: HEK-293T
Description: Human embryonic kidney Fibroblast
Known as: HEK293T, 293T
Method
- To produce rAAV2 vectors, HEK293T cells were transfected with pAAV-ITR-sgRNA-Cas12f1 or pAAV-ITR-sgRNA-SaCas9, pAAVED2/2 and helper plasmid.
- HEK293T cells were cultured in DMEM with 2% FBS.
- Recombinant pseudotyped AAV vector stocks were generated using polyethylenimine coprecipitated with PEIpro (Polyplus transfection) and triple transfection with plasmids at an equal molar ratio in HEK293T cells.
- After 72 h of incubation, cells were lysed and particles were purified by iodixanol step-gradient ultracentrifugation.
- The number of vector genomes was determined by quantitative (q)PCR.
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.