Journal: Molecular Therapy. Nucleic Acids

Article Title: CRISPR-Cas9-mediated reactivation of the uricase pseudogene in human cells prevents acute hyperuricemia

doi: 10.1016/j.omtn.2021.08.002

Figure Lengend Snippet: Schematic for CRISPR-Cas9-mediated knockin of exogenous DNA fragment into human AAVS1 locus in HEK293 cells Top: the human AAVS1 allele. gRNA sequences are designated (g1 and g2) and located upstream of exon 2. Center: donor plasmid is represented schematically. Dashed lines show homology arms (∼800 bp on each side of donor) between genomic locus and plasmid DNA. A splice acceptor sequence is inserted upstream of RFP gene to allow proper RNA splicing using the endogenous transcriptional promoter. T2A sequences are inserted both upstream and between RFP/AncUOX to allow the release of the individual proteins after translation. SV40 poly(A) signal sequence is designated downstream of the donor DNA. Bottom: engineered allele. Post Cas9 cuts at the site corresponding to the gRNA sequences, donor DNA is integrated into the breakpoint through homology directed repair (HDR).

Article Snippet: To construct the pU6-(Bbs1)_CBh-Cas9-T2A-GFP-p2A-Ad4E4orf6 plasmid (CRISPR plasmid), we digested px458 (Addgene, #48138) and pU6-(BbsI)_CBh-Cas9-T2A-mCherry-P2A-Ad4E4orf6 (Addgene, #64222) with FseI/BsrGI (New England Biolabs).

Techniques: CRISPR, Knock-In, Plasmid Preparation, Sequencing