The CRISPR Journal. 2022 Jun 8
S Chen, Z Liu, W Xie, H Yu, L Lai, Z Li
Products used in the paper Details Operation
plasmid construction & AAV vector packaging Construction of AAV–Cje3Cas9 and AAV–8enCje3Cas9 expression cassettes and viral production were conducted by Packgene Biotech (China). Request Quote

Research Field: genome editing

Dose: 2 · 10^11 GCs AAV–Cje3Cas9 targeting Pcsk9-1 site was delivered to an 8-week-old female ICR mouse intravenously through lateral tail vein injection. For AAV–8e-nCje3Cas9 targeting Pcsk9-2 site, the titers used in each mouse were 2 · 10^11 GCs and 4 · 10^12 GCs, respectively.

Routes of Administration: intravenously through lateral tail vein injection

Animal or cell line strain: 8-week-old female ICR mouse


Many therapeutic applications of CRISPR-Cas9 gene editing rely on delivery using the highly versatile adeno-associated virus (AAV) vector. The smallest type II Cas9 ortholog—Cje1Cas9, derived from Campylobacter jejuni with <1,000 amino acids—is particularly attractive for AAV delivery. However, the complex protospacer adjacent motif (PAM) of Cje1Cas9 (N3VRYAC) greatly restricts the density of recognition sequences in human genome. In this study, we identify two compact CjeCas9 orthologs designated as Cje2Cas9 and Cje3Cas9, whose PAM-interacting residues are different from those of the well-known Cje1Cas9. They can induce efficient genome editing in human cells, and their simpler trinucleotide PAM (N4CYA) requirements expand the scope of targeting. Moreover, Cje3Cas9 efficiently disrupts the Tyr gene in mice after being micro-injected into zygotes with the corresponding sgRNA. It also successfully disrupts the Pcsk9 gene in 8-week-old mouse liver after delivery with an sgRNA using an all-in-one AAV delivery vehicle. The gene-edited mice showed lower cholesterol level than wild-type mice. Notably, the 8e-nCje3–ABE and an sgRNA targeting Pcsk9 were successfully packaged into a single AAV vector for genome editing in adult mouse liver, with editing efficiency up to 12%. Thus, simple PAMs and a compact size enable Cje2/3Cas9 to expand the target scope of CRISPR-Cas9 toolsets, exhibiting considerable potential for therapeutic applications.

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