CRISPR-KRISPR: a method to identify on-target and random insertion of donor DNAs and their characterization in knock-in mice

• Published in: Genome Biology Vol. 23; no. 1; pp. 228 - 19
• Main Authors: Tanaka, Masayuki, Yokoyama, Keiko, Hayashi, Hideki, Isaki, Sanae, Kitatani, Kanae, Wang, Ting, Kawata, Hisako, Matsuzawa, Hideyuki, Gurumurthy, Channabasavaiah B, Miura, Hiromi, Ohtsuka, Masato
• Format: Journal Article
• Language: English
• Published: England BioMed Central 25.10.2022; BMC
• Subjects: Animal models; Animals; CIRCLE-seq; Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR; CRISPR-Cas Systems; CRISPR-KRISPR; DNA - genetics; Gene Editing - methods; Gene Knock-In Techniques; Genetic testing; Genome; Genomes; Genomics; Genotypes; Knock-in; Lesions; Method; Methods; Mice; Mutation; Off target; Random insertion; CRISPR-KRISPR; Donor DNA; Random insertion; CRISPR; CIRCLE-seq; Off target; Repeat element; Knock-in; Intronic region
• ISSN: 1474-760X; 1474-7596; 1474-760X
• DOI: 10.1186/s13059-022-02779-8

CRISPR tools can generate knockout and knock-in animal models easily, but the models can contain off-target genomic lesions or random insertions of donor DNAs. Simpler methods to identify off-target lesions and random insertions, using tail or earpiece DNA, are unavailable. We develop CRISPR-KRISPR (CRISPR-Knock-ins and Random Inserts Searching PRotocol), a method to identify both off-target lesions and random insertions. CRISPR-KRISPR uses as little as 3.4 μg of genomic DNA; thus, it can be easily incorporated as an additional step to genotype founder animals for further breeding.