A robust pipeline for efficient knock-in of point mutations and epitope tags in zebrafish using fluorescent PCR based screening

作者：Blake Carrington, Ramanagouda Ramanagoudr‐Bhojappa, Erica Bresciani, Tae‐Un Han, Raman Sood · 发表于：BMC Genomics · 年份：2022 · DOI：10.1186/s12864-022-08971-1 · 被引用次数：15 · 研究领域：CRISPR and Genetic Engineering、Zebrafish Biomedical Research Applications、Genomics and Rare Diseases

BACKGROUND: Genome editing using CRISPR/Cas9 has become a powerful tool in zebrafish to generate targeted gene knockouts models. However, its use for targeted knock-in remains challenging due to inefficient homology directed repair (HDR) pathway in zebrafish, highlighting the need for efficient and cost-effective screening methods. RESULTS: Here, we present our fluorescent PCR and capillary electrophoresis based screening approach for knock-in using a single-stranded oligodeoxynucleotide donor (ssODN) as a repair template for the targeted insertion of epitope tags, or single nucleotide changes to recapitulate pathogenic human alleles. For the insertion of epitope tags, we took advantage of the expected change in size of the PCR product. For point mutations, we combined fluorescent PCR with restriction fragment length polymorphism (RFLP) analysis to distinguish the fish with the knock-in allele. As a proof-of-principle, we present our data on the generation of fish lines with insertion of a FLAG tag at the tcnba locus, an HA tag at the gata2b locus, and a point mutation observed in Gaucher disease patients in the gba gene. Despite the low number of germline transmitting founders (1-5%), combining our screening methods with prioritization of founder fish by fin biopsies allowed us to establish stable knock-in lines by screening 12 or less fish per gene. CONCLUSIONS: We have established a robust pipeline for the generation of zebrafish models with precise integration of small DN...