CRISPR-Cas9-induced gene knockout in zebrafish

  • STAR Protoc. 2022 Oct 26;3(4):101779. doi: 10.1016/j.xpro.2022.101779.
Raghavender Medishetti  1 ,  Keerthana Balamurugan  2 ,  Krishnaveni Yadavalli  3 ,  Rita Rani  3 ,  Aarti Sevilimedu  3 ,  Anil Kumar Challa  3 ,  Kishore Parsa  3 ,  Kiranam Chatti  4
Affiliations
  • 1. Dr. Reddy's Institute of Life Sciences, Hyderabad, Telangana, India; Doctoral Student Registered with Manipal Academy of Higher Education, Manipal, Karnataka, India. Electronic address: [email protected].
  • 2. Dr. Reddy's Institute of Life Sciences, Hyderabad, Telangana, India; Doctoral Student Registered with Manipal Academy of Higher Education, Manipal, Karnataka, India.
  • 3. Dr. Reddy's Institute of Life Sciences, Hyderabad, Telangana, India.
  • 4. Dr. Reddy's Institute of Life Sciences, Hyderabad, Telangana, India. Electronic address: [email protected].
PMID: 36317180 DOI: 10.1016/j.xpro.2022.101779
Abstract

The application of CRISPR has greatly facilitated genotype-phenotype studies of human disease models. In this protocol, we describe CRISPR-Cas9-induced gene knockout in zebrafish, utilizing purified Cas9 protein and in vitro-transcribed sgRNA. This protocol targets the PHLPP1 gene in an Indian wild-caught strain, but is broadly applicable. Major factors influencing protocol success include zebrafish health and fecundity, sgRNA efficiency and specificity, germline transmission, and mutant viability. For complete details on the use and execution of this protocol, please refer to Balamurugan et al. (2022).

Keywords
CRISPR; Genetics; Model organisms; Molecular biology; Sequencing.