Instantaneous visual genotyping and facile site-specific transgenesis via CRISPR-Cas9 and phiC31 integrase

Author:

Ma Junyan12,Zhang Weiting2,Rahimialiabadi Simin3,Ganesh Nikkitha Umesh2,Sun Zhengwang4,Parvez Saba3,Peterson Randall T.3,Yeh Jing-Ruey Joanna2ORCID

Affiliation:

1. 1 Department of Basic Medical Science, Quanzhou Medical College, Quanzhou, Fujian 362011, China

2. 2 Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, MA 02129, USA and Department of Medicine, Harvard Medical School, Boston, MA 02115, USA

3. 3 Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, UT 84112, USA

4. 4 Center for Immunology and Inflammatory Disease, Massachusetts General Hospital, Charlestown, MA 02129, USA

Abstract

ABSTRACT Here, we introduce ‘TICIT’, targeted integration by CRISPR-Cas9 and integrase technologies, which utilizes the site-specific DNA recombinase – phiC31 integrase – to insert large DNA fragments into CRISPR-Cas9 target loci. This technique, which relies on first knocking in a 39-basepair phiC31 landing site via CRISPR-Cas9, enables researchers to repeatedly perform site-specific transgenesis at the exact genomic location with high precision and efficiency. We applied this approach to devise a method for the instantaneous determination of a zebrafish's genotype simply by examining its color. When a zebrafish mutant line must be propagated as heterozygotes due to homozygous lethality, employing this method allows facile identification of a population of homozygous mutant embryos even before the mutant phenotypes manifest. Thus, it should facilitate various downstream applications, such as large-scale chemical screens. We demonstrated that TICIT could also create reporter fish driven by an endogenous promoter. Further, we identified a landing site in the tyrosinase gene that could support transgene expression in a broad spectrum of tissue and cell types. In sum, TICIT enables site-specific DNA integration without requiring complex donor DNA construction. It can yield consistent transgene expression, facilitate diverse applications in zebrafish, and may be applicable to cells in culture and other model organisms.

Funder

NIH

China Scholarship Council

Harvard Medical School; University of Utah

Publisher

The Company of Biologists

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