A CRISPR-Cas9, Crelox, and Flp-FRT Cascade Strategy for the Precise and Efficient Integration of Exogenous DNA into Cellular Genomes

Jixue Li, Yanjie Li, Kevin M. Pawlik, Jill S. Napierala, Marek Napierala

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

We describe a protocol for the precise integration of exogenous DNA into user-defined genomic loci in cultured cells. This strategy first introduces a promoter and a lox site to a specific location via a Cas9-induced double-strand break. Second, a gene of interest (GOI) is inserted into the lox site via Cre-lox recombination. Upon correct insertion, a cis-linked antibiotic resistance gene will be expressed from a promoter introduced into the genome in the first step assuring selection for correct integrants. Last, the selection cassette is excised via a Flp-FRT recombination event, leaving a precisely targeted GOI. This method is broadly applicable to any exogenous DNA to be integrated, choice of integration site, and choice of cell type. The most remarkable aspect of this versatile approach, termed "CasPi"(cascaded precise integration), is that it allows for precise genome targeting with large, frequently complex, and repetitive DNA sequences that do not integrate efficiently or at all with current genome targeting methods.

Original languageEnglish (US)
Pages (from-to)470-486
Number of pages17
JournalCRISPR Journal
Volume3
Issue number6
DOIs
StatePublished - Dec 2020
Externally publishedYes

ASJC Scopus subject areas

  • Genetics
  • Biotechnology

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