Open AccessA CRISPR-Cas9, Cre-lox, and Flp-FRT Cascade Strategy for the Precise and Efficient Integration of Exogenous DNA into Cellular Genomes
Author(s) -
Jixue Li,
Yanjie Li,
Kevin M. Pawlik,
Marek Napierala
Publication year - 2020
Publication title -
the crispr journal
Language(s) - English
Resource type - Journals
eISSN - 2573-1602
pISSN - 2573-1599
DOI - 10.1089/crispr.2020.0042
Subject(s) - crispr , genome , cas9 , biology , genetics , computational biology , dna , gene , genome editing , homologous recombination
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" ( cas caded p recise i ntegration), 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.