Precise and Scarless Insertion of Transposable Elements by Cas9-Mediated Genome Engineering

Abstract

Transposable element insertions can have broad effects on gene expression, ranging from new regulatory functions to pathogenic consequences by transplanting new cis-regulating elements or perturbing existing ones. Genetic manipulation of such DNA sequences can help decipher their mechanism of action. Here, we describe a CRISPR-Cas9-mediated two-step approach to precisely insert transposable elements into into the genome of cultured human cells, without scar or reporter gene. First, a double-selection cassette is inserted into the desired target locus. Once a clone containing a single copy of this cassette has been isolated, a second editing step is performed to exchange the double-selection cassette with a markerless transposable element sequence. More generally, this method can be used for knocking in any large insert without genetic markers.

Keywords: CRISPR-Cas9; Genome editing; Homology-directed repair; Knock-in; L1; LINE-1; Retrotransposon; Thymidine kinase; Transposable element.