Gene Knock-Ins in Using Homology-Independent Insertion of Universal Donor Plasmids

2019_Genetics_Bosch.pdf1.87 MB

Date Published:

2019 Nov 04

Abstract:

Targeted genomic knock-ins are a valuable tool to probe gene function. However, knock-in methods involving homology-directed repair (HDR) can be laborious. Here, we adapt the mammalian CRISPaint homology-independent knock-in method for , which uses CRISPR/Cas9 and non-homologous end joining (NHEJ) to insert universal donor plasmids into the genome. Using this method in cultured S2R+ cells, we efficiently tagged four endogenous proteins with the bright fluorescent protein mNeonGreen, thereby demonstrating that an existing collection of CRISPaint universal donor plasmids is compatible with insect cells. In addition, we inserted the transgenesis marker into seven genes in the fly germ line, producing heritable loss of function alleles that were isolated by simple fluorescence screening. Unlike in cultured cells, indels always occurred at the genomic insertion site, which prevents predictably matching the insert coding frame to the target gene. Despite this effect, we were able to isolate insertions in four genes that serve as in vivo expression reporters. Therefore, homology-independent insertion in is a fast and simple alternative to HDR that will enable researchers to dissect gene function.

Last updated on 11/08/2019