CRISPR-based engineering of gene knockout cells by homology-directed insertion in polyploid Drosophila S2R+ cells
- 21 September 2020
- journal article
- research article
- Published by Springer Science and Business Media LLC in Nature Protocols
- Vol. 15 (10), 3478-3498
- https://doi.org/10.1038/s41596-020-0383-8
Abstract
Precise and efficient genome modifications provide powerful tools for biological studies. Previous CRISPR gene knockout methods in cell lines have relied on frameshifts caused by stochastic insertion/deletion in all alleles. However, this method is inefficient for genes with high copy number due to polyploidy or gene amplification because frameshifts in all alleles can be difficult to generate and detect. Here we describe a homology-directed insertion method to knockout genes in the polyploid Drosophila S2R+ cell line. This protocol allows generation of homozygous mutant cell lines using an insertion cassette which autocatalytically generates insertion mutations in all alleles. Knockout cells generated using this method can be directly identified by PCR without a need for DNA sequencing. This protocol takes 2–3 months and can be applied to other polyploid cell lines or high-copy-number genes.This publication has 29 references indexed in Scilit:
- Small Molecules Enhance CRISPR Genome Editing in Pluripotent Stem CellsCell Stem Cell, 2015
- DNA copy number evolution in Drosophila cell linesGenome Biology, 2014
- A single blastocyst assay optimized for detecting CRISPR/Cas9 system-induced indel mutations in miceBMC Biotechnology, 2014
- Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in DrosophilaProceedings of the National Academy of Sciences of the United States of America, 2014
- Genome engineering using the CRISPR-Cas9 systemNature Protocols, 2013
- RNA-Guided Human Genome Engineering via Cas9Science, 2013
- Multiplex Genome Engineering Using CRISPR/Cas SystemsScience, 2013
- A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial ImmunityScience, 2012
- The transcriptional diversity of 25 Drosophila cell linesGenome Research, 2010
- Gene targeting using a promoterless gene trap vector (“targeted trapping”) is an efficient method to mutate a large fraction of genesProceedings of the National Academy of Sciences of the United States of America, 2005