Abstract
sBackgroundCRISPR/Cas9 system is a powerful tool for knocking out genes in cells. However, genes essential for cell survival cannot be directly knocked out. Traditionally, generation of conditional knockout cells requires multiple steps.ResultsIn this study, we developed an easy and efficient strategy to generate conditional knockout cells by using double episomal vectors – one which expresses gRNA and Cas9 nuclease, and the other expresses an inducible rescue gene. Using this system which we named “krCRISPR” (knockout-rescue CRISPR), we showed that essential genes, HDAC3 and DNMT1, can be efficiently knocked out. When cells reach a desired confluency, the exogenous rescue genes can be silenced by the addition of doxycycline. Furthermore, the krCRISPR system enabled us to study the effects of the essential gene mutations on cells. We showed that the P507L mutation in DNMT1 led to downregulation of global DNA methylation in cells, indicating that it is a disease-causing mutation.ConclusionsThe krCRISPR system offers an easy and efficient platform that facilitates the study of essential genes’ function.
Highlights
IntroductionGenes essential for cell survival cannot be directly knocked out
CRISPR/Cas9 system is a powerful tool for knocking out genes in cells
One plasmid which encodes Cas9 and guide RNA (gRNA) for gene knockout was designated as KO plasmid and the other plasmid which encodes the rescue gene was designated as Rescue plasmid
Summary
Genes essential for cell survival cannot be directly knocked out. Deletion of a target gene in cells and observation of the resulting phenotype is a common strategy to determine the function of a gene in biological research [1,2,3]. Numerous genes that are essential for cell viability result in cell death when they are ablated [3,4,5]. To study essential genes in cells, conditional knockout strategies have been developed. The Cre/loxP recombination system is the most commonly used technique to knockout essential genes. This technique requires insertion of a pair of 34 bp loxP sites flanking the target genes, and expression of Cre recombinase enzyme will cause a deletion of the genes between
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