Abstract
The availability of CRISPR/Cas9 technology has enabled the rapid establishment of gene knockouts in many cell types and even whole organisms. However, conditional inactivation of essential genes remains a challenge. We devised an approach named DECAI (DEgradation based on Cre-regulated- Artificial Intron). It utilizes a small cassette of just 201 nucleotides that is inserted into the coding exon of a target gene using CRISPR/Cas9 technology and homology-directed repair. As its sequence is derived from an artificial intron, the cassette is removed by the splicing machinery and thus leaves no trace in the “off-state”. Upon activation with Cre recombinase (“on-state”), the intron is crippled and the target gene is disrupted by a series of stop codons. We exemplify the utility of this approach on several non-essential and essential human genes. Clones bearing the conditional knockout cassette are recovered at frequencies above 5% and cassette function can be traced at the genomic DNA and the mRNA level. Importantly, cassette activation leads to loss of gene expression as judged by flow cytometry, Western blot or immunofluorescence. Altogether, this highlights the broad utility of the approach for conditional gene inactivation and suggests that this tool could be used to study the loss-of-function phenotypes of essential genes.
Highlights
Genome editing has been revolutionized by the discovery of CRISPR/Cas technology which enables the targeted introduction of double strand breaks into the human genome with an unprecedented precision and efficiency[1,2,3,4]
We aimed to devise an approach that allows the generation of conditional gene knockouts on a large scale with the following features: (i) engineering should be straightforward in diploid or even polyploid cells; (ii) the approach should not require any prior insight into the structure/function of the targeted gene product; (iii) in the “off-state” of the cassette, the activity of the target gene product should be largely unaffected; (iv) cassette activation should trigger a profound decrease in gene expression
We created a novel small cassette that readily allows the tagging of endogenous genes, poising them for conditional gene inactivation
Summary
Genome editing has been revolutionized by the discovery of CRISPR/Cas technology which enables the targeted introduction of double strand breaks into the human genome with an unprecedented precision and efficiency[1,2,3,4]. As non-homologous end joining is by nature quite imprecise, it triggers the accumulation of small insertions or deletions (so called indels)[6] If such indels lie within the coding sequence of a gene, they often disrupt the open reading frame and abrogate the translation (or transcription; as a result of nonsense-mediated decay) of the corresponding gene. To generate conditional knockouts using this strategy, one would first have to flank the target gene or an exon of this gene with loxP sites that enable Cre recombinase-mediated deletion[9]. This would involve the assembly of large homology donors, which could be costly and difficult to synthesize. It would be necessary to screen large numbers of clones until a bi-allelically modified clone is identified or to perform sequential rounds of editing
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