Abstract
BackgroundCRISPR genome-editing technology makes it possible to quickly and cheaply delete non-protein-coding regulatory elements. We present a vector system adapted for this purpose called DECKO (Double Excision CRISPR Knockout), which applies a simple two-step cloning to generate lentiviral vectors expressing two guide RNAs (gRNAs) simultaneously. The key feature of DECKO is its use of a single 165 bp starting oligonucleotide carrying the variable sequences of both gRNAs, making it fully scalable from single-locus studies to complex library cloning.ResultsWe apply DECKO to deleting the promoters of one protein-coding gene and two oncogenic lncRNAs, UCA1 and the highly-expressed MALAT1, focus of many previous studies employing RNA interference approaches. DECKO successfully deleted genomic fragments ranging in size from 100 to 3000 bp in four human cell lines. Using a clone-derivation workflow lasting approximately 20 days, we obtained 9 homozygous and 17 heterozygous promoter knockouts in three human cell lines. Frequent target region inversions were observed. These clones have reductions in steady-state MALAT1 RNA levels of up to 98 % and display reduced proliferation rates.ConclusionsWe present a dual CRISPR tool, DECKO, which is cloned using a single starting oligonucleotide, thereby affording simplicity and scalability to CRISPR knockout studies of non-coding genomic elements, including long non-coding RNAs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2086-z) contains supplementary material, which is available to authorized users.
Highlights
CRISPR genome-editing technology makes it possible to quickly and cheaply delete non-protein-coding regulatory elements
Dual excision CRISPR knockout design CRISPR can be used to delete genomic sequences, by cutting genomic DNA at two sites and relying on nonhomologous end-joining (NHEJ) mechanism to repair the break (Fig. 1a). guide RNAs (gRNAs) are introduced to cells by a plasmid vector, either through transfection or viral infection
To facilitate the disruption of individual long non-coding RNA (lncRNA) and for eventual pooled functional screens of many lncRNAs, we devised a protocol for the simultaneous cloning of two distinct guide RNA sequences into a single lentiviral
Summary
We apply DECKO to deleting the promoters of one protein-coding gene and two oncogenic lncRNAs, UCA1 and the highly-expressed MALAT1, focus of many previous studies employing RNA interference approaches. DECKO successfully deleted genomic fragments ranging in size from 100 to 3000 bp in four human cell lines. Using a clone-derivation workflow lasting approximately 20 days, we obtained 9 homozygous and 17 heterozygous promoter knockouts in three human cell lines. These clones have reductions in steady-state MALAT1 RNA levels of up to 98 % and display reduced proliferation rates
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