DECKO: Single-oligo, dual-CRISPR deletion of genomic elements including long non-coding RNAs. (December 2015)

• Record Type: Journal Article
• Authors: Aparicio-Prat, Estel; Arnan, Carme; Sala, Ilaria; Bosch, Núria; Guigó, Roderic; Johnson, Rory
• Is Part Of: BMC genomics. Volume 16:Number 1(2015)
• Page Start: 1
• Page End: 15
• Publication Date: 2015-12
• Subjects: CRISPR -- Genome editing -- DECKO -- Long non-coding RNA -- lncRNA; Genomes -- Periodicals; Gene mapping -- Periodicals; Genomics -- Periodicals; Base Sequence -- Periodicals; Chromosome Mapping -- Periodicals; Genetic Techniques -- Periodicals; Sequence Analysis, DNA -- Periodicals; 572.8605
• DOI: 10.1186/s12864-015-2086-z
• Languages: English
• ISSNs: 1471-2164

Abstract

Abstract Background CRISPR 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. Results 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. 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. Conclusions We 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.