期刊
BMC GENOMICS
卷 16, 期 -, 页码 -出版社
BIOMED CENTRAL LTD
DOI: 10.1186/s12864-015-2086-z
关键词
CRISPR; Genome editing; DECKO; Long non-coding RNA; lncRNA
资金
- Spanish Ministry of Economy and Competitiveness, 'Centro de Excelencia Severo Ochoa' [SEV-2012-0208]
- Spanish Ministry of Science [CSD2007-00050]
- Catalan Government [SGR-1430]
- European Community financial support under the FP7 [ERC-2011-AdG-294653-RNA-MAPS]
- National Human Genome Research Institute of the National Institutes of Health [R01MH101814]
- Plan Nacional [BIO2011-27220]
- Ramon y Cajal [RYC-2011-08851]
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.
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