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BMC Genomics. 2015 Oct 23;16:846. doi: 10.1186/s12864-015-2086-z.

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

Aparicio-Prat E1,2,3, Arnan C4,5,6, Sala I7,8,9, Bosch N10,11,12, Guigó R13,14,15, Johnson R16,17,18.

Author information

1
Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, 08003, Barcelona, Spain. estel.aparicio@crg.eu.
2
Universitat Pompeu Fabra (UPF), Dr. Aiguader 88, 08003, Barcelona, Spain. estel.aparicio@crg.eu.
3
Institut Hospital del Mar d'Investigacions Mèdiques (IMIM), Dr. Aiguader 88, 08003, Barcelona, Spain. estel.aparicio@crg.eu.
4
Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, 08003, Barcelona, Spain. carme.arnan@crg.eu.
5
Universitat Pompeu Fabra (UPF), Dr. Aiguader 88, 08003, Barcelona, Spain. carme.arnan@crg.eu.
6
Institut Hospital del Mar d'Investigacions Mèdiques (IMIM), Dr. Aiguader 88, 08003, Barcelona, Spain. carme.arnan@crg.eu.
7
Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, 08003, Barcelona, Spain. ilaria.sala@crg.eu.
8
Universitat Pompeu Fabra (UPF), Dr. Aiguader 88, 08003, Barcelona, Spain. ilaria.sala@crg.eu.
9
Institut Hospital del Mar d'Investigacions Mèdiques (IMIM), Dr. Aiguader 88, 08003, Barcelona, Spain. ilaria.sala@crg.eu.
10
Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, 08003, Barcelona, Spain. nuria.bosch@crg.eu.
11
Universitat Pompeu Fabra (UPF), Dr. Aiguader 88, 08003, Barcelona, Spain. nuria.bosch@crg.eu.
12
Institut Hospital del Mar d'Investigacions Mèdiques (IMIM), Dr. Aiguader 88, 08003, Barcelona, Spain. nuria.bosch@crg.eu.
13
Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, 08003, Barcelona, Spain. roderic.guigo@crg.cat.
14
Universitat Pompeu Fabra (UPF), Dr. Aiguader 88, 08003, Barcelona, Spain. roderic.guigo@crg.cat.
15
Institut Hospital del Mar d'Investigacions Mèdiques (IMIM), Dr. Aiguader 88, 08003, Barcelona, Spain. roderic.guigo@crg.cat.
16
Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, 08003, Barcelona, Spain. rory.johnson@crg.eu.
17
Universitat Pompeu Fabra (UPF), Dr. Aiguader 88, 08003, Barcelona, Spain. rory.johnson@crg.eu.
18
Institut Hospital del Mar d'Investigacions Mèdiques (IMIM), Dr. Aiguader 88, 08003, Barcelona, Spain. rory.johnson@crg.eu.

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.

PMID:
26493208
PMCID:
PMC4619085
DOI:
10.1186/s12864-015-2086-z
[Indexed for MEDLINE]
Free PMC Article

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