dc.contributor.author |
Aparicio i Prat, Estel, 1986- |
dc.contributor.author |
Arnan Ros, Carme |
dc.contributor.author |
Sala, Ilaria |
dc.contributor.author |
Bosch Roca, Núria |
dc.contributor.author |
Guigó Serra, Roderic |
dc.contributor.author |
Johnson, Rory |
dc.date |
2015 |
dc.identifier.citation |
Aparicio-Prat E, Arnan C, Sala I, Bosch N, Guigó R, Johnson R. DECKO: Single-oligo, dual-CRISPR deletion of genomic elements including long non-coding RNAs. BMC Genomics. 2015;16:846. |
dc.identifier.citation |
1471-2164 |
dc.identifier.citation |
http://dx.doi.org/10.1186/s12864-015-2086-z |
dc.identifier.uri |
http://hdl.handle.net/10230/25167 |
dc.format |
application/pdf |
dc.language.iso |
eng |
dc.publisher |
BioMed Central |
dc.relation |
BMC Genomics. 2015;16:846 |
dc.relation |
info:eu-repo/grantAgreement/EC/ERC/294653 |
dc.relation |
info:eu-repo/grantAgreement/ES/3PN/SEV2012-0208 |
dc.relation |
info:eu-repo/grantAgreement/ES/3PN/CSD2007-00050 |
dc.relation |
info:eu-repo/grantAgreement/ES/3PN/BIO2011-27220 |
dc.relation |
info:eu-repo/grantAgreement/ES/3PN/RYC2011-08851 |
dc.rights |
info:eu-repo/semantics/openAccess |
dc.rights |
© 2015 Aparicio-Prat et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. |
dc.rights |
http://creativecommons.org/licenses/by/4.0/ |
dc.subject |
Genoma humà |
dc.subject |
CRISPR |
dc.subject |
Genome editing |
dc.subject |
DECKO |
dc.subject |
Long non-coding RNA |
dc.subject |
lncRNA |
dc.title |
DECKO: Single-oligo, dual-CRISPR deletion of genomic elements including long non-coding RNAs |
dc.type |
info:eu-repo/semantics/article |
dc.type |
info:eu-repo/semantics/publishedVersion |
dc.description.abstract |
Erratum note: Unfortunately, the original version of this article [1] contained an error. In the Methods part, in the Design and Cloning of Plasmids section, a sentence was included incorrectly. The correct sentence can be found below/n/n"The Insert-2 sequence was previously assembled from four 5'-phosphorilated oligonucleotides (IDT)"./n/nPlease also note in table S3 The oligo pDECKO_seq_R is lacking one nucleotide. The correct sequence is ATGTCTACTATTCTTTCCCC |
dc.description.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./nResults. 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./nConclusions. 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. |
dc.description.abstract |
We acknowledge support of the Spanish Ministry of Economy and Competitiveness, ‘Centro de Excelencia Severo Ochoa 2013-2017’, SEV-2012-0208. This work was financially supported by the following grants: CSD2007-00050 from the Spanish Ministry of Science, grant SGR-1430 from the Catalan Government, grant ERC-2011-AdG-294653-RNA-MAPS from the European Community financial support under the FP7 and grant R01MH101814 by the National Human Genome Research Institute of the National Institutes of Health, to RG. Ramón y Cajal RYC-2011-08851 and Plan Nacional BIO2011-27220 to RJ. |