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

BMC Genomics

Methodology Article

Roderic Guigó¹  Estel Aparicio-Prat¹  Carme Arnan¹  Ilaria Sala¹  Núria Bosch¹  Rory Johnson¹ 

[1] Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, 08003, Barcelona, Spain;Universitat Pompeu Fabra (UPF), Dr. Aiguader 88, 08003, Barcelona, Spain;Institut Hospital del Mar d’Investigacions Mèdiques (IMIM), Dr. Aiguader 88, 08003, Barcelona, Spain;

关键词: CRISPR;    Genome editing;    DECKO;    Long non-coding RNA;    lncRNA;   

DOI  :  10.1186/s12864-015-2086-z

 received in 2015-09-22, accepted in 2015-10-15,  发布年份 2015

来源: Springer

【 摘 要 】

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.

【 授权许可】

CC BY   
© Aparicio-Prat et al. 2016