DECKO: Single-oligo, dual-CRISPR deletion of genomic elements including long non-coding RNAs.
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.
Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, 08003, Barcelona, Spain. estel.aparicio@crg.eu.|Universitat Pompeu Fabra (UPF), Dr. Aiguader 88, 08003, Barcelona, Spain. estel.aparicio@crg.eu.|Institut Hospital del Mar d'Investigacions Mèdiques (IMIM), Dr. Aiguader 88, 08003, Barcelona, Spain. estel.aparicio@crg.eu.
Estel
E
Aparicio-Prat
Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, 08003, Barcelona, Spain. carme.arnan@crg.eu.|Universitat Pompeu Fabra (UPF), Dr. Aiguader 88, 08003, Barcelona, Spain. carme.arnan@crg.eu.|Institut Hospital del Mar d'Investigacions Mèdiques (IMIM), Dr. Aiguader 88, 08003, Barcelona, Spain. carme.arnan@crg.eu.
Carme
C
Arnan
Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, 08003, Barcelona, Spain. ilaria.sala@crg.eu.|Universitat Pompeu Fabra (UPF), Dr. Aiguader 88, 08003, Barcelona, Spain. ilaria.sala@crg.eu.|Institut Hospital del Mar d'Investigacions Mèdiques (IMIM), Dr. Aiguader 88, 08003, Barcelona, Spain. ilaria.sala@crg.eu.
Ilaria
I
Sala
Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, 08003, Barcelona, Spain. nuria.bosch@crg.eu.|Universitat Pompeu Fabra (UPF), Dr. Aiguader 88, 08003, Barcelona, Spain. nuria.bosch@crg.eu.|Institut Hospital del Mar d'Investigacions Mèdiques (IMIM), Dr. Aiguader 88, 08003, Barcelona, Spain. nuria.bosch@crg.eu.
Núria
N
Bosch
Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, 08003, Barcelona, Spain. roderic.guigo@crg.cat.|Universitat Pompeu Fabra (UPF), Dr. Aiguader 88, 08003, Barcelona, Spain. roderic.guigo@crg.cat.|Institut Hospital del Mar d'Investigacions Mèdiques (IMIM), Dr. Aiguader 88, 08003, Barcelona, Spain. roderic.guigo@crg.cat.
Roderic
R
Guigó
Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, 08003, Barcelona, Spain. rory.johnson@crg.eu.|Universitat Pompeu Fabra (UPF), Dr. Aiguader 88, 08003, Barcelona, Spain. rory.johnson@crg.eu.|Institut Hospital del Mar d'Investigacions Mèdiques (IMIM), Dr. Aiguader 88, 08003, Barcelona, Spain. rory.johnson@crg.eu.
Rory
R
Johnson
D064113:CRISPR-Cas Systems / Q000235:genetics*; D007446:Chromosome Inversion / Q000235:genetics; D005822:Genetic Vectors; D016678:Genome; D023281:Genomics; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*; D062085:RNA, Long Noncoding / Q000235:genetics*; D017384:Sequence Deletion
D000094704:RNA, Guide, CRISPR-Cas Systems; D062085:RNA, Long Noncoding