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

Abstract

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2086-z) contains supplementary material, which is available to authorized users.

Modulating the expression of long non‐coding RNA s for functional studies

“…However, Cas9-mediated mutagenesis through the generation of double-strand breaks and non-homologous end joining is often not suitable for the study of lncRNAs, since by definition they do not produce proteins and therefore may have biological functions that are less likely to be perturbed by small indels that produce frameshift mutations. Double Cas9 excision of DNA sequences that flank lncRNAs are more likely to inactivate lncRNA gene function and has been used to delete up to hundreds of human lncRNAs, revealing the function of previously uncharacterized lncRNA loci [26,88,89]. Engineered Cas9 variants, in particular dCas9 fused to transcriptional activators or repressors, are highly effective for modulating the expression of lncRNAs without alterations to the underlying genomic DNA sequence [90][91][92][93][94][95][96].…”

Long Noncoding RNA NEAT1, Regulated by the EGFR Pathway, Contributes to Glioblastoma Progression Through the WNT/β-Catenin Pathway by Scaffolding EZH2

“…S2A). The CRISPR/ Cas9 system has been reported to successfully knockdown various lncRNAs (26)(27)(28). Therefore, we designed a strategy to knockdown NEAT1 by dual sgRNAs (Supplementary Fig.…”

The emerging molecular biology toolbox for the study of long noncoding RNA biology

“…A further advantage of targeting the promoter is the small size that needs to be deleted, increasing the efficiency of editing. As such, paired guide-based CRISPR deletions of lncRNA promoters have been adapted in large scale studies for screening the effects of lncRNAs [53].…”

“…Quantitative reads of the entire transcriptome [27] CAGE Quantitative reads of the transcriptome by capturing the 5 -end of the transcript [28] 3C (and its derivatives) Mapping of chromosomal interaction for the identification of spatially proximal genes and genetic elements [29][30][31] ChIP-Seq Genome wide mapping and quantification of epigenetic marks and protein on DNA [32] smFISH (and its derivatives) Abundance and subcellular localization of transcripts in single cells [33][34][35][36][37][38][39] Genome and epigenome editing tools Perturbation of lncRNA transcription and position to determine function [44,45,50,51,[53][54][55]58,60] ChIRP Identification of interacting RNA, DNA and protein partners through the capture of the lncRNA transcript [61] Chart Identification of interacting DNA and protein partners through the capture of the lncRNA transcript [62] RAP Identification of interacting RNA, DNA and protein partners through the capture of the lncRNA transcript [20] PAR-CLIP Identification of lncRNAs that are interacting with a particular protein of interest through the immunoprecipitation of the protein [63,64] SHAPE LncRNA secondary structure at single nucleotide resolution [65] the recruitment of proteins, such as PRC2, the entire X-chromosome is epigenetically silenced [20]. Through the use of these molecular tools, these studies have defined the functional mechanism of Xist, which has now been established as a common modality for a subclass of lncRNAs [5,25].…”