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

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.

Fig. 1

Outline of DECKO. a Deletion of genomic elements such as promoters by dual CRISPR targeting. b Structure of pDECKO. Two independent gRNA transgenes are driven by human U6 and human H1 promoter sequences. Variable sequence are highlighted in blue. c Cloning step 1: Synthesised oligonucleotide Insert-1, carrying all variable sequence, is inserted into BsmBI-digested parental plasmid by Gibson assembly. d Cloning step 2: the preassembled universal Insert-2 is inserted into the intermediate plasmid by standard ligation. e Nucleotide sequence of the 165 nt Insert-1. From left to right: Light orange: 5’ Gibson assembly overhang; light blue: targeting region of gRNA1; grey: fragment of first scaffold region; yellow: BsmBI sites; dark blue: target region of gRNA2; dark orange: fragment of H1 promoter; grey: 3’ Gibson assembly overhang

Fig. 2

Targeting promoter regions of genes for knockout. a The structure of the MALAT1 locus showing the major promoter and upstream promoter. The MALAT1 transcript annotations from Gencode version 19 (green) lie on the positive strand. Coloured bars indicate regions targeted by CRISPR deletion in this study. Shown below are ENCODE whole cell polyA+ RNA sequencing read density for three of the cell lines used, showing that the majority of transcription originates at the major promoter. The four primer sets for qRTPCR are shown. b The TFRC gene locus. Coloured bars indicate regions targeted by CRISPR deletion. In black, the amplicon used for qRTPCR

Fig. 3

Expression of gRNAs. a Primer design for specific detection of gRNAs by qRTPCR. The reverse primer is constant for all PCRs while a specific primer is designed for each gRNA. (b and c) Relative expression of transfected gRNAs in HCT116 cells (b) and Hela cells (c). Cells were transfected and selected with puromycin (2ug/mL) for 3 days. Data are normalised to the housekeeping gene GAPDH. Control amplifications were carried out using indicated primers with cDNA template from pDECKO-GFP cells

Fig. 4

Sequence deletion in bulk cells. a Outline of the genomic PCR (gPCR) primer strategy used for genotyping. b Deletion of TFRC promoter (construct B in Fig. 2), as validated by electrophoresis of gPCR products. Wild type gDNA and water templates are used as positive and negative controls, respectively. Green and red arrows indicate the size of PCR products expected from wild type (WT) and deleted alleles. Note that in this and subsequent panels, separated lanes originate from the same original agarose gel, rearranged for clarity. c-e gPCR on bulk cells transfected with the indicated DECKO plasmids targeting (c) TFRC promoter, (d) MALAT1 upstream promoter, (e) MALAT1 major promoter. (f-h) qRTPCR on cell samples shown in (c-e). Control indicates RNA from cells transfected with a DECKO targeting GFP. Levels were normalised to GAPDH. Error bars show the standard deviation of three technical replicates. i Expression of TFRC protein on cell surface, as determined by flow cytometry analysis of antibody-stained cells. Left: histogram of cell fluorescence intensity counts. Right: Calculation of relative stain index, a normalised measure of fluorescence intensity

Fig. 5

Derivation of TFRC cell clones. a Outline of the clone-derivation protocol used for TFRC and MALAT1 knock out cells, indicating approximate time required. b First and c second stage PCRs to genotype clones. Primer combination schemes are indicated below the electrophoresis gels. H: TFRC_B cell clone genotyped as heterozygote; WT, cell clones genotyped as wild type; +, positive control wild type cells; H₂O, water. d qRTPCR for TFRC mRNA, normalised to GAPDH. Error bars indicate the standard deviation of three technical replicates. e Flow cytometry analysis of surface levels of TFRC protein. Left: histogram of cell fluorescence intensity counts. Right: Calculation of relative stain index. f Sequencing analysis of mutant junction of the heterozygous clones. In red, region complementary to the gRNA variable region; Green, PAM sequences; Blue, indel. Expected cut location is marked with vertical bar

Fig. 6

Derivation of MALAT1 knockout clones. a MALAT1 RNA expression levels of three HCT116 clones lacking the upstream promoter, deleted using the MALAT1_A DECKO construct. KO, homozygous knockout. Expression detected using Primer Set 1 (see below) and values are relative to a control cell clone expressing pDECKO GFP gRNAs. Error bars indicate the standard deviation of three technical replicates. b As for (a) in a heterozygous clone for the major promoter, deleted using the DECKO_C construct. Het, heterozygote. c MALAT1 expression in HEK293T cells heterozygous or homozygous for the major promoter, deleted using the DECKO_C & D constructs. Expression detected using Primer Set 1. d As for (c) in HeLa clones, using the DECKO_C & E constructs. e Semi-quantitative PCR carried out on the same HEK293T clones that in Fig. 6c. “RT-“indicates control samples where Reverse Transcriptase was omitted. f-g qRTPCR using three additional downstream primer pairs (see Fig. 2a), illustrating knockdown of MALAT1 RNA throughout the gene, for the HEK293T and HeLa MALAT1_C clones. h Sequence analysis of mutant junctions. MALAT1_A KO 3 contains one extra C in the deletion point. MALAT1_A 1, MALAT1_A 2 and MALAT1_A 4 have the expected deletion, with no indel. Both gRNA are in antisense direction. Expected cut location is marked with vertical bar. i Proliferation assay comparing the growth rates of control GFP and MALAT1 HEK293T knockout and heterozygous clones. Error bars indicate standard deviation of five cell wells. **, P < 0.01/***, P < 0.001 by Student’s one-sided, paired t test