Genome-wide knockout libraries for entire classes of genomic elements in humans and other species.

(A) An example of paired sgRNAs designed against the upstream ultraconserved element (UCE) and promoter of the human IRX3 gene. IRX3 lies on the antisense strand. The exact target regions are shown in black, flanked by the design regions in green. The ten sgRNA pairs for each are denoted by red bars. Integrated chromatin marks from the ENCODE project [26] are displayed below, in addition to PhyloP multispecies conservation scores [33]. Note the region of elevated conservation corresponding to the UCE. (B,C) Summary of paired sgRNA designs targeting entire classes of genomic elements. In each figure, the left scale and grey bars represent the design performance, as in Fig 2. The right scale and black bars indicate the total number of elements in each class. (B) shows a series of genomic element classes for human, while (C) shows designs for the entire set of annotated microRNA genes in five species. Designs were created with default settings; designs using “DECKO” construction method give identical results.</p

Benchmarking and performance.

(A) Genome size and filtered protospacer density for the five species tested. (B) The fraction of protospacers passing filters of off-targeting, efficiency score, and both. The latter are defined as “filtered protospacers”, whose density is shown in (A). Data are displayed as a fraction of the total number of canonical PAM sequences in each genome. (C) The effect on library quality of modifying design variables. Y-axis denotes the percent of target regions, divided by: “successful”, where n = 10 distinct sgRNA pair designs are returned per target; “intermediate” designs, where 0Materials and Methods for details). The first column represents the run performed with default settings, and in each subsequent column one variable is modified (see Table 3 for details).</p

Pre-generated paired CRISPR libraries.

<p>Pre-generated paired CRISPR libraries.</p

Benchmarking results.

<p>Analyses were performed on a set of 7000 regions composed of different human target types (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005341#sec009" target="_blank">Methods</a> for details). % full depth refers to the percent of targets receiving <i>n</i> = 10 sgRNA pair designs. % partial depth refers to targets receiving 0<<i>n</i><10 designs. Designed targets refers to the total number of target features receiving full or partial depth designs.</p

User-defined parameters.

<p>User-defined parameters.</p

Overview of CRISPETa pipeline.

(A) Schematic of CRISPR-mediated genomic deletion. The aim is elimination of the Target region through recruitment of a pair of Cas9 proteins. Red boxes represent protospacers, the 20 bp upstream of a PAM and recognised by the sgRNA. (B) The CRISPETa workflow.</p

Species analysed by CRISPETa and for which off-target databases were compiled.

<p>Filtered protospacers are those passing default off-target and efficiency score cutoffs.</p

Production of truncated <i>MALAT1</i> RNA from mutated alleles.

<p>(A) RT-PCR was performed on RNA from bulk cells where MALAT1 exon region was deleted (sgRNA Pair 1, in two biological replicates), or control cells transfected with pDECKO targeting EGFP. Primers flanking the deleted region were used, and are expected to amplify fragments of the indicated sizes, depending on whether the RNA arises from a wild type or a deleted allele. Specificity was ensured by the exclusion of the reverse transcriptase enzyme in control reactions (“RT-”). (B) Sequencing analysis of mutant junctions of 4 of the colonies after TA cloning of the RT-PCR product. In red, region complementary to the sgRNA variable region; Green, PAM sequences; Blue, indel. Expected cut location is marked with vertical bar.</p

Measuring the deletion efficiency of paired sgRNA designs.

<p>(A) Structure of the pDECKO_mCherry vector. Note that the blue “target” regions contain protospacer sequences, and must be cloned as appropriate for each target region, while the grey “scaffold” regions are constant for all experiments. Note the presence of mCherry and puromycin selection markers. pDECKO_mCherry is compatible with lentivirus production or transfection. (B) Outline of the QC-PCR method for assessing deletion efficiency. Concentration of unmutated, wild-type target sites (using “In” and “Out” primers) is normalised to the reference amplicon, to control for template gDNA concentration. (C) The human <i>MALAT1</i> locus. The <i>MALAT1</i> lncRNA gene, shown in green, lies on the positive strand. The two selected target regions are shown: the conserved upstream enhancer-like region (note the overlap with H3K4Me1 and H3K28Ac modifications), and the exonic region. As before, target regions are shown in black, and sgRNA design regions in green. sgRNA pairs are represented by red bars, and genotyping primers as black arrowheads. (D) Overview of the experimental scheme. (E) Results for Enhancer region (upper panel) and Exon region (lower panel). Left: Agarose gel showing conventional genotyping results from bulk (unsorted) cells, with primers flanking the deleted region (primers out-out). 1–4 designate sgRNA pairs. “WT” indicates control cells transfected with pDECKO_mCherry targeting the EGFP sequence. Numbers on the gel refer to the expected size for the PCR amplicons. Right: QC-PCR results from four independent biological replicates (primers in-out). Y-axis shows the normalised fraction of unmutated, wild-type alleles, using primers amplifying the targeted region (orange/green), or a distal, non-targeted region (grey). Data were normalised to control cells transfected with pDECKO targeting EGFP. Error bars denote the standard deviation of four independent biological replicates. The differences between treated cells and control cells were statistically significant for all four sgRNA pairs, for both target regions (P<0.01, paired <i>t</i> test).</p