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

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

Pre-generated paired CRISPR libraries.

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

Additional file 3 of Paired guide RNA CRISPR-Cas9 screening for protein-coding genes and lncRNAs involved in transdifferentiation of human B-cells to macrophages

Additional file 3: Supplementary Table S2: CRISPETa output lists for controls and targets including final CRISPR 165 oligonucleotide library

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

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

Additional file 1 of Paired guide RNA CRISPR-Cas9 screening for protein-coding genes and lncRNAs involved in transdifferentiation of human B-cells to macrophages

Additional file 1: Supplementary Figure S1. Expression clusters of lncRNAs during transdifferentiation. Supplementary Figure S2. Expression clusters of protein coding genes during transdifferentiation. Supplementary Figure S3. Expression profiles of lncRNAs and protein coding genes during transdifferentiation. Supplementary Figure S4. pDECKO plasmid and sequencing oligos binding scheme. Supplementary Figure S5. FACS sorting of BLaER1-Cas9 library. Supplementary Figure S6. Statistics on quantification of pgRNA representation in the screening. Supplementary Figure S7. Quantification of pgRNA distribution before and during screening. Supplementary Figure S8. Target genes disrupted by CRISPR-Cas9. Supplementary Figure S9. CEBPa and SPI1 validation at genomic level. Supplementary Figure S10. Western blot of CEBPa. Supplementary Figure S11. Individual target validation by flow cytometry. Supplementary Figure S12. lncRNA target sites and individual validations. Supplementary Figure S13. Validation of lncRNAs knockout at genomic level. Supplementary Figure S14: Epigenetic landscape of the candidate lncRNAs. Supplementary Figure S15. FURIN and NFE2 target sites and validationsat genomic level. Supplementary Figure S16. Uncropped western blots