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. 2016 Mar 24;17:55.
doi: 10.1186/s13059-016-0915-2.

CRISPR Library Designer (CLD): Software for Multispecies Design of Single Guide RNA Libraries

Free PMC article

CRISPR Library Designer (CLD): Software for Multispecies Design of Single Guide RNA Libraries

Florian Heigwer et al. Genome Biol. .
Free PMC article


Background: Genetic screens using CRISPR/Cas9 are a powerful method for the functional analysis of genomes.

Results: Here we describe CRISPR library designer (CLD), an integrated bioinformatics application for the design of custom single guide RNA (sgRNA) libraries for all organisms with annotated genomes. CLD is suitable for the design of libraries using modified CRISPR enzymes and targeting non-coding regions. To demonstrate its utility, we perform a pooled screen for modulators of the TNF-related apoptosis inducing ligand (TRAIL) pathway using a custom library of 12,471 sgRNAs.

Conclusion: CLD predicts a high fraction of functional sgRNAs and is publicly available at

Keywords: Bioinformatics; CRISPR/Cas9; Functional genomics; Pooled screens; sgRNA design.


Fig. 1
Fig. 1
CRISPR library designer workflow. CLD is a command line tool, tailored for fast end-to-end design of sgRNA libraries. Its back-end steps are performed by the depicted algorithm: the genome data of the target organism, a gene list of interest, and a parameter file are needed as input files. Each gene sequence is then scanned for the presence of protospacer adjacent motifs (PAM). Valid target sites are evaluated for their annotation, sequence, and off-target characteristics and passed to the library formatting steps. There, target sites are tested for specific restriction sites and then flanked by cloning adapters. A user-defined minimum of best-annotated sgRNAs is selected for each gene. Genes with sgRNA coverage below the defined minimum are discarded. In the end, output files are generated, including a FASTA file containing ready-to-order oligonucleotide sequences
Fig. 2
Fig. 2
A pooled screen for functional validation of CLD. a The screening strategy in SW480 cells. In brief, a pool of mutant SW480 cells harboring 12,471 sgRNA designs against 408 genes was generated by lentiviral infection and antibiotic selection. Fourteen million cells per condition were then treated with PBS (control) or recombinant TRAIL (treatment) for a total of 12 days. Subsequently, the genomic DNA of the samples was extracted and sgRNA composition analyzed by next-generation sequencing (NGS). b Comparison of sgRNA sequence counts between two biological replicates demonstrates high reproducibility (Pearson correlation coefficient ~0.79). c Distribution of sgRNAs targeting positive pathway regulators (CASP8, CASP3, FADD, BAX, BID, TNFRSF10A, TNFRSF10B) in red, negative regulators (XIAP, BCL2L1) in blue, and random, non-targeting sgRNAs in orange between TRAIL (y-axis) and PBS (x-axis) treated cell populations. d Scatter plot showing relative enrichment of genes (y-axis) with their corresponding p value (x-axis). Positive regulators are plotted in red, negative regulators in blue, and random, non-targeting sgRNAs in orange. P values were calculated by Wilcoxon rank sum test between 30 sgRNAs of one gene and 200 random, non-targeting sgRNAs. Log2 fold change is calculated as median log2 ratio between normalized sgRNA count of TRAIL- over PBS-treated populations. The vertical line marks a p value of 0.05. eg Median normalized fold change of all sgRNAs targeting three essential TRAIL pathway components. A total of 100 sgRNAs are depicted for each gene. Enriched sgRNAs are colored in red, depleted sgRNAs in grey. The dashed line represents the median fold change of all sgRNAs of the corresponding gene
Fig. 3
Fig. 3
Impact of sgRNA features on library design. a Scatter plot showing log2 fold change of sgRNAs targeting CASP8 relative to their exon location. The gene models of major transcripts of CASP8 are depicted (ENST00000432109, ENST00000392258, ENST00000323492, ENST00000264275, ENST00000358485). b Box plots showing fold change of all sgRNAs targeting the first exon of CASP8 compared with other targeted exons. sgRNAs against the first exon are less enriched (*p ≤ 0.05, two-sided t-test). c Comparison of sgRNA features between functional and non-functional sgRNAs. All sgRNAs of BAX, FADD, and CASP8 were selected for analysis. Fold changes of individual sgRNAs of each gene were compared with the mean fold change of all sgRNAs of the respective genes. sgRNAs with a z-score >1 were grouped as functional and those with a z-score < −1 were grouped as non-functional. The on-target score by Doench et al. was calculated for both groups (y-axis) and are presented as box plots. Differences between groups were determined by a two-sided t-test. d Line graph showing interdependence between number of targetable human protein coding genes (y-axis), sgRNA coverage per gene (x-axis), and number of off-targets (colored lines). Off-targets are defined as genomic regions with at least 12 bases of homology to the protospacer of the on-target

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