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