To perform CRISPR knockouts on a genome-wide scale, collections of sgRNAs known as sgRNA libraries, or CRISPR knockout libraries, must be generated. The first step in creating a sgRNA library is to identify genomic regions of interest based on known sgRNA targeting rules. For example, sgRNAs are most efficient when targeting the coding regions of genes and not the 5’ and 3’ UTRs. Conserved exons present as attractive targets, and position relative to the transcription start site should be considered. Secondly, all the possible PAM sites are identified and selected for. On- and off-target activity should be analysed, as should GC content, and homopolymer stretches should be avoided. The most commonly used Cas9 endonuclease, derived from Streptococcus pyogenes, recognises a PAM sequence of NGG.
Furthermore, specific nucleotides appear to be favoured at specific locations. Guanine is strongly favoured over cytosine on position 20 right next to the PAM motif, and on position 16 cytosine is preferred over guanine. For the variable nucleotide in the NGG PAM motif, it has been shown that cytosine is preferred and thymine disfavoured. With such criteria taken into account, the sgRNA library is computationally designed around the selected PAM sites.
Multiple sgRNAs (at least 4–6) should be created against every single gene to limit false-positive detection, and negative control sgRNAs with no known targets should be included. The sgRNAs are then created by in situ synthesis, amplified by PCR, and cloned into a vector delivery system.