Computational pipeline for designing guide RNAs for mismatch-CRISPRi

Summary CRISPR interference is an increasingly popular method for perturbing gene expression. Guided by single-guide RNAs (sgRNAs), nuclease-deficient Cas9 proteins bind to specific DNA sequences and hinder transcription. Specificity is achieved through complementarity of the sgRNAs to the DNA. Changing complementarity by introducing single-nucleotide mismatches can be exploited to tune knockdown. Here, we present a computational pipeline to identify sgRNAs targeting specific genes in a bacterial genome, filter them, and titrate their activity by introducing mismatches. For complete details on the use and execution of this protocol, please refer to Hawkins et al. (2020) .