Sequence-specific control of gene expression on a genome-wide scale is an 
important approach for understanding gene functions and for engineering genetic 
regulatory systems. We have recently described an RNA-based method, CRISPR 
interference (CRISPRi), for targeted silencing of transcription in bacteria and 
human cells. The CRISPRi system is derived from the Streptococcus pyogenes 
CRISPR (clustered regularly interspaced palindromic repeats) pathway, requiring 
only the coexpression of a catalytically inactive Cas9 protein and a 
customizable single guide RNA (sgRNA). The Cas9-sgRNA complex binds to DNA 
elements complementary to the sgRNA and causes a steric block that halts 
transcript elongation by RNA polymerase, resulting in the repression of the 
target gene. Here we provide a protocol for the design, construction and 
expression of customized sgRNAs for transcriptional repression of any gene of 
interest. We also provide details for testing the repression activity of CRISPRi 
using quantitative fluorescence assays and native elongating transcript 
sequencing. CRISPRi provides a simplified approach for rapid gene repression 
within 1-2 weeks. The method can also be adapted for high-throughput 
interrogation of genome-wide gene functions and genetic interactions, thus 
providing a complementary approach to RNA interference, which can be used in a 
wider variety of organisms.