The ENCODE4 Functional Characterization Centers conducted 108 CRISPR screens in human cell lines, covering over 540,000 perturbations across 24.85 megabases of the genome. Using 332 functionally confirmed CRE–gene links in K562 cells, the authors established guidelines for screening endogenous noncoding elements with CRISPR interference (CRISPRi), including accurate detection of CREs with variable and often low transcriptional effects. Benchmarking five screen analysis tools, they found that CASA produces the most conservative CRE calls and is robust to artifacts of low-specificity single guide RNAs (sgRNAs). They also uncovered a subtle DNA strand bias for CRISPRi in transcribed regions, which has implications for screen design and analysis. The study provides an accessible data resource, predesigned single guide RNAs for targeting 3,275,697 ENCODE SCREEN candidate CREs with CRISPRi, and screening guidelines to accelerate the functional characterization of the noncoding genome. The results highlight the importance of considering strand specificity in CRISPRi effects within gene bodies and suggest that coding strand-targeting sgRNAs are more effective in these regions.The ENCODE4 Functional Characterization Centers conducted 108 CRISPR screens in human cell lines, covering over 540,000 perturbations across 24.85 megabases of the genome. Using 332 functionally confirmed CRE–gene links in K562 cells, the authors established guidelines for screening endogenous noncoding elements with CRISPR interference (CRISPRi), including accurate detection of CREs with variable and often low transcriptional effects. Benchmarking five screen analysis tools, they found that CASA produces the most conservative CRE calls and is robust to artifacts of low-specificity single guide RNAs (sgRNAs). They also uncovered a subtle DNA strand bias for CRISPRi in transcribed regions, which has implications for screen design and analysis. The study provides an accessible data resource, predesigned single guide RNAs for targeting 3,275,697 ENCODE SCREEN candidate CREs with CRISPRi, and screening guidelines to accelerate the functional characterization of the noncoding genome. The results highlight the importance of considering strand specificity in CRISPRi effects within gene bodies and suggest that coding strand-targeting sgRNAs are more effective in these regions.