CRISPR-based functional screening of noncoding regulatory elements

The vast majority of trait-associated genetic variants reside in noncoding cis-regulatory elements (CREs), yet most regulatory sequences remain functionally uncharacterized. CRISPR-based screens enable direct perturbation of CREs in their native genomic context and have expanded into a diverse toolkit, including CRISPRi/CRISPRa, nuclease-mediated editing, base editing, prime editing, and continuous mutagenesis. Moreover, these approaches can be paired with phenotypic, transcriptomic, or chromatin readouts at single-cell resolution. However, a limitation of conventional pooled CRISPR screens is that cells are assigned by sgRNA identity rather than by the genotype installed. New genotype-resolved approaches address this gap by directly reading the edited endogenous sequence. Because sequence variants in CREs are associated with human disease, continued development of CRISPR-based technologies may help advance new therapies. In this review, we discuss diverse CRISPR-based modalities and their limitations, emerging genotype-resolved techniques, their applications in gene therapy, and computational approaches for predicting the function of regulatory sequences.