CRISPR-Hybrid: Intracellular Selection of CRISPR-Associated Aptamers in Bacteria

CRISPR technologies have evolved from nuclease-based genome editing to programmable systems for transcriptional and epigenetic regulations. Emerging CRISPR systems expand editing versatility by incorporating CRISPR-associated aptamers (CAPs) into single-guide RNAs (sgRNAs), enabling recruitment of RNA-binding proteins (RBPs) fused to diverse effectors. However, the limited availability of orthogonal aptamer-RBP pairs has hindered broad application, as conventional SELEX-based aptamer discovery is time-intensive and often fails to yield aptamers functional in cells. We developed the CRISPR-Hybrid platform, an intracellular selection method that directly evolves CAPs within bacterial cells. This system links aptamer-RBP interactions to a fluorescent reporter readout, allowing fluorescence-activated cell sorting (FACS) to enrich functional variants from libraries exceeding 10^⁷ sequences. Each selection round can be completed in 2 days, enabling rapid enrichment of aptamers that retain activity in both bacterial and mammalian contexts. This protocol details construction of randomized DNA libraries, preparation of host cells, execution of intracellular selection and FACS enrichment, and recovery of aptamer sequences for downstream analysis. By providing a fast, in-cell, and physiologically relevant approach to aptamer discovery, CRISPR-Hybrid expands the repertoire of CAPs available for modular and multiplexed CRISPR editing.