![]() ![]() non-self discrimination during CRISPR RNA-directed immunity. A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors. Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants. The protocol enables comprehensive PAM characterization of dozens or hundreds of Cas enzymes in parallel in <2 weeks. HT-PAMDA does not require specialized equipment or expertise and is cost effective for multiplexed characterization of many enzymes. A distinguishing feature of HT-PAMDA is that the enzymes are expressed in a cell type or organism of interest (e.g., mammalian cells), permitting scalable characterization and comparison of hundreds of enzymes in a relevant setting. ![]() Here, we provide a step-by-step protocol for the method, discuss experimental design considerations, and highlight how the method can be used to profile naturally occurring CRISPR–Cas9 enzymes, engineered derivatives with improved properties, orthologs of different classes (e.g., Cas12a), and even different platforms (e.g., base editors). The high-throughput PAM determination assay (HT-PAMDA) is a method that enables scalable characterization of the PAM preferences of different Cas proteins. One such property is the requirement for Cas proteins to recognize a protospacer-adjacent motif (PAM) in DNA target sites. The continued expansion of the genome-editing toolbox necessitates methods to characterize important properties of CRISPR–Cas enzymes. ![]()
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