Reconstitution and biochemical characterization of the RNA-guided helicase-nuclease protein Cas3 from type I-A CRISPR-Cas system.

Type I is the most prevalent CRISPR system found in nature. It can be further defined into six subtypes, from I-A to I-G. Among them, the Type I-A CRISPR-Cas systems are almost exclusively found in hyperthermophilic archaeal organisms. The system achieves RNA-guided DNA degradation through the concerted action of a CRISPR RNA containing complex Cascade and a helicase-nuclease fusion enzyme Cas3. Here, we summarize assays to characterize the biochemical behavior of Cas3. A steep temperature-dependency was found for the helicase component of Cas3HEL, but not the nuclease component HD. This finding enabled us to establish the correct experimental condition to carry out I-A CRISPR-Cas based genome editing in human cells with extremely high efficiency.