Hands-on CRISPR-Cas9 Experiments for Undergraduate Biochemistry Students: Plasmid DNA Digestion Using the Cas9 Ribonucleoprotein and Restriction Enzymes

Abstract The development of the CRISPR-Cas9 technology has profoundly impacted a wide range of biological fields. Derived from bacteria and archaea, the CRISPR-Cas9 system enables genome editing. It is used as a research tool and has significant promise for the treatment of genetic diseases and for plant engineering. Given the growing number of applications for CRISPR-Cas9 systems, the curriculum for undergraduate biochemistry students must be updated to provide a conceptual understanding and hands-on experience with this technology. Here, we describe a simple CRISPR-Cas9 experiment that can be carried out in a standard biochemistry teaching laboratory. The experiment involves incubation of a target plasmid with Cas9 ribonucleoprotein, the single guide RNA, and a restriction enzyme, resulting in the efficient cleavage of the plasmid, which is then visualized using agarose gel electrophoresis. To simplify Cas9 ribonucleoprotein preparation for instructors, we utilized a dual vector for the expression of both Cas9 and a single guide RNA in Escherichia coli. This laboratory protocol was successfully implemented in our biochemistry laboratory course. The activity enabled students to understand the essential functions and mechanisms of CRISPR-Cas9. Additionally, it provided an opportunity for students to study and discuss recent applications of the technology. The lab could be expanded to include other themes, such as plasmid transformation, protein expression in Escherichia coli, and protein purification.