Light activation and deactivation of Cas9 for DNA repair studies.

ABSTRACT

DNA double-strand breaks in DNA (DSBs) are common yet highly detrimental events in living organisms. To repair the damage, each cell requires a coordinated set of DNA damage response (DDR) proteins that can respond quickly, effectively, and precisely. Better understanding of these processes is therefore essential and would require an effective means of inducing targeted DSBs on demand, but previous methods are hampered by limited control over genomic location, timing, or lesion types. Tight spatiotemporal control of CRISPR-Cas9 activity has potential to overcome these limitations, which led to the development of two methods for rapid activation or deactivation of Cas9 using light. In this chapter, we discuss how control of Cas9 can advance DDR studies, describe protocols to control Cas9 activation and deactivation using this new technology, and finally outline three compatible readouts of DNA damage and the cellular response DSB levels using droplet digital PCR, repair factor localization using ChIP-seq, and insertion-deletion (indel) repair outcomes using Sanger sequencing.