Light, CRISPR and DNA repair*

We present multi-target CRISPR, an approach whereby a degenerate gRNA directs Cas9 to simultaneously target a large number of epigenetically diverse genomic locations. This multiplexing of CRISPR is enabled by targeting the highly abundant Alu repetitive elements. Combined with different next-generation

sequencing readouts, multi-target CRISPR offered new insights into Cas9 activity and the ensuing cellular response. First, by interrogating the same protospacer in different genomic contexts, multi-target CRISPR showed that Cas9 binding is more efficient at open chromatin regions, and Cas9 cleavage is enhanced near transcribed regions. We then combined multi-target CRISPR with ATAC-Seq to unveil an increase in chromatin accessibility around Cas9 DSBs, which spreads over a window of ~1 kb centered at the cut site.

We attribute this chromatin opening to nucleosomes being evicted from the damaged region. Notably, the extent of chromatin opening is independent of the chromatin context in which the lesions occurs, suggesting that nucleosome eviction is a robust part of the DNA damage response. By combining multi-target CRISPR with photo-activatable gRNAs, we determined the characteristic time-scales of DSB-induced chromatin relaxation and found that nucleosome removal occurs within ~30 min. Last, we combined multi-target CRISPR with a photo-deactivatable gRNA to measure chromatin dynamics after DSB repair completion. Once repair is completed, chromatin accessibility levels returned to their original pre-

cleavage state within 1 h, revealing a quick repositioning of nucleosomes around the repaired cut site. Based on these measurements, we propose a model in which chromatin undergoes fast and transient opening in response to Cas9 breaks to facilitate recruitment of repair factors. In conclusion, multi-target CRISPR enables high-throughput studies of Cas9 activity and DNA repair in cellulo, revealing new insights on the interplay between Cas9 activity and chromatin.