{"doi":"10.1101/2022.01.18.476836","title":"Massively parallel genomic perturbations with multi-target CRISPR reveal new insights on Cas9 activity and DNA damage responses at endogenous sites","abstract":"ABSTRACT We present an approach that combines a Cas9 that simultaneously targets hundreds of epigenetically diverse endogenous genomic sites with high-throughput sequencing technologies to measure Cas9 dynamics and cellular responses at scale. This massive multiplexing of CRISPR is enabled by means of novel multi-target gRNAs (mgRNAs), degenerate gRNAs that direct Cas9 to a pre-determined number of well-mapped sites. mgRNAs uncovered generalizable insights into Cas9 binding and cleavage, discovering rapid post-cleavage Cas9 departure and repair factor loading at PAM-proximal genomic DNA. Moreover, by bypassing confounding effects from gRNA sequence, mgRNAs unveiled that Cas9 binding is enhanced at chromatin-accessible regions, and Cas9 cleavage is more efficient near transcribed regions. Combined with light-mediated activation and deactivation of Cas9 activity, mgRNAs further enabled high-throughput study of the cellular response to double strand breaks with high temporal resolution, discovering the presence, extent (under 2 kb), and kinetics (~ 0.5 hr) of reversible DNA damage-induced chromatin decompaction. Altogether, this work establishes mgRNAs as a generalizable platform for multiplexing CRISPR and advances our understanding of intracellular Cas9 activity and the DNA damage response at endogenous loci.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2022,"id":302441,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.953,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2022-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":864357,"name":"Alberto Marín-González","orcid":"0000-0002-9076-1270","position":1,"is_corresponding":false},{"id":949189,"name":"Yang Liu","orcid":"0000-0002-3889-9707","position":2,"is_corresponding":false},{"id":574616,"name":"Hans B. Liu","orcid":"0000-0001-8911-5637","position":3,"is_corresponding":false},{"id":791949,"name":"Leo Shen","orcid":"0000-0002-9563-6276","position":4,"is_corresponding":false},{"id":43303,"name":"Rachel Dveirin","orcid":"0000-0003-0453-503X","position":5,"is_corresponding":false},{"id":864359,"name":"Jay X. J. Luo","orcid":"0000-0002-5111-0192","position":6,"is_corresponding":false},{"id":43302,"name":"Reza Kalhor","orcid":"0000-0002-5558-7545","position":7,"is_corresponding":false},{"id":22168,"name":"Taekjip Ha","orcid":"0000-0003-2195-6258","position":8,"is_corresponding":false},{"id":656706,"name":"Roger S. Zou","orcid":"0000-0003-1338-6398","position":0,"is_corresponding":true}],"reference_count":57,"raw_metadata":null,"created_at":"2026-07-19T00:32:16.279991Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}