{"doi":"10.1371/journal.pone.0232594","title":"TIM, a targeted insertional mutagenesis method utilizing CRISPR/Cas9 in Chlamydomonas reinhardtii","abstract":"Generation and subsequent analysis of mutants is critical to understanding the functions of genes and proteins. Here we describe TIM, an efficient, cost-effective, CRISPR-based targeted insertional mutagenesis method for the model organism Chlamydomonas reinhardtii. TIM utilizes delivery into the cell of a Cas9-guide RNA (gRNA) ribonucleoprotein (RNP) together with exogenous double-stranded (donor) DNA. The donor DNA contains gene-specific homology arms and an integral antibiotic-resistance gene that inserts at the double-stranded break generated by Cas9. After optimizing multiple parameters of this method, we were able to generate mutants for six out of six different genes in two different cell-walled strains with mutation efficiencies ranging from 40% to 95%. Furthermore, these high efficiencies allowed simultaneous targeting of two separate genes in a single experiment. TIM is flexible with regard to many parameters and can be carried out using either electroporation or the glass-bead method for delivery of the RNP and donor DNA. TIM achieves a far higher mutation rate than any previously reported for CRISPR-based methods in C. reinhardtii and promises to be effective for many, if not all, non-essential nuclear genes.","journal":"PLoS ONE","year":2020,"id":55793,"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":90,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9567,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2020-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":289044,"name":"Yuqing Hou","orcid":"0000-0002-7038-675X","position":1,"is_corresponding":false},{"id":289045,"name":"Tomohiro Kubo","orcid":"0000-0002-4341-5631","position":2,"is_corresponding":false},{"id":289046,"name":"Nathan McNeill","orcid":"0000-0001-9688-5728","position":3,"is_corresponding":false},{"id":289047,"name":"Haruaki Yanagisawa","orcid":"0000-0003-0313-2343","position":4,"is_corresponding":false},{"id":289048,"name":"Toshiyuki Oda","orcid":"0000-0001-8090-2159","position":5,"is_corresponding":false},{"id":289049,"name":"George B. Witman","orcid":"0000-0002-9497-9218","position":6,"is_corresponding":false},{"id":289043,"name":"Tyler Picariello","orcid":"0000-0003-0049-1148","position":0,"is_corresponding":true}],"reference_count":48,"raw_metadata":null,"created_at":"2026-07-18T21:05:29.672878Z","pmid":"32401787","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":[]}