{"doi":"10.1101/2021.04.20.440537","title":"Detection of precisely edited CRISPR/Cas9 alleles through co-introduced restriction-fragment length polymorphisms","abstract":"ABSTRACT CRISPR/Cas9 is a powerful tool for producing genomic in sertions and del etions (indels) to interrogate gene function. Modified CRISPR/Cas9 protocols can produce targeted genetic changes that are more precise than indels, but founder recovery is less efficient. Focusing on producing missense mutations in zebrafish using s ingle- s tranded o ligo d eoxy n ucleotide (ssODN) donor templates, we pioneered a strategy of adding synonymous changes to create novel r estriction- e nzyme (RE) sites, allowing detection of rare precise edits in a modified fluorescent-PCR fragment assay. We have named this process TIARS ( t est for i ncorporation of a dded r ecognition s ites). Aided by TIARS, we induced two distinct amino-acid substitutions (T979I and P1387S) in the atp7a gene among somatic tissues of CRISPR-Cas9-treated F 0 zebrafish. One of these F 0s transmitted the allele to atp7a T979I/+ F 1 progeny, and trans-heterozygosity of this allele against a null atp7a allele causes hypopigmentation, consistent with more severe pigment deficits in zebrafish or humans carrying only null mutations in atp7a/ATP7A . Design of ssODNs with novel RE recognition sites is labor-intensive, so we developed an in silico tool, TIARS Designer, and performed bioinformatic validation indicating that TIARS should be generalizable to other genes and experimental systems that employ donor template DNA.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2021,"id":223821,"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":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9538,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2021-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":825991,"name":"Sydney Hertafeld","orcid":null,"position":1,"is_corresponding":false},{"id":825992,"name":"Yvonne Rosario","orcid":null,"position":2,"is_corresponding":false},{"id":263045,"name":"James Iben","orcid":"0000-0002-1130-7660","position":3,"is_corresponding":false},{"id":257816,"name":"Eric H. Chang","orcid":"0000-0001-5592-4276","position":4,"is_corresponding":false},{"id":400570,"name":"Ling Yi","orcid":"0000-0001-5250-214X","position":5,"is_corresponding":false},{"id":396914,"name":"Steven L. Coon","orcid":"0000-0002-5233-0277","position":6,"is_corresponding":false},{"id":333641,"name":"Stephen G. Kaler","orcid":"0000-0002-7522-926X","position":7,"is_corresponding":false},{"id":331664,"name":"Ryan Dale","orcid":"0000-0003-2664-3744","position":8,"is_corresponding":false},{"id":385013,"name":"Benjamin Feldman","orcid":"0000-0003-4838-8641","position":9,"is_corresponding":false},{"id":385012,"name":"Chon‐Hwa Tsai‐Morris","orcid":"0000-0002-8113-0979","position":0,"is_corresponding":true}],"reference_count":22,"raw_metadata":null,"created_at":"2026-07-18T23:54:14.760456Z","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":[]}