{"doi":"10.1093/nar/gkac142","title":"Spacer2PAM: A computational framework to guide experimental determination of functional CRISPR-Cas system PAM sequences","abstract":"<jats:title>Abstract</jats:title><jats:p>RNA-guided nucleases from CRISPR-Cas systems expand opportunities for precise, targeted genome modification. Endogenous CRISPR-Cas systems in many prokaryotes are attractive to circumvent expression, functionality, and unintended activity hurdles posed by heterologous CRISPR-Cas effectors. However, each CRISPR-Cas system recognizes a unique set of protospacer adjacent motifs (PAMs), which requires identification by extensive screening of randomized DNA libraries. This challenge hinders development of endogenous CRISPR-Cas systems, especially those based on multi-protein effectors and in organisms that are slow-growing or have transformation idiosyncrasies. To address this challenge, we present Spacer2PAM, an easy-to-use, easy-to-interpret R package built to predict and guide experimental determination of functional PAM sequences for any CRISPR-Cas system given its corresponding CRISPR array as input. Spacer2PAM can be used in a ‘Quick’ method to generate a single PAM prediction or in a ‘Comprehensive’ method to inform targeted PAM libraries small enough to screen in difficult to transform organisms. We demonstrate Spacer2PAM by predicting PAM sequences for industrially relevant organisms and experimentally identifying seven PAM sequences that mediate interference from the Spacer2PAM-informed PAM library for the type I-B CRISPR-Cas system from Clostridium autoethanogenum. We anticipate that Spacer2PAM will facilitate the use of endogenous CRISPR-Cas systems for industrial biotechnology and synthetic biology.</jats:p>","journal":"Nucleic Acids Research","year":2022,"id":588293,"datarank":0.7732491842462621,"base_score":2.995732273553991,"endowment":2.995732273553991,"self_citation_contribution":0.4493598410330987,"citation_network_contribution":0.3238893432131635,"self_endowment_contribution":0.4493598410330987,"citer_contribution":0.3238893432131635,"corpus_percentile":null,"corpus_rank":null,"citation_count":19,"citer_count":18,"citers_with_citation_signal":13,"citers_with_endowment":13,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":null,"is_data_producer":false,"deposit_databanks":null,"is_oa":false,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":null,"fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1505049,"name":"Nicholas A Fackler","orcid":"0000-0003-3291-7617","position":1,"is_corresponding":false},{"id":268958,"name":"Ashty S. Karim","orcid":"0000-0002-5789-7715","position":2,"is_corresponding":false},{"id":88584,"name":"Michael Köpke","orcid":"0000-0003-0642-1415","position":3,"is_corresponding":false},{"id":242369,"name":"Michael C. Jewett","orcid":"0000-0003-2948-6211","position":4,"is_corresponding":false},{"id":1505048,"name":"Grant A Rybnicky","orcid":"0000-0002-0198-4596","position":0,"is_corresponding":false}],"reference_count":0,"raw_metadata":{"has_enrichment":true,"resolved":true,"title":"Spacer2PAM: A computational framework to guide experimental determination of functional CRISPR-Cas system PAM sequences","abstract":"<jats:title>Abstract</jats:title><jats:p>RNA-guided nucleases from CRISPR-Cas systems expand opportunities for precise, targeted genome modification. Endogenous CRISPR-Cas systems in many prokaryotes are attractive to circumvent expression, functionality, and unintended activity hurdles posed by heterologous CRISPR-Cas effectors. However, each CRISPR-Cas system recognizes a unique set of protospacer adjacent motifs (PAMs), which requires identification by extensive screening of randomized DNA libraries. This challenge hinders development of endogenous CRISPR-Cas systems, especially those based on multi-protein effectors and in organisms that are slow-growing or have transformation idiosyncrasies. To address this challenge, we present Spacer2PAM, an easy-to-use, easy-to-interpret R package built to predict and guide experimental determination of functional PAM sequences for any CRISPR-Cas system given its corresponding CRISPR array as input. Spacer2PAM can be used in a ‘Quick’ method to generate a single PAM prediction or in a ‘Comprehensive’ method to inform targeted PAM libraries small enough to screen in difficult to transform organisms. We demonstrate Spacer2PAM by predicting PAM sequences for industrially relevant organisms and experimentally identifying seven PAM sequences that mediate interference from the Spacer2PAM-informed PAM library for the type I-B CRISPR-Cas system from Clostridium autoethanogenum. We anticipate that Spacer2PAM will facilitate the use of endogenous CRISPR-Cas systems for industrial biotechnology and synthetic biology.</jats:p>","is_dataset_classified":null,"base_score":2.995732273553991,"endowment":2.995732273553991,"datacite_reuse_total":0,"file_count":0,"downloads":0,"views":0,"has_version_chain":false,"is_dataset":false,"is_oa":false,"pmid":"35258601","pmcid":"PMC8990532","openalex_id":"https://openalex.org/W4213036952","authors":[],"funders":[{"funder_name":"Department of Energy","grant_id":"DE-SC0018249","title":null},{"funder_name":"Department of Energy","grant_id":"DE-AC02-05CH11231","title":null},{"funder_name":"Community Science Program","grant_id":"CSP-503280","title":null},{"funder_name":"David and Lucile Packard Foundation","grant_id":"2011-37152","title":null},{"funder_name":"National Science Foundation Graduate Research Fellowship Program","grant_id":"DGE-1842165","title":null},{"funder_name":"National Science Foundation","grant_id":"1842165","title":"Graduate Research Fellowship Program (GRFP)"},{"funder_name":"Camille Dreyfus Teacher-Scholar Program","grant_id":"","title":null}],"total_grants":7,"fwci":1.3317,"citation_percentile":0.79325492,"influential_citations":2,"citation_trend":[{"year":2022,"count":4},{"year":2023,"count":5},{"year":2024,"count":4},{"year":2025,"count":4},{"year":2026,"count":2}],"oa_status":"gold","license":"CC BY NC","oa_locations":[{"url":"https://academic.oup.com/nar/article-pdf/50/6/3523/43246274/gkac142.pdf","host_type":"journal"},{"url":"https://academic.oup.com/nar/article-pdf/50/6/3523/43246274/gkac142.pdf","host_type":"GOLD"},{"url":"https://academic.oup.com/nar/article-pdf/50/6/3523/43246274/gkac142.pdf","host_type":"publisher"},{"url":"https://academic.oup.com/nar/article-pdf/50/6/3523/49563108/gkac142.pdf","host_type":"publisher"},{"url":"https://doi.org/10.1093/nar/gkac142","host_type":"journal"},{"url":"https://pubmed.ncbi.nlm.nih.gov/35258601","host_type":"repository"},{"url":"https://www.osti.gov/biblio/1853842","host_type":"repository"},{"url":"https://www.osti.gov/biblio/1904392","host_type":"repository"},{"url":"https://www.osti.gov/biblio/2298961","host_type":"repository"},{"url":"https://www.ncbi.nlm.nih.gov/pmc/articles/8990532","host_type":"repository"},{"url":"https://europepmc.org/articles/PMC8990532","host_type":"Europe_PMC"},{"url":"https://europepmc.org/articles/PMC8990532?pdf=render","host_type":"Europe_PMC"},{"url":"http://dx.doi.org/10.1093/nar/gkac142","host_type":""}],"fields_of_study":["CRISPR and Genetic Engineering","RNA and protein synthesis mechanisms","Insect symbiosis and bacterial influences","Medicine","Computer Science","Biology","0206 medical engineering","02 engineering and technology","CRISPR-Cas Systems","Clostridium","Computational Biology","Gene Library","Nucleotide Motifs"],"mesh_terms":["Clostridium","Gene Library","Computational Biology","Nucleotide Motifs","CRISPR-Cas Systems"],"keywords":["CRISPR","Computational biology","Biology","Trans-activating crRNA","Effector","CRISPR interference","Genome editing","Synthetic biology","Genetics","Gene","Cell biology","Clostridium","CRISPR-Cas Systems","Nucleotide Motifs","Synthetic Biology and Bioengineering","Gene Library"],"sdg_mappings":[{"sdg_number":0,"sdg_label":"Industry, innovation and infrastructure"}],"linked_datasets":[],"clinical_trials":[],"software_tools":[],"database_accessions":[{"name":"bioproject"}],"source":"live","citation_network_status":"fetched"},"created_at":"2026-07-20T05:16:01.858468Z","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":[]}