{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T01:10:19Z","timestamp":1790298619497,"version":"4.1.0"},"reference-count":44,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,4,14]],"date-time":"2026-04-14T00:00:00Z","timestamp":1776124800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100010269","name":"Wellcome Trust","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100010269","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Cell. Infect. Microbiol."],"abstract":"<jats:p>\n                    <jats:italic>Leishmania mexicana<\/jats:italic>\n                    , like other trypanosomatids, possess a unique kinetochore\u2014the protein complex crucial for chromosome segregation during mitosis. To\u00a0investigate the functional significance of specific phosphorylation sites on essential kinetochore proteins, we adapted a selection-free precision editing strategy using CRISPR-Cas9 in\n                    <jats:italic>Leishmania mexicana<\/jats:italic>\n                    promastigotes. Our method\u00a0targeted genomic DNA with 160-bp double-stranded DNA repair templates and guide RNAs to introduce targeted modifications. We focused on\u00a0six phosphosites within the kinetochore proteins KKT2, KKT4, and KKT7, generating phosphodeficient, phosphomimetic, and synonymous mutants for each site. Across 18 independent transfections, we achieved a successful editing rate of 27.5% as determined by PCR screening, with 30.4% of clones confirmed as edited by Sanger sequencing. A significant portion of these edited clones (22.1%) were homozygous. Despite these precise genomic modifications, none of the phosphosite mutant clones exhibited any apparent growth defects or cell cycle dysregulation, suggesting these phosphorylation sites individually may not be critical for these processes under standard culture conditions. To facilitate higher-throughput precision editing, we developed a Python script that automates the design of the 160 bp repair templates. This script uses a FASTA file, a codon usage table, and a simple configuration file to design templates with a single nonsynonymous mutation and additional synonymous mutations for screening purposes. It also generates a corresponding synonymous-only repair template and primers for both screening and repair template generation, offering a \u201cready-to-go\u201d approach. While designed for\n                    <jats:italic>Leishmania<\/jats:italic>\n                    , this powerful tool is adaptable for use with other kinetoplastids.\n                  <\/jats:p>","DOI":"10.3389\/fcimb.2026.1788564","type":"journal-article","created":{"date-parts":[[2026,4,14]],"date-time":"2026-04-14T05:36:21Z","timestamp":1776144981000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":3,"title":["CRISPR-Cas9 precision editing of kinetochore protein phosphosite codons in Leishmania mexicana"],"prefix":"10.3389","volume":"16","author":[{"given":"Charlotte","family":"McNiven","sequence":"first","affiliation":[{"name":"York Biomedical Research Institute and Department of Biology, University of York","place":["York, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juliana Brambilla","family":"Carnielli Trindade","sequence":"additional","affiliation":[{"name":"York Biomedical Research Institute and Department of Biology, University of York","place":["York, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vincent","family":"Geoghegan","sequence":"additional","affiliation":[{"name":"York Biomedical Research Institute and Department of Biology, University of York","place":["York, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joana R. C.","family":"Faria","sequence":"additional","affiliation":[{"name":"York Biomedical Research Institute and Department of Biology, University of York","place":["York, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jeremy C.","family":"Mottram","sequence":"additional","affiliation":[{"name":"York Biomedical Research Institute and Department of Biology, University of York","place":["York, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,4,14]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"1247","DOI":"10.1016\/J.CELL.2014.01.049","article-title":"Discovery of unconventional kinetochores in kinetoplastids","volume":"156","author":"Akiyoshi","year":"2014","journal-title":"Cell"},{"key":"B2","doi-asserted-by":"publisher","first-page":"e79","DOI":"10.1093\/NAR\/GKAC319","article-title":"Oligo targeting for profiling drug resistance mutations in the parasitic trypanosomatids","volume":"50","author":"Altmann","year":"2022","journal-title":"Nucleic Acids Res."},{"key":"B3","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1111\/MMI.15256","article-title":"Streptococcus pyogenes Cas9 ribonucleoprotein delivery for efficient, rapid and marker-free gene editing in Trypanosoma and Leishmania","author":"Asencio","year":"2024","journal-title":"Mol. 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