{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,5]],"date-time":"2026-10-05T21:42:51Z","timestamp":1791236571971,"version":"4.1.0"},"reference-count":58,"publisher":"American Society for Microbiology","issue":"4","license":[{"start":{"date-parts":[[2015,7,21]],"date-time":"2015-07-21T00:00:00Z","timestamp":1437436800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-sa\/3.0\/"},{"start":{"date-parts":[[2015,9,1]],"date-time":"2015-09-01T00:00:00Z","timestamp":1441065600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["mBio"],"published-print":{"date-parts":[[2015,9]]},"abstract":"ABSTRACT<\/jats:title>\n \n The prokaryotic CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9, an RNA-guided endonuclease, has been shown to mediate efficient genome editing in a wide variety of organisms. In the present study, the CRISPR-Cas9 system has been adapted to\n Leishmania donovani<\/jats:named-content>\n , a protozoan parasite that causes fatal human visceral leishmaniasis. We introduced the Cas9 nuclease into\n L. donovani<\/jats:named-content>\n and generated guide RNA (gRNA) expression vectors by using the\n L. donovani<\/jats:named-content>\n rRNA promoter and the hepatitis delta virus (HDV) ribozyme. It is demonstrated within that\n L. donovani<\/jats:named-content>\n mainly used homology-directed repair (HDR) and microhomology-mediated end joining (MMEJ) to repair the Cas9 nuclease-created double-strand DNA break (DSB). The nonhomologous end-joining (NHEJ) pathway appears to be absent in\n L. donovani<\/jats:named-content>\n . With this CRISPR-Cas9 system, it was possible to generate knockouts without selection by insertion of an oligonucleotide donor with stop codons and 25-nucleotide homology arms into the Cas9 cleavage site. Likewise, we disrupted and precisely tagged endogenous genes by inserting a bleomycin drug selection marker and\n GFP<\/jats:italic>\n gene into the Cas9 cleavage site. With the use of Hammerhead and HDV ribozymes, a double-gRNA expression vector that further improved gene-targeting efficiency was developed, and it was used to make precise deletion of the 3-kb miltefosine transporter gene (\n LdMT<\/jats:italic>\n ). In addition, this study identified a novel single point mutation caused by CRISPR-Cas9 in\n LdMT<\/jats:italic>\n (M381T) that led to miltefosine resistance, a concern for the only available oral antileishmanial drug. Together, these results demonstrate that the CRISPR-Cas9 system represents an effective genome engineering tool for\n L.\u00a0donovani.<\/jats:italic>\n <\/jats:p>\n \n IMPORTANCE<\/jats:bold>\n Leishmania donovani<\/jats:named-content>\n is the causative agent of fatal visceral leishmaniasis. To understand\n Leishmania<\/jats:italic>\n infection and pathogenesis and identify new drug targets for control of leishmaniasis, more-efficient ways to manipulate this parasite genome are required. In this study, we have implemented CRISPR-Cas9 genome-editing technology in\n L. donovani<\/jats:named-content>\n . Both single- and dual-gRNA expression vectors were developed using a strong RNA polymerase I promoter and ribozymes. With this system, it was possible to generate loss-of-function insertion and deletion mutations and introduce drug selection markers and the\n GFP<\/jats:italic>\n sequence precisely into the\n L. donovani<\/jats:named-content>\n genome. These methods greatly improved the ability to manipulate this parasite genome and will help pave the way for high-throughput functional analysis of\n Leishmania<\/jats:italic>\n genes. This study further revealed that double-stranded DNA breaks created by CRISPR-Cas9 were repaired by the homology-directed repair (HDR) pathway and microhomology-mediated end joining (MMEJ) in\n Leishmania<\/jats:italic>\n .\n <\/jats:p>","DOI":"10.1128\/mbio.00861-15","type":"journal-article","created":{"date-parts":[[2015,7,21]],"date-time":"2015-07-21T21:01:19Z","timestamp":1437512479000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":190,"title":["CRISPR-Cas9-Mediated Genome Editing in Leishmania donovani"],"prefix":"10.1128","volume":"6","author":[{"given":"Wen-Wei","family":"Zhang","sequence":"first","affiliation":[{"name":"Department of Microbiology and Immunology, McGill University, Montreal, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Greg","family":"Matlashewski","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, McGill University, 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