CRISPR-Cas9-Mediated Genome Editing in <named-content content-type="genus-species">Leishmania donovani</named-content>

ABSTRACT 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 Leishmania donovani, a prot...

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Autores principales:	Wen-Wei Zhang, Greg Matlashewski
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2015
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/9b09ad8b222043ceaba963c2e604ced7
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spelling	oai:doaj.org-article:9b09ad8b222043ceaba963c2e604ced72021-11-15T15:41:27ZCRISPR-Cas9-Mediated Genome Editing in <named-content content-type="genus-species">Leishmania donovani</named-content>10.1128/mBio.00861-152150-7511https://doaj.org/article/9b09ad8b222043ceaba963c2e604ced72015-09-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00861-15https://doaj.org/toc/2150-7511ABSTRACT 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 Leishmania donovani, a protozoan parasite that causes fatal human visceral leishmaniasis. We introduced the Cas9 nuclease into L. donovani and generated guide RNA (gRNA) expression vectors by using the L. donovani rRNA promoter and the hepatitis delta virus (HDV) ribozyme. It is demonstrated within that L. donovani 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 L. donovani. 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 GFP 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 (LdMT). In addition, this study identified a novel single point mutation caused by CRISPR-Cas9 in LdMT (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 L. donovani. IMPORTANCE Leishmania donovani is the causative agent of fatal visceral leishmaniasis. To understand Leishmania 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 L. donovani. 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 GFP sequence precisely into the L. donovani genome. These methods greatly improved the ability to manipulate this parasite genome and will help pave the way for high-throughput functional analysis of Leishmania 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 Leishmania.Wen-Wei ZhangGreg MatlashewskiAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 6, Iss 4 (2015)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Wen-Wei Zhang Greg Matlashewski CRISPR-Cas9-Mediated Genome Editing in <named-content content-type="genus-species">Leishmania donovani</named-content>
description	ABSTRACT 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 Leishmania donovani, a protozoan parasite that causes fatal human visceral leishmaniasis. We introduced the Cas9 nuclease into L. donovani and generated guide RNA (gRNA) expression vectors by using the L. donovani rRNA promoter and the hepatitis delta virus (HDV) ribozyme. It is demonstrated within that L. donovani 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 L. donovani. 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 GFP 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 (LdMT). In addition, this study identified a novel single point mutation caused by CRISPR-Cas9 in LdMT (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 L. donovani. IMPORTANCE Leishmania donovani is the causative agent of fatal visceral leishmaniasis. To understand Leishmania 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 L. donovani. 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 GFP sequence precisely into the L. donovani genome. These methods greatly improved the ability to manipulate this parasite genome and will help pave the way for high-throughput functional analysis of Leishmania 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 Leishmania.
format	article
author	Wen-Wei Zhang Greg Matlashewski
author_facet	Wen-Wei Zhang Greg Matlashewski
author_sort	Wen-Wei Zhang
title	CRISPR-Cas9-Mediated Genome Editing in <named-content content-type="genus-species">Leishmania donovani</named-content>
title_short	CRISPR-Cas9-Mediated Genome Editing in <named-content content-type="genus-species">Leishmania donovani</named-content>
title_full	CRISPR-Cas9-Mediated Genome Editing in <named-content content-type="genus-species">Leishmania donovani</named-content>
title_fullStr	CRISPR-Cas9-Mediated Genome Editing in <named-content content-type="genus-species">Leishmania donovani</named-content>
title_full_unstemmed	CRISPR-Cas9-Mediated Genome Editing in <named-content content-type="genus-species">Leishmania donovani</named-content>
title_sort	crispr-cas9-mediated genome editing in <named-content content-type="genus-species">leishmania donovani</named-content>
publisher	American Society for Microbiology
publishDate	2015
url	https://doaj.org/article/9b09ad8b222043ceaba963c2e604ced7
work_keys_str_mv	AT wenweizhang crisprcas9mediatedgenomeeditinginnamedcontentcontenttypegenusspeciesleishmaniadonovaninamedcontent AT gregmatlashewski crisprcas9mediatedgenomeeditinginnamedcontentcontenttypegenusspeciesleishmaniadonovaninamedcontent
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CRISPR-Cas9-Mediated Genome Editing in <named-content content-type="genus-species">Leishmania donovani</named-content>

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