The system successfully enabled genome editing in Leishmania donovani, allowing for precise genetic modifications.
L. donovani primarily utilized (HDR) and (MMEJ) for repairing double-strand DNA breaks created by Cas9.
The nonhomologous end-joining (NHEJ) pathway appears to be absent in L. donovani.
Knockouts were achieved without selection by inserting oligonucleotide donors with stop codons into the Cas9 cleavage site.
Endogenous genes were disrupted and tagged with a bleomycin drug selection marker and GFP gene via precise insertions.
A novel point mutation (M381T) in the miltefosine transporter gene (LdMT) was identified, which is associated with resistance to the drug miltefosine.
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UNLABELLED: 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 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 (HDR) and (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.
Key numbers
2 to 6×
Increase in MLF resistance rate
Resistance rates increased after transfection with oligonucleotide donor.
12%
MLF resistance rate for gRNAa
Rate of MLF-resistant cells increased over time.
25 to 50%
MLF resistance rate for gRNA constructs
Resistance rates observed in cells transfected with dual-gRNA constructs.
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