title: Optimized CRISPR-Cas9 Genome Editing forLeishmaniaand Its Use To Target a Multigene Family, Induce Chromosomal Translocation, and Study DNA Break Repair Mechanisms.
abstract: CRISPR-Cas9-mediated genome editing has recently been adapted forLeishmaniaspp. parasites, the causative agents of human leishmaniasis. We have optimized this genome-editing tool by selecting for cells with CRISPR-Cas9 activity through cotargeting the miltefosine transporter gene; mutation of this gene leads to miltefosine resistance. This cotargeting strategy integrated into a triple guide RNA (gRNA) expression vector was used to delete all 11 copies of the A2 multigene family; this was not previously possible with the traditional gene-targeting method. We found that theLeishmania donovanirRNA promoter is more efficient than the U6 promoter in driving gRNA expression, and sequential transfections of the oligonucleotide donor significantly eased the isolation of edited mutants. A gRNA and Cas9 coexpression vector was developed that was functional in all testedLeishmaniaspecies, includingL. donovani,L. major, andL. mexicana. By simultaneously targeting sites from two different chromosomes, all four types of targeted chromosomal translocations were generated, regardless of the polycistronic transcription direction from the parent chromosomes. It was possible to use this CRISPR system to create a single conserved amino acid substitution (A189G) mutation for both alleles ofRAD51, a DNA recombinase involved in homology-directed repair. We found that RAD51 is essential forL. donovanisurvival based on direct observation of the death of mutants with bothRAD51alleles disrupted, further confirming that this CRISPR system can reveal gene essentiality. Evidence is also provided that microhomology-mediated end joining (MMEJ) plays a major role in double-strand DNA break repair inL. donovani.IMPORTANCELeishmaniaparasites cause human leishmaniasis. To accelerate characterization ofLeishmaniagenes for new drug and vaccine development, we optimized and simplified the CRISPR-Cas9 genome-editing tool forLeishmania. We show that co-CRISPR targeting of the miltefosine transporter gene and serial transfections of an oligonucleotide donor significantly eased isolation of edited mutants. This cotargeting strategy was efficiently used to delete all 11 members of the A2 virulence gene family. This technical advancement is valuable, since there are many gene clusters and supernumerary chromosomes in the variousLeishmaniaspecies and isolates. We simplified this CRISPR system by developing a gRNA and Cas9 coexpression vector which could be used to delete genes in variousLeishmaniaspecies. This CRISPR system could also be used to generate specific chromosomal translocations, which will help in the study ofLeishmaniagene expression and transcription control. This study also provides new information about double-strand DNA break repair mechanisms inLeishmania.
authors.affiliation: Department of Microbiology and Immunology, McGill University, Montreal, Canada.
authors.forename: Wen-Wei
authors.initials: WW
authors.lastname: Zhang
authors.affiliation: Department of Microbiology and Immunology, McGill University, Montreal, Canada.
authors.forename: Patrick
authors.initials: P
authors.lastname: Lypaczewski
authors.affiliation: Department of Microbiology and Immunology, McGill University, Montreal, Canada.
authors.forename: Greg
authors.initials: G
authors.lastname: Matlashewski
keywords: CRISPR-Cas9; Leishmania; MMEJ; Rad51; chromosomal translocation; co-CRISPR targeting; double-strand break repair; genome editing; homology-directed repair; microhomology-mediated end joining; miltefosine transporter; multiple gene family