Optimized CRISPR-Cas9 Genome Editing for Leishmania and Its Use To Target a Multigene Family, Induce Chromosomal Translocation, and Study DNA Break Repair Mechanisms

• DOI: 10.1128/msphere.00340-16
• Publication Date: 2017-01-19T02:10:37Z
• Authors: Wen-Wei Zhang; Patrick Lypaczewski; Greg Matlashewski
• Keywords: chromosomal translocation; Leishmania; 0301 basic medicine; co-CRISPR targeting; homology-directed repair; 03 medical and health sciences; MMEJ; CRISPR-Cas9; Microbiology; QR1-502; Research Article; 3. Good health

Leishmania parasites cause human leishmaniasis. To accelerate characterization of Leishmania genes for new drug and vaccine development, we optimized and simplified the CRISPR-Cas9 genome-editing tool for Leishmania . 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 various Leishmania species and isolates. We simplified this CRISPR system by developing a gRNA and Cas9 coexpression vector which could be used to delete genes in various Leishmania species. This CRISPR system could also be used to generate specific chromosomal translocations, which will help in the study of Leishmania gene expression and transcription control. This study also provides new information about double-strand DNA break repair mechanisms in Leishmania .