Developing An Efficient Precision Editing Methodology For Leishmania Mexicana To Investigate The Kinetochore Complex.

Abstract

Leishmania mexicana is a parasitic protozoan, and one of the causative agents of cutaneous leishmaniasis – a skin infection causing large lesions. Leishmania spp. have some unusual biological features, due to their early evolutionary split from other eukaryotes. One is their unique kinetochore complex – the protein complex responsible for binding the chromosomes to microtubules during mitosis. To evaluate the role of specific phosphorylation sites on essential kinetochore proteins, a selection-free precision editing strategy using the CRISPR-Cas9 system was investigated in promastigotes. Genomic DNA was targeted with 120 nt single-stranded oligonucleotide repair DNA to generate 10 unique amino acid substitutions to create phosphosite mutants from kinetochore proteins KKT1, KKT2, KKT4 and KKT7 but was only successful in 2.0% of clones. Comparatively, using 160 bp double-stranded repair DNA targeting 6 phosphosites between KKT2, KKT4 and KKT7 generated phosphodeficient, phosphomimetic and synonymous mutants at each target site tested. Across 18 unique transfections, PCR screening detected integration of the repair template in 24.6% of clones screened. Surprisingly, following Sanger sequencing, it was found that 29.2% of clones screened were in fact edited. Mutant clones were predominantly homozygous (21.7% of clones), including at least one clone per transfection. Kinetochore phosphosite mutant clones were assessed for growth changes and cell cycle dysregulation, but no apparent phenotypes were detected. Lastly, to pave the way for higher-throughput precision editing using this method, a Python script was developed to replicate the design process used to create the 160 bp repair templates. The script uses a FASTA file, codon usage table and a simple Excel spreadsheet configuration file to design the desired repair template with a single nonsynonymous mutation, and additional synonymous mutations for screening purposes. It also generates a corresponding synonymous-mutation only repair template, as well as screening primers and primers to produce the repair templates for a ready-to-go approach.

Metadata

Supervisors:	Mottram, Jeremy and Correia Faria, Joana
Keywords:	Leishmania; gene editing; CRISPR-Cas9; precision editing; kinetochore
Awarding institution:	University of York
Academic Units:	The University of York > Biology (York)
Depositing User:	Miss Charlotte Hughes
Date Deposited:	18 Jun 2025 07:45
Last Modified:	18 Jun 2025 07:45
Open Archives Initiative ID (OAI ID):	oai:etheses.whiterose.ac.uk:36980

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Supplementary Material

Filename: Hughes_202006829_repair_template_input_excel.xlsx

Description: Python script Repair Template Configuration File

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Supplementary Material

Filename: Hughes_202006829_main.py

Description: Python Script - Main Script File

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Supplementary Material

Filename: Hughes_202006829_codon_dataframes.py

Description: Python Script - Codon Dataframes File

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Filename: Hughes_202006829_codon_dictionaries.py

Description: Python Script - Codon Dictionaries File

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Filename: Hughes_202006829_formatting_functions.py

Description: Python Script - Formatting Functions File

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Filename: Hughes_202006829_primer_blast_functions.py

Description: Python Script - Primer Blast Functions File

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Filename: Hughes_202006829_primer_functions.py

Description: Python Script - Primer Functions File

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Filename: Hughes_202006829_reading_input_file.py

Description: Python Script - Reading Input File

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Filename: Hughes_202006829_stitching_functions.py

Description: Python Script - Stitching Functions File

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Filename: Hughes_202006829_validator.py

Description: Python Script - Validator Functions File

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Supplementary Material

Filename: Hughes_202006829_repair_template_input_excel_batch.xlsx

Description: Python script Batch Version Repair Template Configuration File

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Supplementary Material

Filename: Hughes_202006829_repair_template_input_excel_multi_mutant.xlsx

Description: Python script Multi-mutant Repair Template Configuration File

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Supplementary Material

Filename: Hughes_202006829_main_batch.py

Description: Python Script Batch Version - Main Script File

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Supplementary Material

Filename: Hughes_202006829_main_multi_mutant.py

Description: Python Script Multi-mutant Version - Main Script File

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Developing An Efficient Precision Editing Methodology For Leishmania Mexicana To Investigate The Kinetochore Complex.

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