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Bar-seq strategies for the LeishGEdit toolbox.
1Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK.
Molecular and Biochemical Parasitology
|July 14, 2020
Summary
Researchers can now easily perform high-throughput gene function screens in kinetoplastids using CRISPR bar-seq. This new tool, LeishGEdit, enables pooled mutant screens for accelerated genetic analysis in organisms like Leishmania.
Area of Science:
- Genetics and Genomics
- Molecular Biology
- Parasitology
Background:
- The steady increase in sequenced genomes highlights a significant gap in understanding gene function, particularly in kinetoplastids.
- Previous development of the LeishGEdit pipeline facilitated CRISPR-Cas9 gene editing in Leishmania spp. and related organisms.
- The need for high-throughput methods is critical for efficient gene function dissection in these species.
Purpose of the Study:
- To adapt the LeishGEdit pipeline for high-throughput mutant screening using barcoding strategies.
- To develop user-friendly tools for designing CRISPR primers and analyzing barcoded mutants for pooled screens.
- To provide researchers with accessible resources for implementing bar-seq experiments in kinetoplastids.
Main Methods:
- Adaptation of the LeishGEdit CRISPR-Cas9 gene editing pipeline by incorporating unique 17-nucleotide barcodes for mutant identification.
- Development of a standalone software pipeline for designing CRISPR primers compatible with the LeishGEdit toolbox for any target genome.
- Generation and curation of a library of 14,995 unique barcodes and associated oligonucleotide sequences.
Main Results:
- Successful adaptation of the LeishGEdit pipeline to enable barcoded CRISPR screens, allowing loss-of-function analysis in mixed mutant populations.
- A user-friendly primer design and analysis tool is now available, simplifying the implementation of bar-seq strategies.
- A comprehensive library of 14,995 barcodes and oligo sequences has been generated and made accessible online via www.leishgedit.net.
Conclusions:
- The developed tools and barcode library significantly streamline CRISPR bar-seq assays in kinetoplastids.
- This advancement enables pooled mutant screens across the scientific community, accelerating the study of gene function in organisms like Leishmania.
- The accessibility of resources via www.leishgedit.net supports broad adoption and application in TriTrypDB genomes.
