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Utilising polymorphisms to achieve allele-specific genome editing in zebrafish
Samuel J Capon1, Gregory J Baillie1, Neil I Bower1
1Division of Genomics of Development and Disease, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia.
Biology Open
|November 30, 2016
Summary
Single nucleotide polymorphisms (SNPs) enhance CRISPR/Cas9 genome editing efficiency in zebrafish. By exploiting sequence variations, researchers can achieve allele-specific editing and improve control over gene editing outcomes in this model organism.
Area of Science:
- Genetics
- Molecular Biology
- Developmental Biology
Background:
- CRISPR/Cas9 is a powerful genome editing tool widely used in genetic research.
- Understanding the selectivity of CRISPR/Cas9, particularly concerning sequence variations, is crucial for optimizing its application.
- The zebrafish model offers a versatile platform for studying genome editing in vivo.
Purpose of the Study:
- To investigate the impact of single base pair mismatches on CRISPR/Cas9 genome editing efficiency in zebrafish.
- To explore the utility of exploiting natural genetic polymorphisms for allele-specific editing and phenotype control.
- To assess how single nucleotide polymorphisms (SNPs) influence CRISPR/Cas9-mediated gene targeting, deletion formation, and sequence integration.
Main Methods:
- Utilized two deep-sequenced zebrafish strains with identified polymorphisms in CRISPR/Cas9 target sites.
- Generated heterozygous zebrafish by crossing strains and injected CRISPR/Cas9 complexes designed to complement specific alleles.
- Analyzed targeted sites via sequencing to determine editing bias and assessed phenotypic outcomes, deletion frequencies, and loxP integration patterns.
Main Results:
- Demonstrated allele-specific CRISPR/Cas9 editing favoring the perfectly complementary sequence in the majority of tested cases (14/19).
- Observed reduced frequency of gene-knockout phenotypes in heterozygotes compared to homozygous targets when using polymorphisms.
- Showed biased chromosome selection for deletions and preferential integration of loxP sequences in perfectly complementary alleles.
Conclusions:
- Single nucleotide polymorphisms (SNPs) can be leveraged to enhance the efficiency and specificity of in cis genome editing with CRISPR/Cas9 in zebrafish.
- Exploiting SNPs provides a strategy for internal control of gene editing and associated phenotypes in zebrafish models.
- This approach offers improved precision for genome editing applications in zebrafish research.
