Utilising polymorphisms to achieve allele-specific genome editiing in zebrafish

Capon, Samuel James, Baillie, Gregory, Bower, Neil, Da Silva, Jason Andrew, Paterson, Scott, Hogan, Ben, Simons, Cas and Smith, Kelly (2017) Utilising polymorphisms to achieve allele-specific genome editiing in zebrafish. Biology Open, 6 1: 125-131. doi:10.1242/bio.020974


Author Capon, Samuel James
Baillie, Gregory
Bower, Neil
Da Silva, Jason Andrew
Paterson, Scott
Hogan, Ben
Simons, Cas
Smith, Kelly
Title Utilising polymorphisms to achieve allele-specific genome editiing in zebrafish
Journal name Biology Open   Check publisher's open access policy
ISSN 2046-6390
Publication date 2017-01-01
Year available 2017
Sub-type Article (original research)
DOI 10.1242/bio.020974
Open Access Status DOI
Volume 6
Issue 1
Start page 125
End page 131
Total pages 7
Place of publication Cambridge, United Kingdom
Publisher The Company of Biologists
Language eng
Subject 1300 Biochemistry, Genetics and Molecular Biology
1100 Agricultural and Biological Sciences
Abstract The advent of genome editing has significantly altered genetic research, including research using the zebrafish model. To better understand the selectivity of the commonly used CRISPR/Cas9 system, we investigated single base pair mismatches in target sites and examined how they affect genome editing in the zebrafish model. Using two different zebrafish strains that have been deep sequenced, CRISPR/Cas9 target sites containing polymorphisms between the two strains were identified. These strains were crossed (creating heterozygotes at polymorphic sites) and CRISPR/Cas9 complexes that perfectly complement one strain injected. Sequencing of targeted sites showed biased, allele-specific editing for the perfectly complementary sequence in the majority of cases (14/19). To test utility, we examined whether phenotypes generated by F0 injection could be internally controlled with such polymorphisms. Targeting of genes bmp7a and chordin showed reduction in the frequency of phenotypes in injected 'heterozygotes' compared with injecting the strain with perfect complementarity. Next, injecting CRISPR/Cas9 complexes targeting two separate sites created deletions, but deletions were biased to selected chromosomes when one CRISPR/Cas9 target contained a polymorphism. Finally, integration of loxP sequences occurred preferentially in alleles with perfect complementarity. These experiments demonstrate that single nucleotide polymorphisms (SNPs) present throughout the genome can be utilised to increase the efficiency of in cis genome editing using CRISPR/Cas9 in the zebrafish model.
Formatted abstract
The advent of genome editing has significantly altered genetic research, including research using the zebrafish model. To better understand the selectivity of the commonly used CRISPR/Cas9 system, we investigated single base pair mismatches in target sites and examined how they affect genome editing in the zebrafish model. Using two different zebrafish strains that have been deep sequenced, CRISPR/Cas9 target sites containing polymorphisms between the two strains were identified. These strains were crossed (creating heterozygotes at polymorphic sites) and CRISPR/Cas9 complexes that perfectly complement one strain injected. Sequencing of targeted sites showed biased, allele-specific editing for the perfectly complementary sequence in the majority of cases (14/19). To test utility, we examined whether phenotypes generated by F0 injection could be internally controlled with such polymorphisms. Targeting of genes bmp7a and chordin showed reduction in the frequency of phenotypes in injected “heterozygotes” compared with injecting the strain with perfect complementarity. Next, injecting CRISPR/Cas9 complexes targeting two separate sites created deletions, but deletions were biased to selected chromosomes when one CRISPR/Cas9 target contains a polymorphism. Finally, integration of loxP sequences occurred preferential in alleles with perfect complementarity. These experiments demonstrate that SNPs present throughout the genome can be utilised to increase the efficiency of in cis genome editing using CRISPR/Cas9 in the zebrafish model.
Keyword Biology
Life Sciences & Biomedicine - Other Topics
Q-Index Code C1
Q-Index Status Provisional Code
Grant ID FT100100165
1083811
1046028
Institutional Status UQ

 
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Created: Fri, 06 Jan 2017, 21:03:31 EST by Susan Allen on behalf of Institute for Molecular Bioscience