Utilising polymorphisms to achieve allele-specific genome editing in zebrafish.

Samuel J Capon,Jason A Da Silva,Kelly A Smith,Cas Simons,Neil I Bower,Benjamin M Hogan,Gregory J Baillie,Scott Paterson

doi:10.1242/bio.020974

Abstract

ABSTRACTThe 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.

Highlights

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 (Cas9) system has recently emerged as the method of choice for genome editing in a wide variety of systems and organisms
A number of studies have demonstrated targeting of variant or disease alleles by CRISPR/Cas9 genome editing, followed by variant repair achieved through homologydirected repair (HDR) (Schwank et al, 2013; Smith et al, 2015; Wu et al, 2013; Yoshimi et al, 2014)
We find that single nucleotide polymorphisms (SNPs) within the target site insulate genome editing, biasing editing in favour of perfect complementarity between guide RNA molecules (gRNAs) and target site in the majority of examples

Summary

Introduction

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 (Cas9) system has recently emerged as the method of choice for genome editing in a wide variety of systems and organisms. To take advantage of the site specificity of CRISPR/Cas9 targeting to build more capabilities in zebrafish genome editing, we have used two whole genome sequenced zebrafish strains, for which we have established strain-specific reference genomes, and tested the utility of polymorphism directed targeting. We conducted a genome-wide survey to identify putative CRISPR/Cas9 target sites that contain strain-specific single nucleotide polymorphisms (SNPs).

Results

Conclusion