1Bowes, J., Budu-Aggrey, A., Huffmeier, U., Uebe, S., Steel, K., Hebert, H. L., ⦠Barton, A. (2015). Dense genotyping of immune-related susceptibility loci reveals.
Genome engineering with the CRISPR Cas9 system in Psoriatic Arthritis and other autoimmune diseases 1 P.Roidos ,
A.
2 Adamson ,
J.
1 Bowes ,
1 Eyre
S.
1Arthritis
Research UK Centre for Genetics and Genomics, Manchester, Academic Health Science Centre, University of Manchester, Manchester, UK, 2Transgenic Unit, Faculty of Life Sciences, University of Manchester, Manchester, UK
Introduction
CRISPR Cas9 genome editing
A major challenge of disease genetic research is to unravel the connection between genotype and
G e n o m e e d i t i n g is the ability to insert, remove or even
Clone selected gRNA sequences into px458
phenotype. GWAS have been successful in narrowing the region of association and pinpointing the
edit DNA fragments in an easy and precise way in living
gRNA T2A
variants that are responsible for the increased risk in complex diseases, including psoriatic arthritis (PsA).
cells. CRISPR all it does is induce a simple double-strand
The majority of associated variants are in non-coding DNA regions, where the lead associated variant, due
break (DSB) at the genome.
to inheritance patters, exhibits linkage disequilibrium with a number of equally implicated SNPs.
U6
SpCas9
CAGG
eGFP
Design gRNAs targeted to stop codon of gene of interest
Application of Genome editing for target validation: 1. Generate knock in reporter cell line for gene of
The aim of this research is to
interest by CRISPR mediated HDR using verified
• describe how CRISPR Cas9 system can be utilized to identify which genes
gene of interest
gRNAs.
• and which SNPs are responsible for the increased risk in regions associated with PsA.
STOP
2. Validate SNP function with respect to gene activation
3’ Exon
using dCas9 variants targeted to SNPs, or generate
From GWAS to functional target validation
SNPs by ssDNA HDR.
Potential gRNAs
Tr a n s f e c t a n d S c r e e n g R N A a c t i v i t y i n v i t r o
3. Potential to multiplex gRNAs.
5q31
3’ UTR
No guide
gRNA1
gRNA2
gRNA3
4. Quantitative measurement.
1
• The 5q31 region harbours genes encoding IL-4, IL-5 and IL-13. • The non-coding SNP (rs715285) is associated with PsA.
Fluorescent repor ter system with HDR repair cassette
1. Optimize transfection for cell type of choice 2. Optimize delivery components
Sort GFP+ cells
1
CRISPR Cas9 system will be applied to • change the index SNP and measure the effect on local gene expression (eQTL).
Sequence insertions can be used to fuse functional domains, such as fluorescent proteins, to endogenous gene products allowing a fluorescence reporter system to be established and used to quantitatively characterize gene expression.
Recover 2-3 days, extract genomic DNA, PCR and perform T7E1 assay to select most efficient gRNA
gene of interest DNA
IL-23R
HDR cassette encompassing DsRedxp gene
2
The IL-23 - TH 17 pathway is crucial in many inflammatory diseases. • The associated SNP (rs12044149) for PsA is in the promoter region of the IL23R gene. • The polymorphism of IL23R implicates a large number of equally ‘credible SNPs, potentially encompassing a number of enhancers. CRISPR Cas9 system will be applied to • identify the causal variant and mechanism by blocking groups of SNPs in enhancers
Combine gRNA/Cas9 and dsDNA HDR cassette and recover population 2-3 days
Stop Codon
Linker
DsRedxp 3’ homology arm
5’ homology arm PAM shield mutation
deadCas9 Sort clonal line positive for targeted knock in of fluorophore with tagged gene of interest
The dCas9 binds DNA but does not cut. By fusing the dCas9 to a further molecule (such as VPR or KRAB) the complex can take on the activity of that molecule. VPR is a transcriptional enhancer, and KRAB a transcriptional repressor.
Enhance
Repress
Target Validation
DsRedxp SNP
gene of interest
SNP
gene of interest
Generate the specific SNP v i a c u t a n d r e p a i r m e ch a n i s m
DsRedxp
5’ H
GWAS
CRISPR
Target Validation
3’ H
Measure reporter expression
Desired point mutation
Conclusions Construct fluorescence reporter system
Use catalytically inactive dCas9 for disease risk enhancer modification
Regulation of gene expression with genotype (eQTL)
There is the requirement to understand the disease mechanism by distinguishing which genes or SNPs are
Physical chromatin interaction (3C)
implicated cell lines, like T-lymphocytes, we demonstrate that genomic DNA can be precisely altered,
Primary T-lymphocytes Incorporate single basepair change
involved in the complex disease of interest. By creating these novel model systems in autoimmune
candidate genes can be fluorescently tagged and enhancers can be up or down regulated in order to interrogate a number of regions that emerge from the GWAS efforts. This research aims to shed light to pathways that are affected by genes of interest, and potentially to the discovery of new therapeutic targets.
References Clonal cell line for multiple assays targeting gene of interest associated SNPs
1Bowes,
J., Budu-Aggrey, A., Huffmeier, U., Uebe, S., Steel, K., Hebert, H. L., … Barton, A. (2015). Dense genotyping of immune-related susceptibility loci reveals
new insights into the genetics of psoriatic arthritis. Nature Communications, 6, 6046. http://doi.org/10.1038/ncomms7046 2Budu-Aggrey A,
Bowes J, Lohr S. et al. Replication of a distinct psoriatic arthritis risk variant at the IL23R locus. Ann Rheum Dis 2016 (in submission)
