Abstract
Purpose :
Mutations of TGFBI cause a family of inherited corneal dystrophies. To date 60 different missense mutations have been described. Since haplosufficiency of TGFBI has previously been demonstrated, an allele-specific targeting strategy for the TGFBI cornel dystrophies holds potential as a gene therapy. CRISPR/Cas9 induction of the non-homologous end joining (NHEJ) DNA repair pathway may be used to achieve mutant gene disruption. In addition to a lack of sufficient specificity for the mutant allele, less than half of the TGFBI mutations can be directly targeted by CRISPR/Cas9. Here we present a novel approach to target all TGFBI mutations, coined ASNIP CRISPR: allele-specific, SNP-derived, novel PAM, in cis, personalised CRISPR.
Methods :
Analysis of the TGFBI gene and 50kb upstream and downstream flanking regions was perfomed to identify non-disease causing SNPs with a minor allele frequency (MAF) of >10% in which only one allele represents a Spy Cas9 PAM site. Phased sequencing was performed on a patient with granular corneal dystrophy type 2 in whom an R124H TGFBI mutation had been identified, to determine PAM-generating SNPs in cis with her mutation; sgRNAs were designed to target Spy Cas9 to these sites. Peripheral blood mononuclear cells were isolated from the patient’s whole blood sample and transformed with Epstein Barr Virus to generate a proliferating lymphocyte cell line (LCL). Ribonucleoprotein (RNP) complexes of sgRNA and Cas9 were introduced into the LCLs by nucleofection. Allele-specific gene disruption by NHEJ was determined by TIDE (tracking of indels by decomposition) analysis and deep sequencing of PCR amplicons.
Results :
The degree of specificity and efficiency of mutant allele targeting achieved varied depending on the guide sequence utilised and PAM site targeted. A streamlined guide design process was developed that allowed improved mutant allele-specificity to be achieved by implementing the ASNIP CRISPR approach. Genome wide off-target screening revealed minimal off target events when meticulous guide design was employed.
Conclusions :
ASNIP CRISPR offers a novel, highly specific, personalised gene editing strategy to treat autosomal dominant disease, specifically corneal dystrophies. An optimised guide design workflow allows confident selection of commonly occurring SNPs associated with highly specific guides.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.