Contracting CAG/CTG repeats using the CRISPR-Cas9 nickase

systemJohanna Martin

Professor Vincent Dion, Supervisor: Cinzia Cinesi

SUR Programme, Centre for Integrative Genomics, UNIL

Trinucleotide repeat disorders

At least 13 neurological diseasesknown to be caused by unstableCAG repeats

Huntington disease

CAG repeat at the N-terminus ofthe huntingtin protein

Normal is 3-36 repeats

Abnormal is 36-121

Trinucleotide repeat disorders

At least 13 neurological diseasesknown to be caused by unstableCAG repeats

Huntington disease

CAG repeat at the N-terminus ofthe huntingtin protein

Normal is 3-36 repeats

Abnormal is 36-121

"The biological significance of these sequences is not known." Ishino et al., 1987

Clustered regular interspaced shortpalindromic repeats (CRISPR)

RNA-guided DNA cleaving enzyme(Cas9)

Introduces double stranded breaks(DSBs)

Using Cas9 D10A mutant to introduceSSBs

DOUDNA, J.A. and CHARPENTIER, E., 2014. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science (New York, N.Y.), 346(6213), pp. 1258096.

The DNA gap model

Cinesi C., et al., 2016. Submitted. Contracting CAG/CTG repeats using the CRISPR-Cas9 nickase system

The effect of CAG repeat length and ability to cause contraction

Transfection of HEK293 Flp-In Rexcells

Inducible promoter before the GFPmini gene, in which CAG repeatsinserted within intron

GFP-based fluorescence assay

PCR amplification and sequenceanalysis CINESI, C., et al., 2016. Submitted. Contracting

CAG/CTG repeats using the CRISPR-Cas9 nickasesystem

Aims

What is the minimum CAG repeat size that is a substrate of Cas9 nickase?

What is the effect of double strand breaks within the repeat track using twodifferent gRNAs along with Cas9 nickase?