This document discusses using the CRISPR-Cas9 nickase system to contract unstable trinucleotide repeats that cause neurological diseases like Huntington's disease. It introduces trinucleotide repeat disorders and describes how the CRISPR-Cas9 system can introduce single-stranded breaks instead of double-stranded breaks to contract repeats. The document outlines experiments transfecting cells with inducible GFP genes containing variable CAG repeat lengths to determine the minimum repeat size contractible by Cas9 nickase and whether double-stranded breaks from two gRNAs improve contraction efficiency.

1. Contracting CAG/CTG repeats
using the CRISPR-Cas9 nickase
system
Johanna Martin
Professor Vincent Dion, Supervisor: Cinzia Cinesi
SUR Programme, Centre for Integrative Genomics, UNIL

2. Trinucleotide repeat disorders
 At least 13 neurological diseases
known to be caused by unstable
CAG repeats
 Huntington disease
 CAG repeat at the N-terminus of
the huntingtin protein
 Normal is 3-36 repeats
 Abnormal is 36-121

3. Trinucleotide repeat disorders
 At least 13 neurological diseases
known to be caused by unstable
CAG repeats
 Huntington disease
 CAG repeat at the N-terminus of
the huntingtin protein
 Normal is 3-36 repeats
 Abnormal is 36-121

4. "The biological significance of these
sequences is not known." Ishino et al., 1987
 Clustered regular interspaced short
palindromic repeats (CRISPR)
 RNA-guided DNA cleaving enzyme
(Cas9)
 Introduces double stranded breaks
(DSBs)
 Using Cas9 D10A mutant to introduce
SSBs
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.

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

6. The effect of CAG repeat length and ability
to cause contraction
 Transfection of HEK293 Flp-In Rex
cells
 Inducible promoter before the GFP
mini gene, in which CAG repeats
inserted within intron
 GFP-based fluorescence assay
 PCR amplification and sequence
analysis CINESI, C., et al., 2016. Submitted. Contracting
CAG/CTG repeats using the CRISPR-Cas9 nickase
system

7. 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 two
different gRNAs along with Cas9 nickase?