PT  - JOURNAL ARTICLE
AU  - Yu-Chih Tsai
AU  - David Greenberg
AU  - James Powell
AU  - Ida Höijer
AU  - Adam Ameur
AU  - Maya Strahl
AU  - Ethan Ellis
AU  - Inger Jonasson
AU  - Ricardo Mouro Pinto
AU  - Vanessa C. Wheeler
AU  - Melissa L. Smith
AU  - Ulf Gyllensten
AU  - Robert Sebra
AU  - Jonas Korlach
AU  - Tyson A. Clark
TI  - Amplification-free, CRISPR-Cas9 Targeted Enrichment and SMRT Sequencing of Repeat-Expansion Disease Causative Genomic Regions
AID  - 10.1101/203919
DP  - 2017 Jan 01
TA  - bioRxiv
PG  - 203919
4099  - http://biorxiv.org/content/early/2017/10/16/203919.short
4100  - http://biorxiv.org/content/early/2017/10/16/203919.full
AB  - Targeted sequencing has proven to be an economical means of obtaining sequence information for one or more defined regions of a larger genome. However, most target enrichment methods require amplification. Some genomic regions, such as those with extreme GC content and repetitive sequences, are recalcitrant to faithful amplification. Yet, many human genetic disorders are caused by repeat expansions, including difficult to sequence tandem repeats.We have developed a novel, amplification-free enrichment technique that employs the CRISPR-Cas9 system for specific targeting multiple genomic loci. This method, in conjunction with long reads generated through Single Molecule, Real-Time (SMRT) sequencing and unbiased coverage, enables enrichment and sequencing of complex genomic regions that cannot be investigated with other technologies. Using human genomic DNA samples, we demonstrate successful targeting of causative loci for Huntington’s disease (HTT; CAG repeat), Fragile X syndrome (FMR1; CGG repeat), amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (C9orf72; GGGGCC repeat), and spinocerebellar ataxia type 10 (SCA10) (ATXN10; variable ATTCT repeat). The method, amenable to multiplexing across multiple genomic loci, uses an amplification-free approach that facilitates the isolation of hundreds of individual on-target molecules in a single SMRT Cell and accurate sequencing through long repeat stretches, regardless of extreme GC percent or sequence complexity content. Our novel targeted sequencing method opens new doors to genomic analyses independent of PCR amplification that will facilitate the study of repeat expansion disorders.Guide RNA SequencesC9orf72a5’-GCAAUUCCACCAGUCGCUAG-3’C9orf72b5’-GCAUGAUCUCCUCGCCGGCA-3’FMR15’-AGAGGCCGAACUGGGAUAAC-3’HTT5’-AGCGGGCCCAAACUCACGG U-3’ATXN105’-AUACAAAGGAUCAGAAUCCC-3’