Targeted, amplification-free DNA sequencing using CRISPR/Cas9

Traditional amplification-based targeted sequencing approaches can be limited by a number of factors, including base composition (e.g. GC-rich content) and bias (e.g. allele bias). In addition, while long-range PCR approaches can, with careful optimisation, generate fragments in the region of 20 kb, such fragment sizes may not cover all genes or regions of interest and do not fully exploit the ultralong read length capabilities provided by nanopore sequencing. Furthermore, the process of amplification removes all information on base modifications, thereby losing a potentially informative source of variation. In order to address these challenges, researchers are now investigating the potential of CRISPR/ Cas9 techniques to enrich for specific regions of interest.

At Johns Hopkins University, USA, Timothy Gilpatrick is researching the methylation profiles of known cancer driver genes¹. In order to cost-effectively target specific regions of the genome whilst maintaining epigenetic marks, Timothy employed a CRISPR/Cas9 enrichment methodology together with nanopore sequencing. Following a successful pilot study in E. coli, where 20,000-fold coverage of a 5 kb targeted fragment was observed, Timothy moved on to human DNA where he targeted the hTERT gene, which encodes a core protein component of the telomerase complex. Telomerase, which acts to maintain the telomeric sequence at the ends of chromosomes, is inactive in most somatic cells; however in cancer cells, telomerase activity is commonly turned on. This telomerase activity may be associated with methylation of the hTERT gene promoter.

The repetitive nature and high GC content of the hTERT gene region makes it difficult to analyse using conventional PCR amplicons. Using the CRISPR/Cas9 approach on a thyroid cancer cell line, Timothy was able to demonstrate 50-fold increase in coverage of the targeted 2.4 kb region. Subsequent analysis of the nanopore sequencing data allowed identification of the epigenetic modification 5-methylcytosine with high concordance to alternative bisulfite sequencing approaches. A further benefit of the long nanopore reads is the facility for phasing and the identification of methylation patterns across both parental alleles.

Figure 1: Distal, allele-specific DNA methylation patterns in the TERT locus of a BCPAP thyroid cancer cell line identified using nanopore sequencing. Black lines indicate points of CpG methylation. The alleles are distinguishable by a point mutation (green line).

The team at Johns Hopkins are also examining the potential of CRISPR/Cas9 enrichment to analyse the methylation status of a panel of gene promoters, which, they believe, may lead to diagnostic, prognostic and therapeutic applications.

At Tel Aviv University, Israel, researchers are applying Cas9-assisted targeting of chromosome segments (CATCH) to characterise the entire BRCA1 gene². Mutations in BRCA1 are associated with significantly increased risk of breast, ovarian and other cancers. Due to the large size of this gene (~80 kb), current sequencing methodologies focus on the coding sequence, neglecting potentially important intronic and regulatory regions. In addition, the BRCA1 genomic region is also highly repetitive (50%), which contributes to genetic instability and genomic rearrangements, and can be difficult to analyse using short-read sequencing.

A 200 kb region containing the entire 80 kb BRCA1 gene was enriched.

In brief, the CATCH methodology uses Cas9 to excise the targeted region, which is then separated using pulsed field gel electrophoresis, prior to amplification and sequencing. Cas9 allows enrichment of a target region without prior knowledge of its sequence. Only the sequence of flanking regions needs to be known. Applying this technique, the team enriched a 200 kb region containing the entire 80 kb BRCA1 gene, regulatory elements and non-coding regions. The target was enriched 237-fold and sequenced at up to 70x coverage on a single MinION flow cell. The data revealed a deletion of three di-nucleotide blocks in a 44 bp repeat array which was not detected using short-read sequencing. In addition, the read-lengths provided by nanopore sequencing were sufficient for analysis of structural variation.

The team commented that this approach: ‘…may shed light on the mechanisms of disease onset and progression’ ². They are now investigating the potential for direct sequencing without the requirement for amplification, in order to retain and study epigenetic marks.

This case study is taken from the human white paper.