Going Deeper into Long Reads: Oxford Nanopore Licenses CRISPR-Cas9 IP from Caribou

CRISPR-Cas9 is a customizable tool that lets scientists cut and insert small pieces of DNA at precise areas along a DNA strand. The tool is composed of two basic parts: the Cas9 protein, which acts like the wrench, and the specific RNA guides, CRISPRs, which act as the set of different socket heads. These guides direct the Cas9 protein to the correct gene, or area on the DNA strand, that controls a particular trait. This lets scientists study our genes in a specific, targeted way and in real-time.

The British sequencing company famous for producing ultra-long reads, Oxford Nanopore Technologies announced today that have entered into a non-exclusive license agreement with Caribou Biosciences for CRISPR-Cas9 enrichment for nanopore sequencing. Caribou has granted Oxford Nanopore a worldwide, non-exclusive license under foundational CRISPR-Cas9 intellectual property controlled by Caribou for nanopore sequencing.

“The Cas9 technique will enable users to select and isolate the regions of the genome they are most interested in, including those not available to existing methods, ready for rapid analysis using our long-read, real-time sequencing technology”, said Gordon Sanghera, PhD, CEO of Oxford Nanopore.

Caribou Biosciences, co-founded by Jennifer Doudna, Ph.D., Martin Kinek, PhD, and Rachel Haurwitz, PhD in 2011, carries a broad, foundational CRIPSR-Cas9 IP portfolio. Oxford Nanopore is known in the next-generation sequencing space for their technology that produces ultra-long reads—up to 2 Mb—in platforms that are small and portable. “While all sequencing platforms should be able to benefit from this method of enrichment, the simplicity and rapidity could pair especially well with the handheld MinION system” notes Shawn Baker, PhD a consultant at SanDiegOmics.com who has spent his career entrenched in the world of genomic sequencing.

The coupling of these two technologies will, Baker adds, “allow for the development of rapid field-based assays.” Indeed, Oxford Nanopore notes that “the entire library preparation process takes less than two hours.” “If combined with our portable sequencer MinION” Oxford Nanopore asserts, “this has the potential to open up fast-turnaround, near-sample testing in new ways.”

CRISPR-Cas9-mediated enrichment for nanopore sequencing enables targeted sequencing of long regions of interest, without the need for amplification—read lengths of over 100Kb have been observed to date. The technique opens up regions of the genome previously only accessible with long read whole genome sequencing.

“This is a nice example of using CRISPR technology not for editing, which tends to get all the press, but for its ability to rapidly isolate specific DNA sequences from a complex mixture” notes Baker. For example, the method is suitable for characterizing repeat expansions, Single nucleotide variants and structural variants, whilst retaining the methylation status of the native molecule‑information about which is provided as standard with the latest basecalling software. It can also be used to sequence many targets simultaneously, allowing users to build their own panels or look across larger regions.

Earlier this year, a team from Oxford Nanopore in collaboration with colleagues at Johns Hopkins University and Baylor College of Medicine, published a bioRxivpreprint introducing a technique called ‘nanopore Cas9 Targeted-Sequencing’ (nCATS). By developing this method, the researchers demonstrated that Cas9 enrichment used with nanopore sequencing allows for enhanced analysis of genomic regions, including, they note SNPs, structural variation at known hot spots, and methylation patterns at cancer driver genes. Specifically, they “generated a median 165X coverage at 10 genomic loci with a median length of 18kb, representing a several hundred-fold improvement over the 2-3X coverage achieved without enrichment” using the Oxford Nanopore MinION flow cell. The authors note that the method “has extensive clinical applications for assessing medically relevant genes and has the versatility to be a rapid and comprehensive diagnostic tool.”

In addition, a team from the University of Leeds, St. James’s University Hospital published data this past summer in Laboratory Investigationusing the method in to decipher two challenging clinical cases,both of whom presented with neurodevelopmental problems. The team describesin the paper entitled, “Cas9-based enrichment and single-molecule sequencing for precise characterization of genomic duplications” that they were able to use a Cas9 enrichment strategy to describe the nucleotide-level resolution of the two cases in which duplications had been identified by array comprehensive genomic hydridisation (CGH), but the precise breakpoints at nucleotide resolution had remained elusive.

Not surprisingly, Oxford Nanopore plans to release a Cas9 Sequencing Kit later in the year. For now, researchers can access a protocol and bioinformatics tutorialthat can be accessed on the website with a login.

Given the early success of this approach, this announcement may be the first of many more to follow. Baker tells Clinical OMICS, “I expect to start seeing CRISPR used for targeted assays on a variety of sequencing platforms as well for non-NGS based diagnostics.”

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