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European Biopharmaceutical Review

The Solution to Complex CRISPR Modifications: Droplet Digital PCR

Development of the CRISPR-Cas system has unlocked new and improved opportunities in biological and genetic research, food production, and, increasingly, in the development of gene therapy and cell-based therapeutics. While the system was initially developed as a method to knock out target genes in functional studies, recent technical advances have extended this remit to selectively activate or repress target genes, purify specific regions of DNA, image DNA in live cells, and precisely edit DNA and RNA sequences.

However, as with all new developments, the full potential of this technology will only be reached if it is scientifically valuable, commercially transferable, cost-effective, and user friendly. While some applications of the conventional CRISPR-Cas system have achieved this, complex gene editing applications are still limited, including simultaneous multiple gene knock-out (KO), knock-in (KI) of longer length DNA fragments, and single-nucleotide modification (SNP) (1).

The chance of achieving complex CRISPR-Cas modifications can be enhanced by the addition of a Droplet Digital polymerase chain reaction (ddPCR). Like conventional PCR, ddPCR uses Taq polymerase to amplify target sequences using a validated primer, but with one key difference: the PCR mix is portioned into thousands of nanolitre-sized droplets. Such partitioning of the sample enables the screening of thousands of nucleic acid-containing droplets at once (see Figure 1) (2).

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Dr Karen McAulay began her career as a research scientist at the MRC Institute of Virology before moving to Edinburgh University, UK, to undertake her PhD thesis and develop a research portfolio investigating the immune response to Epstein–Barr virus (EBV) infection. In 2010, she moved to Glasgow University, UK, as a research fellow, with a focus on risk factors for EBV-associated disease. Currently, Karen is the Clinical Alliance Manager for REPROCELL (Biopta), with responsibility for maintaining the REPROCELL global tissue procurement network.

Zara Puckrin graduated last year with a first-class honors degree in Cell and Molecular Biology. During her studies, she was awarded a £2,000 vacation scholarship and the prize for the best academic poster in her graduate year. After undertaking part-time administrative work with REPROCELL (Biopta) in her final years of study, Zara is now working full time as Marketing Manager for the EMEA and US branches of the company.

Dr Robert Annand is a drug discovery scientist with more than 20 years’ experience in the field. After graduating with a PhD in Medicinal Chemistry and Biochemistry, he pursued a variety of research and management roles in the scientific and technical industry. Currently, Robert is Senior Technical Product Manager at REPROCELL (Stemgent).
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Dr Karen McAulay
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Zara Puckrin
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Dr Robert Annand
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