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, 12, 85-90

The History and Market Impact of CRISPR RNA-guided Nucleases

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The History and Market Impact of CRISPR RNA-guided Nucleases

Paul Bg van Erp et al. Curr Opin Virol.

Abstract

The interface between viruses and their hosts' are hot spots for biological and biotechnological innovation. Bacteria use restriction endonucleases to destroy invading DNA, and industry has exploited these enzymes for molecular cut-and-paste reactions that are central to many recombinant DNA technologies. Today, another class of nucleases central to adaptive immune systems that protect bacteria and archaea from invading viruses and plasmids are blazing a similar path from basic science to profound biomedical and industrial applications.

Figures

Figure 1
Figure 1. Cas9 delivery and repair of the targeted DNA
Cas9 and a single-guide RNA (sgRNA) have been delivered to eukaryotic cells by several methods: transient transfections (expression vectors or purified Cas9/sgRNA complex), cytoplasmic or nuclear injections (expression vectors, mRNA or purified Cas9/sgRNA) and by transduction (Lentiviruses or Adeno-Associated Virus). Cas9 identifies its target by protein mediated PAM (yellow) recognition and base pairing between the sgRNA and the DNA target. Target recognition activates the nuclease sites (red triangles), resulting in double stranded breaks (DSBs) 3–4 nucleotides downstream from the PAM. DSBs can be repaired by non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ results in insertion or deletions (indels), which often results in a frameshift mutation. HDR relies on homologous recombination with a donor DNA molecule. This donor DNA can be used to specifically insert desired sequences.

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