Brake System for Gene Drive

Sanjay Basu (webpage photo)

Sanjay Basu, Ph.D., Postdoctoral Researcher, The Pirbright Institute, Woking, UK
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Wu et al (2016) in February’s issue of Nature describe how they have developed a brake for a Cas9 gene drive system. This is an interesting correspondence reflecting how the field has shifted from proof-of-principle of Cas9 gene drive systems to now also considering mechanisms that might prevent or contain an unwanted gene drive system.

CRISPR-Cas9 gene drives have advanced rapidly and have been demonstrated in cage experiments in number of different insects of interest including Anopheles stephensi  and An. gambiae. One of the underlying concepts involved in CRISPR-Cas9 gene drive is homology-directed repair (HDR) involving the repair of a double-stranded DNA break by copying the corresponding allele. In consideration of gene drives, this results in copying over the CRISPR-Cas9 cassette within the germline and subsequently causing super-Mendelian inheritance within the progeny.

CATCHA 1

The CATCHA transgene ubiquitously expresses a guide strand (gRNA) that targets the Cas9 DNA sequence. The homology arms of the donor plasmid are recognised by the HDR pathway and copy the transgene into the unwanted Cas9 sequence causing disruption of the coding sequence.

In this study the authors employ HDR as the underlying principle as a mechanism to stop a CRISPR-Cas9 gene drive within Drosophila melanogaster. The basic principle uses a guide strand component targeting Cas9 DNA sequence that is also flanked by Cas9 DNA sequence corresponding to the cleavage point. The unwanted Cas9 endonuclease combines with the guide strand causing cleavage of its own coding DNA sequence. During the repair of the break, the HDR pathway copies the Cas9-triggered chain ablation (CATCHA) transgene into the unwanted Cas9 sequence thereby disrupting the drives’ crucial component.

CATCHA 2

The CATCHA transgene ubiquitously expresses a guide strand that targets the Cas9 DNA sequence. The HDR pathway now recognises the homologous chromosome and copies the CATCHA transgene into the unwanted Cas9 sequence causing disruption of the coding sequence.

The authors demonstrate this mechanism initially by an injected plasmid to express the guide strand (figure 1) then from a genomic transgenic insertion (figure 2). Their specific CATCHA construct utilises a U6:2 promoter to express the guide strand and 1Kbp homology arms. To experimentally characterise CATCHA they use a gene target that would cause a lethal phenotype (Wg), therefore when the unwanted Cas9 is disrupted this prevents the lethal guide strand from causing death. Their results show a CATCHA conversion rate of 57%.

Following establishment of the CATCHA transgene as a stock, the F1 females were crossed to males that express the ebony guide strand. In 93.4% of the F2 progeny the ebony gene was disrupted. Sequencing ebony from individuals of one group showed indels typical of non-homologous end joining meaning that the CATCHA did not copy.

As a proof of principle, the experimental design demonstrates the technique and the authors do acknowledge factors that would need to be considered if this technique might be advanced to a practicable scenario such as target sequence mutation and modelling of the population dynamics.

Wu, B., Luo, L. & Gao, X. J. Cas9-triggered chain ablation of cas9 as a gene drive brake. Nat Biotechnol 34, 137-138, doi:10.1038/nbt.3444 (2016)

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