Cas9 Expression Patterns Impact on Resistance Evolution in Different Genetic Backgrounds

Gene drive systems are sensitive to the evolution of resistance in the form of polymorphisms in the guide RNA’s target DNA sequence.  Champer et al. (2017) have recently reported on their exploration of how Cas9 expression patterns as well as different genetic backgrounds impact the evolution of resistance to a gene drive system in Drosophila melanogaster.

Homing-based gene drive systems depend upon a high rate of Homology-Directed-DNA-Repair (HDR) following target site cleavage.  When and where Cas9 cleavage occurs can impact the proportion of repair events that result from Non Homologous End Joining (NHEJ) and HDR.  For example, if cleavage and repair occur in gametes then one would expect NHEJ-based repair to occur since there would be no homologous chromosome upon which HDR would depend.

Champer et al. created two Cas9-based drive systems; one in which Cas9 expression was regulated by the promoter from vasa and the other in which Cas9 expression was regulated by the promoter from nanos.  Both genes are known to be expressed largely or exclusively in the germline.  In both cases the drive systems were targeting the yellow locus; either the open reading frame or the promoter.

The systems in this case enabled the authors to estimate gene drive conversion rates as well as the rates of resistance allele formation and the mechanisms by which those resistance alleles arose.

Image result for nanos Drosophila

Expression pattern of nanos and nanos mRNA localization in Drosophila

In the case of the nanos-based drive system 62% of the wild-type yellow alleles in a female heterozygous for the drive element were converted to drive-containing alleles.  The corresponding rate in the vasa-based drive system was 52%.  Unlike a previous study in which a vasa-based drive system was introduced into D. melanogaster, Champer et al. found there was no significant conversion of wild-type alleles in embryos.  The basis of these differences is not yet clear.

The authors also estimated rates of resistance formation.  For the nanos drive system 29% of the wild-type yellow alleles in females heterozygous for the drive element were converted to resistant alleles.  Consequently, some 95% of the wild-type alleles in females heterozygous for the nanos drive system were converted either to resistant alleles or drive-containing alleles.

In the case of the vasa-system about 48% of the wild-type alleles in a drive-heterozygote were converted to resistance alleles.

Image result for gene drive resistance

Gene drive with 100% conversion in a heterozygote.

Some resistance alleles were created in the embryo (post fertilization) suggesting the Cas9 expressed prior to meiosis can persist in embryos (or Cas9 was expressed after meiosis).

A particularly interesting and novel set of experiments reported in this paper examined the impact of differing genetic backgrounds on conversion rates and resistance evolution.  The authors introduced their nanos drive system into five lines from the Global Diversity Lines, a set of lines established from D. melanogaster collected from various locations around the world.  Drive conversion rates and resistance alleles showed modest variation among the lines. What did vary by over an order of magnitude were the rates of post fertilization resistance evolution.

Related imageThe largest unknown at this point regarding the use of gene drive systems as tools for population suppression or modification is whether resistance evolution can be managed so that the effectiveness of these tools will persist long enough to affect the desired changes.

There are lots of interesting data and analyses in this paper that are worth considering more fully.

Champer J, Reeves R, Oh SY, Liu C, Liu J, et al. (2017) Novel CRISPR/Cas9 gene drive constructs reveal insights into mechanisms of resistance allele formation and drive efficiency in genetically diverse populations. PLOS Genetics 13(7): e1006796. https://doi.org/10.1371/journal.pgen.1006796

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