Cas9 Gene Drive, Sex-Conversion and Evolved Resistance

Anna Buchman, Ph.D. is a postdoctoral researcher at the University of California, San Diego. She is interested in engineering gene drive systems in economically and medically significant insects. My current projects include developing gene drive systems such as Medea in Spotted Wing Drosophila, a major crop pest. MORE ABOUT THE AUTHOR

In a recent manuscript, KaramiNejadRanjbar et al. demonstrate the development of a proof of principle Cas9-based suppression gene drive in D. melanogaster that can be applied to pest insects, and discuss the implications of resistance allele formation for practical use of such a drive system.

The use of CRISPR-Cas9 technology to develop homing based gene drive systems has attracted significant interest in the fields of insect pest and disease vector control, as homing CRISPR/Cas9 gene-drive (HCGD) systems may be greatly useful for manipulating and suppressing wild populations of harmful insects. Such systems typically contain a CRISPR/Cas9 homing element (CHE) comprised of a Cas9-coding sequence and a guide RNA (gRNA) integrated in the host genome at the gRNA target site, and can convert their corresponding allele on the opposite chromosome into a copy of themselves, resulting in super-Mendelian CHE inheritance. HCGD systems can theoretically drive any desired trait through a population (given certain fitness cost limitations), and, depending on the specific gRNA target site selected, can be used to bring about population replacement/modification or population suppression.

C. capitata sex determination seems to be based on a Drosophila partially conserved sex determination cascade. In this comparative scheme an hypothetical C. capitata sex determination cascade is depicted. 

Sex determination in Drosophila and Ceratitis showing the he central role of tra. Image from: Saccone G1, Pane A, Polito LC. (2002). Sex determination in flies, fruitflies and butterflies. Genetica. 2002 Sep;116(1):15-23

Non-Homologous End Joining following cleavage by a Cas9/gRNA complex often results in mutagenesis and loss of the gRNA target site resulting in the creation of an allele resistant to the original Cas9/gRNA complex.

In the present study, KaramiNejadRanjbar et al. describe the development of a sex-conversion suppression HCGD that targets transformer (tra), a pivotal female sex determination gene in Dipterans and other insects. In the Mediterranean fruit fly, Ceratitis capitata (medfly), which is a major agricultural pest, tra-knockdown XY males develop normally, while XX individuals develop as fertile males. Therefore, XX males carrying a tra-targeting CHE could spread the CHE, resulting in an effective gene drive that would skew sex ratios towards males and eventually lead to an all-male population collapse. Conversely, in D. melanogaster, tra-mutant XX individuals develop into infertile pseudomales and a tra-targeting CHE is therefore not capable of gene drive, allowing for a level of biological containment that facilitates safe study of such a system.

KaramiNejadRanjbar et al. therefore developed several tra-targeting CHEs in D. melanogaster, and demonstrated that one such CHE was capable of bringing about both super-Mendelian inheritance (with an average homing rate of 56%) and efficient sex conversion (with an average male/pseudomale rate of 89%). However, in characterizing the functional tra-CHE, KaramiNejadRanjbar and colleagues observed that cleavage drive resistant tra alleles evolved at a rapid rate, largely as a result of in-frame indel mutations in the tra locus resulting from NHEJ events induced by the CHE itself. Population cage experiments indicated that such resistance alleles were constantly created, heritable, and increased in frequency over time, causing a restoration of an almost regular 1:1 sex ratio within 15 generations. Simulations of the performance of such a tra-CHE system in the medfly indicated that it would not work to cause a population collapse, even if multiple releases were implemented. Importantly, however, further simulations demonstrated that using multiple gRNAs to reduce the generation rate of in-frame resistance alleles greatly increases the effectiveness of this system and enables it to cause a population collapse, especially if heterozygous CHE/+ XX males are fertile (as they are expected to be in the medfly system).

Medfly, Ceratitis capitata
By Alvesgaspar – https://commons.wikimedia.org/w/index.php?curid=5027949

Altogether, these results provide a path forward for the development of effective sex-conversion suppression HCGDs in the medfly and in other insect pests and disease vectors. Additionally, the observed rapid generation and spread of HCGD-induced resistance alleles are consistent with results from other CHE studies, and suggest that simple HCGD designs will not work unless resistance-mitigating design elements such as multiple gRNAs are incorporated.

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