Novel CRISPR/Cas9 gene drive constructs reveal insights into mechanisms of resistance allele formation and drive efficiency in genetically diverse populations

Abstract

A functioning gene drive system could fundamentally change our strategies for the control of vector-borne diseases by facilitating rapid dissemination of transgenes that prevent pathogen transmission or reduce vector capacity. CRISPR/Cas9 gene drive promises such a mechanism, which works by converting cells that are heterozygous for the drive construct into homozygotes, thereby enabling super-Mendelian inheritance. Although CRISPR gene drive activity has already been demonstrated, a key obstacle for current systems is their propensity to generate resistance alleles, which cannot be converted to drive alleles. In this study, we developed two CRISPR gene drive constructs based on the nanos and vasa promoters that allowed us to illuminate the different mechanisms by which resistance alleles are formed in the model organism Drosophila melanogaster. We observed resistance allele formation at high rates both prior to fertilization in the germline and post-fertilization in the embryo due to maternally deposited Cas9. Assessment of drive activity in genetically diverse backgrounds further revealed substantial differences in conversion efficiency and resistance rates. Our results demonstrate that the evolution of resistance will likely impose a severe limitation to the effectiveness of current CRISPR gene drive approaches, especially when applied to diverse natural populations.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated 9‐mediated mutagenesis of the multiple edematous wings gene induces muscle weakness and flightlessness in Bactrocera dorsalis (Diptera: Tephritidae)

“…S6). Noteworthily, there is some evidence to show that the occurrence frequency of HDR is not absolutely 100% and that NHEJ occurred in some cases when double-strand DNA breaks were repaired (Champer et al, 2017). Many breaks are repaired by NHEJ, which would prevent the gene drive DNA from transferring to the new allele and subsequently generating a gene drive resistance insect (Bull and Malik, 2017).…”

MGDrivE: A modular simulation framework for the spread of gene drives through spatially-explicit mosquito populations

“…The stochastic implementation is more relevant to population suppression as it can capture rare resistant allele generation and the possibility of population extinction. In both cases, we include the generation of in-frame and out-of-frame or otherwise costly resistant alleles [28,51] and parameterize the gene drive model based on recently engineered constructs [7,30].…”

“…90% of wild-type (h) alleles are converted to homing (H) alleles in the germline of Hh males, and 50% of h alleles are converted to H alleles in the germline of Hh females. A third of the remaining h alleles in Hh individuals are converted to in-frame resistant alleles (R), and the remainder are converted to out-of-frame or otherwise costly resistant alleles (B) due to error-prone copying during the homing process [51]. Female fecundity and male mating fitness are reduced by 25% per H or R allele and by 50% per B allele.…”

“…The field of gene drive has been moving extremely quickly, especially since the discovery of CRISPR-based gene editing, and we intend the MGDrivE package to provide a flexible tool capable of modeling novel inheritance-modifying constructs as they are proposed and become available. Future functionality that we intend to incorporate into the software includes: a) "shadow drive", in which the Cas9 enzyme is passed on to the offspring even if the gene expressing it is not [51], b) life history models encompassing a more diverse range of insect disease vectors and agricultural pests, and c) populations that vary in size periodically or in response to environmental input variables. We are also developing a corresponding individual-based model that is capable of modeling multi-locus systems for which the number of possible genotypes exceeds the number of individuals in the population.…”

Evaluating the Probability of CRISPR-based Gene Drive Contaminating Another Species

“…So far, CRISPR-based gene drives have only been tested in laboratories or in large indoor cages. They have been shown to efficiently boost their own transmission in yeasts (DiCarlo et al 2015) , Drosophila flies (Gantz and Bier 2015;Champer et al 2017;KaramiNejadRanjbar et al 2018) , mosquitoes Hammond et al 2016;Kyrou et al 2018) , the pathogenic fungus Candida albicans (Shapiro et al 2018) and mice (Grunwald et al 2019) .…”