Overcoming evolved resistance to population-suppressing homing-based gene drives

Area of Science:

• Genetics
• Molecular Biology
• Vector Control

Background:

• CRISPR-Cas9 gene drive systems offer a novel approach for controlling populations of disease vectors, such as Anopheles gambiae mosquitoes.
• Current CRISPR-Cas9 homing systems face challenges due to the rapid emergence of homing-resistant alleles, which possess a fitness advantage and can lead to population rebound.
• The rate of homing-resistant allele generation in existing systems is a critical factor limiting their effectiveness for population suppression.

Purpose of the Study:

• To develop a mathematical model to determine the acceptable rates of homing-resistant allele generation for effective wild population suppression.
• To theoretically investigate a multiplexed guide RNA (gRNA) homing system architecture to enhance gene drive efficacy and mitigate resistance.
• To demonstrate the in vivo proof-of-principle for multiplexed gRNAs in inducing mutations for potential application in stable gene drives.

Main Methods:

• Development of a mathematical model to estimate tolerable rates of homing-resistant allele generation based on wild population size.
• Theoretical exploration of a multiplexed gRNA homing system to increase homing rates and decrease resistant allele generation rates.
• In vivo experimental validation using multiplexed ribozyme-flanked gRNAs to induce mutations in Drosophila melanogaster.

Main Results:

• Mathematical modeling indicates that tolerable rates of resistance allele generation must be orders of magnitude lower than currently observed for effective population suppression.
• Multiplexing gRNAs exponentially increases the potential population size that can be suppressed.
• A system with four multiplexed gRNAs could potentially suppress mosquito populations on a continental scale; proof-of-principle demonstrated in Drosophila.

Conclusions:

• Current CRISPR-Cas9 gene drive designs require significant improvement in resistance allele management for successful population suppression.
• Multiplexed gRNA homing systems represent a promising strategy to overcome resistance and achieve large-scale vector control.
• The developed multiplexed gRNA strategy is adaptable for engineering stable homing-based drives in various target organisms.