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Reducing resistance allele formation in CRISPR gene drive.

Proc. Natl. Acad. Sci. U.S.A.2018 May 22;115(21):5522-5527. Epub 2018 May 07
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摘要


CRISPR homing gene drives can convert heterozygous cells with one copy of the drive allele into homozygotes, thereby enabling super-Mendelian inheritance. Such a mechanism could be used, for example, to rapidly disseminate a genetic payload in a population, promising effective strategies for the control of vector-borne diseases. However, all CRISPR homing gene drives studied in insects thus far have produced significant quantities of resistance alleles that would limit their spread. In this study, we provide an experimental demonstration that multiplexing of guide RNAs can both significantly increase the drive conversion efficiency and reduce germline resistance rates of a CRISPR homing gene drive in Drosophila melanogaster We further show that an autosomal drive can achieve drive conversion in the male germline, with no subsequent formation of resistance alleles in embryos through paternal carryover of Cas9. Finally, we find that the nanos promoter significantly lowers somatic Cas9 expression compared with the vasa promoter, suggesting that nanos provides a superior choice in drive strategies where gene disruption in somatic cells could have fitness costs. Comparison of drive parameters among the different constructs developed in this study and a previous study suggests that, while drive conversion and germline resistance rates are similar between different genomic targets, embryo resistance rates can vary significantly. Taken together, our results mark an important step toward developing effective gene drives capable of functioning in natural populations and provide several possible avenues for further control of resistance rates.

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