Division of Biological Sciences, Section of Cell and Developmental Biology, University of California San Diego, La Jolla, CA, 92093, USA.
Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA, 92093, USA.
Nat Commun. 2022 May 9;13(1):2595. doi: 10.1038/s41467-022-29868-3.
Homing CRISPR gene drives could aid in curbing the spread of vector-borne diseases and controlling crop pest and invasive species populations due to an inheritance rate that surpasses Mendelian laws. However, this technology suffers from resistance alleles formed when the drive-induced DNA break is repaired by error-prone pathways, which creates mutations that disrupt the gRNA recognition sequence and prevent further gene-drive propagation. Here, we attempt to counteract this by encoding additional gRNAs that target the most commonly generated resistance alleles into the gene drive, allowing a second opportunity at gene-drive conversion. Our presented "double-tap" strategy improved drive efficiency by recycling resistance alleles. The double-tap drive also efficiently spreads in caged populations, outperforming the control drive. Overall, this double-tap strategy can be readily implemented in any CRISPR-based gene drive to improve performance, and similar approaches could benefit other systems suffering from low HDR frequencies, such as mammalian cells or mouse germline transformations.
归巢 CRISPR 基因驱动器可以帮助控制媒介传播疾病和作物害虫以及入侵物种的种群，因为其遗传率超过了孟德尔定律。然而，该技术在驱动诱导的 DNA 断裂通过易错途径修复时会产生抗性等位基因，这会产生突变，破坏 gRNA 识别序列，并阻止进一步的基因驱动传播。在这里，我们试图通过将额外的靶向最常见产生的抗性等位基因的 gRNA 编码到基因驱动器中，来对抗这种情况，从而为基因驱动转换提供第二次机会。我们提出的“双点击”策略通过回收抗性等位基因提高了驱动效率。双点击驱动在笼养种群中也能有效地传播，性能优于对照驱动。总的来说，这种双点击策略可以很容易地应用于任何基于 CRISPR 的基因驱动中，以提高性能，类似的方法也可以使其他系统受益，例如低频的 HDR，如哺乳动物细胞或小鼠生殖系转化。
