Enhanced Golic+: highly effective CRISPR gene targeting and transgene HACKing in Drosophila.

Hui-Min Chen, Xiaohao Yao, Qingzhong Ren, Chuan-Chie Chang, Ling-Yu Liu, Rosa Linda Miyares, Tzumin Lee

Development (Cambridge, England) 2020 Jun 11

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Gene targeting is an incredibly valuable technique. Sometimes, however, it can also be extremely challenging for various intrinsic reasons (e.g. low target accessibility or nature/extent of gene modification). To bypass these barriers, we designed a transgene-based system in Drosophila that increases the number of independent gene targeting events while at the same time enriching for correctly targeted progeny. Unfortunately, with particularly challenging gene targeting experiments, our original design yielded numerous false positives. Here, we deliver a much-improved technique, named Enhanced Golic+ (E-Golic+). E-Golic+ incorporates genetic modifications to tighten lethality-based selection while simultaneously boosting efficiency. With E-Golic+, we easily achieve previously unattainable gene targeting. Additionally, we built an E-Golic+-based, high-efficiency genetic pipeline for transgene swapping. We demonstrate its utility by transforming GAL4 enhancer-trap lines into tissue-specific Cas9-expressing lines. Given the superior efficiency, specificity and scalability, E-Golic+ promises to expedite development of additional sophisticated genetic/genomic tools in Drosophila. © 2020. Published by The Company of Biologists Ltd.

Citation

Hui-Min Chen, Xiaohao Yao, Qingzhong Ren, Chuan-Chie Chang, Ling-Yu Liu, Rosa Linda Miyares, Tzumin Lee. Enhanced Golic+: highly effective CRISPR gene targeting and transgene HACKing in Drosophila. Development (Cambridge, England). 2020 Jun 11;147(11)