@article {65622, title = {Enhanced Golic+: Highly effective CRISPR gene targeting and transgene HACKing in .}, journal = {Development}, year = {2020}, month = {2020 May 28}, abstract = {

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 .

}, issn = {1477-9129}, doi = {10.1242/dev.181974}, author = {Chen, Hui-Min and Yao, Xiaohao and Ren, Qingzhong and Chang, Chuan-Chie and Liu, Ling-Yu and Miyares, Rosa Linda and Lee, Tzumin} } @article {47307, title = {Transcriptomes of lineage-specific Drosophila neuroblasts profiled by genetic targeting and robotic sorting.}, journal = {Development (Cambridge, England)}, volume = {143}, year = {2016}, month = {2016 Feb 1}, pages = {411-21}, abstract = {

A brain consists of numerous distinct neurons arising from a limited number of progenitors, called neuroblasts in Drosophila. Each neuroblast produces a specific neuronal lineage. To unravel the transcriptional networks that underlie the development of distinct neuroblast lineages, we marked and isolated lineage-specific neuroblasts for RNA sequencing. We labeled particular neuroblasts throughout neurogenesis by activating a conditional neuroblast driver in specific lineages using various intersection strategies. The targeted neuroblasts were efficiently recovered using a custom-built device for robotic single-cell picking. Transcriptome analysis of mushroom body, antennal lobe and type II neuroblasts compared with non-selective neuroblasts, neurons and glia revealed a rich repertoire of transcription factors expressed among neuroblasts in diverse patterns. Besides transcription factors that are likely to be pan-neuroblast, many transcription factors exist that are selectively enriched or repressed in certain neuroblasts. The unique combinations of transcription factors present in different neuroblasts may govern the diverse lineage-specific neuron fates.

}, issn = {1477-9129}, doi = {10.1242/dev.129163}, author = {Yang, Ching-Po and Fu, Chi-Cheng and Sugino, Ken and Liu, Zhiyong and Ren, Qingzhong and Liu, Ling-Yu and Yao, Xiaohao and Lee, Luke P and Lee, Tzumin} } @article {46471, title = {An enhanced gene targeting toolkit for Drosophila: golic+.}, journal = {Genetics}, volume = {199}, year = {2015}, month = {2015 Mar}, pages = {683-94}, abstract = {

Ends-out gene targeting allows seamless replacement of endogenous genes with engineered DNA fragments by homologous recombination, thus creating designer "genes" in the endogenous locus. Conventional gene targeting in Drosophila involves targeting with the preintegrated donor DNA in the larval primordial germ cells. Here we report G: ene targeting during O: ogenesis with L: ethality I: nhibitor and C: RISPR/Cas (Golic+), which improves on all major steps in such transgene-based gene targeting systems. First, donor DNA is integrated into precharacterized attP sites for efficient flip-out. Second, FLP, I-SceI, and Cas9 are specifically expressed in cystoblasts, which arise continuously from female germline stem cells, thereby providing a continual source of independent targeting events in each offspring. Third, a repressor-based lethality selection is implemented to facilitate screening for correct targeting events. Altogether, Golic+ realizes high-efficiency ends-out gene targeting in ovarian cystoblasts, which can be readily scaled up to achieve high-throughput genome editing.

}, issn = {1943-2631}, doi = {10.1534/genetics.114.173716}, author = {Chen, Hui-Min and Huang, Yaling and Pfeiffer, Barret D and Yao, Xiaohao and Lee, Tzumin} } @article {47126, title = {Opposing intrinsic temporal gradients guide neural stem cell production of varied neuronal fates.}, journal = {Science (New York, N.Y.)}, volume = {350}, year = {2015}, month = {2015 Oct 16}, pages = {317-20}, abstract = {

Neural stem cells show age-dependent developmental potentials, as evidenced by their production of distinct neuron types at different developmental times. Drosophila neuroblasts produce long, stereotyped lineages of neurons. We searched for factors that could regulate neural temporal fate by RNA-sequencing lineage-specific neuroblasts at various developmental times. We found that two RNA-binding proteins, IGF-II mRNA-binding protein (Imp) and Syncrip (Syp), display opposing high-to-low and low-to-high temporal gradients with lineage-specific temporal dynamics. Imp and Syp promote early and late fates, respectively, in both a slowly progressing and a rapidly changing lineage. Imp and Syp control neuronal fates in the mushroom body lineages by regulating the temporal transcription factor Chinmo translation. Together, the opposing Imp/Syp gradients encode stem cell age, specifying multiple cell fates within a lineage.

}, issn = {1095-9203}, doi = {10.1126/science.aad1886}, author = {Liu, Zhiyong and Yang, Ching-Po and Sugino, Ken and Fu, Chi-Cheng and Liu, Ling-Yu and Yao, Xiaohao and Lee, Luke P and Lee, Tzumin} }