@article {65371, title = {CAMIO: a transgenic CRISPR pipeline to create diverse targeted genome deletions in Drosophila.}, journal = {Nucleic Acids Research}, year = {2020}, month = {2020 Mar 18}, abstract = {

The genome is the blueprint for an organism. Interrogating the genome, especially locating critical cis-regulatory elements, requires deletion analysis. This is conventionally performed using synthetic constructs, making it cumbersome and non-physiological. Thus, we created Cas9-mediated Arrayed Mutagenesis of Individual Offspring (CAMIO) to achieve comprehensive analysis of a targeted region of native DNA. CAMIO utilizes CRISPR that is spatially restricted to generate independent deletions in the intact Drosophila genome. Controlled by recombination, a single guide RNA is stochastically chosen from a set targeting a specific DNA region. Combining two sets increases variability, leading to either indels at 1-2 target sites or inter-target deletions. Cas9 restriction to male germ cells elicits autonomous double-strand-break repair, consequently creating offspring with diverse mutations. Thus, from a single population cross, we can obtain a deletion matrix covering a large expanse of DNA at both coarse and fine resolution. We demonstrate the ease and power of CAMIO by mapping 5{\textquoteright}UTR sequences crucial for chinmo{\textquoteright}s post-transcriptional regulation.

}, issn = {1362-4962}, doi = {10.1093/nar/gkaa177}, author = {Chen, Hui-Min and Marques, Jorge Garcia and Sugino, Ken and Wei, Dingjun and Miyares, Rosa Linda and Lee, Tzumin} } @article {65382, title = {Conservation and divergence of related neuronal lineages in the central brain.}, journal = {eLife}, volume = {9}, year = {2020}, month = {2020 Apr 07}, abstract = {

Wiring a complex brain requires many neurons with intricate cell specificity, generated by a limited number of neural stem cells. central brain lineages are a predetermined series of neurons, born in a specific order. To understand how lineage identity translates to neuron morphology, we mapped 18 central brain lineages. While we found large aggregate differences between lineages, we also discovered shared patterns of morphological diversification. Lineage identity plus Notch-mediated sister fate govern primary neuron trajectories, whereas temporal fate diversifies terminal elaborations. Further, morphological neuron types may arise repeatedly, interspersed with other types. Despite the complexity, related lineages produce similar neuron types in comparable temporal patterns. Different stem cells even yield two identical series of dopaminergic neuron types, but with unrelated sister neurons. Together, these phenomena suggest that straightforward rules drive incredible neuronal complexity, and that large changes in morphology can result from relatively simple fating mechanisms.

}, issn = {2050-084X}, doi = {10.7554/eLife.53518}, author = {Lee, Ying-Jou and Yang, Ching-Po and Miyares, Rosa L and Huang, Yu-Fen and He, Yisheng and Ren, Qingzhong and Chen, Hui-Min and Kawase, Takashi and Ito, Masayoshi and Otsuna, Hideo and Sugino, Ken and Aso, Yoshi and Ito, Kei and Lee, Tzumin} } @article {68341, title = {Dense reconstruction of elongated cell lineages: overcoming suboptimum lineage encoding and sparse cell sampling}, journal = {bioRxiv}, year = {2020}, month = {07/2020}, abstract = {

Acquiring both lineage and cell-type information during brain development could elucidate transcriptional programs underling neuronal diversification. This is now feasible with single-cell RNA-seq combined with CRISPR-based lineage tracing, which generates genetic barcodes with cumulative CRISPR edits. This technique has not yet been optimized to deliver high-resolution lineage reconstruction of protracted lineages. Drosophila neuronal lineages are an ideal model to consider, as multiple lineages have been morphologically mapped at single-cell resolution. Here we find the parameter ranges required to encode a representative neuronal lineage emanating from 100 stem cell divisions. We derive the optimum editing rate to be inversely proportional to lineage depth, enabling encoding to persist across lineage progression. Further, we experimentally determine the editing rates of a Cas9-deaminase in cycling neural stem cells, finding near ideal rates to map elongated Drosophila neuronal lineages. Moreover, we propose and evaluate strategies to separate recurring cell-types for lineage reconstruction. Finally, we present a simple method to combine multiple experiments, which permits dense reconstruction of protracted cell lineages despite suboptimum lineage encoding and sparse cell sampling.Competing Interest StatementThe authors have declared no competing interest.

}, doi = {10.1101/2020.07.27.223321}, url = {https://www.biorxiv.org/content/early/2020/07/29/2020.07.27.223321}, author = {Sugino, Ken and Miyares, Rosa L. and Espinosa-Medina, Isabel and Chen, Hui-Min and Potter, Christopher J and Lee, Tzumin} } @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 {64849, title = {Neurotransmitter identity is acquired in a lineage-restricted manner in the Drosophila CNS.}, journal = {Elife}, volume = {8}, year = {2019}, month = {2019 Mar 26}, abstract = {

The vast majority of the adult fly ventral nerve cord is composed of 34 hemilineages, which are clusters of lineally related neurons. Neurons in these hemilineages use one of the three fast-acting neurotransmitters (acetylcholine, GABA, or glutamate) for communication. We generated a comprehensive neurotransmitter usage map for the entire ventral nerve cord. We did not find any cases of neurons using more than one neurotransmitter, but found that the acetylcholine specific gene ChAT is transcribed in many glutamatergic and GABAergic neurons, but these transcripts typically do not leave the nucleus and are not translated. Importantly, our work uncovered a simple rule: All neurons within a hemilineage use the same neurotransmitter. Thus, neurotransmitter identity is acquired at the stem cell level. Our detailed transmitter- usage/lineage identity map will be a great resource for studying the developmental basis of behavior and deciphering how neuronal circuits function to regulate behavior.

}, issn = {2050-084X}, doi = {10.7554/eLife.43701}, author = {Lacin, Haluk and Chen, Hui-Min and Long, Xi and Singer, Robert H and Lee, Tzumin and Truman, James W} } @article {48478, title = {Genetic tools to study juvenile hormone action in Drosophila.}, journal = {Scientific Reports}, volume = {7}, year = {2017}, month = {2017 May 18}, pages = {2132}, abstract = {

The insect juvenile hormone receptor is a basic helix-loop-helix (bHLH), Per-Arnt-Sim (PAS) domain protein, a novel type of hormone receptor. In higher flies like\ Drosophila, the ancestral receptor\ germ cell-expressed\ (gce) gene has duplicated to yield the paralog\ Methoprene-tolerant\ (Met). These paralogous receptors share redundant function during development but play unique roles in adults. Some aspects of JH function apparently require one receptor or the other. To provide a foundation for studying JH receptor function, we have recapitulated endogenous JH receptor expression with single cell resolution. Using Bacteria Artificial Chromosome (BAC) recombineering and a transgenic knock-in, we have generated a spatiotemporal expressional atlas of\ Metand\ gce\ throughout development. We demonstrate JH receptor expression in known JH target tissues, in which temporal expression corresponds with periods of hormone sensitivity. Larval expression largely supports the notion of functional redundancy. Furthermore, we provide the neuroanatomical distribution of JH receptors in both the larval and adult central nervous system, which will serve as a platform for future studies regarding JH action on insect behavior.

}, doi = {10.1038/s41598-017-02264-4}, author = {Baumann, Aaron A and Texada, Michael J and Chen, Hui-Min and Etheredge, Jack N and Miller, D L and Picard, Serge and Warner, Raechel D and Truman, James W and Riddiford, Lynn M} } @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 {45063, title = {Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {111}, year = {2014}, month = {2014 Jul 22}, pages = {E2967-76}, abstract = {

The type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system has emerged recently as a powerful method to manipulate the genomes of various organisms. Here, we report a toolbox for high-efficiency genome engineering of Drosophila melanogaster consisting of transgenic Cas9 lines and versatile guide RNA (gRNA) expression plasmids. Systematic evaluation reveals Cas9 lines with ubiquitous or germ-line-restricted patterns of activity. We also demonstrate differential activity of the same gRNA expressed from different U6 snRNA promoters, with the previously untested U6:3 promoter giving the most potent effect. An appropriate combination of Cas9 and gRNA allows targeting of essential and nonessential genes with transmission rates ranging from 25-100\%. We also demonstrate that our optimized CRISPR/Cas tools can be used for offset nicking-based mutagenesis. Furthermore, in combination with oligonucleotide or long double-stranded donor templates, our reagents allow precise genome editing by homology-directed repair with rates that make selection markers unnecessary. Last, we demonstrate a novel application of CRISPR/Cas-mediated technology in revealing loss-of-function phenotypes in somatic cells following efficient biallelic targeting by Cas9 expressed in a ubiquitous or tissue-restricted manner. Our CRISPR/Cas tools will facilitate the rapid evaluation of mutant phenotypes of specific genes and the precise modification of the genome with single-nucleotide precision. Our results also pave the way for high-throughput genetic screening with CRISPR/Cas.

}, issn = {1091-6490}, doi = {10.1073/pnas.1405500111}, author = {Port, Fillip and Chen, Hui-Min and Lee, Tzumin and Bullock, Simon L} }