@article {68342, title = {TEMPO: A system to sequentially label and genetically manipulate vertebrate cell lineages}, journal = {bioRxiv}, year = {2021}, month = {10/2021}, abstract = {

During development, regulatory factors appear in a precise order to determine cell fates over time. To investigate complex tissue development, one should not just label cell lineages but further visualize and manipulate cells with temporal control. Current strategies for tracing vertebrate cell lineages lack genetic access to sequentially produced cells. Here we present TEMPO (Temporal Encoding and Manipulation in a Predefined Order), an imaging-readable genetic tool allowing differential labelling and manipulation of consecutive cell generations in vertebrates. TEMPO is based on CRISPR and powered by a cascade of gRNAs that drive orderly activation/inactivation of reporters/effectors. Using TEMPO to visualize zebrafish and mouse neurogenesis, we recapitulated birth-order-dependent neuronal fates. Temporally manipulating cell-cycle regulators in mouse cortex progenitors altered the proportion and distribution of neurons and glia, revealing the effects of temporal gene perturbation on serial cell fates. Thus, TEMPO enables sequential manipulation of molecular factors, crucial to study cell-type specification.One-Sentence Summary Gaining sequential genetic access to vertebrate cell lineages.Competing Interest StatementThe authors have declared no competing interest.

}, doi = {10.1101/2021.10.27.466134}, url = {https://www.biorxiv.org/content/early/2021/10/28/2021.10.27.466134}, author = {Espinosa-Medina, Isabel and Feliciano, Daniel and Belmonte-Mateos, Carla and Garcia-Marques, Jorge and Foster, Benjamin and Miyares, Rosa Linda and Pujades, Cristina and Koyama, Minoru and Lee, Tzumin} } @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 {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 {49547, title = {Temporal control of Drosophila central nervous system development.}, journal = {Current Opinion in Neurobiology}, volume = {56}, year = {2018}, month = {2018 Nov 27}, pages = {24-32}, abstract = {

A complex nervous system requires precise numbers of various neuronal types produced with exquisite spatiotemporal control. This striking diversity is generated by a limited number of neural stem cells (NSC), where spatial and temporal patterning intersect. Drosophila is a genetically tractable model system that has significant advantages for studying stem cell biology and neuronal fate specification. Here we review the latest findings in the rich literature of temporal patterning of neuronal identity in the Drosophila central nervous system. Rapidly changing consecutive transcription factors expressed in NSCs specify short series of neurons with considerable differences. More slowly progressing changes are orchestrated by NSC intrinsic temporal factor gradients which integrate extrinsic signals to coordinate nervous system and organismal development.

}, issn = {1873-6882}, doi = {10.1016/j.conb.2018.10.016}, author = {Miyares, Rosa Linda and Lee, Tzumin} }