An Enhanced Gene Targeting Toolkit for<i>Drosophila</i>: Golic+

Chen, Hui Min; Huang, Ya-Ling; Pfeiffer, Barret D.; Yao, Xiaohao; Lee, Tzumin

doi:10.1534/genetics.114.173716

Cited by 30 publications

(41 citation statements)

References 58 publications

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“…To analyze Vnd + NBs, we created a GAL4 under the control of endogenous vnd regulatory sequences via gene targeting [21]. To immortalize the NB expression of Vnd into the neuronal progeny, we derived a Vnd-specific, lineage-restricted LexA driver using a cascade of sitespecific recombinases.…”

Section: Mapping 18 Neuronal Lineages Concurrently With Vnd-gal4mentioning

confidence: 99%

Conservation and Divergence of Related Neuronal Lineages in theDrosophilaCentral Brain

Lee

Yang

Huang

et al. 2019

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Wiring a complex brain requires enormous cell specificity. This specificity is laid out via a developmental process where neural stem cells produce countless diverse neurons. To help elucidate this process and resolve the considerable dynamic specificity, we need to observe the development of multiple neuronal lineages. Drosophila central brain lineages are predetermined, comprised of a fixed set of neurons born in pairs in a specific order. To reveal specific roles of lineage identity, Notch-dependent sister fate specification, and temporal patterning in morphological diversification, we mapped approximately one quarter of the Drosophila central brain lineages. While we found large aggregate differences, we also discovered similar patterns of morphological specification and diversification. Lineage identity plus Notch state govern primary neuronal trajectories, whereas temporal fates diversify terminal elaborations in target-specific manners. In addition, we identified 'related' lineages of analogous neuron types produced in similar temporal patterns. Two stem cells even yield identical series of dopaminergic neuron types, but with completely disparate sister neurons. These phenomena suggest that large changes in morphological diversity can be the consequence of relatively small differences in lineage fating. Taken together, this large-scale lineage mapping study reveals that relatively simple rules drive incredible neuronal complexity.

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Section: Mapping 18 Neuronal Lineages Concurrently With Vnd-gal4mentioning

confidence: 99%

Conservation and Divergence of Related Neuronal Lineages in theDrosophilaCentral Brain

Lee

Yang

Huang

et al. 2019

Preprint

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“…Here we describe a new high-throughput technology for deletion analysis called CAMIO (Cas9-mediated Arrayed Mutagenesis of Individual Offspring). We built a CRISPR-based mutagenesis pipeline in Drosophila male germ cells, to achieve massive production of independent indels in targeted loci with germline transmission (26,27). We further created a transgenic system for targeting multiple sites simultaneously with an array of gRNAs.…”

Section: Introductionmentioning

confidence: 99%

CAMIO for deletion analysis of endogenous DNA sequences in multicellular organisms

Chen

Marqués

Sugino

et al. 2019

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19The genome is the blueprint for an organism. Interrogating the genome, 20 especially locating critical cis-regulatory elements, requires deletion analysis. 21 This is conventionally performed using synthetic constructs, making it 22 cumbersome and non-physiological. Thus, we created Cas9-mediated Arrayed 23Mutagenesis of Individual Offspring (CAMIO) to achieve high-throughput analysis 24 of native DNA. CAMIO utilizes CRISPR that is spatially restricted to generate 25 independent deletions. Controlled by recombination, a single guide RNA is 26 stochastically chosen from a set targeting a specific DNA region. Combining two 27 sets increases variability, leading to either indels at 1-2 target sites or inter-target 28 deletions. Cas9 restriction to male germ cells elicits autonomous double-strand-29 break repair, consequently creating offspring with diverse mutations. Thus, from 30 a single population cross, we can obtain a deletion matrix covering a large 31 expanse of DNA at both coarse and fine resolution. We demonstrate the ease 32 and power of CAMIO by mapping 5'UTR sequences crucial for chinmo's post-33 transcriptional regulation. 34 35 36 non-physiological, prompting the search for tailor-made sequence-specific DNA 63 nucleases that permit targeted mutagenesis of native sequences. 64 65In the early 2000s, zinc-finger nucleases (ZFNs) 6 , the first of the tailored 66 nucleases, were heavily adopted 7,8 . Although it seemed a promising strategy for 67 genome editing, major setbacks, especially off-target toxicity limited its utility 9 . 68 Therefore, the enthusiasm for ZFNs declined after the arrival of transcription 69 activator-like effector nucleases (TALENs) 10-12 . The higher specificity of TALENs 70 led to fewer off-target disruptions, and hence less cytotoxicity 13 . However, 71 constructing a TALEN is technically challenging because the homologous 72 sequences encoding the TALEN repeats are prone to recombine with each 73 other 9 . 74 75The newest technology, CRISPR (clustered regularly interspaced short 76 palindromic repeats), was first exploited for targeted mutagenesis 14 and then 77 swiftly adopted to edit genomes of diverse organisms 15-20 . CRISPR's popularity 78 lies in its simplicity: a Cas9 nuclease plus an easily made guide RNA (gRNA) 79 induces a double-strand break (DSB) targeted by DNA-RNA base pairing 14,16 . 80Cells repair the DSBs either through nonhomologous end joining (NHEJ) 21 , 81 leading to insertion or deletion (indel), or through homology-directed repair 82 (HDR) 22 which replaces discontinuous sequences with an available template. 83The fact that CRISPR can produce targeted DNA modifications with near 84 unlimited specificity has ensured its rapid expansion throughout the biological 85 7 132 Figure 1. bamP-Cas9 induces efficient CRISPR targeted mutagenesis in male germ 133 cells. (A) Ebony transcript shown with UTRs in turquoise. U6 drives guide RNA (gRNA-134 e), which targets 5' end of ebony coding sequence. (B) Percent of progeny with ebony 135 loss-of-function (LOF) mutati...

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“…To recover rare gene-targeting events in those challenging cases 71 requires (1) generation of numerous offspring, each with independent trials, and (2) 72 enrichment of offspring with correct gene targeting (especially those with decreased 73 viability) by selection against 'unperturbed' progeny. 74 75 Golic+ is a transgene-based gene targeting system designed to achieve the above 76 two objectives (Chen et al 2015). First, it employs a bam promoter to confine gene 77 targeting to germ cells rather than germline stem cells (Chen and McKearin 2003;78 Lehmann 2012).…”

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confidence: 99%