Cas9 fusions for precision<i>in vivo</i>editing

Richardson, Ryan R.; Steyert, Marilyn; Inen, Jeffrey; Khim, Saovleak; Romanowski, Andrea J.; Altas, Bekir; Poulopoulos, Alexandros

doi:10.1101/2020.07.15.199620

Cited by 3 publications

(2 citation statements)

References 41 publications

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“…pFCaGW is a lentiviral vector that expresses EGFP (G) under the CaMKII promoter (Ca). The pCaMKII-EGFP transcriptional unit was assembled into a custom backbone (derived from (57), a gift from Alexandros Poulopoulos, and the pEGFP-N1 backbone (Clontech)) by Golden Gate Assembly (NEB), with the CaMKII promoter from mCh-GluA1-CIB (a gift from Matthew Kennedy (Addgene plasmid # 89444; http://n2t.net/addgene:89444; RRID:Addgene_89444)), and EGFP from pEGFP-N1. pCaMKII-EGFP was inserted by NEB HIFI Assembly into the PacI site of a modified pFUGW vector (pFW), where the ubiquitin promoter and EGFP were deleted by KLD mutagenesis (NEB), yielding pFCaGW.…”

Section: Methodsmentioning

confidence: 99%

Differential nanoscale organization of excitatory synapses onto excitatory vs inhibitory neurons

Dharmasri,

Levy,

Blanpied

2023

Preprint

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A key feature of excitatory synapses is the existence of subsynaptic protein nanoclusters whose precise alignment across the cleft in a trans-synaptic nanocolumn influences the strength of synaptic transmission. However, whether nanocolumn properties vary between excitatory synapses functioning in different cellular contexts is unknown. We used a combination of confocal and DNA-PAINT super-resolution microscopy to directly compare the organization of shared scaffold proteins at two important excitatory synapses - those forming onto excitatory principal neurons (Ex→Ex synapses) and those forming onto parvalbumin-expressing interneurons (Ex→PV synapses). As in Ex→Ex synapses, we find that in Ex→PV synapses presynaptic Munc13-1 and postsynaptic PSD-95 both form nanoclusters that demonstrate alignment, underscoring synaptic nanostructure and the trans-synaptic nanocolumn as conserved organizational principles of excitatory synapses. Despite the general conservation of these features, we observed specific differences in the characteristics of pre- and postsynaptic Ex→PV nanostructure. Ex→PV synapses contained larger PSDs with fewer PSD-95 NCs when accounting for size than Ex→Ex synapses. Furthermore, the PSD-95 NCs were larger and denser. The identity of the postsynaptic cell also had a retrograde impact on Munc13-1 organization, as Ex→PV synapses hosted larger Munc13-1 puncta that contained less dense but larger and more numerous Munc13-1 NCs. Moreover, we measured the spatial variability of trans- synaptic alignment in these synapse types, revealing protein alignment in Ex→PV synapses over a distinct range of distances compared to Ex→Ex synapses. We conclude that while general principles of nanostructure and alignment are shared, cell-specific elements of nanodomain organization likely contribute to functional diversity of excitatory synapses. Understanding the rules of synapse nanodomain assembly, which themselves are cell-type specific, will be essential for illuminating brain network dynamics.

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Section: Methodsmentioning

confidence: 99%

Differential nanoscale organization of excitatory synapses onto excitatory vs inhibitory neurons

Dharmasri,

Levy,

Blanpied

2023

Preprint

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“…Alternative labeling approaches will be necessary to preserve endogenous mTOR localization while achieving single-cell labeling to directly visualize the behavior of mTOR outposts. Approaches such as fluorescent intracellular nanobodies (Gross et al, 2013 ) or sparse in vivo knockin (Mikuni et al, 2016 ; Richardson et al, 2020 ) have the potential to overcome these limitations.…”

Section: Discussionmentioning

confidence: 99%

Neuronal mTOR Outposts: Implications for Translation, Signaling, and Plasticity

Altas

Romanowski

Bunce

et al. 2022

Front. Cell. Neurosci.

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The kinase mTOR is a signaling hub for pathways that regulate cellular growth. In neurons, the subcellular localization of mTOR takes on increased significance. Here, we review findings on the localization of mTOR in axons and offer a perspective on how these may impact our understanding of nervous system development, function, and disease. We propose a model where mTOR accumulates in local foci we term mTOR outposts, which can be found in processes distant from a neuron’s cell body. In this model, pathways that funnel through mTOR are gated by local outposts to spatially select and amplify local signaling. The presence or absence of mTOR outposts in a segment of axon or dendrite may determine whether regional mTOR-dependent signals, such as nutrient and growth factor signaling, register toward neuron-wide responses. In this perspective, we present the emerging evidence for mTOR outposts in neurons, their putative roles as spatial gatekeepers of signaling inputs, and the implications of the mTOR outpost model for neuronal protein synthesis, signal transduction, and synaptic plasticity.

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Unravelling roles of error-prone DNA polymerases in shaping cancer genomes

Vaziri

Rogozin

et al. 2021

Oncogene

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Mutagenesis is a key hallmark and enabling characteristic of cancer cells, yet the diverse underlying mutagenic mechanisms that shape cancer genomes are not understood. This review will consider the emerging challenge of determining how DNA damage response pathways—both tolerance and repair—act upon specific forms of DNA damage to generate mutations characteristic of tumors. DNA polymerases are typically the ultimate mutagenic effectors of DNA repair pathways. Therefore, understanding the contributions of DNA polymerases is critical to develop a more comprehensive picture of mutagenic mechanisms in tumors. Selection of an appropriate DNA polymerase—whether error-free or error-prone—for a particular DNA template is critical to the maintenance of genome stability. We review different modes of DNA polymerase dysregulation including mutation, polymorphism, and over-expression of the polymerases themselves or their associated activators. Based upon recent findings connecting DNA polymerases with specific mechanisms of mutagenesis, we propose that compensation for DNA repair defects by error-prone polymerases may be a general paradigm molding the mutational landscape of cancer cells. Notably, we demonstrate that correlation of error-prone polymerase expression with mutation burden in a subset of patient tumors from The Cancer Genome Atlas can identify mechanistic hypotheses for further testing. We contrast experimental approaches from broad, genome-wide strategies to approaches with a narrower focus on a few hundred base pairs of DNA. In addition, we consider recent developments in computational annotation of patient tumor data to identify patterns of mutagenesis. Finally, we discuss the innovations and future experiments that will develop a more comprehensive portrait of mutagenic mechanisms in human tumors.

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Cas9 fusions for precisionin vivoediting

Cited by 3 publications

References 41 publications

Differential nanoscale organization of excitatory synapses onto excitatory vs inhibitory neurons

Differential nanoscale organization of excitatory synapses onto excitatory vs inhibitory neurons

Neuronal mTOR Outposts: Implications for Translation, Signaling, and Plasticity

Unravelling roles of error-prone DNA polymerases in shaping cancer genomes

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