An optimized CRISPR/Cas9 approach for precise genome editing in neurons

The efficient knock-in of large DNA fragments to label endogenous proteins remains especially challenging in non-dividing cells such as neurons. We developed Targeted Knock-In with Two (TKIT) guides as a novel CRISPR/Cas9 based approach for efficient, and precise, genomic knock-in. Through targeting non-coding regions TKIT is resistant to INDEL mutations. We demonstrate TKIT labeling of endogenous synaptic proteins with various tags, with efficiencies up to 42% in mouse primary cultured neurons. Utilizing in utero electroporation or viral injections in mice TKIT can label AMPAR subunits with Super Ecliptic pHluorin, enabling visualization of endogenous AMPARs in vivo using two-photon microscopy. We further use TKIT to assess the mobility of endogenous AMPARs using fluorescence recovery after photobleaching. Finally, we show that TKIT can be used to tag AMPARs in rat neurons, demonstrating precise genome editing in another model organism and highlighting the broad potential of TKIT as a method to visualize endogenous proteins.

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An Optimized CRISPR/Cas9 Approach for Precise Genome Editing in Neurons

10.1101/2020.11.30.402883 ◽

2020 ◽

Author(s):

Huaqiang Fang ◽

Alexei M. Bygrave ◽

Richard H. Roth ◽

Richard C. Johnson ◽

Richard L. Huganir

Keyword(s):

Genome Editing ◽

Model Organism ◽

Synaptic Proteins ◽

Two Photon Microscopy ◽

Two Photon ◽

Coding Regions ◽

Dividing Cells ◽

Endogenous Proteins ◽

Super Ecliptic Phluorin

AbstractThe efficient knock-in of large DNA fragments to label endogenous proteins remains especially challenging in non-dividing cells such as neurons. We developed Targeted Knock-In with Two-guides (TKIT) as a novel CRISPR/Cas9 based approach for efficient, and precise, genomic knock-in. Through targeting non-coding regions TKIT is resistant to INDEL mutations. We demonstrate TKIT labelling of endogenous synaptic proteins with various tags, with efficiencies up to 42% in mouse primary cultured neurons. Utilizing in utero electroporation or viral injections in mice TKIT can label AMPAR subunits with Super Ecliptic pHluorin, enabling visualization of endogenous AMPARs in vivo using two-photon microscopy. We further use TKIT to assess the mobility of endogenous AMPARs using fluorescence recovery after photobleaching. Finally, we show that TKIT can be used to tag AMPARs in rat neurons, demonstrating precise genome editing in another model organism and highlighting the broad potential of TKIT as a method to visualize endogenous proteins.

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In vivo imaging of liver injury and regeneration by functional two-photon microscopy

Zeitschrift für Gastroenterologie ◽

10.1055/s-0036-1597342 ◽

2016 ◽

Vol 54 (12) ◽

pp. 1343-1404

Author(s):

A Ghallab ◽

R Reif ◽

R Hassan ◽

AS Seddek ◽

JG Hengstler

Keyword(s):

Liver Injury ◽

In Vivo Imaging ◽

Two Photon Microscopy ◽

Two Photon

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Sensory Transmission is Bi-directionally Modulated by Astrocytic Ca2+ in Barrel Cortex of Behaving Mice

10.21203/rs.3.rs-199750/v1 ◽

2021 ◽

Author(s):

Simeng Gu ◽

Wei Wang ◽

Kuan Zhang ◽

Rou Feng ◽

Naling Li ◽

...

Keyword(s):

Sensory Input ◽

Barrel Cortex ◽

Synaptic Function ◽

Metabolic Clearance ◽

Sleep State ◽

Two Photon Microscopy ◽

Two Photon ◽

Sensory Transmission

Abstract Different effects of astrocyte during sleep and awake have been extensively studied, especially for metabolic clearance by the glymphatic system, which works during sleep and stops working during waking states. However, how astrocytes contribute to modulation of sensory transmission during sleep and awake animals remain largely unknown. Recent advances in genetically encoded Ca2+ indicators have provided a wealth of information on astrocytic Ca2+, especially in their fine perisynaptic processes, where astrocytic Ca2+ most likely affects the synaptic function. Here we use two-photon microscopy to image astrocytic Ca2+ signaling in freely moving mice trained to run on a wheel in combination with in vivo whole-cell recordings to evaluate the role of astrocytic Ca2+ signaling in different behavior states. We found that there are two kinds of astrocytic Ca2+ signaling: a small long-lasting Ca2+ increase during sleep state and a sharp widespread but short-long-lasting Ca2+ spike when the animal was awake (fluorescence increases were 23.2 ± 14.4% for whisker stimulation at sleep state, compared with 73.3 ± 11.7% for at awake state, paired t-test, p < 0.01). The small Ca2+ transients decreased extracellular K+, hyperpolarized the neurons, and suppressed sensory transmission; while the large Ca2+ wave enhanced sensory input, contributing to reliable sensory transmission in aroused states. Locus coeruleus activation works as a switch between these two kinds of astrocytic Ca2+ elevation. Thus, we show that cortical astrocytes play an important role in processing of sensory input. These two types of events appear to have different pharmacological sources and may play a different role in facilitating the efficacy of sensory transmission.

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