رؤى - Neurobiology - # Conditional gene manipulation of chemical transmission genes

Conditional Chemoconnectomics: A Highly Efficient Toolkit for Targeted Manipulation of Chemical Transmission Genes in Drosophila

Q: How could the conditional CCTomics toolkit be further expanded or optimized to enable more comprehensive and efficient manipulation of neural circuits?

The conditional CCTomics toolkit can be expanded and optimized in several ways to enhance the manipulation of neural circuits. One approach could involve the development of additional conditional gene manipulation strategies beyond Flp-out/GFPi or CRISPR/Cas9. For example, incorporating inducible systems like the Tet-On or Tet-Off system could provide temporal control over gene expression or knockout. This would allow researchers to investigate the dynamic changes in neural circuits over time. Furthermore, optimizing the efficiency and specificity of the CRISPR/Cas9 system by fine-tuning the sgRNA design and Cas9 variants could improve the accuracy of gene manipulation. Utilizing advanced technologies such as base editing or prime editing could enable precise modifications without inducing double-strand breaks, reducing the risk of off-target effects. Expanding the CCTomics toolkit to include a wider range of genes related to neural circuitry and behavior would also be beneficial. Creating libraries of UAS-sgRNAs targeting a broader set of genes involved in different neural processes could facilitate more comprehensive studies of neural circuits and their functions.

Q: What other behavioral or physiological processes beyond circadian rhythms could be investigated using the conditional CCTomics approach?

The conditional CCTomics approach can be applied to investigate a wide range of behavioral and physiological processes beyond circadian rhythms. One potential area of study could be the regulation of learning and memory processes in the brain. By manipulating genes related to synaptic plasticity, neurotransmitter signaling, or neuronal development in specific brain regions, researchers could uncover the molecular mechanisms underlying learning and memory formation. Additionally, the conditional CCTomics toolkit could be used to explore the neural circuits involved in sensory processing, such as olfaction, vision, or touch. By targeting genes associated with sensory perception and signal transduction, researchers could elucidate how sensory information is processed and integrated in the brain to generate appropriate behavioral responses. Furthermore, the conditional manipulation of genes involved in stress responses, social behaviors, or motor coordination could provide insights into the neural circuits underlying these complex behaviors. By selectively modulating gene expression in specific neuronal populations, researchers can dissect the neural circuits that regulate these behaviors and identify potential therapeutic targets for related disorders.

Q: Could the chromatin-modulating Cas9 variants developed in this study be applied to gene manipulation in other model organisms or cell types to enhance editing efficiency?

Yes, the chromatin-modulating Cas9 variants developed in this study could be applied to gene manipulation in other model organisms or cell types to enhance editing efficiency. The modified Cas9 variants, such as Cas9.M6 and Cas9.M9, have shown improved gene disruption efficiency compared to the unmodified Cas9.HC in Drosophila. These variants could be utilized in other model organisms like mice, zebrafish, or C. elegans to enhance the precision and efficacy of gene editing. In cell culture studies, the chromatin-modulating Cas9 variants could be valuable for manipulating gene expression in specific cell types or studying the regulation of gene networks. By incorporating these variants into CRISPR/Cas9 systems, researchers can achieve more targeted and efficient gene editing, leading to a better understanding of cellular processes and disease mechanisms. Overall, the application of chromatin-modulating Cas9 variants in other model systems or cell types has the potential to advance research in various fields, including developmental biology, neuroscience, and disease modeling. Their enhanced efficiency and specificity make them valuable tools for precise genetic manipulation and functional genomics studies.

المفاهيم الأساسية

The authors have developed highly efficient conditional gene manipulation systems, including GFP-RNAi/Flp-out and CRISPR/Cas9-based approaches, to target all chemical transmission (chemoconnectome, CCT) genes in Drosophila. These tools enable detailed dissection of neural circuits underlying specific behaviors.

الملخص

The authors have developed two conditional gene manipulation systems, cCCTomics and C-cCCTomics, to target all chemical transmission (CCT) genes in Drosophila.

cCCTomics utilizes GFP-RNAi and Flp-out strategies to achieve near-complete disruption of target CCT genes. This system leverages previously generated CCT knockin lines, where the GFP coding sequence is fused to the 3' end of each gene and the gene span is flanked by FRT sequences. Expressing GFP-RNAi or Flp recombinase can efficiently eliminate GFP signals, indicating effective gene disruption.

To further simplify the conditional manipulation of CCT genes, the authors developed C-cCCTomics, a CRISPR/Cas9-based system. They generated UAS-sgRNA transgenic lines targeting all 209 defined CCT genes and combined them with UAS-Cas9 variants. The authors found that Cas9 variants fused with chromatin-modulating peptides, Cas9.M6 and Cas9.M9, exhibited significantly higher gene disruption efficiency compared to the unmodified Cas9.

The authors then applied these conditional gene manipulation tools to dissect the chemoconnectome of Drosophila clock neurons. Intersecting Clk856-Gal4 with CCT gene knockin lines, they identified 43 CCT genes expressed in various subsets of clock neurons.

Further functional analysis using C-cCCTomics revealed that the neuropeptide CNMa and its receptor CNMaR play an antagonistic role with PDF-PDFR signaling in regulating morning anticipation behavior. Specifically, knockout of CNMa or CNMaR in clock neurons, particularly the PDF-PDFR co-expressing DN1p neurons, led to advanced morning activity.

These results demonstrate the effectiveness of the authors' conditional CCTomics toolkit and its application in dissecting neural circuits underlying complex behaviors.

تخصيص الملخص

إعادة الكتابة بالذكاء الاصطناعي

إنشاء الاستشهادات

ترجمة المصدر

إلى لغة أخرى

إنشاء خريطة ذهنية

من محتوى المصدر

زيارة المصدر

biorxiv.org

الإحصائيات

The authors report that pan-neuronal expression of GFP-RNAi or Flp recombinase in cCCT flies almost completely eliminated GFP signals, indicating near-complete disruption of target genes.
Cas9.M6 and Cas9.M9 exhibited significantly higher gene disruption efficiency (87.51±2.24% and 89.59±2.39%, respectively) compared to unmodified Cas9.HC (70.72±3.82%) when targeting 14 additional CCT genes.
Conditional knockout of nAChRβ2 or nAChRα2 using Cas9.M9 significantly decreased fly sleep, similar to their mutant phenotypes.

اقتباسات

"Knocking out of Pdf or Pdfr in clock neurons phenocopied their mutants with lower MAI, advanced evening activity, low power, high arrhythmic rate and shorter period."
"Only the Pdf and Pdfr clock-neuron knockout flies showed positive EAPIs, indicating an advanced evening activity."
"CNMa knockout in Type I or Type II neurons (GMR51H05-GAL4, GMR91F02-GAL4, and GMR79A11-GAL4) all reproduced the MAPI-increased phenotype of clk856 specific CNMa knockout."

الرؤى الأساسية المستخلصة من

Conditional Chemoconnectomics (cCCTomics): Conditional Targeting of Chemical Transmission Efficiently

by Mao,R., Yu,J... في www.biorxiv.org 09-27-2023

https://www.biorxiv.org/content/10.1101/2023.09.26.559642v2

استفسارات أعمق

How could the conditional CCTomics toolkit be further expanded or optimized to enable more comprehensive and efficient manipulation of neural circuits?

The conditional CCTomics toolkit can be expanded and optimized in several ways to enhance the manipulation of neural circuits. One approach could involve the development of additional conditional gene manipulation strategies beyond Flp-out/GFPi or CRISPR/Cas9. For example, incorporating inducible systems like the Tet-On or Tet-Off system could provide temporal control over gene expression or knockout. This would allow researchers to investigate the dynamic changes in neural circuits over time.
Furthermore, optimizing the efficiency and specificity of the CRISPR/Cas9 system by fine-tuning the sgRNA design and Cas9 variants could improve the accuracy of gene manipulation. Utilizing advanced technologies such as base editing or prime editing could enable precise modifications without inducing double-strand breaks, reducing the risk of off-target effects.
Expanding the CCTomics toolkit to include a wider range of genes related to neural circuitry and behavior would also be beneficial. Creating libraries of UAS-sgRNAs targeting a broader set of genes involved in different neural processes could facilitate more comprehensive studies of neural circuits and their functions.

What other behavioral or physiological processes beyond circadian rhythms could be investigated using the conditional CCTomics approach?

The conditional CCTomics approach can be applied to investigate a wide range of behavioral and physiological processes beyond circadian rhythms. One potential area of study could be the regulation of learning and memory processes in the brain. By manipulating genes related to synaptic plasticity, neurotransmitter signaling, or neuronal development in specific brain regions, researchers could uncover the molecular mechanisms underlying learning and memory formation.
Additionally, the conditional CCTomics toolkit could be used to explore the neural circuits involved in sensory processing, such as olfaction, vision, or touch. By targeting genes associated with sensory perception and signal transduction, researchers could elucidate how sensory information is processed and integrated in the brain to generate appropriate behavioral responses.
Furthermore, the conditional manipulation of genes involved in stress responses, social behaviors, or motor coordination could provide insights into the neural circuits underlying these complex behaviors. By selectively modulating gene expression in specific neuronal populations, researchers can dissect the neural circuits that regulate these behaviors and identify potential therapeutic targets for related disorders.

Could the chromatin-modulating Cas9 variants developed in this study be applied to gene manipulation in other model organisms or cell types to enhance editing efficiency?

Yes, the chromatin-modulating Cas9 variants developed in this study could be applied to gene manipulation in other model organisms or cell types to enhance editing efficiency. The modified Cas9 variants, such as Cas9.M6 and Cas9.M9, have shown improved gene disruption efficiency compared to the unmodified Cas9.HC in Drosophila. These variants could be utilized in other model organisms like mice, zebrafish, or C. elegans to enhance the precision and efficacy of gene editing.
In cell culture studies, the chromatin-modulating Cas9 variants could be valuable for manipulating gene expression in specific cell types or studying the regulation of gene networks. By incorporating these variants into CRISPR/Cas9 systems, researchers can achieve more targeted and efficient gene editing, leading to a better understanding of cellular processes and disease mechanisms.
Overall, the application of chromatin-modulating Cas9 variants in other model systems or cell types has the potential to advance research in various fields, including developmental biology, neuroscience, and disease modeling. Their enhanced efficiency and specificity make them valuable tools for precise genetic manipulation and functional genomics studies.