Applications of CRISPR-Cas Technologies to Proteomics

Dolgalev, Georgii; Poverennaya, Ekaterina V.

doi:10.3390/genes12111790

Cited by 8 publications

(5 citation statements)

References 165 publications

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“…This approach can be extended to other targets and species of interest, for example genes important for aquaculture traits other than salinity tolerance and candidate genes other than myca, including those identi ed by previous GWAS and SNP analyses in multiple aquaculture sh species (Omeka et al 2022;Zhou et al 2022;Tsai et al 2015). These ko cell lines allow for deep functional analyses that associate speci c genotypes with complex phenotypes, including systems level molecular phenotypes revealed by transcriptomics (Xie et al 2020) and proteomics (Dolgalev and Poverennaya 2021).…”

Section: Interpretation Of Gene Editing Results By Tide and Ice Analysesmentioning

confidence: 99%

Removal of evolutionarily conserved functional MYC domains in a tilapia cell line using a vector-based CRISPR/Cas9 system

Kim

Cnaani

Kültz

2022

Preprint

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MYC transcription factors have critical roles in facilitating a variety of cellular functions that have been highly conserved among species during evolution. However, despite circumstantial evidence for an involvement of MYC in animal osmoregulation, mechanistic links between MYC function and osmoregulation are missing. Mozambique tilapia (Oreochromis mossambicus) represents an excellent model system to study these links because it is highly euryhaline and highly tolerant to osmotic (salinity) stress at both the whole organism and cellular levels of biological organization. Here, we utilize an O. mossambicus brain cell line (OmB cells) and an optimized vector-based CRISPR/Cas9 system to functionally knockout MYC in the tilapia genome and to establish causal links between MYC and cell function, including cellular osmoregulation. A cell isolation and dilution strategy yielded polyclonal MYC (myca) knockout cell pools with low genetic variability and high gene editing efficiencies (as high as 98.2 %). Further isolation and dilution of cells from these pools yielded a monoclonal myca ko cell line harboring a 1-bp deletion that caused a frameshift mutation. This frameshift functionally inactivated the transcriptional regulatory and DNA-binding domains predicted by bioinformatics and structural analyses. Both the polyclonal and monoclonal myca ko cell lines were viable, propagated well in standard medium, and differed from wild-type cells in morphology. As such, they represent a new tool for causally linking myca to cellular osmoregulation and other cell functions.

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Section: Interpretation Of Gene Editing Results By Tide and Ice Analysesmentioning

confidence: 99%

Removal of evolutionarily conserved functional MYC domains in a tilapia cell line using a vector-based CRISPR/Cas9 system

Kim

Cnaani

Kültz

2022

Preprint

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show abstract

“…This approach can be extended to other targets and species of interest, for example genes important for aquaculture traits other than salinity tolerance and candidate genes other than myca , including those identified by previous GWAS and SNP analyses in multiple aquaculture fish species 79 – 81 . These ko cell lines allow for deep functional analyses that associate specific genotypes with complex phenotypes, including systems level molecular phenotypes revealed by transcriptomics 82 and proteomics 83 .…”

Section: Discussionmentioning

confidence: 99%

Removal of evolutionarily conserved functional MYC domains in a tilapia cell line using a vector-based CRISPR/Cas9 system

Kim

Cnaani

Kültz

2023

Sci Rep

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MYC transcription factors have critical roles in facilitating a variety of cellular functions that have been highly conserved among species during evolution. However, despite circumstantial evidence for an involvement of MYC in animal osmoregulation, mechanistic links between MYC function and osmoregulation are missing. Mozambique tilapia (Oreochromis mossambicus) represents an excellent model system to study these links because it is highly euryhaline and highly tolerant to osmotic (salinity) stress at both the whole organism and cellular levels of biological organization. Here, we utilize an O. mossambicus brain cell line and an optimized vector-based CRISPR/Cas9 system to functionally disrupt MYC in the tilapia genome and to establish causal links between MYC and cell functions, including cellular osmoregulation. A cell isolation and dilution strategy yielded polyclonal myca (a gene encoding MYC) knockout (ko) cell pools with low genetic variability and high gene editing efficiencies (as high as 98.2%). Subsequent isolation and dilution of cells from these pools produced a myca ko cell line harboring a 1-bp deletion that caused a frameshift mutation. This frameshift functionally inactivated the transcriptional regulatory and DNA-binding domains predicted by bioinformatics and structural analyses. Both the polyclonal and monoclonal myca ko cell lines were viable, propagated well in standard medium, and differed from wild-type cells in morphology. As such, they represent a new tool for causally linking myca to cellular osmoregulation and other cell functions.

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“…Currently, quantitative proteomics is used to study the effect of protein knockout using CRISPR-Cas [ 194 ]. During genome editing, CRISPR-Cas technology uses many proteomic methods to study protein–protein and protein–chromatin interactions [ 195 ]. As an example, affinity purification with MS (AP-MS) protein–protein interactions can be studied precisely and on a large scale [ 196 ].…”

Section: Limitations and Potential Of Proteomics For Abiotic Stress T...mentioning

confidence: 99%

“…As an example, affinity purification with MS (AP-MS) protein–protein interactions can be studied precisely and on a large scale [ 196 ]. Combining CRISPR-Cas and AP-MS solves the challenges of mislocalization of proteins and their non-endogenous binding during genome editing [ 195 ]. In wheat, subcellular protein identification has been explored by combining CRISPR-Cas as it allows the direct insertion of fluorescent tags into the protein-coding gene frame [ 191 ].…”

Section: Limitations and Potential Of Proteomics For Abiotic Stress T...mentioning

confidence: 99%

Wheat Proteomics for Abiotic Stress Tolerance and Root System Architecture: Current Status and Future Prospects

et al. 2022

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Wheat is an important staple cereal for global food security. However, climate change is hampering wheat production due to abiotic stresses, such as heat, salinity, and drought. Besides shoot architectural traits, improving root system architecture (RSA) traits have the potential to improve yields under normal and stressed environments. RSA growth and development and other stress responses involve the expression of proteins encoded by the trait controlling gene/genes. Hence, mining the key proteins associated with abiotic stress responses and RSA is important for improving sustainable yields in wheat. Proteomic studies in wheat started in the early 21st century using the two-dimensional (2-DE) gel technique and have extensively improved over time with advancements in mass spectrometry. The availability of the wheat reference genome has allowed the exploration of proteomics to identify differentially expressed or abundant proteins (DEPs or DAPs) for abiotic stress tolerance and RSA improvement. Proteomics contributed significantly to identifying key proteins imparting abiotic stress tolerance, primarily related to photosynthesis, protein synthesis, carbon metabolism, redox homeostasis, defense response, energy metabolism and signal transduction. However, the use of proteomics to improve RSA traits in wheat is in its infancy. Proteins related to cell wall biogenesis, carbohydrate metabolism, brassinosteroid biosynthesis, and transportation are involved in the growth and development of several RSA traits. This review covers advances in quantification techniques of proteomics, progress in identifying DEPs and/or DAPs for heat, salinity, and drought stresses, and RSA traits, and the limitations and future directions for harnessing proteomics in wheat improvement.

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Applications of CRISPR-Cas Technologies to Proteomics

Cited by 8 publications

References 165 publications

Removal of evolutionarily conserved functional MYC domains in a tilapia cell line using a vector-based CRISPR/Cas9 system

Removal of evolutionarily conserved functional MYC domains in a tilapia cell line using a vector-based CRISPR/Cas9 system

Removal of evolutionarily conserved functional MYC domains in a tilapia cell line using a vector-based CRISPR/Cas9 system

Wheat Proteomics for Abiotic Stress Tolerance and Root System Architecture: Current Status and Future Prospects

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