{"id":6616,"date":"2015-12-09T07:01:22","date_gmt":"2015-12-09T12:01:22","guid":{"rendered":"http:\/\/admin.acceleratingscience.com\/?p=6616"},"modified":"2016-08-30T20:29:58","modified_gmt":"2016-08-30T20:29:58","slug":"snip-snip-crispr-cas9-genomic-editing-characterizes-the-prion-protein-knockout-proteome","status":"publish","type":"post","link":"https:\/\/www.thermofisher.com\/blog\/proteomics\/snip-snip-crispr-cas9-genomic-editing-characterizes-the-prion-protein-knockout-proteome\/","title":{"rendered":"Snip Snip! CRISPR-Cas9 Genomic Editing Characterizes the Prion Protein Knockout Proteome"},"content":{"rendered":"<p><img loading=\"lazy\" decoding=\"async\" style=\"float: left; margin: 10px;\" src=\"http:\/\/admin.acceleratingscience.com\/proteomics\/wp-content\/uploads\/sites\/2\/2015\/12\/shutterstock_102810086.jpg\" alt=\"Human prion protein. Image: molekuul.be\/Shutterstock.om\" width=\"330\" height=\"219\" \/>Although prion proteins are widely implicated in the pathogenesis of several neurodegenerative diseases, including Alzheimer&#8217;s in animals and humans, their normal physiological function is still relatively unknown. However, Mehrabian et al. (2014) recently published a cell-specific deep proteome analysis that gives the first clues into prion protein function. Using <a href=\"https:\/\/www.thermofisher.com\/us\/en\/home\/life-science\/genome-editing\/geneart-crispr\/crispr-cas9-based-genome-editing.html\">CRISPR-Cas9 technology<\/a>, the researchers created knockout cell clones and then examined changes in the proteome associated with deleting PrP<sup>C<\/sup>, the cellular form of prion protein.<\/p>\n<p>In order to uncover the physiological effects of deleting PrP<sup>C <\/sup>from the cell, Mehrabian and colleagues used CRISPR-Cas9 genomic editing to establish a knockout clone using various murine cell lines. These included the N2a neuroblastoma cells, C2C12 myoblasts and NMuMG epithelial cells. Although the researchers noted that knockout mouse models do exist, there have been no studies of the effects of deleting <em>Prnp <\/em>in single populations of cells. The researchers thought that by studying cells in isolation, they could avoid potential masking through the compensatory actions of surrounding tissues.<\/p>\n<p>First, the researchers created the knockout clone line, devising a successful CRISPR-Cas9 strategy and then applying the methodology to the three different cell lines. They confirmed successful deletion in each before continuing the investigation using the NMuMG epithelial cells.<\/p>\n<p>As controls, the team used both wild type NMuMG cells and another clone stably transfected with a PrP shRNA that reduced protein expression by 75%. Following cell culture, the team harvested the cells and lysed them before digesting the proteins using trypsin. They then used<a href=\"https:\/\/www.thermofisher.com\/ca\/en\/home\/life-science\/protein-biology\/protein-mass-spectrometry-analysis\/protein-quantitation-mass-spectrometry\/tandem-mass-tag-systems.html\" target=\"_blank\"> isobaric tandem mass tagging (TMT) techniques<\/a> (Thermo Scientific) for multiplex labeling of the peptide preparation. Mehrabian et al. performed a deep proteomic analysis of the labeled cell digests using an EASY-nLC 1100 system coupled with an <a href=\"http:\/\/www.thermoscientific.com\/en\/product\/orbitrap-fusion-tribrid-mass-spectrometer.html\" target=\"_blank\">Orbitrap Fusion Tribrid mass spectrometer <\/a>(both Thermo Scientific). They used <a href=\"http:\/\/www.thermoscientific.com\/en\/product\/proteome-discoverer-software.html\" target=\"_blank\">Proteome Discover software (revision 1.4 <\/a>Thermo Scientific) for spectral data analysis.<\/p>\n<p>The proteomic analysis revealed 201 proteins with changed abundance relative to wild type in the knockout cells and 200 proteins affected in the stable transfection control group. O<span class=\"thread\">f<\/span> these, around 120 proteins showed consistent alterations, both upregulation and downregulation, in each of the experimental cell lines with reduced or absent PrP abundance. Mehrabian et al. note that the changes observed are modest, with nothing greater than a three-fold change in abundance recorded for the proteomic sets.<\/p>\n<p>The research used gene ontology annotations to examine biological processes affected by PrP<sup>C<\/sup> deficiency. They found that PrP<sup>C<\/sup> deletion\/reduction altered the abundance of proteins found in the extracellular region, at cell junctions and within the cytoskeleton. Pathways associated with these altered proteins included those involved in adhesion and differentiation.<\/p>\n<p>Recognizing the advantages in their mass spectrometry approach to quantifying proteome changes, Mehrabian et al. conclude that CRISPR-Cas9 genome editing offers a useful complementary approach to existing in vivo and in vitro models examining PrP<sup>C<\/sup> knockout or interference. The team suggests that offering a single cell population approach could benefit research into downstream PrP<sup>C<\/sup> signaling events, enabling a more detailed oversight of prion cellular biology and its role in neurodegenerative disease pathogenesis.<\/p>\n<p><strong>Reference<\/strong><\/p>\n<p>1. Mehrabian, M., et al. (2014) &#8220;<a href=\"http:\/\/www.ncbi.nlm.nih.gov\/pubmed\/25490046\" target=\"_blank\">CRISPR-Cas9-based knockout of the prion protein and its effect on the proteome<\/a>,&#8221; PLoS ONE, 9(12), e114594. <a href=\"http:\/\/journals.plos.org\/plosone\/article?id=10.1371\/journal.pone.0114594\" target=\"_blank\">doi:10.1371\/journal.pone.0114594<\/a><\/p>\n<p><strong>\u00a0<\/strong><\/p>\n","protected":false},"excerpt":{"rendered":"<p>Although prion proteins are widely implicated in the pathogenesis of several neurodegenerative diseases, including Alzheimer&#8217;s in animals and humans, their normal physiological function is still relatively unknown. However, Mehrabian et al. (2014) recently published a cell-specific deep proteome analysis that gives the first clues into prion protein function. Using CRISPR-Cas9 technology, the researchers created knockout<\/p>\n","protected":false},"author":21,"featured_media":6777,"comment_status":"open","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"_monsterinsights_skip_tracking":false,"_genesis_hide_title":false,"_genesis_hide_breadcrumbs":false,"_genesis_hide_singular_image":false,"_genesis_hide_footer_widgets":false,"_genesis_custom_body_class":"","_genesis_custom_post_class":"","_genesis_layout":"","_jetpack_newsletter_access":"","_jetpack_dont_email_post_to_subs":false,"_jetpack_newsletter_tier_id":0,"_jetpack_memberships_contains_paywalled_content":false,"_jetpack_memberships_contains_paid_content":false,"footnotes":""},"categories":[131],"tags":[604,605,606,396,607],"division":[],"class_list":{"0":"post-6616","1":"post","2":"type-post","3":"status-publish","4":"format-standard","5":"has-post-thumbnail","7":"category-genetics","8":"tag-crispr-cas9","9":"tag-genomic-editing","10":"tag-prion-protein","11":"tag-proteome","12":"tag-prp","13":"entry"},"_selected_authors":"","_selected_reviewers":"","acf":[],"yoast_head":"<!-- This site is optimized with the Yoast SEO Premium plugin v27.8 (Yoast SEO v27.8) - https:\/\/yoast.com\/product\/yoast-seo-premium-wordpress\/ -->\n<title>Genomic Editing Characterizes the Prion Protein Knockout Proteome<\/title>\n<meta name=\"description\" content=\"Using CRISPR-Cas9 genomic editing, researchers create knockout cell populations to study prion protein deletion and its effect on the proteome.\" \/>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/www.thermofisher.com\/blog\/proteomics\/snip-snip-crispr-cas9-genomic-editing-characterizes-the-prion-protein-knockout-proteome\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Snip Snip! 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