{"id":87,"date":"2015-04-11T16:53:07","date_gmt":"2015-04-11T23:53:07","guid":{"rendered":"https:\/\/blogs.ubc.ca\/ryanyenn\/?p=87"},"modified":"2015-04-11T16:53:07","modified_gmt":"2015-04-11T23:53:07","slug":"speed-dating-crispr-cas","status":"publish","type":"post","link":"https:\/\/blogs.ubc.ca\/ryanyenn\/assignments\/speed-dating-crispr-cas\/","title":{"rendered":"Speed Dating: CRISPR-Cas"},"content":{"rendered":"

TECHNIQUES SPEED-DATING PRESENTATIONS: WRITE-UP<\/strong>
\nNames and contributions of group members:
\nMandy Feng: in-depth research for mini report, prop preparation, final editing
\nJordan Henriksen: group coordination, in-depth research for mini report, prop preparation
\nPhillip Chau: in-depth research for mini report, final editing
\nAbhijit Parolia: in-depth research for mini report, final editing
\nRyan Yen: in-depth research for mini report, final editing<\/p>\n

Video: CRISPR-Cas Technique<\/strong>
\nhttps:\/\/www.youtube.com\/watch?v=MXDwHBMDq8g<\/a><\/p>\n

Technique chosen:<\/strong>
\nCRISPR-Cas Technology: Clustered, Regularly Interspaced, Short Palindromic Repeats-CRISPR Associated<\/p>\n

What does this technique \u2018do\u2019?<\/strong>
\nIntroducing double-stranded breaks in DNA in a sequence-specific manner<\/p>\n

What applications is this technique employed for?<\/strong>
\nThe central purpose of employing this technique is to study gene function in\u00a0physiological and diseased states by one of the following methods:
\n1. Simultaneous editing of multiple genes mediated by exogenous small guide RNAs\u00a0(sgRNA) and Cas9 nuclease complex (Pennisi, 2013).<\/p>\n

-Wang et al. (2013) verified mutations in all of the 5 targeted gene\u00a0sequences in a single \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 eukaryotic cell using gene-specific sgRNAs.
\n2. Reversible gene knockdowns, which is a complementary technique to RNA\u00a0interference (Pennisi, 2013).<\/p>\n

-In a prokaryotic cell, Qi et al. (2013) demonstrated ~100-fold\u00a0downregulation of a reporter gene using \u2018dead\u2019 Cas9 (dCas9):sgRNA\u00a0complex directed to the -35 box, upstream of the coding sequence.
\n-In an eukaryotic system, which involves more robust regulatory control\u00a0mechanisms, this approach is enhanced by fusion of dCas9 to a\u00a0mammalian transcriptional repressor domain. (Gilbert et al., 2013).
\n3. Activation of specific genes by delivery of synthetic transcriptional activators to\u00a0promoter sequences (Gilbert et al., 2013).
\n-Gilbert et al. (2013) demonstrated that fusion of dCas9 (still directed to the\u00a0gene promoter) with transcriptional activators effectively resulted in target\u00a0gene up-regulation, in some cases by >25-fold.<\/p>\n

Some additional applications of this technology include:<\/strong>
\n\u2022 Generating gRNA libraries
\n\u2022 Labeling specific chromosomal loci (Sander & Joung, 2014).<\/p>\n

What questions (give a couple of examples) relating to gene regulation and\/or\u00a0<\/strong>development can be addressed using this technique?<\/strong>
\n\u2022 Loss of Function Experiments
\no Is this gene\/protein necessary for a particular function?
\no Enables targeted genome editing by insertion or deletion of DNA\u00a0sequences.
\n\u00a7 If you simultaneously disrupt an enhancer in an ELP4 intron and\u00a0the enhancer SIMO using the CRISPR-Cas technique, how is\u00a0PAX6 expression affected (Bhatia & Kleinjan, 2014)?
\n\u2022 Gain of Function Experiments
\no Is this gene\/protein sufficient to cause a change?
\no Allows for over-expression analysis through mutation or the delivery of\u00a0synthetic transcription factors.
\n\u00a7 If you use the CRISPR-Cas technique to induce a mutation in an\u00a0enhancer that increases the binding affinity of a particular\u00a0transcription factor for that enhancer, how does this affect\u00a0development?<\/p>\n

What critical reagents are required to use this technique?<\/strong>
\nBased on the paper by Sander & Joung (2014), the following reagents are required:
\n\u2022 RNA-guided nuclease
\no Cas enzyme specific for required function
\n\u2022 Specific sequence guide RNA (gRNA)
\no CRISPR RNA (crRNA) and transactivating CRISPR RNA (tracrRNA)
\n\u2022 Non-replicating plasmid
\no For expression of Cas enzyme and gRNA
\n\u2022 Transfection reagents
\no Such as electroporation, nucleofection, or Lipofectamine.<\/p>\n

What critical information is required to be able to employ this technique?<\/strong>
\n1. DNA sequence of the gene-of-interest – The \u2018seed\u2019 region of the sgRNA is\u00a0restricted by mandatory presence of a protospacer adjacent motif (5\u2019 NGG) for the\u00a0Cas9 nuclease activity to occur (Pennisi, 2013).
\n2. Choice of promoter of the transgenic sgRNA and Cas9 genes – for instance, it\u00a0needs to be compatible with the eukaryotic transcriptional machinery (Sander &\u00a0Joung, 2014).
\n3. Empirically established regulatory protein domains that can affect expression of\u00a0the target gene – for example, it is critical to know if VP16 can act as a\u00a0transcriptional activation domain for the gene in question (Gilbert et al., 2013).
\n4. Configuration of the viral capsid to allow specific targeting of cells – more so in\u00a0the case of in vivo application (Sander & Joung, 2014).<\/p>\n

References:<\/strong>
\nBhatia, S., Kleinjan, D. A. (2014). Disruption of long-range gene regulation in human\u00a0genetic disease: a kaleidoscope of general principles, diverse mechanism and\u00a0unique phenotypic consequences. Hum Genet 133, 815-845.<\/p>\n

Gilbert, L. A., Larson, M. H., Morsut, L., Liu, M., Brar, G. A., Torres, S. E., Stern-Ginossar, N\u2026 Qi, L. (2013). CRISPR-Mediated Modular RNA-Guided\u00a0Regulation of Transcription in Eukaryotes. Cell 154, 442-451.<\/p>\n

Pennisi, E. (2013). The CRISPR Craze. Science 341, 833-836.<\/p>\n

Qi, L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A., Weissman, J. S., Arkin, A. P.,\u00a0Lim, W. A. (2013). Repurposing CRISPR as an RNA-Guided Platform for\u00a0Sequence-Specific Control of Gene Expression. Cell 152(5), 1173-1183.<\/p>\n

Sander, J. D., & Joung, J. K. (2014). CriSPr-Cas systems for editing, regulating and\u00a0targeting genomes. Nature Biotechnology, 32(4), 347-355.<\/p>\n

Wang, H. (2013). One-Step Generation of Mice Carrying Mutations in Multiple Genes\u00a0by CRISPR\/Cas-Mediated Genome Engineering. Cell 153, 910-918.<\/p>\n","protected":false},"excerpt":{"rendered":"

TECHNIQUES SPEED-DATING PRESENTATIONS: WRITE-UP Names and contributions of group members: Mandy Feng: in-depth research for mini report, prop preparation, final editing Jordan Henriksen: group coordination, in-depth research for mini report, prop preparation Phillip Chau: in-depth research for mini report, final editing Abhijit Parolia: in-depth research for mini report, final editing Ryan Yen: in-depth research for […]<\/p>\n","protected":false},"author":28747,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[98],"tags":[],"class_list":["post-87","post","type-post","status-publish","format-standard","hentry","category-assignments"],"_links":{"self":[{"href":"https:\/\/blogs.ubc.ca\/ryanyenn\/wp-json\/wp\/v2\/posts\/87","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/blogs.ubc.ca\/ryanyenn\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/blogs.ubc.ca\/ryanyenn\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/blogs.ubc.ca\/ryanyenn\/wp-json\/wp\/v2\/users\/28747"}],"replies":[{"embeddable":true,"href":"https:\/\/blogs.ubc.ca\/ryanyenn\/wp-json\/wp\/v2\/comments?post=87"}],"version-history":[{"count":1,"href":"https:\/\/blogs.ubc.ca\/ryanyenn\/wp-json\/wp\/v2\/posts\/87\/revisions"}],"predecessor-version":[{"id":88,"href":"https:\/\/blogs.ubc.ca\/ryanyenn\/wp-json\/wp\/v2\/posts\/87\/revisions\/88"}],"wp:attachment":[{"href":"https:\/\/blogs.ubc.ca\/ryanyenn\/wp-json\/wp\/v2\/media?parent=87"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/blogs.ubc.ca\/ryanyenn\/wp-json\/wp\/v2\/categories?post=87"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/blogs.ubc.ca\/ryanyenn\/wp-json\/wp\/v2\/tags?post=87"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}