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 mini report, final editing
Video: CRISPR-Cas Technique
https://www.youtube.com/watch?v=MXDwHBMDq8g
Technique chosen:
CRISPR-Cas Technology: Clustered, Regularly Interspaced, Short Palindromic Repeats-CRISPR Associated
What does this technique ‘do’?
Introducing double-stranded breaks in DNA in a sequence-specific manner
What applications is this technique employed for?
The central purpose of employing this technique is to study gene function in physiological and diseased states by one of the following methods:
1. Simultaneous editing of multiple genes mediated by exogenous small guide RNAs (sgRNA) and Cas9 nuclease complex (Pennisi, 2013).
-Wang et al. (2013) verified mutations in all of the 5 targeted gene sequences in a single eukaryotic cell using gene-specific sgRNAs.
2. Reversible gene knockdowns, which is a complementary technique to RNA interference (Pennisi, 2013).
-In a prokaryotic cell, Qi et al. (2013) demonstrated ~100-fold downregulation of a reporter gene using ‘dead’ Cas9 (dCas9):sgRNA complex directed to the -35 box, upstream of the coding sequence.
-In an eukaryotic system, which involves more robust regulatory control mechanisms, this approach is enhanced by fusion of dCas9 to a mammalian transcriptional repressor domain. (Gilbert et al., 2013).
3. Activation of specific genes by delivery of synthetic transcriptional activators to promoter sequences (Gilbert et al., 2013).
-Gilbert et al. (2013) demonstrated that fusion of dCas9 (still directed to the gene promoter) with transcriptional activators effectively resulted in target gene up-regulation, in some cases by >25-fold.
Some additional applications of this technology include:
• Generating gRNA libraries
• Labeling specific chromosomal loci (Sander & Joung, 2014).
What questions (give a couple of examples) relating to gene regulation and/or development can be addressed using this technique?
• Loss of Function Experiments
o Is this gene/protein necessary for a particular function?
o Enables targeted genome editing by insertion or deletion of DNA sequences.
§ If you simultaneously disrupt an enhancer in an ELP4 intron and the enhancer SIMO using the CRISPR-Cas technique, how is PAX6 expression affected (Bhatia & Kleinjan, 2014)?
• Gain of Function Experiments
o Is this gene/protein sufficient to cause a change?
o Allows for over-expression analysis through mutation or the delivery of synthetic transcription factors.
§ If you use the CRISPR-Cas technique to induce a mutation in an enhancer that increases the binding affinity of a particular transcription factor for that enhancer, how does this affect development?
What critical reagents are required to use this technique?
Based on the paper by Sander & Joung (2014), the following reagents are required:
• RNA-guided nuclease
o Cas enzyme specific for required function
• Specific sequence guide RNA (gRNA)
o CRISPR RNA (crRNA) and transactivating CRISPR RNA (tracrRNA)
• Non-replicating plasmid
o For expression of Cas enzyme and gRNA
• Transfection reagents
o Such as electroporation, nucleofection, or Lipofectamine.
What critical information is required to be able to employ this technique?
1. DNA sequence of the gene-of-interest – The ‘seed’ region of the sgRNA is restricted by mandatory presence of a protospacer adjacent motif (5’ NGG) for the Cas9 nuclease activity to occur (Pennisi, 2013).
2. Choice of promoter of the transgenic sgRNA and Cas9 genes – for instance, it needs to be compatible with the eukaryotic transcriptional machinery (Sander & Joung, 2014).
3. Empirically established regulatory protein domains that can affect expression of the target gene – for example, it is critical to know if VP16 can act as a transcriptional activation domain for the gene in question (Gilbert et al., 2013).
4. Configuration of the viral capsid to allow specific targeting of cells – more so in the case of in vivo application (Sander & Joung, 2014).
References:
Bhatia, S., Kleinjan, D. A. (2014). Disruption of long-range gene regulation in human genetic disease: a kaleidoscope of general principles, diverse mechanism and unique phenotypic consequences. Hum Genet 133, 815-845.
Gilbert, L. A., Larson, M. H., Morsut, L., Liu, M., Brar, G. A., Torres, S. E., Stern-Ginossar, N… Qi, L. (2013). CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes. Cell 154, 442-451.
Pennisi, E. (2013). The CRISPR Craze. Science 341, 833-836.
Qi, L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A., Weissman, J. S., Arkin, A. P., Lim, W. A. (2013). Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell 152(5), 1173-1183.
Sander, J. D., & Joung, J. K. (2014). CriSPr-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology, 32(4), 347-355.
Wang, H. (2013). One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering. Cell 153, 910-918.