Comments and Questions:


Comments:

As we know, CRISPR/Cas 9 system is a magic genetic scissor, which executes its gene-editing functionsin other words, repairing the mutant genes and inserting the ideal genes in vivo or in vitro. Recently, researchers have observed that the CRISPR/Cas 9 system can binds to not only the target gene locus, but also the non-specific homologous gene loci. To lessen the unexpectable and non-ideal occurrence, several ways have been carried out to identify which are the preferred ones. Finally, the authors mentioned the application of CRISPR/Cas 9 system, both in genome and epigenome area.

Questions:

  1. Which optimizing method is preferred for us to knock down or silence the expression of oxytocin gene in the brain cells of Spraque Dawley rats?

  2. How can we design the single guide RNA by using the genomic sequence from Human Genome Project?

  3. There exist several nice lab websites for most for us to seek the ideal gRNA.(Cited from DXY, their website: http://www.dxy.cn/)

    Target Finder: http://crispr.mit.edu/

    E-CRISPR: http://www.e-crispr.org/E-CRISPR/designcrispr.html

    CasFinder: http://arep.med.harvard.edu/CasFinder

    CRISPR Optimal Target Finder: http://tools.flycrispr.molbio.wisc.edu/targetFinder/

  4. How can we make sure that only the oxytocin gene is silenced rather than other homologous genes?

  5. As the authors mentioned, they use the directly intravenous injection to lessen the non-specific gene manipulation. Therefore, what is the most suitable definite time span between transfecting injection in vivo and decapitation (here , we mainly focus on the oxytocin gene in brain cells of SD rats)?

  6. If we just want to rectify the mutant genes or the “wrong” ones and do not decapitate the rats, how to ensure that the CRISPR/Cas 9 system injection of virus or plasmid cannot recognize the non-specific gene loci during the future life span of rats.


2018年06月21日

2018-5-18 Literature Analysis

Alexis C. Komor1,2,3, Ahmed H. Badran1,2,3, and David R. Liu1,2,3,* CRISPR-based technologies for the manipulation of eukaryotic genomes. Cell 2017 January 12; 168(1-2): 20–36. doi:10.1016/j.cell.2016.10.044. Presented by Haitao Liu,Dongyang Li, Yang liu and Dan Cui. Edited by Xiaoran Wang

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