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2

Whatever sequence (plus direction) is target, so enzyme will cut. In your example "correct" and "antiparallel" sequences are two different sites. Actually, your example of 5'-CTTAAG-3' is cut by AflII enzyme.


-1

I found my answer here ISSUES RELATED TO PARTICULAR CLASSES OF VECTORS FOR GENE THERAPY http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/CellularandGeneTherapy/ucm072987.htm#vi


3

You can do with a single gRNA. All that CRISPR-Cas, ZFN or TALEN systems do is to introduce a double strand break at a specific site. The DNA gets repaired via two mechanisms — non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ is error prone and it may introduce indels that can compromise with gene function (frameshifts etc). While HR ...


1

The thing we must remember is that during evolution, there occurs many events that may result in genomic reshuffling. Considering your example of MHC, when we look into the evolution of the complexes The classical human MHC contains 224 genes, .... Antibody and T cell mediated immune responses against invading pathogens are initiated through MHC class ...


7

It is called conditional mutation. You flox (put lox sites around) gene of interest and express Cre recombinase driven by tissue-of-interest-specific promoter. Illustration from here: Using chemically-activatable variant of Cre recombinase (cre-ER) you can create knock-out in some cells of tissue of interest, not every. Addition: a bit weird but still ...


7

To be specific: I am talking about adult, somatic gene therapy here, and germline gene therapy experiments is still a landmine when considering ethical reasons. The defective gene codes for a defective protein, that usually plays a part in pathways. Since the protein is also defective, that pathway is also rendered defective because of this protein, and ...



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