Question

1. Explain the difference in the inhibitory mechanisms between small interfering RNA (siRNA) and Dominant negative. Also, explain how you can detect their inhibitory effect (i.e. quantitative PCR, Western-blotting using what type of antibody).

2. Explain/draw how CRISPR/Cas9 generate homozygote of the mutated gene. Use terms: double-strand break, guide RNA

please keep it simple so i can understand (:

Answer 1

1. Small interfering RNA (siRNA) or silencing RNA is double stranded RNA with 20-25 base pairs.They are introduced into the cell by transfection. siRNA prevents translation by gene silencing.

siRNA is formed by the cleavage of long dsRNA by an endo-ribonuclease called Dicer , after that the siRNA enters the cell. Then the functional siRNAs are recognized by the Ago2/RISC complex(RNA-Induced Silencing Complex) which degrades one strand and the other strand guides the inhibitory complex to complementary mRNA.Then the targeted mRNAs are degraded through cleavage by the RISC complex. As a result the cut mRNA is recognised as abnormal and no translation occurs. This is the gene silencing method by siRNA.

Dominant negative inhibition is commonly seen when a a mutant subunit of a multisubunit protein is coexpressed with the wild-type protein. As a result the functional oligomer is silenced. In other words we can say that the function of a wild type gene product is silenced by the coexpressed mutant variant of the same gene product. This type of inhibition is fragment-specific. if an inhibitory fragment binds with the intact enzyme (mutated) it forms a complex which will leads to enzyme inactivation. And this complex will interrupt the folding pathway of our wild type enzyme.

Detection of this type of inhibition is done by  Immunoprecipitation. The inhibitory fragment is transferred into a plasmid and label it with some epitope tags at the C terminal. This is done with the help of a PCR . The coexpressing genes are also transformed. After that cell extracts are taken and immunoprecipitation is carried out with the appropriate materials. The immunoprecipitated sample is washed and electrophoresed on a SDS polyacrylamide gel. Then the proteins in the gel are electroblotted onto polyvinylidene difluoride membranes and detect using some chemiluminescence system.

2. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-associated (Cas) genes are used to repair mutations. CRISPR is designed accordingly.

DNA from viruses is cut into small fragments and they are incorporated into CRISPR (a series of short repeats). Then these genes are transcribed , after that the transcripts are processed to generate small RNAs called crRNA (CRISPR RNA). This new crRNA  will guide the effector endonucleases to target DNA based on sequence complementarity . The endonucleases will cause a double strand break at the site of mutation and the donor DNA that contained a synonymous codon for the correct amino acid which will repair the cut with the correct sequence.