Virology Report: CRISPR/Cas9 Gene Silencing in Dengue Virus Research

This virology report details research on the Dengue virus, focusing on the identification of prohibitin-2 (PHB-2) as a receptor in insects. The study employs CRISPR/Cas9 technology to knockout and silence the PHB-2 gene, aiming to understand virus-host interactions and potentially control the spread of Dengue. The research design involves cloning, transfection, clonal selection, and molecular techniques like PCR and DNA sequencing to identify mutations (indels). Chromatogram analysis and tools like CRISPR-ID and ExPASy Translate are utilized. The report outlines the experimental workflow, including the creation of mono-allelic and bi-allelic knockout cells, and the use of immune fluorescence assays and western blots to confirm gene silencing. The ultimate goal is to understand how to prevent the virus from entering insect cells and potentially control the vector.

Running Head: Virology
VIROLOGY

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Aims and objectives
Several stringent cellular and molecular techniques have been used to identify and portray the prohibitin-2
as a receptor of infection of Dengue in a bug. In order to study the virus namely the Host interaction and
thereby proving that PHB-2 is a receptor of Dengue virus in the cells of the insects CRISPR/Cas9 is being
used to knockout the prohibitin-2 gene. Likewise in order to manipulate the PHB-2 gene, which would
result in the mutation of genomic sequence, there has been an aim to prove that PHB-2 gene could also be
silenced just to stop the functional roles with the help of CRISPR/Cas9 tools. Bi-allelic mutation must
happen to accomplish the effective gene function silencing. For stopping the virus namely Dengue to enter
the insect cells will definitely silence the PHB-2. Thus, the infection of dengue would be reasonable
because of controlling of the vector being spread.
Research Design Overview:
Dr. Shin-Wan Chan was the person to start this project, and this project is for the characterization as well
as identification with the help of few techniques. For providing an overview of the project workflow, the
study has been designed in three parts namely- parts done by Dr. Shiu-Wan and few of the stuents pursuing
Masters. The part one of the study includes- Firstly, plasmid vector namely (E-coli) cloned into SgRNA.
Puromycin resistance gene is the vector that is encoded Cas9 which act as a selectable marker for the
indication of transfection. On the second hand, Using lipid by the transfection of plasmid vector. These
help in the continuous growth and helps the cells maintain the selection pressure thereby making the
puromycin in the growth medium. Puromycin helps in the killing of large quantity of cells thereby
indicating that the plasmid is lost with puromycin resistance gene, this takes places after one to two weeks
of the first step. Retention of the plasmid is through the cells that remain growing in the puromycin
containing medium and this may have stably integrated into the genome of the targeted cells.

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For the third time, selection of clonal populations with the help of clonal selection of cells by way of
transferring single clump of isolated cells (the clonal ancestor as well as derived from it) into a twenty four
well plate for repeating five to ten clonal populations.
The fourth step is the identification of single clone with the help of limited expansion and dilution. Not
much of the dilutions has ever been used with cells whether with or without selection of puromycin for the
isolation of each carrying INDELS with their placement at low cell densities approximately less than 1 well
per well in ninety six well plates and thus expanding the colonies from those single cells in different wells
i.e. 24 wells then six wells plate.
The second generation of mono-allelic knockout cells is created. For the creation of bi-allelic knockout
cells from the mono-allelic knockout cells a second CRISPR has been performed. The research is basically
for the screening of cloned cells i.e. to say the third generation cells with the help of using the molecular
and cellular techniques which further includes the culture of cells, extraction and DNA lysate, gel
electrophoresis and PCR along with DNA sequencing for the bi-allelic knockout. For the success of the last
part second part would be used which is further used in techniques such as immune fluorescence essay and
western blot for confirming the gene silencing and proving the hypothesis.
Chromatogram:
The presence of mutation i.e. indels in one gene without the correct positioning indentification is
chromatogram. Further identification of indels becomes difficult for describing the peaks which are
overlapping. Hence forth, two methods can be used for the identification of indels i.e. the prediction of
CRISPR-ID web based application thereby needing confirmation by cloning and the second method is

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experimenting by cloning mixture allele into bacterial vector and further sequencing several colonies as
well as sequence of every allele individually.
CRISPR-ID:
For directing analysing Odd3-4E4 sequence mixture allele, CRISP-ID has been used which therby provides
two sequences, referencing and allelic sequencing thus using the alignments together.
ExPASy Translate:
To determine the correct open reading frame i.e. ORF, ExPASy translate is used which is further used to
translate the nucleotide sequence to a protein sequence and the process starts with the initiation of codon atg
(Methionine) and it ends with a stop codon (tag, taa or tga). There are six possible reading frames in every
region of DNA and in every direction. Typically, in a translating gene only one reading frame of 5’3’ frame 3
is used. Then the DNA i.e. open reading frame can be translated in corresponding sequence of amino acid.