Dengue Virus Receptor Research Report

This report documents a research project focused on identifying and characterizing prohibitin-2 (PHB-2) as a receptor for the Dengue virus in insects. The study employed CRISPR/Cas9 gene editing technology to create PHB-2 knockout insect cells. The process involved cloning sgRNA into a plasmid vector, transfecting the vector into insect cells, and selecting clonal populations. Mono-allelic knockouts were generated initially, followed by a second round of CRISPR editing to achieve bi-allelic knockouts. The report details the molecular and cellular techniques used, including cell culture, DNA extraction, PCR, gel electrophoresis, DNA sequencing, western blotting, and immunofluorescence. Challenges encountered included indel identification from chromatograms, which were addressed using both experimental cloning and the CRISPR-ID web application. ExPASy Translate was used for protein sequence determination. The project aimed to demonstrate that PHB-2 silencing prevents Dengue virus entry into insect cells, contributing to Dengue virus control by managing vector spread. The report provides a detailed overview of the methodology and results, highlighting the progress made and suggesting future research directions.

Aims and objectives
We are aiming to identify and characterise prohibitin-2 as a receptor of Dengue virus in insect via
several stringent molecular and cellular techniques. CRISPR/Cas9 has been used to Knockout the
prohibitin-2 gene to study the virus-Host interaction and prove that PHB-2 is a receptor for Dengue virus in
insect cells. Also, we are aiming to prove that PHB-2 gene can be silenced to stop their functional role by
using CRISPR/Cas9 tools to manipulate the PHB-2 gene, resulting in mutation in the genomic sequence.
To achieve successful gene function silencing, bi- allelic mutation must occur. Successful PHB-2 silencing
in insect cells will stop Dengue virus entering insect cells. Consequently, Dengue virus will be manageable
as a result of controlling of the vector spreading.
Research Design Overview:
This project has been started by Dr Shiu-Wan Chan before we begin, which we participate in the
identification and characterization by using few techniques and we did not finish the whole project due to
dissertation project time. To provide an overview of project workflow (Figure), the study design is
described in three parts: part done by DR Shiu-Wan, by Master students as well as future suggestion. The
first part includes the following techniques and methods:
Firstly, Cloning of SgRNA into plasmid Vector (E. coli). The vectors encode Puromycin resistance gene
(as A selectable marker to indicate the success of a transfection), cas9. Secondly, Transfection of plasmid
vector by using lipid. Continue to grow and passage the cells maintaining selection pressure by keeping
puromycin in the growth medium (with and without puromycin). After 1-2 weeks, a large number of the
cells will be killed by the puromycin, indicating that they did not take up or have lost the plasmid with the
puromycin resistance gene. The cells that remain growing in the puromycin-containing medium have
retained the expression plasmid, which may have stably integrated into the genome of the targeted cells.
Thirdly, Clonal selection Select clonal populations of cells by transferring a well-isolated single clump
of cells (the clonal ancestor and cells derived from it) into a well of a 24 well plate; repeat to select 5-10
clonal populations. Fourthly, Identify single cloned by limited dilution and expansion. Limited dilution has
been used with cells with and without puromycin selection to isolate each cell that carries INDELS by
placing them at very low cell densities (< 1 well per well in 96 well plates), and expand colonies from
those single cells in separate wells (24 wells then six wells plate).

Mono-allelic knockout cells have been created (2nd generation). A second CRISPR knockout has been
performed on these mono-allelic knockout cells tocreate bi-allelic knockout. This project is toscreen the
cloned cells, 3rd generation cells, using cellular and molecular techniques including Cell culture, DNA
lysate and extraction, PCR and gel electrophoresis as well as DNA sequencing for the bi-allelic
knockout.Last Part of the study depends on the success of the second part, which is used techniques such as
western blot and immunofluorescence assay to confirm the gene silencing as well as prove the hypothesis.
Chromatogram:
chromatogram below shows the presence of a mutation (indels) in one gene without specific position
identification. Additionally, indels identification is difficult to describe with overlapped peaks. Thus, two
methods can be used to identify the indels, either experimentally by cloning mixture alleleinto bacterial
vector and then sequence several colonies as well as sequence every allele individually, or using prediction
CRISPR-ID web-based application, which need confirmation by cloning
CRISPR-ID:
CRISP-ID has been used to direct analyse ofOdd3-4E4 sequence mixture allele, which provide two
sequences, reference and allelic sequences that used to alignments together.
ExPASy Translate:
ExPASy Translatehas been usedto translate the nucleotide sequence to a protein sequence todetermine the
correct open reading frame (ORF), which starts with initiating codon atg (Methionine) and ends with a stop
codon (taa, tag or tga). Every region of DNA has six possible reading frames, three in each direction.
Typically, only one reading frame is used in translating a gene, and this is 5'3' Frame 3. Once the open
reading frame is known the DNA sequence can be translated into its corresponding amino acid sequence.