Use the template provided to: 2) give your proposal a title and state the object

Question

Use the template provided to: 2) give your proposal a title and state the objective of making the mutation. 3) State two hypotheses that make predictions for the consequences of the mutation. One hypothesis should be for a consequence at the cellular level. The second hypothesis should be for a consequence at the level of an individual. 4) Write enough background to support your hypothesis; to make it clear why you are making the prediction. Include a figure that shows both the normal interface and the immediate molecular consequence of your proposed mutant protein. 5) State the significance of your proposed mutation experiment. You may need to include some more background to support why the experiment is worth doing. You do NOT need to provide references for your statements.

Explanation / Answer

Title: Investigating the effect of hotspot and non-hotspot mutations in TP53 and CASP8 genes in oral cancer cell lines using CRISPR-Cas9 mediated genome editing.

Objectives:

1)To introduce mutations in TP53 and CASP8 in oral cancer cell lines by CRISPR-Cas9 mediated gene editing technology.

2) To investigate the functional consequence of hotspot vs. non-hotspot mutations in TP53 and CASP8 genes by studying various cell proliferation and migration assays.

Background:

Oral squamous cell carcinoma (OSCC) is the most common malignant tumor of the neck and head. Although OSCC has a striking incidence worldwide it is highly prevalent in countries where tobacco chewing is common. The occurrence of OSCC in India is seen to be higher in men and is estimated to be the 8th most common cancer worldwide. The use of tobacco, betel quid, areca-nut and heavy drinking of alcoholic beverages are the major risk factors for OSCC. Diets deficient in vegetables and fruits, and infections with Human papilloma virus (HPV) have also been implicated to be among the other causes of OSCC. Several cellular signalling pathways are dysregulated in different subtypes of OSCC due to genetic and epigenetic factors that govern tumour initiation, progression and maintaince. Two studies on mutational landscape of head and neck squamous cell carcinoma (HNSCC) identified frequent mutations in TP53, CDKN2A, PIK3CA, HRAS and NOTCH1 using exome sequencing. Another study from India Project Team of the International Cancer Genome Consortium (ICGC) identified mutations in USP9X, MLL4, ARID2, UNC13C, TRPM3, TP53, FAT1, CASP8, HRAS and NOTCH1 in 50 Indian patients with gingivo-buccal oral squamous cell carcinoma (OSCC-GB). Among these genes, TP53 and CASP8 have been selected as candidate genes based on their involvement in cancer, when mutated.

Hypothesis:

TP53 is a prominent tumour-suppressor gene extensively evaluated for its role in causation of cancer while the Caspase family (CASP8) genes play a pivotal role in the execution phase of programmed cell death or apoptosis. The India Project Team of ICGC found frequent hotspot mutations in TP53 and CASP8. The functional impacts of alterations in these genes are unknown and the studies on hotspot mutations have been limited. This research project will help in assessing the severity of hotspot and non-hotspot mutations in oral cancer cell lines and aid in functional validation of the disease variants.

This study will be using CRISPR-Cas9 (Clustered regularly interspaced short palindromic repeats) system to introduce mutations (small nucleotide variations or SNVs) in these two genes and further investigate the effect of hotspot and non-hotspot mutations in TP53 and CASP8 by performing colony forming assay, soft agar assay, Invasion assay, BRDU cell proliferation assay and scratch wound healing assay in appropriate oral cancer cell lines.

Significance:

1) Introduction of mutations in TP53 and CASP8 in oral cancer cell lines by CRISPR-Cas9 mediated gene editing

technology will help in assessing the severity of hotspot and non-hotspot mutations in these genes by studying various cell proliferation and migration assays.

2) Testing the effect of specific hotspot mutations in oral cancer cell lines will help in tailoring personalized therapeutic drugs or therapies.

3) The gene edited oral cancer cell lines generated by CRISPR-Cas9 will further be used in screening compounds or small molecules that can lay a foundation for eventual drug discovery to treat patients with specific hotspot mutations.

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