Team:TzuChiU Formosa/Project

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Background

RNAi RNAi, also known as RNA interference is a type of method that uses small fragments of RNA to interfere the molecular expression of mRNA. When inside an eukaryotic organism (RNAi), it transcribes a pre-shRNA (a strand of RNA that forms a hairpin when folded) and is then cleaved via a dicer (removing the loop) to form smaller fragments of double-stranded 20 bps RNA. The RNAi function is then performed through the use of RISC(RNA-induced silencing complex). [Through the use of RISC (RNA-induced silencing complex) to recognize the complimentary mRNA, the function of RNAi is then performed]. Through the artificial modifications, the 20 bps of RNA is then sent into the cell to achieve the function where we may control the expression of gene translation. When RNAi recognizes its complimentary strand, three things may happen: 1. mRNA simply gets degraded, 2. Blocks mRNA transcription, 3. It controls the promoter and related enzymes after this stand of RNA is transcribed. The main advantage of RNAi is that it is very easy and handy to use. You do not need to go through transgenic procedures to achieve gene regulation. The cons of RNAi on the other hand is that it “knocks down” a gene instead of “knockout” hence, meaning that it cannot completely suppress certain genes. The mechanism of RNA interference is probably originated form an organism that is infected by a virus. The RNA of the virus enters into the cell and is then recognized and cleaved by the dicer into small fragments. These small fragments possess the function of RNAi hence, inhibiting the original physiological mechanism of the cell.

Overview

RNAi does not possess a RISC system inside a prokaryotic cell. In place of the RISC is its peculiar CRISPR system. CRISPR is an immunization gene in a prokaryotic cell that fights against the exogenous DNA. CRISPR is divided into two major components namely the CAS gene and the repeat-spacer array. When an exogenous DNA enters into the prokaryotic cell, the CAS protein captures part of this exogenous DNA and inserts a section of this DNA into the repeat-spacer array to perform transcription. We will then end up having a strand of repetitive RNA with partial sections being the complimentary fragments of the exogenous gene. CAS ll will then cleave these repetitive RNA fragments and form many small fragments with the antisense RNA. Lastly, CAS lll will carry these fragments and recognize the complimentary exogenous DNA therefore, decreasing / suppressing the function of the exogenous gene. We aim to us this mechanism as a basis and send our desired RNA sequence into the cell, to activate the CRISPR system in order to interfere specific genes with our fragments. Our ultimate goal is to use the technique of RNAi, interfering the antibiotic resistance gene of the bacteria. This would then result in the bacteria losing the function of being resistant to antibiotics.