Team:Duke/Project

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Background Information

CRISPR/Cas9 System

Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) are a form of bacterial defense against foreign invaders. When foreign (viral) DNA enters the cell, its DNA is incorporated into the spaces between the CRISPRs. This DNA is transcribed into a long RNA piece. "CRISPR-associated" (Cas) riboendonucleases cleave (cut) the RNA at the repeat sites to form CRISPR RNAs, or (crRNA). The crRNAs guide the Cas protein to to the target foreign DNA (aka the protospacer), which then uses its endonuclease activity to cleave the DNA and make it inactive.

The type of Cas protein we use is called Cas9. It cleaves both strands of DNA, but it requires two conditions for it to work:

  1. The guide crRNA must match the sequence of the protospacer
  2. presence of a protospacer-adjacent motif (PAM) downstream of the protospacer

dCas9 is a form of the Cas9 protein that lacks endonuclease capability. Instead, it just sits on the target site of the DNA. This blocks transcription of the DNA by RNA polymerase, thereby inhibiting gene expression. This is a way of modifying gene expression without modifying the genome by utilizing machinery that is already present in the bacteria.

Project Summary

Project Details

Materials and Methods

The Experiments

Results

Data analysis

Conclusions