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Team:Duke/Project
From 2014.igem.org
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Background Information
CRISPR/Cas9 System
Clustered Regularly Interspersed Short Palindromic Repeats (CRISPRs) 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:
- The guide crRNA must match the sequence of the protospacer
- 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
The goal of this year’s project is to produce an ultrasensitive response. Ultrasensitive responses are defined as such because the level of response is highly dependent on the amount and concentration of the inducer, rather than other variables. Ultrasensitivity has implications for several biological applications, including bistable biological “toggle switches” within the genome.
There are two approaches we are taking to achieve ultrasensitivity.
- We are designing a 2-reporter construct, where induction of one reporter triggers repression of the other. We hypothesize that the number of crRNA repeats in the construct will trigger the ultrasensitive response
- Molecular titration: By adding anti-tracrRNA, we would like to see if the complementary strand to the tracrRNA sequence will sequester tracrRNA and inactivate the Cas9 repression system, thereby causing a previously repressed reporter to become active again. Ultrasensitivity in this case would depend on the concentration of anti-tracrRNA.
