* Our project will have several stages, all pursuant to the general investigation and modularization of the CRISPR pathway:
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:* Proof of concept targeting reporters such as GFP, eventually creating a CRISPR biobrick
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:* Investigate CRISPR system dynamics based on factors such as degradation of self-targeting sequences and maintenance of the array.
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:* Target genes such as NDM-1 or other clinically relevant pathways.
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'''The CRISPR Mechanism '''
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CRISPR ('''C'''lustered '''R'''egularly '''I'''nterspaced '''S'''hort '''P'''alindromic '''R'''epeats) is a genomic feature of some prokaryotes and most archea. A CRISPR locus consists of a set of CAS (CRISPR associated) genes, a leader, or promoter, sequence, and an array. This array consists of repeating elements along with "spacers". These spacer regions direct the CRISPR machinery to degrade or otherwise inactivate a complementary sequence in the cell.
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The CRISPR / CAS pathway can be viewed as a prokaryotic immune system or as an analogue to eukaryotic RNAi. This mechanism of bacterial survival can in theory be directed to silence a gene of interest, which affords us an interesting method to tackle the aforementioned problem. CRISPR gene loci have been demonstrated to equip both prokaryotes and archaea with a defense mechanism against exogenous DNA and RNA sequences <sup>[[Team:Arizona State/Project/References#ref2|[2]]], [[Team:Arizona State/Project/References#ref10|[10]]]</sup>, usually targeting invading viruses. The transcripts from the spacer/repeat region undergo hair pinning due to the palindromic sequence structure. The peptide products of the CAS genes work cooperatively with crRNA to silence a complimentary target [[#diagram1| (Diagram 1)]] <sup>[[Team:Arizona State/Project/References#ref4|[4]]]</sup>. The function is a prokaryotic analog to both RNA interference and immunity. CRISPR presents it self as a potentially useful tool in prokaryotic gene manipulation. Our goal as ASU’s first iGEM team is to develop a CRISPR plasmid that contains elements to target and silence the NDM‐1 gene sequence [[#diagram2| (Diagram 2)]]. While our final goal is the targeting of NDM‐1, we recognize that CRISPR can potentially target any gene of interest. We will develop a robust, modular platform for gene silencing based on CRISPR. The final product of this project will be a fully functioning CRISPR array that will be submitted to the Standard Registry of Biological Parts, an open‐source collection of DNA building blocks, as a BioBrick, a modular component for genetic engineering [[#diagram3| (Diagram 3)]].
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