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Cas9

Cas9 is a protein originally from within the Streptococcus pyogenes immune response system. Cas9, with the Clustered Regularly Interspersed Palindromic Repeats (CRISPR) and other CRISPR-associated proteins were initially used to respond to known pathogenic genetic information. In its native form, it will cut the invasive protein at recognized spots before it can incorporate into the bacteria. Fragments of new viral DNA is saved into a library of DNA fragments which is the CRISPR, named after the repeated loop structure between each fragment. Once these are transcribed and processed, the construct of copied nucleic acid sequence and loop is known as a short guide RNA, or gRNA.

However, because of the ease and predictability of the targeting, the protein has been used as a useful and controllable DNA targeting construct. This level of control has been honed in by modifying the protein to only cut one strand. These nickases require two successful Cas9 landings to fully cut the DNA. Additionally, functional groups such as transcription activators and repressors have been attached to make a deactivated Cas9 protein (dCas9) a programmable activator protein. Because the wide variety of CRISPR systems in bacteria, multiple independent systems can be implemented in the same biological chassis.

dCas9 Repression

From this, Duke iGEM has had an interest in a phenomemon associated with dCas9 called CRISPR interference, where the presence of the large dCas9 protein blocks the RNA polymerase from continuing, knocking down transcription rates.[1] Our project incorporates many of the findings present in this paper. The paper finds that multiple binding sites have between additive and multiplicative, while the farther down the gene from the promoter, the weaker the repression.