User:Ali 4192/sandbox
CRISPR/Cas9 often employs a plasmid to transfect the target cells. The main components of this plasmid are displayed in the image and listed in the table. The crRNA needs to be designed for each application as this is the sequence that Cas9 uses to identify and directly bind to the cell's DNA. The crRNA must bind only where editing is desired. After crRNA binds, Cas9 cuts the DNA in that binding region. The cell then attempts to repair the cut either by inserting random nucleotides or by utilizing a repair template [1]. The repair template must also be designed for each application, as it must overlap with the sequences on either side of the cut and code for the insertion sequence.
Multiple crRNA's and the tracrRNA can be packaged together to form a single-guide RNA (sgRNA). This sgRNA can be joined together with the Cas9 gene and made into a plasmid in order to be transfected into cells (see image for overview).
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- ^ Ramanan, Vyas; Shlomai, Amir; Cox, David B.T.; Schwartz, Robert E.; Michailidis, Eleftherios; Bhatta, Ankit; Scott, David A.; Zhang, Feng; Rice, Charles M. (2015-06-02). "CRISPR/Cas9 cleavage of viral DNA efficiently suppresses hepatitis B virus". Scientific Reports. 5. doi:10.1038/srep10833. ISSN 2045-2322. PMC 4649911. PMID 26035283.