Part:BBa_K2200000
Guide RNA (gRNA) is a specific molecule that guides the Cas nuclease to a targeted dsDNA sequence.
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Usage and Biology
A well-designed gRNA is able to highly enhance the specificity with Cas9 nickase. The specificity of the CRISPR system is determined in large part by how specific the gRNA targeting sequence is for the genomic target compared to the rest of the genome.
We choose the nickase system for highly specific gene editing. Cas9 nickase binds DNA based on gRNA specificity, only resulting in “nicks”, or single strand breaks, instead of double strand breaks (DSB).DNA nicks are rapidly repaired by homology directed repair (HDR) using the intact complementary DNA strand as the template.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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Functional Parameters
In order to confirm the function of our CRISPR/Cas9 system, we performed our functional experiments in two different human melanoma cell lines (A375 and G361) with BRAF V600E mutation. Plasmid pHS-ACR-ZQ170 which encoded Cas9 and sgRNA was constructed for this substantiation.
We transfected A375 and G361 cells with Plasmid pHS-ACR-ZQ170 and utilized the Cell Counting Kit (CCK-8) assay to analyze the quantity of cancer cells by adding CCK solution to each distinguished two-line group every 24 hours. Growth of both cell lines has proved to be inhibited, while the result of A375 was not as obvious as that of G361.
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