Difference between revisions of "Part:BBa K2200000"

Revision as of 10:39, 31 October 2017

Guide RNA (gRNA) is a specific molecule that guides the Cas nuclease to a targeted dsDNA sequence.

Usage and Biology

Single-guide RNA (SgRNA) is an artificial RNA which is designed to bind a certain DNA sequence. It can combine with Cas9 protein to play a role in the cleavage of target DNA.It is much shorter but have the same function as the original RNAs in CRISPR-Cas-9 system.

We design the gRNA for BRAF V600E to specifically target this mutant gene in melanoma cells while it has no effect on normal cells.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Functional Parameters

In order to demonstrate our CRISPR/Cas9 system in melanoma cell lines, we extracted genome of two different human melanoma cell lines (A375 and G361) with BRAF V600E mutation after transfection. A375 is a melanoma cell line with BRAF V600E homozygous mutation while G361 is with heterozygous mutation and comparison can made between the outcome of them.

The cell genome was treated with TA cloning Assay. Twenty-one single clones from the transfected cells were selected for DNA sequencing and result showed that BRAF gene was cleaved in three of them and the wild-type BRAF wasn’t cleaved in two of them. Results showed that the CRISPR/Cas9 system specifically cleaved the mutant, but not wide-type BRAF gene in melanoma cells.

Figure 1: The mutated BRAF gene was specifically cleaved in melanoma cells.A. the Chromatogram of DNA squencing. B. DNA squence comparison. Our CRISPR/CAS9 system specifically cleaved the mutated BRAF gene in Malenoma cells, resulting in insertion, deletion and frameshift mutation on the gene.

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 https://static.igem.org/mediawiki/2017/thumb/2/23/Demonstrate-sequencing-ATCG.PNG/800px-Demonstrate-sequencing-ATCG.PNG.jpeg
-<p>Fig.1. The mutated BRAF gene was specifically cleaved in Malenoma cells. A. the Chromatogram of DNA squencing. B. DNA squence comparison. Our CRISPR/CAS9 system specifically cleaved the mutated BRAF gene in Malenoma cells, resulting in insertion, deletion and frameshift mutation on the gene. </p>
+<small><p><b>Figure 1: The mutated BRAF gene was specifically cleaved in melanoma cells.A.</b> the Chromatogram of DNA squencing. <b>B.</b> DNA squence comparison. Our CRISPR/CAS9 system specifically cleaved the mutated BRAF gene in Malenoma cells, resulting in insertion, deletion and frameshift mutation on the gene.</p></small>
 <partinfo>BBa_K2200000 parameters</partinfo>