Difference between revisions of "Part:BBa K2200000"
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===Usage and Biology===
 
===Usage and Biology===
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<p>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.</p>
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<p>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.</p>
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<p>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.</p>
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<p>We design the gRNA for BRAF V600E to specifically target this mutant gene in melanoma cells while it has no effect on normal cells. </p>
  
  
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===Functional Parameters===
 
===Functional Parameters===
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<p>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. </p>
+
<p>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.</p>
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<p>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. </p>
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<p>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.</p>
 
<partinfo>BBa_K2200000 parameters</partinfo>
 
<partinfo>BBa_K2200000 parameters</partinfo>

Revision as of 05:55, 27 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.