Difference between revisions of "Part:BBa K2200010"
 
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===Usage and Biology===
 
===Usage and Biology===
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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.
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We design the gRNA for BRAF V600E to specifically target this mutant gene in melanoma cells while it has no effect on normal cells.  
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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 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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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2200010 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K2200000 SequenceAndFeatures</partinfo>
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Uncomment this to enable Functional Parameter display
 
 
===Functional Parameters===
 
===Functional Parameters===
 
<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 be made between the outcome of them.</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 be made between the outcome of them.</p>
 
<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>
 
<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>
  
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<small><p><b>Figure 1: The mutant BRAF gene was specifically cleaved in melanoma cells. A</b>, the Chormatogram of DNA sequencing.<b>B</b>, DNA sequence comparison. Our CRISPR/Cas9 system specifically cleaved the mutanted BRAF gene in melanoma cells, resulting in insertion, deletion and frameshift mutation on the gene.<p></small>
 
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<partinfo>BBa_K2200010 parameters</partinfo>
 
<partinfo>BBa_K2200010 parameters</partinfo>

Latest revision as of 14:42, 1 November 2017


It's a composite part encoding our gRNA promoted by Human U6 promoter.

The composite part encodes our specific gRNA promoted by Human U6 promoter. It is utilized to guide the Cas9 protein to the BRAF V600E mutation.


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 be 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.

746px-Sfls-demonstrate-sequencing_final.jpg