Difference between revisions of "Part:BBa K2060000:Design"
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<p>This guide sequence was added to the conserved DNA scaffold sequence that will interact with bacterial Cas9: GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT
 
<p>This guide sequence was added to the conserved DNA scaffold sequence that will interact with bacterial Cas9: GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT
  
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<p>This was synthesised as a single fragment and directly cloned into pSB1C3 using EcoRI and PstI.  
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<p>This was synthesised by <a href="https://www.idtdna.com/pages/products/genes/gblocks-gene-fragments">IDT gBlock<a/> as a single fragment together with the biobrick Prefix and Suffix and directly cloned into pSB1C3 using EcoRI and PstI.  
  
 
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Revision as of 17:03, 17 October 2016


CRISPR-Cas9 guide RNA targeting to 16S RNA


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 48
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

One of the Cardiff_Wales iGEM instructors Daniel Pass, designed a bioinformatic toolfor the selection of appropriate guide RNAs from E.coli ribosomal 16S RNA that adhered to the following rules:

- The 3' end of the DNA target sequence must have a proto-spacer adjacent motif (PAM) sequence (5'-NGG-3').

- The 20 nucleotides upstream of the PAM sequence will be your targeting sequence (crRNA) and Cas9 nuclease will cleave approximately 3 bases upstream of the PAM.

- The PAM sequence itself is absolutely required for cleavage, but it is NOT part of the sgRNA sequence and therefore should not be included in the sgRNA.

- The target sequence can be on either DNA strand.

From this programwe identified the the following 'R1' guide sequence: GGTGGGGTAACGGCTCACCA

This guide sequence was added to the conserved DNA scaffold sequence that will interact with bacterial Cas9: GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT

This was synthesised by IDT gBlock as a single fragment together with the biobrick Prefix and Suffix and directly cloned into pSB1C3 using EcoRI and PstI.

Source

E.coli 16S ribosomal RNA

References