Composite

Part:BBa_K2060000:Design

Designed by: Cardiff_Wales Team, Geraint Parry   Group: iGEM16_Cardiff_Wales   (2016-09-23)
Revision as of 14:57, 17 October 2016 by Gparry75 (Talk | contribs) (→‎Design Notes)


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 tool for the selection of appropriate guide RNAs that adhered to the following rules:

Use the following guidelines to select a genomic DNA region that corresponds to the crRNA sequence of the sgRNA: <p> - 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. The sequence is below has the Forward guide sequence and the General Scaffold sequences highlighted. This was synthesised as a single fragment and we aimed to directly clone this fragment into pSB1C3 using EcoRI and PstI. However we were unable to identify a clone with the correct sequence. Therefore this aspect of the project will continue during future related research. GTCATCCTCTCAGACCAGCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT GGTGGGGTAACGGCTCACCA Used standard design considerations for guide RNAs

Source

E.coli 16S ribosomal RNA

===References===