RNA

Part:BBa_K2515002:Design

Designed by: Slavil Peykov   Group: iGEM17_Bulgaria   (2017-10-27)
Revision as of 18:43, 1 November 2017 by Slavil (Talk | contribs)

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gRNA expression vector for use with Cas9 or dCas9


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 8
    Illegal NheI site found at 31
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 64
    Illegal BsaI.rc site found at 38


Design Notes

This part was inspired by the following publication: "CRISPR interference (CRISPRi) for sequence-specific control of gene expression" doi:10.1038/nprot.2013.132 This part is BioBrick compatible so you can assemble gRNA arrays using the standart types of BioBrick assembly (assuming you gRNA sequences do not contain any of the restriction sites needed (EcoRi, XbaI, SpeI or PstI). If you want to have this synthesized by IDT, order the following sequence: 

gRNA cloning cassette
GAATTCGCGGCCGCTTCTAGAGTTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCAGAGACCA
ACTTTCAGTTTAGCGGTCTGGTCTCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGT
CCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGAACTGCCTACTAGTAGCGGC
CGCTGCAG

and then clone it in a pSB vector of your choice using the following primers:

C_BioBrick_Prefix-F: TATGAATTCGCGGCCGCTTCTAG

C_BioBrick_Suffix-R: TATCTGCAGCGGCCGCTACTAGTA

Note: C means cloning - these primers have 3 extra bases at their 5' ends so the EcoRI and PstI can cut efficiently. Do not use the regular BioBrick_Prefix-F and R pair instead - it won't work!



To clone gRNAs you need to:

1) Determine the 20 bp sequence of your gRNA (check our Wiki or read "CRISPR interference (CRISPRi) for sequence-specific control of gene expression" doi:10.1038/nprot.2013.132 for some good recommendations regarding this point).

2) Order 2 oligos like normal primers from IDT (or any other supplier)

F: (5’-TAGCNNNNNNNNNNNNNNNNNNNN.........-3’)
R: (3’-.........NNNNNNNNNNNNNNNNNNNNCAAA-5’)

3) Digest this vector with Eco31I.

4) Anneal and ligate the 2 oligos (check our Wiki for a detailed protocols) 5) Check your clones via colony PCR. You can use the F or the R gRNA Oligo as one of your primers. The other primer should be located on the vector backbone. Avoid usage of VF2 or VR - the product gets pretty small (approx. 200 bp) and can be confused with primer dimers. 6) Test your gRNA for functional activity - see the Experience page of this part.

Source

This part was made as gBlock fragment by IDT.

References