Coding

Part:BBa_K2130013

Designed by: Ooi Kean Hean   Group: iGEM16_NTU-Singapore   (2016-10-24)
Revision as of 18:03, 25 October 2020 by Chenyiling (Talk | contribs)


SpCas9


This past is a human condon optimised SpCas9. SpCas9 recognises an 5'-NGG-3' PAM at the 3' end of the target sequence.

BNU-China 2020 - Contribution Contribution：First, we expressed this protein in Cryptococcus neoformans and demonstrated that it can function well. Second, we use a model to predict the expression speed of Cas9. Third, we found an article about Cas9 studied the speed of searching targets. Characterization in Cryptococcus neoformans We used this human condon optimised SpCas9(BBa_K2130013) and sgRNA(BBa_K3506050) in Cryptococcus neoformans. sgRNA was designed to target the ADE2 gene. This gene encoding a phosphoribosylaminoimidazole carboxylase in the biosynthetic pathway of adenine. A loss-of-function mutation in ADE2 results in an adenine auxotroph that forms pink colonies on culture plates that contain a low level of adenine, thereby enabling a visual evaluation of the action of CRISPR-Cas9. Upon transforming the linearized vectors carrying both the Cas9 and the sgDNA cassettes into 4500FOA, a large proportion of URA5-positive transformants formed on the YNBA plates. Then we transferred them to a 4℃ refrigerator. Red colonies were selected and inoculated into YPD medium, then placed it in 30℃ incubator for days, pink colonies grew, indicating that SpCas9(BBa_K2130013) successful targeted at the ADE2 locus in Cryptococcus neoformans. Predict the expression speed of Cas9 Example(PAM in brackets): 5'-NNNN...NNN(NGG)-3' Evaluation studies from our team have compared the editing efficiency of this part compared to other Cas9/Cpf1.
Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 341
    Illegal BglII site found at 1136
    Illegal BamHI site found at 1430
    Illegal XhoI site found at 1936
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1168
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 259
    Illegal BsaI.rc site found at 1501
    Illegal BsaI.rc site found at 2857
    Illegal BsaI.rc site found at 3280
    Illegal BsaI.rc site found at 3292
    Illegal BsaI.rc site found at 4159
    Illegal SapI.rc site found at 2964
    Illegal SapI.rc site found at 3546
    Illegal SapI.rc site found at 3561

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Categories
Parameters
None