Part:BBa_K3506050
gRNA targets ADE2 gene
A guide RNA (gRNA) is an artificial RNA that is designed to bind a certain DNA sequence. It can combine with Cas9 protein to play a role in the cleavage of targeting DNA. We design a gRNA that targets ADE2 gene to specifically knock this gene in Cryptococcus neoformans.
Properties
We electroporated this part and spCas9 (BBa_K2130013) into Cryptococcus neoformans 4500FOA as the experimental group. Use 4500FOA as the control group. gRNA was designed to target the ADE2 gene. A loss-of-function mutation in ADE2 results in an adenine auxotroph that forms pink colonies on culture plates containing a low level of adenine, therefore enabling a visual evaluation of the action of CRISPR-Cas9. In our result, pink colonies grew on the YNBA plates (Figure 1), indicating that gRNA was successfully targeted the ADE2 locus in Cryptococcus neoformans.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Expenrimental approach
1.Construct recombinant plasmid. Insert spacer sequence of gRNA on PRH003 plasmid.
2.Transform the product (2.5μL) into DH5α competent cells（50μL）, grow cells on LB-amphenicol medium. Incubate at 37°C overnight. Monoclones are selected by colony PCR. Expanding culture colonies at 37℃ 200rpm, then extracting plasmids and sequencing.
3.Use Kpn1 enzyme to linearise the plasmids and transform them into Cryptococcus neoformans 4500FOA by electroporation.
4.The Cryptococcus neoformans is spread on YNBA selection medium, and the transformants grow after being cultured in an incubator kept at 30℃ for 4 days. Then the culture is transferred to a refrigerator at 4℃.
5.Pink colonies are selected and inoculated into YPD medium, then placed in an incubator kept at 30℃ for 4 days. Finally it is kept at 4℃ refrigerator.
6.Comparing the experimental group with the control group, we evaluate whether this system work well according to the color of colonies.
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