Part:BBa_K3506050
gRNA targets ADE2 gene
A guide RNA (gRNA) is an artificial RNA which is designed to bind a certain DNA sequence. It can combine with Cas9 protein to play a role in the cleavage of target DNA. We design a gRNA that targeted on ADE2 gene to specifically knock this gene in Cryptococcus neoformans.
Properties
We electroporated this part and spCas9(BBa_K2130013) into Cryptococcus neoformans 4500FOA as experimental group. Use 4500FOA as 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, indicating that gRNA successful targeted at 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 C. neoformans<i> 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.Red colonies are selected and inoculated into YPD medium, then place 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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