Difference between revisions of "Part:BBa K2130013"

Revision as of 18:01, 25 October 2020

SpCas9

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

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

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