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    Results

    Overview


    On this page, we will focus on the final results and statistical data of each experimental part. It consists of the following main parts:
    1. We have constructed a multi-site editing system, which consists of ultra-long gRNA array and CBE. In addition, plasmids containing pictures(Micro Nuwa) and music(Ode to Joy) data were also successfully constructed.
    2. We also verified the editing efficiency of Micro nuwa through the cell phenotype at the colony level.
    3. It has proved that Micro nuwa successfully edited the data sequence of pictures (Micro Venus) and music (Ode to Joy).
    4. The editing effect of Micro Nuwa editing system is verified by sanger sequencing. Micro Nuwa. We have re-edited 33 out of 36 sites, and up to 27 sites in a single colony were re-edited simultaneously.
    In the following subsections, we will analyze all the experimental results in detail.

    Design and Construct Micro Nuwa Multi-site Editing System


    We designed and constructed a 30-gRNA array, and transferred it into Saccharomyces cerevisiae yHX0362, which integrates the base editor with galactose-inducible promoter in the genome, to construct the engineering strains of our Micro Nuwa editing system.



    Figure 1: Ultra -long gRNA arrays.




    Figure 2: The structure of nCDA1Δ198-BE3


    In addition, we constructed the data plasmid pRS-hyg-joy. The method of constructing plasmid is seamless cloning, and the method of verifying whether succeed is PCR and sequencing of the two junctions. The recombinant plasmid was transformed into a strain containing the multi-site editing system, and the strains yNuwa002 and yNuwa003 were obtained.



    Figure 3: The connection verification of the junctions (Marker: Trans2K PlusⅡDNA Marker, Odd numbers are the results of PCR of junction1, Even numbers are the results of PCR of junction2 )


    Measurement of Micro nuwa editing system


    We transferred Micro Nuwa editing system into the strain yHX0378 expressing violacetin, in which gRNA was designed to target five genes of VIO pathway. We verified the multi-site editing ability of the Micro Nuwa editing system through the cell phenotype at the colony level and sanger sequencing. Among the 25 editing sites, 23 sites were edited. Up to 20 sites were edited in single colony. The average number of edited sites is 17.25.



    Figure 4: Editing situation of VIO pathway. The edited sites are arranged in horizontal rows. The 8 colonies sequenced were arranged in columns. The sequencing results validated the editing capability of our 30-gRNA array.


    Editing of picture (Micro Venus) and music (Ode to Joy) data sequences


    We induced yNuwa002 and yNuwa003 with galactose at 30 ℃ for 24h. Under the condition of OD600=0.3, SC-Leu-Ura+1/3ade+hyg nutrient deficient medium is used for screening.



    Figure 5: Multi-site editing results. (A) Average editing efficiency 58.83%. (B) Average editing efficiency 53.53%.


    Verifying editing result via Sanger Sequencing


    We obtained the data sequences in pRS - hyg – joy and pRS - hyg – GIF plasmids through PCR, and then carried out agarose gel electrophoresis on the PCR products. We split the GIF data sequence into three fragments for PCR. The length of the sequence was verified by electrophoresis.



    Figure 6: Electrophoresis Results of Segmented Information Fragments(Marker: Trans2K PlusⅡDNA Marker, NO.1 to 8 are the results of PCR of fragments)


    The sequencing result of GIF


    After obtaining the sequence from GENEWIZ, we compared the sequencing file with the original file and analyzed the editing results. In the pRS - hyg – GIF plasmid, we designed 36 editing sites, including: 33 sites were edited, and up to 27 sites were edited in the colony, with an average of 13.8 sites edited.


    Figure 7: The number of colonies with editing events at each site.



    Figure 8: The number of editing events per colony.


    The editing event on B-7, B-12 and C-5 did not occur. The C-G content of these gRNA sequences is high, which may form a secondary structure after being released. This will affect the binding of dCas9 and sgRNA.





    Figure 9: All sites in yNuwa002

    When the sequence is input into the decoding program, the actual GIF can be obtained, and the proof of concept can be done.



    The sequencing result of Ode to Joy


    After obtaining the sequence from GENEWIZ, we compared the sequencing file with the original file and analyzed the editing results. In the pRS - hyg – JOY plasmid, we designed 9 editing sites, including: 8 sites were edited, and up to 7 sites were edited in the colony, with an average of 3.2 sites edited.


    Figure 10: The number of colonies with editing events at each site.


    Figure 11: The number of editing events per colony.




    Figure 12: All sites in yNuwa003.

    When the sequence is input into the decoding program, the actual piece can be obtained, and the proof of concept can be done.