Team:BNU-China/Engineering

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  Overview Cas9 Folding Rate Predictions of Diversity of Barcode Selection of Degradation Tags Tracing Generations Number of hgRNA Required
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Overview

    This year, we built the ABC (Automatic Barcode Creator) system and set a fundamental paradigm in yeast. This system can automatically label each cell with different barcodes and trace their relationships by single cell RNA sequencing. Our engineering can be divided into three modules: label each cell automatically and prolong the tracing generations, increase the variants of barcodes, and read barcodes together with transcriptomic information. Delightfully, we have validated these modules successfully by carrying out experiments, modeling and literature review.

Label each cell automatically and prolong the tracing generations

   In order to achieve the automatic addition of barcodes and prolong the tracing generations, we used CLB2 promoter to express Cas9 periodically instead of constitutive expression. We constructed the recombinant plasmid containing CLB2 promoter (BBa_K3506007) and Cas9 gene (BBa_K2130013).

   After transforming it into Saccharomyces cerevisiae BY4741, the result of colony PCR showed that we successfully integrated the target fragment into the yeast genome (Figure 1).

Figure 1. Gel electrophoresis results of colony PCR. Lane 1: Marker; Lane 2-5: PCR product of the upstream of integration site (1069bp); Lane 6-9: PCR production of the downstream of integration site (2862bp).

   Then, we synchronized Saccharomyces cerevisiae to the G1 phase. After releasing, we extracted total protein every 10 minutes for 100 minutes. Western Blot showed that the expression level of Cas9 was fairly high in G2/M phase but fell sharply in other phases (as is shown in Figure 2), suggesting that we could use it to label cells as we planned.

Figure 2. Level of Cas9 and GAPDH protein. Lane 1: block cell cycle of Saccharomyces cerevisiae at early S phase; Lane 2-10: sample every ten minutes after synchronization release

   In order to degrade Cas9 timely, we planned to create a fusion protein of Cas9 and Clb2 protein, so that Cas9 can be broken down together with Clb2. Then we found in literature that the first 124 amino acids of Clb2 also has degradation ability. In order to evaluate its degradation effect, we used GAL1 promoter to express the fusion protein that contains GFP (BBa_E0040) and the degradation tag (BBa_K3506036).

   We transformed the recombinant plasmid into Saccharomyces cerevisiae and used fluorescence microscopy and flow cytometer to observe the degradation process. After being induced by galactose, green fluorescence was observed. After removing the induction, the fluorescence intensity of the experimental group showed a significant decrease compared to the control group (Figure 3), indicating that the first 124 amino acids of Clb2 can degrade Cas9 effectively.

Figure 3. The fluorescence intensity of experiment group and control group by time

   We also predicted the degradation effect for the full-length Clb2 by modeling.Considering the concentration and degradation rate of Cas9, we chose the first 124 amino acids of Clb2.
   Through modeling,we proved that expressing Cas9 periodically does prolong the generations that could be traced (about 1.5 times as much as before).

Increase the variants of barcodes

   Through modeling,we concluded that the total number of variants one hgRNA can produce is about 1,400. This strongly proved that compared with sgRNA, hgRNA can be used to label more cells at any one time point.

Read barcodes together with transcriptomic information

   In order to obtain the lineage information in hgRNA out of the transcriptomic information, we designed a constitutive double promoter module (BBa_K3506095) and an inducible double promoter module (BBa_K3506022).



   We tested these modules by two steps.
   First step: to test whether GAPDH promoter will affect the function of gRNA. We put gRNA targeting ADE2 gene downstream of U6 promoter in both the experimental and control groups, and put GAPDH promoter upstream of U6 promoter only in the experimental group. A loss-of function mutation in ADE2 can result in an adenine auxotroph that forms pink colonies on YNBA plates containing low level of adenine, thus enabling a visual evaluation of the action of CRISPR-Cas9. Results showed that colonies of both two groups turn red (Figure 4), which indicates GAPDH promoter won’t affect the function of gRNA.

Figure 4. A. control group(pU6-gDNA);
B. experimental group(pGAP-pU6-gDNA);
C and D. 4500FOA (the recipient strain).

   Second step: to test whether gRNA can be captured by oligo dT. For both experimental groups and control groups, we extracted the total RNA of red colonies by Trizol. The RNA was reverse transcribed using oligo dT as a primer. PCR and sequencing were performed later. A band with the correct size (208bp) (Figure 5) and the anticipated sequencing showed that gRNA can be captured by oligo dT successfully.

Figure 5. Gel electrophoresis results of control group(pU6-gDNA) and experimental group(pGAP-pU6-gDNA). Lane 1: Marker; Lane 2 and Lane 3: RT-PCR product of control group(pU6-gDNA); Lane 4 and Lane 5: RT-PCR product of experimental group(pGAP-pU6-gDNA) (208 bp).

   Considering that the information of barcodes only needs to be read at specific time, we replaced GAPDH promoter with GAL7 promoter and constructed a different inducible double promoter module (BBa_K3506022). As shown in Figure 6, colonies of both the experimental and control groups turned red. PCR and sequencing were performed. Results showed that gRNA can be captured by oligo dT successfully(Figure 6 and Figure 7) .
   This result suggests that both the constitutive double promoter module (BBa_K3506095) and the inducible double promoter module (BBa_K3506022) can work successfully.

Figure 6. A. control group(pU6-gDNA); B. experimental group(pGAL7-pU6-gDNA); C. and D. 4500FOA (the recipient strain).

Figure 7. Gel electrophoresis results of control group(pU6-gDNA) and experimental group(pGAL7-pU6-gRNA). Lane 1: Marker; Lane 2 and Lane 3: RT-PCR product of control group(pU6-gDNA); Lane 4 and Lane 5: RT-PCR product of experimental group(pGAL7-pU6-gDNA) (208 bp).

   To conclude, we demonstrated the engineering success of our three modules through experiments, modeling and literature review.