Team:BNU-China/Poster

Poster: BNU-China

Automatic Barcode Creator

Automatic Barcode Creator

Presented by Team BNU-China 2020

Team Member: Yiling Chen, Ran Yan, Yiying Yu, Fanmeng Zhang, Aoyu Ma, Xinwen Dong, Xiaoxue Zhao, Jingzi Wang, Yuxin Wang, Minglei Zhang, Jiangyue Zhang, Yunhan Jiao, Xinmiao Li, Mingyue Hu, Xinyue Shan, Yuzhen Chen, Chang Gao, Xinyao Pan, Yumo Li, Xinrui Li, Lin Xu, Xiaoyu Ye, Li'ang Li, Nianci Jiang, Yuchen Feng, Dake Gao, Shibei Meng, Jingyuan Wang, Qing Hu, Huilin Chen, Pengbo Liu.

Abstract

Early cell lineage tracing techniques used dyes or fluorescent markers which helped resolve major questions in cell development. In recent years, the method combining CRISPR/Cas9 and barcode, a DNA sequence as genetic marker, has drawn great attention. However, the total number of barcodes is limited and the constitutively expressed Cas9 consumes barcodes quickly.

To solve this problem, we built an inducible expression module of Cas9 in association with cell division to label each cell automatically. What's more, we used homing guide RNA (hgRNA) to replace the single guide RNA (sgRNA), so that simplify the system by using hgDNA as the barcode and enable retargeting and evolvability of barcodes. Furthermore, we designed the double promoter module, which allows barcodes to be read at RNA level.

Introduction

lt is an amazing process that fertilized eggs become colorful life forms by continuous division and differentiation. Many researchers try to trace the whole process by giving each cell a unique biological label, which is called "lineage tracing".

ln recent years, the method combining CRISPR/Cas9 and barcode, a DNA sequence as a genetic marker, has drawn great attention. However, the total number of barcodes is insufficient and the constitutively expressed Cas9 consumes barcodes quickly.

These make barcodes inadequate for tracing purpose after several generations, which give a biased view on organ complexity and limits comprehensive understanding of global stem cell dynamics within tissues. These problems puzzled scientists a lot and blocked further research. So this year, BNU-China designed the Automatic Barcode Creator system, which can label each cell uniquely and automatically for many generations.

Inspiration

An article published in Cell on June 17, 2020_[2] established an inducible lineage tracing model in mouse based on CRISPR/Cas9 (as shown in Figure1 below).

A tetO-operons was added in the upstream of Cas9.

However, the induction of Cas9 still needs the addition of exogenous substances and has not yet reached automation.

Figure 1. Schematic of the model (Bowling, S et al., 2020)

Goal

• Express Cas9 periodically to  prolong the tracing generations

• Replace sgRNA with hgRNA to increase the tracing number of cells in every generation

• Read hgRNA at RNA level to obtain the lineage tracing information together with transcriptomic information

Design & Experiments

Our project aims to label each cell uniquely and automatically, prolong the tracing generations and increase the tracing number of cells in every generation. Furthermore, we want to read hgRNA at RNA level.

♣ To label each cell automatically and prolong the traced generations, we built a periodic expression and degradation module of Cas9.

♣ To increase the tracing number of cells in one generation, we replaced single guide RNA with homing guide RNA to increase the variants of barcode.

♣ Furthermore, considering the lineage information in hgRNA can't be read at RNA level, we built double promoter systems to obtain the lineage information together with transcriptomic information.

Periodic Expression of Cas 9

Use CLB2 promoter to initiate the expression of Cas9.

Design:

According to the literature review, under the control of CLB2 promoter, the downstream gene starts to transcribe in the S phase, reaching a peak at the G2/M transition (Figure 1). So, we put CLB2 promoter in the upstream of Cas9.

Figure 1. DOA1 mRNA expressed from the CLB2 promoter (Trcek et al., 2011)

Experiments:

We constructed a periodic expression module of Cas9.
Then we synchronized the yeast and performed Western Blot to detect the protein level of Cas9 in cell cycle.

Use a degradation tag to degrade Cas9 timely.

We considered both the full-length Clb2 and the first 124 amino acids of Clb2.

The full-length Clb2:

We evaluated its efficiency in degrading Cas9 through modeling.

Results showed that Cas9 degrades quickly before the components stabilize and the relative concentration of Cas9 has been lower than 5%.

The first 124 amino acids of Clb2:

Due to the lacking of data, we designed experiments to verify the degradation ability of Clb2 N124aa.

We constructed this module, detected the changes of fluorescence intensity and calculated its degradation rate through modeling.

Increase the Variants of Barcodes

We replaced sgRNA with hgRNA and developed models to estimate the total number of variants.

Literature data was used for reference and #22 hgRNA was selected as an example.

♠ Fitting function 1:

Figure 1. The frequency and count data of 22hgRNA mutant

Result: Barcode number: 1363

♠ Fitting function 2:

Result: Barcode number: 1401

Since both of the fitting functions showed the similar results (the total number of variants produced by one hgRNA is about 1400), the superiority of our project was proved.

Read hgRNA at RNA Level

Constitutive double promoter module ：

Design:

U6 promoter is used to to express the functional gRNA and GAPDH promoter is used to drive the transcription of another gRNA and add the polyA tail. So hgRNA can be captured by oligo dT in single cell RNA sequencing.

Experiments:

We tested the ability of gRNA by targeting ADE2 gene: If ADE2 gene was mutated, colonies would turn pink.

Then we captured gRNA by using oligo dT when performing RT-PCR.

Inducible double promoter module:

Considering that the lineage information may only need to be read at specific time, we replaced GAPDH promoter with GAL7 promoter for further testing.

Results

Couple the expression of Cas9 with cell cycle——> Label each cell uniquely and automatically

We constructed a periodic expression module of Cas9 in Saccharomyces cerevisiae and verified the degradation effect of the first 124 amino acids of Clb2.

Figure 1. Gel electrophoresis results of colony PCR

Figure 2. Fitting curve of the relationship between GFP concentration and time

A previous study has demonstrated that CLB2 promoter can regulate the transcription of its downstream gene periodically, which suggested the feasibility of our design.

Increase the variants of barcodes ——> label more cells and more generations

Through modeling, we concluded that the total number of variants that one hgRNA can produce is about 1,400.

Add a polyA tail to hgRNA——>Read barcodes together with transcriptomic information

The experiments showed that:

(1) U6 promoter can transcribe gRNA normally (if gRNA successfully targeted the ADE2 gene, the colonies will turn pink).

(2) GAPDH promoter/GAL7 promoter can add a polyA tail to hgRNA, and it can be captured by oligo dT through RT-PCR.

Figure 3. Verification of the double promoter module.

Future Direction

• We will try to apply our ABC system in mammals. It can contribute to researches on some proposed applications like cancer or stem cell therapy.

• We will strive for suitable promoters and hgRNAs to make it more compatible with various species. We will maximize activity and minimize the off-target effect of Cas9 from three aspects: rationally designing hgRNA, switching to high-fidelity Cas9 and changing the concentration of Cas9/hgRNA.

• We will construct kits convenient for users.

Reference

[1] Trcek, T., Larson, D. R., Moldón, A., Query, C. C., & Singer, R. H. (2011). Single-molecule mRNA decay measurements reveal promoter- regulated mRNA stability in yeast. Cell, 147(7), 1484–1497.

[2] Bowling, S., Sritharan, D., Osorio, F. G., Nguyen, M., Cheung, P., Rodriguez-Fraticelli, A., Patel, S., Yuan, W. C., Fujiwara, Y., Li, B. E., Orkin, S. H., Hormoz, S., & Camargo, F. D. (2020). An Engineered CRISPR-Cas9 Mouse Line for Simultaneous Readout of Lineage Histories and Gene Expression Profiles in Single Cells. Cell, 181(7), 1693–1694.

[3] Kalhor, R., Mali, P., & Church, G. M. (2017). Rapidly evolving homing CRISPR barcodes. Nature methods, 14(2), 195–200. (Kalhor, R., et al, 2017)