Team:BNU-China/Design
In recent years, the method combining CRISPR/Cas9 and barcode, a DNA sequence as genetic marker, has drawn great attention [1]. However, the constitutively expressed Cas9 consumes barcodes quickly and the total number of barcodes is limited. Our project aims to label cells automatically, prolong the tracing generations and increase the tracing number of cells in every generation. To label each cell automatically and prolong the traced generations, we built a periodic expression and degradation module of Cas9. To increase the number of traced cells in one generation, we replaced single guide RNA with homing guide RNA. To prolong the tracing generations, we build a periodic expression module of Cas9 in association with cell division to label each cell automatically. Meanwhile, we also have to degrade Cas9 at a proper speed, which needs to be degraded before early G1 phase.
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 and degradation of Cas9
In order to control the expression of Cas9 and achieve the automatic addition of barcodes in each cell cycle, we selected the promoter of the G2 / mitotic-specific Cyclin B2 to initiate the expression of Cas9. In Saccharomyces cerevisiae, the homologous gene is CLB2. Previous studies have revealed that Clb2 promoter can regulate the transcription of its downstream gene periodically. 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) [2].
Thus, we built the system and used CLB2 promoter to express Cas9 periodically.
Figure 1. Percent of cells with a CLB2 transcription site per cell cycle phase
In order to degrade Cas9 timely, we used the first 124 amino acids of Clb2 as a degradation tag. Research shows that it contains D-Box and KEN Box (Figure 2), which are the recognition site of E3 ubiquitin ligase APC (Anaphase-promoting complex), which initiates the ubiquitin-proteasome proteolytic. To evaluate the degradation effect mediated by the first 124 amino acids of Clb2, we fused it to GFP and put them downstream the galactose-activated promoter GAL1. Through detecting the changes of fluorescence intensity and modeling, we can calculate the degradation rate of the first 124 amino acids of Clb2.
Figure 2. A schematic diagram of the Clb2 protein sequence
hgRNA module
The CRISPR/Cas9 mediated lineage tracing system involves three components: the sgRNA, the Cas9 protein, and barcodes. The sgRNA consists of a constant scaffold which interacts with the Cas9 protein and a variable spacer whose sequence determines the target site. The target site includes a specific protospacer adjacent motif (PAM) that is directly recognized by the Cas9 protein. To increase the number of traced cells in one generation, we replaced single guide RNA(sgRNA) with homing guide RNA (hgRNA). Different from sgRNA, the homing guide RNA (hgRNA) itself contains a PAM sequence which is immediately downstream its spacer, guiding the Cas9: hgRNA complex to its own locus.
In our project, we use hgDNA as barcode. When it transcribes and produces hgRNA, the Cas9: hgRNA complex will cleave itself and change its sequence. After cleaving, new hgDNA produce new hgRNA, match with hgDNA and cleave over and over again, producing a great amout of barcodes.
Double promoter module
In order to better serve academic and industry laboratories engaged in related research, we interviewed companies focused on single cell sequencing. We learned that the 10X Genomics platform is currently the most commonly used transcriptome sequencing platform and it uses primers with oligo dT to capture all kinds of mRNA. If we want to effectively utilize the 10X Genomics system, a poly A tail is required. However, the addition of a polyA tail will disable hgRNA.
Based on this, we use two promoters and put GAPDH promoter in the upstream of U6 promoter. U6 promoter is used to express the functional hgRNA. GAPDH promoter is used to drive the transcription of barcode and add the polyA tail. In this way, we can read the lineage information out of transcriptomic information. Considering that the lineage information may only need to be read at specific time, we also replaced GAPDH promoter with GAL7 promoter for further testing.
We learned that the 10X Genomics platform is currently the most commonly used transcriptome sequencing platform and it uses primers with oligo dT to capture all kinds of mRNA. In order to read the lineage information in hgRNA together with transcriptomic information, we designed the double promoter module. We put GAPDH promoter in the upstream of U6 promoter. The U6 promoter is used to transcribe hgRNA constitutively, and the GAPDH promoter is used to add the polyA tail to hgRNA, which enables it to be captured by oligo dT in single cell RNA sequencing. we also designed an inducible double promoter module by replaced GAPDH promoter with GAL7 promoter for further testing.
To test whether the first promoter will affect the function of gRNA, we put the double promoter upstream of gRNA targeting ADE2 gene. A loss-of-function mutation in ADE2 can result in an adenine auxotroph that forms pink colonies on YNBA plates containing a low level of adenine, thus enabling a visual evaluation of the action of CRISPR/Cas9.
Further, we use oligo dT as primer to reverse transcribe gRNA to test whether gRNA can be captured by 10X Genomics platform.
Postcode:100875
Email：igem2020bnu_china@163.com
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