Contribution

In this project, the cytosine base editor (CBE for short) is at the core[1]. It is composed of four parts: dCas9 protein, gRNA, cytosine deaminase and uracil DNA glycosylase inhibitor (UGI). We have previously described the use of plasmids containing gRNA tandem arrays. Through our experiments, 36 targets can be edited simultaneously through this array.

In addition, It is inefficient and costly to send all the colonies to be sequenced, we need a mechanism to know the editing effect in advance. Advisors suggested that we could reserve three gRNAs targeting the Ade2 gene of Saccharomyces cerevisiae while targeting the information sequence,which is a marker gene commonly used. In our project, the working principle is that the editing of C to T bases is able to form the stop codon of TAA, which terminates the translation process of ADE2 prematurely, and the deletion of ADE2 in Saccharomyces cerevisiae leads to the accumulation of the intermediate phosphoriboglutaminosazole in the cell, which forms a red pigment after oxidation, causing yeast to fall from white to red on the medium lacking adenines. By looking at the color of these colonies, we can make a preliminary judgment about the success of the base editing, after which these red colonies are sequenced.

The selection of gRNA sequences is also one of our important contributions. For the 24 different gRNA sequences we use, their editing ability and editing efficiency are not the same. Most of the gRNA sequences we use can achieve the goal.

After experimental verification, we are glad to notice other teams that the following gRNA sequences work very well.

At the same time, we regret to point out that the editing effect of the following gRNA sequences is not ideal. In our several rounds of experiments, they had not been edited or the editing success rate was less than 20%.