MolecularCloud August Newsletter: Glycosylase base editors enable C-to-A and C-to-G base changes

The CRISPR–Cas9 system is able to recognize target sequences based purely on nucleotide sequences, and it has been adapted for genome editing in both eukaryotes and prokaryotes. Recently, a new family of CRISPR-based genome-editing methods, named base-editor techniques, was developed. Because current base-editing techniques can only facilitate C-to-T editing and A-to-G editing, a complete base-editing technique for converting any base to any other base is highly desirable.

In the paper published in Nature Biotechnology by Dr. Changhao Bi’s team and Dr. Xueli Zhang’s team at Tianjin Institute of Industrial Biotechnology on July 20, 2020, they present BEs that cause C-to-A transversions in Escherichia coli and C-to-G transversions in mammalian cells. All plasmids of this article have been deposited on MolecularCloud (Dongdong Zhao, et al., 2020). 

These glycosylase base editors (GBEs) consist of a Cas9 nickase, a cytidine deaminase and a uracil-DNA glycosylase (Ung). Ung excises the U base created by the deaminase, forming an apurinic/apyrimidinic (AP) site that initiates the DNA repair process.

In E. coli, they used activation-induced cytidine deaminase (AID) to construct AID-nCas9-Ung and found that it converts C to A with an average editing specificity of 93.8% ± 4.8% and editing efficiency of 87.2% ± 6.9%. When used in mammalian cells, they replaced AID with rat APOBEC1 (APOBEC-nCas9-Ung). They tested APOBEC-nCas9-Ung at 30 endogenous sites, and they observed C-to-G conversions with a high editing specificity at the sixth position of the protospacer between 29.7% and 92.2% and an editing efficiency between 5.3% and 53.0%. APOBEC-nCas9-Ung supplements the current adenine and cytidine BEs (ABE and CBE, respectively) and could be used to target G/C disease-causing mutations.

Table 1. Plasmids from this article

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