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

MC Cat No.

Plasmid Name

Description

MC_0101154

pAPOBEC_nCas9_Ung

HEK293T expression of APOBEC_nCas9_Ung

MC_0101155

pRNF2-site2-gRNA

HEK293T expression, gRNAs targeting RNF2-site2

MC_0101156

pRNF2-site3-gRNA

HEK293T expression, gRNAs targeting RNF2-site3

MC_0101157

pEMX1-site1-gRNA

HEK293T expression, gRNAs targeting EMX1-site1

MC_0101158

pEMX1-site4-gRNA

HEK293T expression, gRNAs targeting EMX1-site4

MC_0101159

pEMX1-site5-gRNA

HEK293T expression, gRNAs targeting EMX1-site5

MC_0101160

pEMX1-site6-gRNA

HEK293T expression, gRNAs targeting EMX1-site6

MC_0101162

pFANCF-gRNA-site4

HEK293T expression, gRNAs targeting FANCF-site4

MC_0101163

pRP11-177B4-gRNA-1

HEK293T expression, gRNAs targeting RP11-177B4-1

MC_0101164

pRP11-177B4-gRNA-2

HEK293T expression, gRNAs targeting RP11-177B4-2

MC_0101165

pRP11-177B4-gRNA-3

HEK293T expression, gRNAs targeting RP11-177B4-3

MC_0101166

pRP11-177B4-gRNA-4

HEK293T expression, gRNAs targeting RP11-177B4-4

MC_0101167

pPSMB2-gRNA-1

HEK293T expression, gRNAs targeting PSMB2-1

MC_0101168

pPSMB2-gRNA-2

HEK293T expression, gRNAs targeting PSMB2-2

MC_0101169

pVISTAhs267-gRNA

HEK293T expression, gRNAs targeting VISTAhs267

MC_0101170

pKLF4-gRNA-1

HEK293T expression, gRNAs targeting KLF4-1

MC_0101171

pKLF4-gRNA-2

HEK293T expression, gRNAs targeting KLF4-2

MC_0101172

pVEGFA-gRNA-3

HEK293T expression, gRNAs targeting VEGFA-3

MC_0101173

pDNMT3B-OTS1-gRNA

HEK293T expression, gRNAs targeting DNMT3B-OTS1

MC_0101174

pHEK2 OT-gRNA-1

HEK293T expression, gRNAs targeting HEK2 OT1

MC_0101175

pHEK2 OT-gRNA-2

HEK293T expression, gRNAs targeting HEK2 OT2

MC_0101176

pHEK2 OT-gRNA-3

HEK293T expression, gRNAs targeting HEK2 OT3

MC_0101177

pHEK4 OT-gRNA-1

HEK293T expression, gRNAs targeting HEK4 OT1

MC_0101178

pHEK4 OT-gRNA-2

HEK293T expression, gRNAs targeting HEK4 OT2

MC_0101179

pHIRA-gRNA-1

HEK293T expression, gRNAs targeting HIRA-1

MC_0101180

pHIRA-gRNA-2

HEK293T expression, gRNAs targeting HIRA-2

MC_0101181

pNIBAN1-gRNA

HEK293T expression, gRNAs targeting NIBAN1

MC_0101182

pRP1-97D16-gRNA

HEK293T expression, gRNAs targeting RP1-97D16

MC_0101183

pnCas9_AID

E.coli expression of nCas9_AID

MC_0101184

pUng-nCas9-AID

E.coli expression of Ung-nCas9-AID

MC_0101185

pgRNA_lacZ1444-1463

E.coli expression, gRNAs targeting lacZ1444-1463apramycin resistance


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