Engineered APOBEC3C Sequencing Enables Bisulfite-Free and Direct Detection of DNA Methylation at a Single-Base Resolution

Abstract

DNA methylation (5-methylcytosine, 5mC) is the most important epigenetic modification in mammals. Deciphering the roles of 5mC relies on the quantitative detection of 5mC at the single-base resolution. Bisulfite sequencing (BS-seq) is the most often employed technique for mapping 5mC in DNA. However, bisulfite treatment may cause serious degradation of input DNA due to the harsh reaction conditions. Here, we engineered the human apolipoprotein B mRNA-editing catalytic polypeptide-like 3C (A3C) protein to endow the engineered A3C (eA3C) protein with differential deamination activity toward cytosine and 5mC. By the virtue of the unique property of eA3C, we proposed an engineered A3C sequencing (EAC-seq) method for the bisulfitefree and quantitative mapping of 5mC in DNA at the single-base resolution. In EAC-seq, the eA3C protein can deaminate C but not 5mC, which is employed to differentiate C and 5mC in sequencing. Using the EAC-seq method, we quantitatively detected 5mC in genomic DNA of lung cancer tissue. In contrast to the harsh reaction conditions of BS-seq, which could lead to significant degradation of DNA, the whole procedure of EAC-seq is carried out under mild conditions, thereby preventing DNA damage. Taken together, the EAC-seq approach is bisulfite-free and straightforward, making it an invaluable tool for the quantitative detection of 5mC in limited DNA at the single-base resolution.

The role of imprinting genes’ loss of imprints in cancers and their clinical implications

“…Chemical enzymes compensate for this defect ( 143 ). For instance, a combination of chemical enzymes such as APOBEC3A (A3A) or engineered APOBEC3C (eA3C) and sequencing technologies has achieved consistent and reliable results ( 144 , 145 ). This highlights the potential of multidisciplinary combinations to lead to new approaches.…”

Simultaneous detection of 5-methylcytosine and 5-hydroxymethylcytosine at specific genomic loci by engineered deaminase-assisted sequencing

“…† The synthesis of DNA-C1, DNA-5mC1, and DNA-5hmC1 followed the protocols outlined in our previous report. 37 For the preparation of DNA-C2, 0.5 ng of synthetic DNA from Takara was used as a template for PCR amplification. The PCR was performed in a 50 μL mixture containing 1 unit of Q5 High-Fidelity DNA polymerase, 4 μL of 2.5 mM dNTPs, 5 μL of 10× reaction buffer, 2 μL of 10 μM forward primer, and 2 μL of 10 μM reverse primer (Table S3 † ).…”

Recent Advances in Deciphering the Mechanisms and Biological Functions of DNA Demethylation

“…5mC on genomes can undergo deamination by activation‐induced cytidine deaminase (AID) or apolipoprotein B mRNA editing enzyme catalytic subunit (APOBEC) family enzymes. [ 57‐64 ] This deamination can result in the production of thymine in genomes, leading to T:G mismatches that can be repaired by subsequent TDG‐ or methyl‐CpG binding domain IV (MBD4)‐BER machinery to restore unmodified cytosine (Figure 5A). [ 65 ] However, 5mC is abundant in genomes, and deamination of 5mC could lead to a large number of DNA strand breaks, resulting in high gene mutations.…”