Biology subjects

Zeng, R.

Publications and source records attributed to Zeng, R..

22 records · Page 2Linked to original sources

bioRxiv2020

High-fidelity base editor with no detectable genome-wide off-target effects

Base editors hold promise for correcting pathogenic mutations, while substantial single nucleotide variations (SNVs) on both DNA and RNA were generated by cytosine base editors (CBEs). Here we examined possibilities to reduce off-target effects by engineering cytosine deaminases. By screening 24 CBEs harboring various rAPOBEC1 (BE3) or human APOBEC3A (BE3-hA3A) mutations on the ssDNA or RNA binding domain, we found 8 CBE variations could maintain high on-target editing efficiency. Using Genome-wide Off-target analysis by Two-cell embryo Injection (GOTI) method and RNA sequencing analysis, we found DNA off-target SNVs induced by BE3 could be completely eliminated in BE3R126E but the off-target RNA SNVs was only slightly reduced. By contrast, BE3-hA3AY130F abolished the RNA off-target effects while could not reduce the DNA off-target effects. Notably, BE3R132E, BE3W90Y+R126E and BE3W90F+R126E achieved the elimination of off-target SNVs on both DNA and RNA, suggesting the feasibility of engineering base editors for high fidelity deaminases.

genomics↗

bioRxiv2019

Suppressor mutations in Mecp2-null mice reveal that the DNA damage response is key to Rett syndrome pathology

Mutations in X-linked methyl-CpG-binding protein 2 (MECP2) cause Rett syndrome (RTT). We carried out a genetic screen for secondary mutations that improved phenotypes in Mecp2/Y mice after mutagenesis with N-ethyl-N-nitrosourea (ENU), aiming to identify potential therapeutic entry points. Here we report the isolation of 106 founder animals that show suppression of Mecp2-null traits from screening 3,177 Mecp2/Y genomes. Using exome sequencing, genetic crosses and association analysis, we identify 33 candidate genes in 30 of the suppressor lines. A network analysis shows that 61% of the candidate genes cluster into the functional categories of transcriptional repression, chromatin modification or DNA repair, delineating a pathway relationship with MECP2. Many mutations lie in genes that are predicted to modulate synaptic signaling or lipid homeostasis. Surprisingly, mutations in genes that function in the DNA damage response (DDR) also improve symptoms in Mecp2/Y mice. The combinatorial effects of multiple loci can be resolved by employing association analysis. One line, which was previously reported to carry a suppressor mutation in a gene required for cholesterol synthesis, Sqle, carries a second mutation in retinoblastoma binding protein 8 (Rbbp8 or CtIP), which regulates a DDR choice in double stranded break (DSB) repair. Cells from Mecp2/Y mice have increased DSBs, so this finding suggests that the balance between homology directed repair and non-homologous end joining is important for neuronal cells. In this and other lines, the presence of two suppressor mutations confers better symptom improvement than one locus alone, suggesting that combination therapies could be effective in RTT.

genetics↗

bioRxiv2018

Data-Independent Acquisition Mass Spectrometry to Localize Phosphosites

In conventional data-dependent acquisition (DDA) mode used for phosphoproteome analysis by mass spectrometry, a considerable number of phosphosites are localized ambiguously. We found that data-independent acquisition (DIA) method could localize phosphosite accurately and confidently due to its acquisition nature of repeated and unbiased mode. Here, we present a robust and promising DIA workflow for modification sites determination. Through dedicated collection and analyses of site-determining ions following their chromatographic profilings, phosphosites could be assigned or rectified unambiguously.

bioinformatics↗

bioRxiv2018

A novel amidase signature family amidase from the marine actinomycete Salinispora arenicola CNS-205

We cloned a new gene from the amidase signature (AS) family, designated am, from the marine actinomycete Salinispora arenicola CNS-205. As indicated by bioinformatics analysis and site-directed mutagenesis, the AM protein belonged to the AS family. AM was expressed, purified, and characterised in Escherichia coli BL21 (DE3), and the AM molecular mass was determined to be 51 kDa. The optimal temperature and pH were 40 {degrees}C and pH 8.0, respectively. AM exhibited a wide substrate spectrum and showed amidase, aryl acylamidase, and acyl transferase activities. AM had high activity towards aromatic and aliphatic amides. The AM substrate specificity for anilides was very narrow; only propanil could be used as an effective substrate. The extensive substrate range of AM indicates it may have broad potential applications in biosynthetic processes and biodegradation.

molecular biology↗