Biology subjects

Yang, X.-J.

Publications and source records attributed to Yang, X.-J..

8 recordsLinked to original sources

bioRxiv2025

P3a site-specific or cassette mutagenesis for seamless protein and plasmid engineering

Site-directed mutagenesis is a basic molecular tool required for protein, RNA and plasmid engineering. For mutagenesis methods, an ideal goal is to reach the efficiency of 100%. Towards this goal, we have recently taken the first step by adopting an innovative strategy via primer pairs with 3'-overhangs and developing P3 site-directed mutagenesis, with an average efficiency of [~]50%. As the second step towards the ideal goal, we report here P3a site-directed mutagenesis with an efficiency reaching [~]100%. We systematically evaluated this new method by engineering >100 point mutations and small deletions (or insertions) on >20 mammalian expression vectors encoding various epigenetic regulators and the spike protein of SARS-CoV-2. As all known mutagenesis methods are limited to point mutations and small deletions/insertions (up to a dozen nucleotides), a technical problem is how to carry out cassette mutagenesis for replacement, deletion or insertion of large DNA fragments. The high efficiency of P3a mutagenesis and the handshaking feature of primer pairs with 3'-overhangs inspired us to adapt this new method for seamless cassette mutagenesis, including highly efficient epitope tagging and untagging, deletion of small or large DNA fragments (up to 5 kb) and insertion of gene fragments (up to [~]0.4 kb), LoxP sites and sequences encoding degrons, sgRNA and tigRNA. Thus, this new site-specific and cassette mutagenesis method is highly efficient, fast and versatile, likely resulting in its wide use for typical biomedical research, as well as for engineering and refining synthetic or mutant proteins from AI-assisted design.

molecular biology↗

bioRxiv2024

P3 site-directed mutagenesis: An efficient method based on primer pairs with 3-prime overhangs

Site-directed mutagenesis is a fundamental tool indispensable for protein and plasmid engineering. An important technological question is how to achieve the efficiency at the ideal level of 100%. Based on complementary primer pairs, the QuickChange method has been widely used, but it requires significant improvements due to its low efficiency and frequent unwanted mutations. An alternative and innovative strategy is to utilize primer pairs with 3-prime overhangs, but this approach has not been fully developed. As the first step towards reaching the efficiency of 100%, we have optimized this approach systematically (such as use of newly designed short primers, test of different Pfu DNA polymerases and modification of PCR parameters) and evaluated the resulting method extensively with over 100 mutations on 12 mammalian expression vectors, ranging from 7.0-13.4 kb in size and encoding ten epigenetic regulators with links to cancer and neurodevelopmental disorders. We have also tested the new method with two expression vectors for the SARS-COV-2 spike protein. Compared to the QuickChange method, the success rate has increased substantially, with an average efficiency of [~]50%, with some at or close to 100 percent, and requiring much less time for engineering various mutations. Therefore, we have developed a new site-directed mutagenesis method for efficient and economical generation of various mutations. Notably, the method failed with a human KAT2B expression plasmid that possesses extremely GC-rich sequences. Thus, this study also sheds light on how to improve the method for developing ideal mutagenesis methods with the efficiency at [~]100% for a wide spectrum of plasmids.

molecular biology↗

bioRxiv2022

Omicron variant of SARS-COV-2 gains new mutations in New Zealand and Hong Kong

Disclaimer statementThe authors have withdrawn this manuscript because an issue was raised about the SARS- COV-2 genome sequence data used in the study. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

genomics↗

bioRxiv2021

CNTF enhances aerobic glycolysis and anabolic activities in degenerating retinas

Ciliary neurotrophic factor (CNTF) acts as a potent neuroprotective cytokine in multiple models of retinal degeneration. To understand mechanisms underlying its broad neuroprotective effects, we have investigated the influence of CNTF on metabolism in a mouse model of photoreceptor degeneration. CNTF treatment improves the morphology of photoreceptor mitochondria, but also leads to reduced oxygen consumption and suppressed respiratory chain activities. Molecular analyses show elevated glycolytic pathway gene transcripts and active enzymes. Metabolomics analyses detect significantly higher levels of ATP and the energy currency phosphocreatine, elevated glycolytic pathway metabolites, increased TCA cycle metabolites, lipid biosynthetic pathway intermediates, nucleotides, and amino acids. Moreover, CNTF treatment restores the key antioxidant glutathione to the wild type level. Therefore, CNTF significantly impacts the metabolic status of degenerating retinas by promoting aerobic glycolysis and augmenting anabolic activities. These findings reveal cellular mechanisms underlying enhanced neuronal viability and suggest potential therapies for treating retinal degeneration.

neuroscience↗

bioRxiv2021

δ1 variant of SARS-COV-2 acquires spike V1176F and yields a highly mutated subvariant in Europe

Genomic surveillance of SARS-COV-2 has revealed that in addition to many variants of interests, this virus has yielded four variants of concern, , {beta}, {gamma} and {delta}, as designated by the World Health Organization. {delta} variant has recently become the predominant pandemic driver around the world and yielded four different subvariants ({delta}1, {delta}2, {delta}3 and {delta}4). Among them, {delta}1 has emerged as the key subvariant that drives the pandemic in India, Europe and the USA. A relevant question is whether {delta}1 subvariant continues to evolve and acquires additional mutations. Related to this, this subvariant has acquired spike V1176F, a signature substitution of {gamma} variant, and yielded a new sublineage, {delta}1F. The substitution alters heptad repeat 2 of spike protein and is expected to improve interaction with heptad repeat 1 and enhance virus entry. Moreover, there are {delta}1F sublineages encoding spike N501Y, A783S, Q836E and V1264L. While N501Y is a signature substitution shared by , {beta} and {gamma} variants, V1264L is a key substitution in a {delta}1 sublineage that is a major pandemic driver in Southeast Asia. The Q836E-encoding lineage carries an average of 50 mutations per genome, making it the most mutated variant identified so far. Similar to {delta}1 subvariant, {delta}2 subvariant has also acquired spike V1176F and yielded new sublineages. Together, these results suggest that V1176F is a recurrent spike substitution that is frequently acquired by SARS-COV-2 variants to improve viral fitness. It is thus important to track the evolutionary trajectory of related variants for considering and instituting the most effective public health measures.

evolutionary biology↗

bioRxiv2021

SARS-COV-2 γ variant acquires spike P681H or P681R for improved viral fitness

Severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) evolves and generates different variants through a continuously branching model. Four variants of concern have been the major pandemic drivers around the world. One important question is how they may evolve and generate subvariants, some of which may be even more virulent and drive the pandemic further. While investigating how {gamma} (or P.1) variant has been evolving, I noticed the spike substitution P681H in a group of genomes encoding a new subvariant, which has been designated P.1.7. This subvariant has become the dominant P.1 sublineage in Brazil, Italy, Spain and Peru, supporting that P681H confers evolutionary advantage to P.1.7. In Brazil and Peru, P.1.7 was still responsible for ~30% and ~40% cases, respectively, in August 2021. However, it has been competed out by {delta}1 (a {delta} subvariant) in both countries, Italy and Spain, suggesting that P.1.7 is not as virulent as {delta}1. In addition, 160 P.1 genomes possess a related substitution, P681R, and 120 of them encode a new subvariant, designated P.1.8. This P.1 subvariant carries two additional spike substitutions, T470N and C1235F, located at the receptor-binding pocket and cytoplasmic tail of spike protein, respectively. More P.1.8 genomes have been identified than P.1 genomes that encode P681R but not T470N and C1235F, suggesting that these two substitutions improve virulence of P.1.8 subvariant. Some P.1 genomes carry other substitutions (such as N679K, V687L and C1250F) that affect the furin cleavage site or cytoplasmic tail of spike protein. Thus, to improve viral fitness and expand its evolutionary cage, {gamma} variant acquires mutations to finetune the furin cleavage site and cytoplasmic tail of spike protein.

evolutionary biology↗

bioRxiv2021

SARS-COV-2 C.1.2 variant is highly mutated but may possess reduced affinity for ACE2 receptor

SARS-COV-2 evolution generates different variants and drives the pandemic. As the current main driver, delta variant bears little resemblance to the other three variants of concern (alpha, beta and gamma), raising the question what features the future variants of concern may possess. To address this important question, I searched through the GISAID database for potential clues. While investigating how beta variant has been evolving in South Africa, I noticed a small group of genomes mainly classified as C.1.2 variant, with one-year old boy identified in March 2021 being the index case. Over 80% patients are younger than 60. At the average, there are 46-47 mutations per genome, making this variant one of the most mutated lineages identified. A signature substitution is spike Y449H. Like beta and gamma variants, C.1.2 possesses E484K and N501Y. The genomes are heterogenous and encode different subvariants. Like alpha variant, one such subvariant encodes the spike substitution P681H at the furin cleavage site. In a related genome, this substitution is replaced by P681R, which is present in delta variant. In addition, similar to this variant of concern, three C.1.2 subvariants also encode T478K. Mechanistically, spike Y449 recognizes two key residues of the cell-entry receptor ACE2 and Y449H is known to impair the binding to ACE2 receptor, so C.1.2 variant may show reduced affinity for this receptor. If so, this variant needs other mutations to compensate for such deficiency. These results raise the question whether C.1.2 variant is as virulent as suggested by its unexpected high number of mutations.

evolutionary biology↗

bioRxiv2020

Single cell transcriptomic analysis reveals the impact of elevating neurogenic factor expression on human retinal organoid development

The developing retina expresses multiple bHLH transcription factors. Their precise functions and interactions in uncommitted retinal progenitors remain to be fully elucidated. Here, we investigate the roles of bHLH factors ATOH7 and Neurog2 in developing human ES cell-derived 3D retinal organoids. Single cell transcriptome analyses identify three states of proliferating retinal progenitors: pre-neurogenic, neurogenic, and cell cycle-exiting progenitors. Each shows different expression profile of bHLH factors. The distinct cell cycle-exiting progenitors feed into a postmitotic heterozygous neuroblast pool that gives rise to early born neuronal lineages. Elevating ATOH7 or Neurog2 expression accelerates the transition from the pre-neurogenic to the neurogenic state, and expands the exiting progenitor and neuroblast populations. In addition, ATOH7 and Neurog2 significantly, yet differentially, enhance retinal ganglion cell and cone photoreceptor production. Moreover, single cell transcriptome analyses reveal that ATOH7 and Neurog2 assert positive autoregulation, suppress key bHLH factors associated with the neurogenic progenitors, and elevate bHLH factors expressed by exiting progenitors and differentiating neuroblasts. This study thus provides novel insight regarding how ATOH7 and Neurog2 impact human retinal progenitor behaviors and neuroblast fate choices.

developmental biology↗