Biology subjects

Zuo, E.

Publications and source records attributed to Zuo, E..

7 recordsLinked to original sources

bioRxiv2025

Sequential Amino Acid Mutagenesis-Driven De Novo Evolution of Adenine Deaminases Enables Efficient In Vivo Base Editing in Primate

Base editing allows for the precise modification of genetic information, providing new avenues for treating diseases1. The adenine base editor ABE8e is currently the most efficient and widely used tool for adenine base editing. ABE8e was developed through multiple rounds of directed evolution of Escherichia coli tRNA adenine deaminase, including phage-assisted continuous evolution (PACE)2,3. While PACE is highly effective, it is a complex system that poses challenges for implementation4. Despite its high efficiency, ABE8e is associated with limitations such as relatively higher bystander editing effects and elevated off-target activity5, which need to be addressed to further enhance its precision and safety. Here, we developed a novel method for the de novo discovery of evolved ABE components, particularly adenine deaminases. This process involves identifying candidate proteins through AI-based structural prediction and clustering, followed by the enhancement of deaminase editing activity through screening libraries created by sequential amino acid saturation mutagenesis. This evolutionary strategy simplifies the approach by employing saturation mutagenesis libraries tailored to specific segments, thereby enabling exploration of an expanded sequence space and increasing the likelihood of discovering adenine deaminases with superior capabilities. The newly developed hpABE5.20 here demonstrates a more refined editing window, reduced DNA off-target effects that are both sgRNA-dependent and -independent, and minimized RNA off-target activity, while maintaining robust editing efficiency relative to ABE8e. Furthermore, hpABE5.20 has been successfully applied for precise and effective therapeutic adenine base editing in cellular disease models, humanized mice, and non-human primates.

bioengineering↗

bioRxiv2023

Discovery of new deaminase functions by structure-based protein clustering

The elucidation of protein function and its exploitation in bioengineering have greatly contributed to the development of the life sciences. Existing protein mining efforts generally rely on amino acid sequences rather than protein structures due to technical difficulties in structural elucidation. We describe here for the use of AlphaFold2 to predict and subsequently cluster an entire protein family based on predicted structure similarities. We selected the deaminase family of proteins to analyze and through this approach identified many previously unknown deaminase properties. We applied these new deaminases to the development of new cytosine base editors with distinct features. Although we found many new double-stranded DNA deaminases from the DddA-like protein clade, we were surprised to find that most of the proteins in this family were not actually double-stranded DNA cytidine deaminases. From this protein clade, we engineered the smallest single-strand specific cytidine deaminase, which facilitates the first efficient cytosine base editor to be packaged into a single AAV vector. Importantly, we also profiled a deaminase from this clade that edits robustly in soybean plants, which previously suffered from poor editing by cytosine base editors. These newly discovered deaminases based on AI-assisted structural predictions greatly expand the utility of base editors for therapeutic and agricultural applications.

molecular biology↗

bioRxiv2021

THEMIS is a priming substrate of non-receptor tyrosine phosphatase PTPN6/SHP1 and plays dual roles during T cell development

THEMIS plays an indispensable role in T cells, but its mechanism of action is highly controversial. Using the systematic proximity labeling methodology PEPSI, we identified THEMIS as an uncharacterized substrate for the phosphatase SHP1. Saturated mutagenesis analysis revealed that THEMIS phosphorylation at the evolutionally conserved Tyr34 residue was oppositely regulated by SHP1 and the kinase LCK. Like THEMIS-/- mice, THEMISY34F/Y34F knock-in mice showed a significant decrease in CD4 thymocytes and mature CD4 T cells, but a normal thymic development and peripheral homeostasis of CD8 T cells. Mechanistically, phosphorylated THEMIS induced by TCR activation acts as a "priming substrate" to bind SHP1 and convert its phosphatase activity from basal level to nearly fully activated level, ensuring an appropriate negative regulation of TCR signaling. However, cytokine signaling in CD8 T cells failed to elicit THEMIS Y34 phosphorylation, revealing both phosphorylation-dependent and -independent roles of THEMIS in controlling T cell maturation and expansion.

cell biology↗

bioRxiv2021

Develop an efficient and specific AAV-based labeling system for Muller glia in mice

Cell degeneration in the retina leads to several ocular diseases and vision loss. Considerable research efforts focus on reprogramming Muller glia (MG) into functional cells to rescue vision as a promising therapeutic strategy, although whether MG can convert into functional bipolar cells, retinal ganglia cells (RGCs), rods or cones in mammals remains controversial. The broad applicability of tracking MG differentiation thus presents a need for improved labeling efficiency and specificity. Here, we compared AAV-based labeling strategies with conventional lineage-tracking by crossing transgenic mouse lines. We found that reporter expression was weak and not MG-specific in mGFAP-Cre transgenic mice. Different AAV serotypes showed a range of efficiency and specificity in labeling MG, leading us to optimize a human GFAP-Cre reporter system packaged in the AAV9 serotype with the WPRE (WPRE, woodchuck hepatitis virus post-transcriptional regulatory element) removed. The hGFAP-Cre- {Delta}WPRE reporter could label 20-73.8% MGs, with non-specific RGC labeling rates ranging from 0-0.08% at doses of 1 x 108 to 1010 vector genomes (vg) per eye, an approximate 40-fold reduction compared with the AAV9-hGFAP-Cre-WPRE labeling system. Furthermore, we validated the availability of the label system to trace MG reprogramming and also detected false-positive results reported previously. The AAV9-hGFAP-Cre-{Delta}WPRE system thus represents a highly efficient and specific labeling system for MG, providing a valuable tool for tracking cell fate in vivo.

neuroscience↗

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

Chromosome-scale de novo assembly and phasing of a Chinese indigenous pig genome

Chinese indigenous pigs differ significantly from Western commercial pig breeds in phenotypic and genomic characteristics. Thus, building a high-quality reference genome for Chinese indigenous pigs is pivotal to exploring gene function, genome evolution and improving genetic breeding in pigs. Here, we report an ultrahigh-quality phased chromosome-scale genome assembly for a male Luchuan pig, a representative Chinese domestic breed, by generating and combining data from PacBio Sequel reads, Illumina paired-end reads, high-throughput chromatin conformation capture and BioNano optical map. The primary assembly is [~] 2.58 Gb in size with contig and scaffold N50s of 18.03 Mb and 140.09 Mb, respectively. Comparison between primary assembly and alternative haplotig reveals numerous haplotype-specific alleles, which provide a rich resource to study the allele-specific expression, epigenetic regulation, genome structure and evolution of pigs. Gene enrichment analysis indicates that the Luchuan-specific genes are predominantly enriched in Gene Ontology terms for phosphoprotein phosphatase activity, signaling receptor activity and phosphatidylinositol binding. We provide clear molecular evolutionary evidence that the divergence time between Luchuan and Duroc pigs is dated back to about 1.7 million years ago. Meanwhile, Luchuan exhibits fewer events of gene family expansion and stronger gene family contraction than Duroc. The positively selected genes (PSGs) in Luchuan pig significantly enrich for protein tyrosine kinase activity, microtubule motor activity, GTPase activator activity and ubiquitin-protein transferase activity, whereas the PSGs in Duroc pig enrich for G-protein coupled receptor activity. Overall, our findings not only provide key benchmark data for the pig genetics community, but also pave a new avenue for utilizing porcine biomedical models to study human health and diseases.

genomics↗

bioRxiv2018

Base editing generates substantial off-target single nucleotide variants

Genome editing tools including CRISPR/Cas9 and base editors hold great promise for correcting pathogenic mutations. Unbiased genome-wide off-target effects of the editing in mammalian cells is required before clinical applications, but determination of the extent of off-target effects has been difficult due to the existence of single nucleotide polymorphisms (SNPs) in individuals. Here, we developed a method named GOTI (Genome-wide Off-target analysis by Two-cell embryo Injection) to detect off-target mutations without interference of SNPs. We applied GOTI to both the CRISPR-Cas9 and base editing (BE3) systems by editing one blastomere of the two-cell mouse embryo and then compared whole genome sequences of progeny-cell populations at E14.5 stage. Sequence analysis of edited and non-edited cell progenies showed that undesired off-target single nucleotide variants (SNVs) are rare (average 10.5) in CRISPR-edited mouse embryos, with a frequency close to the spontaneous mutation rate. By contrast, BE3 editing induced over 20-fold higher SNVs (average 283), raising the concern of using base-editing approaches for biomedical application.

genomics↗