Biology subjects

huo, y.

Publications and source records attributed to huo, y..

5 recordsLinked to original sources

bioRxiv2024

Chrom-pro: A User-Friendly Toolkit for De-novo Chromosome Assembly and Genomic Analysis

Chromosome-level genome assembly is fundamental to current genomic and post-genomic research, however, the process remains complicated and challenging due to the lack of a standardized automatic workflow. The frequently-used method for high-quality genome assembly generally employs second-generation sequencing (SGS) low error reads, third-generation sequencing (TGS) long reads and Hi-C reads. In this study, we developed a multifunctional toolkit called Chrom-pro that integrated commonly-used algorithms for de novo chromosome-level genome assembly with above three data sets into a user-friendly, automatic workflow. Besides chromosome assembly, Chrom-pro also encompasses multiple functionalities for genome quality assessment, comparative genomic analysis, and structural variant detection, which offers substantial support for downstream research. To evaluate the performance of Chrom-pro software, we tested the software with publicly available sequencing data of mango, pufferfish, and plum, and the excellence was confirmed by achieving a BUSCO completeness score of over 95% as well as high collinearity with the reference genome. Furthermore, we applied Chrom-pro to investigating the impact of different internal algorithm options on the accuracy of chromosome assembly, providing guidance for advancing relevant research in the future. Overall, the development of Chrom-pro will significantly improve the efficiency and quality of chromosome assembly and contributing to the advancement of genomic research.

bioinformatics↗

bioRxiv2022

A tRNA Modification-based strategy for Identifying amiNo acid Overproducers (AMINO)

Amino acids have a multi-billion-dollar market with rising demand, prompting the development of high-performance microbial factories. However, a general screening strategy applicable to all proteinogenic and non-proteinogenic amino acids is still lacking. Modification of the critical structure of tRNA could decrease the aminoacylation level of tRNA catalyzed by aminoacyl-tRNA synthetases. Involved in a two-substrate sequential reaction, amino acids with increased concentration could elevate the reduced aminoacylation rate caused by specific tRNA modification. Here, we developed a selection system for overproducers of specific amino acids using corresponding engineered tRNAs and reporter genes. As a proof-of-concept, overproducers of five amino acids such as L-tryptophan were screened out by growth-based and/or fluorescence-activated cell sorting (FACS)-based screening from random mutation libraries of Escherichia coli and Corynebacterium glutamicum, respectively. This study provided a universal strategy that could be applied to screen overproducers of proteinogenic and non-proteinogenic amino acids in amber-stop-codon-recoded or non-recoded hosts.

synthetic biology↗

bioRxiv2021

Genomic analysis based on chromosome-level genome assembly reveals an expansion of terpene biosynthesis of Azadirachta indica

Azadirachta indica (neem), an evergreen tree of the Meliaceae family, is a source of the potent biopesticide azadirachtin. The lack of a chromosome-level assembly impedes the understanding of in-depth genomic architecture and the comparative genomic analysis of A. indica. Here, a high-quality genome assembly of A. indica was constructed using a combination of data from Illumina, PacBio, and Hi-C technology, which is the first chromosome-scale genome assembly of A. indica. The genome size of A. indica is 281 Mb anchored to 14 chromosomes (contig N50=6 Mb and scaffold N50=19 Mb). The genome assembly contained 115 Mb repetitive elements and 25,767 protein-coding genes. Evolutional analysis revealed that A. indica didnt experience any whole-genome duplication (WGD) event after the core eudicot {gamma} event, but some genes and genome segment might undergo recent duplications. The secondary metabolite clusters, TPS genes, and CYP genes were also identified. Comparative genomic analysis revealed that most of the A. indica-specific TPS genes and CYP genes were located on the terpene-related clusters on chromosome 13. It is suggested that chromosome 13 may play an important role in the specific terpene biosynthesis of A. indica. And the gene duplication events may be responsible for the terpene biosynthesis expansion in A. indica. This will shed light on terpene biosynthesis in A. indica and facilitate comparative genomic research of the family Meliaceae.

genomics↗

bioRxiv2019

Reversed paired-gRNA plasmid cloning strategy for efficient genome editing in Escherichia coli

A growing number of CRISPR-Cas9 associated applications require co-expression of two distinct gRNAs. However, coexpressing paired gRNAs under the driving of independent but identical promoters in the same direction triggers plasmid instability, due to the presence of direct repeats (DRs). In this study, deletion between DRs occurred with high frequencies during plasmid construction and duplication processes, when three DRs-involved paired-gRNA plasmids cloning strategies were tested. This recombination phenomenon was RecA-independent, in agreement with the replication slippage model. To completely eliminate the DRs-induced plasmid instability, a reversed paired-gRNA plasmids (RPGPs) cloning strategy was developed by converting DRs to the more stable invert repeats (IRs). Using RPGPs, we achieved a rapid deletion of chromosome fragments up to 100 kb with high efficiency of 83.33% in Escherichia coli. This study provides general solutions to construct stable plasmids containing short DRs, which can improve the performances of CRISPR systems that relied on paired gRNAs, and also facilitate other applications involving repeated genetic parts.

microbiology↗

bioRxiv2019

CRISPR-Cas9-assisted native end-joining editing offers a simple strategy for efficient genetic engineering in Escherichia coli

Unlike eukaryotes, bacteria are less proficient in homologous recombination (HR) and non-homologous end joining (NHEJ). All existing genomic editing methods for Escherichia coli rely on exogenous HR or NHEJ systems to repair DNA double-strand breaks (DSBs). Although an E. coli native end-joining (ENEJ) system has been reported, its potential in chromosomal engineering has not yet been explored. Here, we present a CRISPR-Cas9-assisted native end-joining editing and show that ENEJ-dependent DNA repair can be used to conduct rapid and efficient knocking-out of E. coli genomic sequence of up to 83 kb. Moreover, the positive rate and editing efficiency is independent of high-efficiency competent cells. The method requires neither exogenous DNA repair systems nor introduced editing template. The Cas9 complex is the only foreign element in this method. This study is the first successful engineering effort to utilize ENEJ mechanism in genomic editing and provides an effective strategy for genetic engineering in bacteria that are inefficient in HR and NHEJ.\n\nSignificanceThe application in prokaryotes is difficult because of the weak homologous recombination and non-homologous end joining systems. E. coli, as the most-used prokaryote in metabolic engineering, has no NHEJ system. All existing genomic editing methods for E. coli rely on exogenous HR or NHEJ systems to repair double-strand breaks introduced by CRISPR/Cas9. In this report, we firstly demonstrate that the weak and previously ignored end-joining mechanism in E. coli can be used for efficient large-scale genetic engineering assisted by CRISPR/Cas9. Our efforts greatly simplify the genomic editing procedure of E. coli and provide an effective strategy for genetic engineering in bacteria that are inefficient in HR and NHEJ.

bioengineering↗