Genomics, Proteomics & Bioinformatics最新文献

{"title":"Sequence-based Functional Metagenomics Reveals Novel Natural Diversity of Functional CopA in Environmental Microbiomes","authors":"Wenjun Li ,&nbsp;Likun Wang ,&nbsp;Xiaofang Li ,&nbsp;Xin Zheng ,&nbsp;Michael F. Cohen ,&nbsp;Yong-Xin Liu","doi":"10.1016/j.gpb.2022.08.006","DOIUrl":"10.1016/j.gpb.2022.08.006","url":null,"abstract":"<div><div>Exploring the <strong>natural diversity</strong> of functional genes/proteins from environmental DNA in high throughput remains challenging. In this study, we developed a sequence-based <strong>functional metagenomics</strong> procedure for mining the diversity of copper (Cu) resistance gene <strong><em>copA</em></strong> in global microbiomes, by combining the metagenomic assembly technology, local BLAST, <strong>evolutionary trace analysis</strong> (ETA), chemical synthesis, and conventional functional genomics. In total, 87 metagenomes were collected from a public database and subjected to <em>copA</em> detection, resulting in 93,899 hits. Manual curation of 1214 hits of high confidence led to the retrieval of 517 unique CopA candidates, which were further subjected to ETA. Eventually, 175 novel <em>copA</em> sequences of high quality were discovered. Phylogenetic analysis showed that almost all these putative CopA proteins were distantly related to known CopA proteins, with 55 sequences from totally unknown species. Ten novel and three known <em>copA</em> genes were chemically synthesized for further functional genomic tests using the Cu-sensitive <em>Escherichia coli</em> (Δ<em>copA</em>). The growth test and Cu uptake determination showed that five novel clones had positive effects on host <strong>Cu resistance</strong> and uptake. One recombinant harboring <em>copA</em>-like 15 (<em>copAL15</em>) successfully restored Cu resistance of the host with a substantially enhanced Cu uptake. Two novel <em>copA</em> genes were fused with the <em>gfp</em> gene and expressed in <em>E. coli</em> for microscopic observation. Imaging results showed that they were successfully expressed and their proteins were localized to the membrane. The results here greatly expand the diversity of known CopA proteins, and the sequence-based procedure developed overcomes biases in length, screening methods, and abundance of conventional functional metagenomics.</div></div>","PeriodicalId":12528,"journal":{"name":"Genomics, Proteomics & Bioinformatics","volume":"21 6","pages":"Pages 1182-1194"},"PeriodicalIF":11.5,"publicationDate":"2023-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11082258/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"33458568","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"High Sensitivity of Shotgun Metagenomic Sequencing in Colon Tissue Biopsy by Host DNA Depletion","authors":"Wing Yin Cheng ,&nbsp;Wei-Xin Liu ,&nbsp;Yanqiang Ding ,&nbsp;Guoping Wang ,&nbsp;Yu Shi ,&nbsp;Eagle S.H. Chu ,&nbsp;Sunny Wong ,&nbsp;Joseph J.Y. Sung ,&nbsp;Jun Yu","doi":"10.1016/j.gpb.2022.09.003","DOIUrl":"10.1016/j.gpb.2022.09.003","url":null,"abstract":"<div><div>The high host genetic background of tissue biopsies hinders the application of <strong>shotgun metagenomic sequencing</strong> in characterizing the tissue microbiota. We proposed an optimized method that removed host DNA from colon biopsies and examined the effect on metagenomic analysis. Human or mouse colon biopsies were divided into two groups, with one group undergoing <strong>host DNA depletion</strong> and the other serving as the control. Host DNA was removed through differential lysis of mammalian and bacterial cells before sequencing. The impact of host DNA depletion on microbiota was compared based on phylogenetic diversity analyses and regression analyses. Removing host DNA enhanced bacterial sequencing depth and improved species discovery, increasing bacterial reads by 2.46 ± 0.20 folds while reducing host reads by 6.80% ± 1.06%. Moreover, 2.40 times more of bacterial species were detected after host DNA depletion. This was confirmed from mouse <strong>colon tissues</strong>, increasing bacterial reads by 5.46 ± 0.42 folds while decreasing host reads by 10.2% ± 0.83%. Similarly, significantly more bacterial species were detected in the mouse colon tissue upon host DNA depletion (<em>P</em> &lt; 0.001). Furthermore, an increased microbial richness was evident in the host DNA-depleted samples compared with non-depleted controls in human colon biopsies and mouse colon tissues (<em>P</em> &lt; 0.001). Our optimized method of host DNA depletion improves the sensitivity of shotgun metagenomic sequencing in bacteria detection in the biopsy, which may yield a more accurate taxonomic profile of the tissue microbiota and identify bacteria that are important for disease initiation or progression.</div></div>","PeriodicalId":12528,"journal":{"name":"Genomics, Proteomics & Bioinformatics","volume":"21 6","pages":"Pages 1195-1205"},"PeriodicalIF":11.5,"publicationDate":"2023-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11082407/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40382461","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"T2T-YAO Reference Genome of Han Chinese — New Step in Advancing Precision Medicine in China","authors":"Xue Zhang","doi":"10.1016/j.gpb.2023.09.001","DOIUrl":"10.1016/j.gpb.2023.09.001","url":null,"abstract":"","PeriodicalId":12528,"journal":{"name":"Genomics, Proteomics & Bioinformatics","volume":"21 6","pages":"Pages 1083-1084"},"PeriodicalIF":11.5,"publicationDate":"2023-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11082255/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41109240","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The Proteome Landscape of Human Placentas for Monochorionic Twins with Selective Intrauterine Growth Restriction","authors":"Xin-Lu Meng ,&nbsp;Peng-Bo Yuan ,&nbsp;Xue-Ju Wang ,&nbsp;Jing Hang ,&nbsp;Xiao-Ming Shi ,&nbsp;Yang-Yu Zhao ,&nbsp;Yuan Wei","doi":"10.1016/j.gpb.2023.03.002","DOIUrl":"10.1016/j.gpb.2023.03.002","url":null,"abstract":"<div><div>In perinatal medicine, intrauterine growth restriction (IUGR) is one of the greatest challenges. The etiology of IUGR is multifactorial, but most cases are thought to arise from placental insufficiency. However, identifying the placental cause of IUGR can be difficult due to numerous confounding factors. Selective IUGR (sIUGR) would be a good model to investigate how impaired placentation affects fetal development, as the growth discordance between monochorionic twins cannot be explained by confounding genetic or maternal factors. Herein, we constructed and analyzed the placental proteomic profiles of IUGR twins and normal cotwins. Specifically, we identified a total of 5481 proteins, of which 233 were differentially expressed (57 up-regulated and 176 down-regulated) in IUGR twins. Bioinformatics analysis indicates that these differentially expressed proteins (DEPs) are mainly associated with cardiovascular system development and function, organismal survival, and organismal development. Notably, 34 DEPs are significantly enriched in <strong>angiogenesis</strong>, and diminished placental angiogenesis in IUGR twins has been further elaborately confirmed. Moreover, we found decreased expression of <strong>metadherin</strong> (MTDH) in the <strong>placentas</strong> of IUGR twins and demonstrated that MTDH contributes to placental angiogenesis and fetal growth <em>in vitro</em>. Collectively, our findings reveal the comprehensive proteomic signatures of placentas for sIUGR twins, and the DEPs identified may provide in-depth insights into the pathogenesis of placental dysfunction and subsequent impaired fetal growth.</div></div>","PeriodicalId":12528,"journal":{"name":"Genomics, Proteomics & Bioinformatics","volume":"21 6","pages":"Pages 1246-1259"},"PeriodicalIF":11.5,"publicationDate":"2023-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11082409/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9375205","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comprehensive Characterization and Global Transcriptome Analysis of Human Fetal Liver Terminal Erythropoiesis","authors":"Yongshuai Han ,&nbsp;Shihui Wang ,&nbsp;Yaomei Wang ,&nbsp;Yumin Huang ,&nbsp;Chengjie Gao ,&nbsp;Xinhua Guo ,&nbsp;Lixiang Chen ,&nbsp;Huizhi Zhao ,&nbsp;Xiuli An","doi":"10.1016/j.gpb.2023.07.001","DOIUrl":"10.1016/j.gpb.2023.07.001","url":null,"abstract":"<div><div>The fetal liver (FL) is the key erythropoietic organ during fetal development, but knowledge on human FL erythropoiesis is very limited. In this study, we sorted primary erythroblasts from FL cells and performed RNA sequencing (RNA-seq) analyses. We found that temporal gene expression patterns reflected changes in function during primary human FL <strong>terminal erythropoiesis</strong>. Notably, the expression of genes enriched in proteolysis and autophagy was up-regulated in orthochromatic erythroblasts (OrthoEs), suggesting the involvement of these pathways in <strong>enucleation</strong>. We also performed RNA-seq of <em>in vitro</em> cultured erythroblasts derived from FL CD34⁺ cells. Comparison of <strong>transcriptomes</strong> between the primary and cultured erythroblasts revealed significant differences, indicating impacts of the culture system on gene expression. Notably, the expression of lipid metabolism-related genes was increased in cultured erythroblasts. We further <strong>immortalized erythroid cell lines</strong> from FL and cord blood (CB) CD34⁺ cells (FL-iEry and CB-iEry, respectively). FL-iEry and CB-iEry were immortalized at the proerythroblast stage and can be induced to differentiate into OrthoEs, but their enucleation ability was very low. Comparison of the transcriptomes between OrthoEs with and without enucleation capability revealed the down-regulation of pathways involved in chromatin organization and mitophagy in OrthoEs without enucleation capacity, indicating that defects in chromatin organization and mitophagy contribute to the inability of OrthoEs to enucleate. Additionally, the expression of <em>HBE1</em>, <em>HBZ</em>, and <em>HBG2</em> was up-regulated in FL-iEry compared with CB-iEry, and such up-regulation was accompanied by down-regulated expression of <em>BCL11A</em> and up-regulated expression of <em>LIN28B</em> and <em>IGF2BP1</em>. Our study provides new insights into human FL erythropoiesis and rich resources for future studies.</div></div>","PeriodicalId":12528,"journal":{"name":"Genomics, Proteomics & Bioinformatics","volume":"21 6","pages":"Pages 1117-1132"},"PeriodicalIF":11.5,"publicationDate":"2023-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11082260/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10135733","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Superior Fidelity and Distinct Editing Outcomes of SaCas9 Compared with SpCas9 in Genome Editing","authors":"Zhi-Xue Yang ,&nbsp;Ya-Wen Fu ,&nbsp;Juan-Juan Zhao ,&nbsp;Feng Zhang ,&nbsp;Si-Ang Li ,&nbsp;Mei Zhao ,&nbsp;Wei Wen ,&nbsp;Lei Zhang ,&nbsp;Tao Cheng ,&nbsp;Jian-Ping Zhang ,&nbsp;Xiao-Bing Zhang","doi":"10.1016/j.gpb.2022.12.003","DOIUrl":"10.1016/j.gpb.2022.12.003","url":null,"abstract":"<div><div>A series of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9) systems have been engineered for genome editing. The most widely used Cas9 is <strong>SpCas9</strong> from <em>Streptococcus pyogenes</em> and <strong>SaCas9</strong> from <em>Staphylococcus aureus</em>. However, a comparison of their detailed gene editing outcomes is still lacking. By characterizing the editing outcomes of 11 sites in human induced pluripotent stem cells (iPSCs) and K562 cells, we found that SaCas9 could edit the genome with greater efficiencies than SpCas9. We also compared the effects of <strong>spacer lengths</strong> of single-guide RNAs (sgRNAs; 18–21 nt for SpCas9 and 19–23 nt for SaCas9) and found that the optimal spacer lengths were 20 nt and 21 nt for SpCas9 and SaCas9, respectively. However, the optimal spacer length for a particular sgRNA was 18–21 nt for SpCas9 and 21–22 nt for SaCas9. Furthermore, SpCas9 exhibited a more substantial bias than SaCas9 for nonhomologous end-joining (NHEJ) +1 insertion at the fourth nucleotide upstream of the protospacer adjacent motif (PAM), indicating a characteristic of a staggered cut. Accordingly, editing with SaCas9 led to higher efficiencies of NHEJ-mediated double-stranded oligodeoxynucleotide (dsODN) insertion or homology-directed repair (HDR)-mediated adeno-associated virus serotype 6 (AAV6) donor knock-in. Finally, GUIDE-seq analysis revealed that SaCas9 exhibited significantly reduced <strong>off-target</strong> effects compared with SpCas9. Our work indicates the superior performance of SaCas9 to SpCas9 in transgene integration-based therapeutic gene editing and the necessity to identify the optimal spacer length to achieve desired editing results.</div></div>","PeriodicalId":12528,"journal":{"name":"Genomics, Proteomics & Bioinformatics","volume":"21 6","pages":"Pages 1206-1220"},"PeriodicalIF":11.5,"publicationDate":"2023-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11082263/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10419418","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cancer Is A Survival Process under Persistent Microenvironmental and Cellular Stresses","authors":"Renbo Tan ,&nbsp;Yi Zhou ,&nbsp;Zheng An ,&nbsp;Ying Xu","doi":"10.1016/j.gpb.2022.03.002","DOIUrl":"10.1016/j.gpb.2022.03.002","url":null,"abstract":"","PeriodicalId":12528,"journal":{"name":"Genomics, Proteomics & Bioinformatics","volume":"21 6","pages":"Pages 1260-1265"},"PeriodicalIF":11.5,"publicationDate":"2023-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11082257/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40150620","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Novel Time-dependent Multi-omics Integration in Sepsis-associated Liver Dysfunction","authors":"Ann-Yae Na ,&nbsp;Hyojin Lee ,&nbsp;Eun Ki Min ,&nbsp;Sanjita Paudel ,&nbsp;So Young Choi ,&nbsp;HyunChae Sim ,&nbsp;Kwang-Hyeon Liu ,&nbsp;Ki-Tae Kim ,&nbsp;Jong-Sup Bae ,&nbsp;Sangkyu Lee","doi":"10.1016/j.gpb.2023.04.002","DOIUrl":"10.1016/j.gpb.2023.04.002","url":null,"abstract":"<div><div>The recently developed technologies that allow the analysis of each <strong>single omics</strong> have provided an unbiased insight into ongoing disease processes. However, it remains challenging to specify the study design for the subsequent integration strategies that can associate sepsis pathophysiology and clinical outcomes. Here, we conducted a time-dependent <strong>multi-omics</strong> integration (TDMI) in a <strong>sepsis-associated liver dysfunction</strong> (SALD) model. We successfully deduced the relation of the Toll-like receptor 4 (TLR4) pathway with SALD. Although TLR4 is a critical factor in sepsis progression, it is not specified in single-omics analyses but only in the TDMI analysis. This finding indicates that the TDMI-based approach is more advantageous than single-omics analyses in terms of exploring the underlying pathophysiological mechanism of SALD. Furthermore, TDMI-based approach can be an ideal paradigm for insightful biological interpretations of multi-omics datasets that will potentially reveal novel insights into basic biology, health, and diseases, thus allowing the identification of promising candidates for therapeutic strategies.</div></div>","PeriodicalId":12528,"journal":{"name":"Genomics, Proteomics & Bioinformatics","volume":"21 6","pages":"Pages 1101-1116"},"PeriodicalIF":11.5,"publicationDate":"2023-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11082264/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9422024","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Protein Lactylation and Metabolic Regulation of the Zoonotic Parasite Toxoplasma gondii","authors":"Deqi Yin ,&nbsp;Ning Jiang ,&nbsp;Chang Cheng ,&nbsp;Xiaoyu Sang ,&nbsp;Ying Feng ,&nbsp;Ran Chen ,&nbsp;Qijun Chen","doi":"10.1016/j.gpb.2022.09.010","DOIUrl":"10.1016/j.gpb.2022.09.010","url":null,"abstract":"<div><div>The biology of <strong><em>Toxoplasma gondii</em></strong>, the causative pathogen of one of the most widespread parasitic diseases (toxoplasmosis), remains poorly understood. Lactate, which is derived from glucose <strong>metabolism</strong>, is not only an energy source in a variety of organisms<strong>,</strong> including <em>T</em>. <em>gondii</em>, but also a regulatory molecule that participates in gene activation and protein function. Lysine <strong>lactylation</strong> (Kla) is a type of post-translational modifications (PTMs) that has been recently associated with chromatin remodeling; however, Kla of histone and non-histone proteins has not yet been studied in <em>T</em>. <em>gondii</em>. To examine the prevalence and function of lactylation in <em>T</em>. <em>gondii</em> parasites, we mapped the lactylome of proliferating tachyzoite cells and identified 1964 Kla sites on 955 proteins in the <em>T</em>. <em>gondii</em> RH strain. Lactylated proteins were distributed in multiple subcellular compartments and were closely related to a wide variety of biological processes, including mRNA splicing, glycolysis, aminoacyl-tRNA biosynthesis, RNA transport<strong>,</strong> and many signaling pathways. We also performed a chromatin immunoprecipitation sequencing (<strong>ChIP-seq</strong>) analysis using a lactylation-specific antibody and found that the histones H4K12la and H3K14la were enriched in the promoter and exon regions of <em>T</em>. <em>gondii</em> associated with microtubule-based movement and cell invasion. We further confirmed the delactylase activity of histone deacetylases TgHDAC2–4, and found that treatment with anti-histone acetyltransferase (TgMYST-A) antibodies profoundly reduced protein lactylation in <em>T</em>. <em>gondii</em>. This study offers the first dataset of the global lactylation proteome and provides a basis for further dissecting the functional biology of <em>T</em>. <em>gondii</em>.</div></div>","PeriodicalId":12528,"journal":{"name":"Genomics, Proteomics & Bioinformatics","volume":"21 6","pages":"Pages 1163-1181"},"PeriodicalIF":11.5,"publicationDate":"2023-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11082259/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"33497560","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"GREPore-seq: A Robust Workflow to Detect Changes After Gene Editing Through Long-range PCR and Nanopore Sequencing","authors":"Zi-Jun Quan ,&nbsp;Si-Ang Li ,&nbsp;Zhi-Xue Yang ,&nbsp;Juan-Juan Zhao ,&nbsp;Guo-Hua Li ,&nbsp;Feng Zhang ,&nbsp;Wei Wen ,&nbsp;Tao Cheng ,&nbsp;Xiao-Bing Zhang","doi":"10.1016/j.gpb.2022.06.002","DOIUrl":"10.1016/j.gpb.2022.06.002","url":null,"abstract":"<div><div>To achieve the enormous potential of gene-editing technology in clinical therapies, one needs to evaluate both the on-target efficiency and unintended editing consequences comprehensively. However, there is a lack of a pipelined, large-scale, and economical workflow for detecting genome editing outcomes, in particular insertion or deletion of a large fragment. Here, we describe an approach for efficient and accurate detection of multiple <strong>genetic changes</strong> after <strong>CRISPR/Cas9</strong> editing by pooled <strong>nanopore sequencing</strong> of barcoded <strong>long-range PCR</strong> products. Recognizing the high error rates of Oxford nanopore sequencing, we developed a novel pipeline to capture the barcoded sequences by grepping reads of nanopore amplicon sequencing (<strong>GREPore-seq</strong>). GREPore-seq can assess nonhomologous end-joining (NHEJ)-mediated double-stranded oligodeoxynucleotide (dsODN) insertions with comparable accuracy to Illumina next-generation sequencing (NGS). GREPore-seq also reveals a full spectrum of homology-directed repair (HDR)-mediated large gene knock-in, correlating well with the fluorescence-activated cell sorting (FACS) analysis results. Of note, we discovered low-level fragmented and full-length plasmid backbone insertion at the CRISPR cutting site. Therefore, we have established a practical workflow to evaluate various genetic changes, including quantifying insertions of short dsODNs, knock-ins of long pieces, plasmid insertions, and large fragment deletions after CRISPR/Cas9-mediated editing. GREPore-seq is freely available at GitHub (<span><span>https://github.com/lisiang/GREPore-seq</span><svg><path></path></svg></span>) and the National Genomics Data Center (NGDC) BioCode (<span><span>https://ngdc.cncb.ac.cn/biocode/tools/BT007293</span><svg><path></path></svg></span>).</div></div>","PeriodicalId":12528,"journal":{"name":"Genomics, Proteomics & Bioinformatics","volume":"21 6","pages":"Pages 1221-1236"},"PeriodicalIF":11.5,"publicationDate":"2023-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11082256/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40398962","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}