首页 > 最新文献

ACS Synthetic Biology最新文献

英文 中文
Simultaneous In Vivo Assembly and Targeted Genome Integration of Gene Clusters in Trichoderma reesei.
IF 3.7 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-02-06 DOI: 10.1021/acssynbio.4c00810
Yu Fang, Xiangfeng Meng, Lin Liu, Zhongye Li, Kaili Jia, Weifeng Liu

The saprophytic filamentous fungus Trichoderma reesei represents one of the most prolific cellulase producers and also has the potential to be developed into a tractable fungal host for biosynthesizing secondary metabolite products. To expedite the genetic engineering of filamentous fungi, efficient DNA assembly processes that can facilitate the transfer of large-sized DNA to fungal hosts, including T. reesei, are still in demand. Here, we developed a method for the simultaneous in vivo assembly and targeted genome integration of multiple DNA fragments (SATIMD) in T. reesei. While efficient orderly DNA end fusions were achieved by homologous recombination (HR) with various lengths of sequence overlaps (100-500 bp), the assembled DNA was also precisely integrated into a specific locus when combined with CRISPR/Cas9-mediated genome cutting. Specifically, we have used this method to achieve the assembly and functional expression of T. reesei key transcriptional activator Xyr1 for cellulase genes. Moreover, fusions and targeted integration of up to 10 different DNA fragments comprising the 32.7 kb sorbicillinoids biosynthetic gene cluster via a single-step transformation was demonstrated. We envision that SATIMD is a powerful tool not only useful for direct large heterologous gene cluster assembly in T. reesei but also can facilitate large-scale fungal strain genetic engineering.

{"title":"Simultaneous <i>In Vivo</i> Assembly and Targeted Genome Integration of Gene Clusters in <i>Trichoderma reesei</i>.","authors":"Yu Fang, Xiangfeng Meng, Lin Liu, Zhongye Li, Kaili Jia, Weifeng Liu","doi":"10.1021/acssynbio.4c00810","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00810","url":null,"abstract":"<p><p>The saprophytic filamentous fungus <i>Trichoderma reesei</i> represents one of the most prolific cellulase producers and also has the potential to be developed into a tractable fungal host for biosynthesizing secondary metabolite products. To expedite the genetic engineering of filamentous fungi, efficient DNA assembly processes that can facilitate the transfer of large-sized DNA to fungal hosts, including <i>T. reesei</i>, are still in demand. Here, we developed a method for the simultaneous <i>in vivo</i> assembly and targeted genome integration of multiple DNA fragments (SATIMD) in <i>T. reesei</i>. While efficient orderly DNA end fusions were achieved by homologous recombination (HR) with various lengths of sequence overlaps (100-500 bp), the assembled DNA was also precisely integrated into a specific locus when combined with CRISPR/Cas9-mediated genome cutting. Specifically, we have used this method to achieve the assembly and functional expression of <i>T. reesei</i> key transcriptional activator Xyr1 for cellulase genes. Moreover, fusions and targeted integration of up to 10 different DNA fragments comprising the 32.7 kb sorbicillinoids biosynthetic gene cluster via a single-step transformation was demonstrated. We envision that SATIMD is a powerful tool not only useful for direct large heterologous gene cluster assembly in <i>T. reesei</i> but also can facilitate large-scale fungal strain genetic engineering.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143363267","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
rmCombi-OGAB for the Directed Evolution of a Biosynthetic Gene Cluster toward Productivity Improvement.
IF 3.7 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-02-05 DOI: 10.1021/acssynbio.4c00734
Naoki Miyamoto, Kentaro Hayashi, Naohisa Ogata, Naoyuki Yamada, Kenji Tsuge

Combinatorial Ordered Gene Assembly in Bacillus subtilis (Combi-OGAB) enables construction of combinatorial libraries of various genetic elements, such as promoters in a biosynthetic gene cluster (BGC), and screening of highly productive combinations from the library. The combinations are limited by the library design, and the selectable productivity is defined within the combination. To refine the selected BGC using conventional Combi-OGAB with expanded diversity, we devised a directed evolutionary method called as random mutagenesis with Combi-OGAB (rmCombi-OGAB), which includes random mutagenesis by error-prone PCR and Combi-OGAB. In the present study, Gramicidin S (GS)-producing plasmids were used to examine the utility of rmCombi-OGAB. GS plasmids, originally generated using conventional Combi-OGAB, were successfully evolved using rmCombi-OGAB. B. subtilis carrying the evolved plasmid with unpredictable mutations showed a 1.5-fold improvement in the GS productivity. We thus expect that rmCombi-OGAB can be applied to various BGCs for useful products, such as antibiotics, to improve their productivity.

{"title":"<i>rm</i>Combi-OGAB for the Directed Evolution of a Biosynthetic Gene Cluster toward Productivity Improvement.","authors":"Naoki Miyamoto, Kentaro Hayashi, Naohisa Ogata, Naoyuki Yamada, Kenji Tsuge","doi":"10.1021/acssynbio.4c00734","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00734","url":null,"abstract":"<p><p><u><b>Combi</b></u>natorial <u><b>O</b></u>rdered <u><b>G</b></u>ene <u><b>A</b></u>ssembly in <u><b><i>B</i></b></u><i>acillus subtilis</i> (Combi-OGAB) enables construction of combinatorial libraries of various genetic elements, such as promoters in a biosynthetic gene cluster (BGC), and screening of highly productive combinations from the library. The combinations are limited by the library design, and the selectable productivity is defined within the combination. To refine the selected BGC using conventional Combi-OGAB with expanded diversity, we devised a directed evolutionary method called as <u><b>r</b></u>andom <u><b>m</b></u>utagenesis with <u><b>Combi-OGAB</b></u> (<i>rm</i>Combi-OGAB), which includes random mutagenesis by error-prone PCR and Combi-OGAB. In the present study, Gramicidin S (GS)-producing plasmids were used to examine the utility of <i>rm</i>Combi-OGAB. GS plasmids, originally generated using conventional Combi-OGAB, were successfully evolved using <i>rm</i>Combi-OGAB. <i>B. subtilis</i> carrying the evolved plasmid with unpredictable mutations showed a 1.5-fold improvement in the GS productivity. We thus expect that <i>rm</i>Combi-OGAB can be applied to various BGCs for useful products, such as antibiotics, to improve their productivity.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143254160","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Combinatorial Nonribosomal Peptide Synthetase Libraries Using the SEAM-Combi-OGAB Method.
IF 3.7 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-02-05 DOI: 10.1021/acssynbio.4c00671
Varada Jagadeesh, Nobuyuki Okahashi, Fumio Matsuda, Kenji Tsuge, Akihiko Kondo

To overcome the difficulty of building large nonribosomal peptide synthetase (NRPS) gene cluster libraries, an efficient one-pot method using Bacillus subtilis was developed. This new method, named Seamed Express Assembly Method (SEAM)-combi-Ordered Gene Assembly in Bacillus subtilis (OGAB), combines the SEAM-OGAB approach for NRPS gene cluster construction with the combi-OGAB method for combinatorial DNA library construction to randomly swap DNA fragments for NRPS modules. In this study, NRPS gene clusters of plipastatin and gramicidin S were used as the starting material. The full length of each gene cluster was prepared as plasmid DNA by introducing restriction enzyme SfiI sites into the module border according to SEAM-OGAB. These two plasmids were mixed, digested with SfiI, ligated in a tandem repeat form, and used to transform B. subtilis according to the combi-OGAB method. While 64 of all the possible combinations were used in the calculation, 32 types of plasmid DNA were obtained from 50 randomly selected transformants. These transformants produced at least 30 types of peptides, including cyclic and linear variations with lengths ranging from 5 to 10 amino acids. Thus, this method enabled an efficient construction of NRPS gene cluster libraries with more than five module members, making it advantageous for applications in peptide libraries.

为了克服构建大型非核糖体肽合成酶(NRPS)基因簇文库的困难,研究人员利用枯草芽孢杆菌开发了一种高效的一步法。这种新方法被命名为Seamed Express Assembly Method (SEAM)-combi-Ordered Gene Assembly in Bacillus subtilis (OGAB),它将用于构建NRPS基因簇的SEAM-OGAB方法与用于构建组合DNA文库的combi-OGAB方法相结合,以随机交换NRPS模块的DNA片段。在这项研究中,我们使用了普立司他丁和蒿甲素 S 的 NRPS 基因簇作为起始材料。根据 SEAM-OGAB 方法,在模块边界引入限制性酶 SfiI 位点,将每个基因簇的全长制备成质粒 DNA。将这两个质粒混合,用 SfiI 消化,以串联重复的形式连接,并按照 combi-OGAB 方法用于转化枯草杆菌。计算中使用了所有可能组合中的 64 种,从 50 个随机选择的转化子中获得了 32 种质粒 DNA。这些转化子产生了至少 30 种肽,包括长度为 5 至 10 个氨基酸的环状和线性变体。因此,这种方法可以高效地构建具有五个以上模块成员的 NRPS 基因簇文库,在多肽文库的应用方面具有优势。
{"title":"Combinatorial Nonribosomal Peptide Synthetase Libraries Using the SEAM-Combi-OGAB Method.","authors":"Varada Jagadeesh, Nobuyuki Okahashi, Fumio Matsuda, Kenji Tsuge, Akihiko Kondo","doi":"10.1021/acssynbio.4c00671","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00671","url":null,"abstract":"<p><p>To overcome the difficulty of building large nonribosomal peptide synthetase (NRPS) gene cluster libraries, an efficient one-pot method using <i>Bacillus subtilis</i> was developed. This new method, named <u>S</u>eamed <u>E</u>xpress <u>A</u>ssembly <u>M</u>ethod (SEAM)-combi-<u>O</u>rdered <u>G</u>ene <u>A</u>ssembly in <i>Bacillus subtilis</i> (OGAB), combines the SEAM-OGAB approach for NRPS gene cluster construction with the combi-OGAB method for combinatorial DNA library construction to randomly swap DNA fragments for NRPS modules. In this study, NRPS gene clusters of plipastatin and gramicidin S were used as the starting material. The full length of each gene cluster was prepared as plasmid DNA by introducing restriction enzyme SfiI sites into the module border according to SEAM-OGAB. These two plasmids were mixed, digested with SfiI, ligated in a tandem repeat form, and used to transform <i>B. subtilis</i> according to the combi-OGAB method. While 64 of all the possible combinations were used in the calculation, 32 types of plasmid DNA were obtained from 50 randomly selected transformants. These transformants produced at least 30 types of peptides, including cyclic and linear variations with lengths ranging from 5 to 10 amino acids. Thus, this method enabled an efficient construction of NRPS gene cluster libraries with more than five module members, making it advantageous for applications in peptide libraries.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143187543","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Synthetic Dual-Input Hybrid Riboswitches─Optimized Genetic Regulators in Yeast.
IF 3.7 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-02-04 DOI: 10.1021/acssynbio.4c00660
Daniel Kelvin, Janette Arias Rodriguez, Ann-Christin Groher, Kiara Petras, Beatrix Suess

Synthetic riboswitches, genetic regulatory elements composed entirely of RNA, have been engineered to control a variety of mechanisms at the level of both transcription and translation in all domains of life. The efficiency of riboswitch regulation can be increased by inserting two of them into an mRNA sequence in close proximity, resulting in a tandem riboswitch. The tandem state results in improved regulation beyond that of a single riboswitch by allowing both binding pockets to contribute to a higher dynamic range. The focus of this study was to create a novel tandem riboswitch design by integrating the binding pockets of two different riboswitches into one continuous structure, thereby creating a dual-input hybrid riboswitch. These hybrids remain compact in size with a shorter sequence length compared to a tandem riboswitch, while taking advantage of the binding pockets and scaffold sequences provided by both parental riboswitches. Through rational design, hybrid constructs derived from the combination of tetracycline-, tobramycin-, neomycin-, and paromomycin-binding riboswitches were engineered that significantly increase the dynamic range (e.g., from 14- to 36-fold for tobramycin) while increasing their expression levels in the absence of ligand (e.g., 28% to 68% expression for tetracycline). This study expands the toolbox of synthetic riboswitches and establishes general design guidelines applicable to similar riboswitches. Additionally, the dual-input state makes hybrid riboswitches an interesting target for the design of genetic regulators following Boolean logic.

合成核糖开关是完全由 RNA 组成的基因调控元件,已被设计用于控制生命各领域转录和翻译水平的各种机制。通过在一个 mRNA 序列中插入两个相邻的核糖开关,形成串联核糖开关,可以提高核糖开关的调控效率。串联状态使两个结合口袋都能产生更大的动态范围,从而提高了调节效率,超过了单个核糖开关。本研究的重点是将两个不同核糖开关的结合口袋整合到一个连续结构中,从而创造出一种新型串联核糖开关设计。与串联式核糖开关相比,这些混合型核糖开关体积小巧,序列长度较短,同时利用了两个亲代核糖开关提供的结合口袋和支架序列。通过合理的设计,从四环素、妥布霉素、新霉素和对霉素结合的核糖开关组合中衍生出了杂交构建物,这些构建物显著提高了动态范围(例如,妥布霉素的动态范围从 14 倍提高到 36 倍),同时在没有配体的情况下提高了表达水平(例如,四环素的表达水平从 28% 提高到 68%)。这项研究拓展了合成核糖开关的工具箱,并建立了适用于类似核糖开关的一般设计准则。此外,双输入状态使混合核糖开关成为按照布尔逻辑设计遗传调节器的一个有趣目标。
{"title":"Synthetic Dual-Input Hybrid Riboswitches─Optimized Genetic Regulators in Yeast.","authors":"Daniel Kelvin, Janette Arias Rodriguez, Ann-Christin Groher, Kiara Petras, Beatrix Suess","doi":"10.1021/acssynbio.4c00660","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00660","url":null,"abstract":"<p><p>Synthetic riboswitches, genetic regulatory elements composed entirely of RNA, have been engineered to control a variety of mechanisms at the level of both transcription and translation in all domains of life. The efficiency of riboswitch regulation can be increased by inserting two of them into an mRNA sequence in close proximity, resulting in a tandem riboswitch. The tandem state results in improved regulation beyond that of a single riboswitch by allowing both binding pockets to contribute to a higher dynamic range. The focus of this study was to create a novel tandem riboswitch design by integrating the binding pockets of two different riboswitches into one continuous structure, thereby creating a dual-input hybrid riboswitch. These hybrids remain compact in size with a shorter sequence length compared to a tandem riboswitch, while taking advantage of the binding pockets and scaffold sequences provided by both parental riboswitches. Through rational design, hybrid constructs derived from the combination of tetracycline-, tobramycin-, neomycin-, and paromomycin-binding riboswitches were engineered that significantly increase the dynamic range (e.g., from 14- to 36-fold for tobramycin) while increasing their expression levels in the absence of ligand (e.g., 28% to 68% expression for tetracycline). This study expands the toolbox of synthetic riboswitches and establishes general design guidelines applicable to similar riboswitches. Additionally, the dual-input state makes hybrid riboswitches an interesting target for the design of genetic regulators following Boolean logic.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143187547","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
DRIMS: A Synthetic Biology Platform that Enables Deletion, Replacement, Insertion, Mutagenesis, and Synthesis of DNA.
IF 3.7 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-02-04 DOI: 10.1021/acssynbio.4c00649
Leidy D Caraballo G, Inci Cevher Zeytin, Purva Rathi, Che-Hsing Li, Ai-Ni Tsao, Yaery J Salvador L, Manish Ranjan, Brendan Magee Traynor, Andras A Heczey

DNA modification and synthesis are fundamental to genetic engineering, and systems that enable time- and cost-effective execution of these processes are crucial. Iteration of genetic construct variants takes significant time, cost and effort to develop new therapeutic strategies to treat diseases including cancer. Thus, decreasing cost and enhancing simplicity while accelerating the speed of advancement is critical. We have developed a PCR-based platform that allows for deletion, replacement, insertion, mutagenesis, and synthesis of DNA (DRIMS). These modifications rely on the recA-independent recombination pathway and are carried out in a single amplification step followed by DpnI digestion and transformation into competent cells. DNA synthesis is accomplished through sequential PCR amplification reactions without the need for a DNA template. Here, we provide proof-of-concept for the DRIMS platform by performing four deletions within DNA fragments of various sizes, sixty-four replacements of DNA binding sequences that incorporate repeat sequences, four replacements of chimeric antigen receptor components, fifty-one insertions of artificial microRNAs that form complex tertiary structures, five varieties of point mutations, and synthesis of eight DNA sequences including two with high GC content. Compared to other advanced cloning methods including Gibson and "in vivo assembly", we demonstrate the significant advantages of the DRIMS platform. In summary, DRIMS allows for efficient modification and synthesis of DNA in a simple, rapid and cost-effective manner to accelerate the synthetic biology field and development of therapeutics.

DNA 修饰和合成是基因工程的基础,而能够以经济高效的方式及时执行这些过程的系统则至关重要。基因构建变体的迭代需要花费大量的时间、成本和精力,才能开发出治疗癌症等疾病的新疗法。因此,在加快进度的同时降低成本和提高简便性至关重要。我们开发了一种基于 PCR 的平台,可以进行 DNA 的删除、替换、插入、诱变和合成(DRIMS)。这些修饰依赖于不依赖 recA 的重组途径,在一个扩增步骤中完成,然后进行 DpnI 消化并转化为合格细胞。DNA 合成是通过连续的 PCR 扩增反应完成的,无需 DNA 模板。在这里,我们对 DRIMS 平台进行了概念验证,对不同大小的 DNA 片段进行了四次缺失,对包含重复序列的 DNA 结合序列进行了六十四次替换,对嵌合抗原受体成分进行了四次替换,对形成复杂三级结构的人工 microRNA 进行了五十一次插入,进行了五种点突变,并合成了八种 DNA 序列,其中包括两种高 GC 含量的 DNA 序列。与其他先进的克隆方法(包括 Gibson 和 "体内组装")相比,我们展示了 DRIMS 平台的显著优势。总之,DRIMS 允许以简单、快速和经济高效的方式高效修饰和合成 DNA,从而加速合成生物学领域和治疗药物的开发。
{"title":"DRIMS: A Synthetic Biology Platform that Enables Deletion, Replacement, Insertion, Mutagenesis, and Synthesis of DNA.","authors":"Leidy D Caraballo G, Inci Cevher Zeytin, Purva Rathi, Che-Hsing Li, Ai-Ni Tsao, Yaery J Salvador L, Manish Ranjan, Brendan Magee Traynor, Andras A Heczey","doi":"10.1021/acssynbio.4c00649","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00649","url":null,"abstract":"<p><p>DNA modification and synthesis are fundamental to genetic engineering, and systems that enable time- and cost-effective execution of these processes are crucial. Iteration of genetic construct variants takes significant time, cost and effort to develop new therapeutic strategies to treat diseases including cancer. Thus, decreasing cost and enhancing simplicity while accelerating the speed of advancement is critical. We have developed a PCR-based platform that allows for <b>d</b>eletion, <b>r</b>eplacement, <b>i</b>nsertion, <b>m</b>utagenesis, and <b>s</b>ynthesis of DNA (DRIMS). These modifications rely on the recA-independent recombination pathway and are carried out in a single amplification step followed by DpnI digestion and transformation into competent cells. DNA synthesis is accomplished through sequential PCR amplification reactions without the need for a DNA template. Here, we provide proof-of-concept for the DRIMS platform by performing four deletions within DNA fragments of various sizes, sixty-four replacements of DNA binding sequences that incorporate repeat sequences, four replacements of chimeric antigen receptor components, fifty-one insertions of artificial microRNAs that form complex tertiary structures, five varieties of point mutations, and synthesis of eight DNA sequences including two with high GC content. Compared to other advanced cloning methods including Gibson and \"in vivo assembly\", we demonstrate the significant advantages of the DRIMS platform. In summary, DRIMS allows for efficient modification and synthesis of DNA in a simple, rapid and cost-effective manner to accelerate the synthetic biology field and development of therapeutics.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143121669","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Characterization of Rationally Designed CRISPR/Cas9-Based DNA Methyltransferases with Distinct Methyltransferase and Gene Silencing Activities in Human Cell Lines and Primary Human T Cells.
IF 3.7 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-02-03 DOI: 10.1021/acssynbio.4c00569
Rosa Selenia Guerra-Resendez, Samantha LeGoff Lydon, Alex J Ma, Guy C Bedford, Daniel R Reed, Sunghwan Kim, Erik R Terán, Tomoki Nishiguchi, Mario Escobar, Andrew R DiNardo, Isaac B Hilton

Nuclease-deactivated Cas (dCas) proteins can be used to recruit epigenetic effectors, and this class of epigenetic editing technologies has revolutionized the ability to synthetically control the mammalian epigenome and transcriptome. DNA methylation is one of the most important and well-characterized epigenetic modifications in mammals, and while many different forms of dCas-based DNA methyltransferases (dCas-DNMTs) have been developed for programmable DNA methylation, these tools are frequently poorly tolerated and/or lowly expressed in mammalian cell types. Further, the use of dCas-DNMTs has largely been restricted to cell lines, which limits mechanistic insights in karyotypically normal contexts and hampers translational utility in the longer term. Here, we extend previous insights into the rational design of the catalytic core of the mammalian DNMT3A methyltransferase and test three dCas9-DNMT3A/3L variants across different human cell lines and in primary donor-derived human T cells. We find that mutations within the catalytic core of DNMT3A stabilize the expression of dCas9-DNMT3A/3L fusion proteins in Jurkat T cells without sacrificing DNA methylation or gene-silencing performance. We also show that these rationally engineered mutations in DNMT3A alter DNA methylation profiles at loci targeted with dCas9-DNMT3A/3L in cell lines and donor-derived human T cells. Finally, we leverage the transcriptionally repressive effects of dCas9-DNMT3A/3L variants to functionally link the expression of a key immunomodulatory transcription factor to cytokine secretion in donor-derived T cells. Overall, our work expands the synthetic biology toolkit for epigenetic editing and provides a roadmap for the use of engineered dCas-based DNMTs in primary mammalian cell types.

{"title":"Characterization of Rationally Designed CRISPR/Cas9-Based DNA Methyltransferases with Distinct Methyltransferase and Gene Silencing Activities in Human Cell Lines and Primary Human T Cells.","authors":"Rosa Selenia Guerra-Resendez, Samantha LeGoff Lydon, Alex J Ma, Guy C Bedford, Daniel R Reed, Sunghwan Kim, Erik R Terán, Tomoki Nishiguchi, Mario Escobar, Andrew R DiNardo, Isaac B Hilton","doi":"10.1021/acssynbio.4c00569","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00569","url":null,"abstract":"<p><p>Nuclease-deactivated Cas (dCas) proteins can be used to recruit epigenetic effectors, and this class of epigenetic editing technologies has revolutionized the ability to synthetically control the mammalian epigenome and transcriptome. DNA methylation is one of the most important and well-characterized epigenetic modifications in mammals, and while many different forms of dCas-based DNA methyltransferases (dCas-DNMTs) have been developed for programmable DNA methylation, these tools are frequently poorly tolerated and/or lowly expressed in mammalian cell types. Further, the use of dCas-DNMTs has largely been restricted to cell lines, which limits mechanistic insights in karyotypically normal contexts and hampers translational utility in the longer term. Here, we extend previous insights into the rational design of the catalytic core of the mammalian DNMT3A methyltransferase and test three dCas9-DNMT3A/3L variants across different human cell lines and in primary donor-derived human T cells. We find that mutations within the catalytic core of DNMT3A stabilize the expression of dCas9-DNMT3A/3L fusion proteins in Jurkat T cells without sacrificing DNA methylation or gene-silencing performance. We also show that these rationally engineered mutations in DNMT3A alter DNA methylation profiles at loci targeted with dCas9-DNMT3A/3L in cell lines and donor-derived human T cells. Finally, we leverage the transcriptionally repressive effects of dCas9-DNMT3A/3L variants to functionally link the expression of a key immunomodulatory transcription factor to cytokine secretion in donor-derived T cells. Overall, our work expands the synthetic biology toolkit for epigenetic editing and provides a roadmap for the use of engineered dCas-based DNMTs in primary mammalian cell types.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143078034","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Harnessing Non-standard Nucleic Acids for Highly Sensitive Icosaplex (20-Plex) Detection of Microbial Threats for Environmental Surveillance.
IF 3.7 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-02-03 DOI: 10.1021/acssynbio.4c00619
Hinako Kawabe, Luran Manfio, Sebastian Magana Pena, Nicolette A Zhou, Kevin M Bradley, Cen Chen, Chris McLendon, Steven A Benner, Karen Levy, Zunyi Yang, Jorge A Marchand, Erica R Fuhrmeister

Environmental surveillance and clinical diagnostics heavily rely on the polymerase chain reaction (PCR) for target detection. A growing list of microbial threats warrants new PCR-based detection methods that are highly sensitive, specific, and multiplexable. Here, we introduce a PCR-based icosaplex (20-plex) assay for detecting 18 enteropathogen and two antimicrobial resistance genes. This multiplexed PCR assay leverages the self-avoiding molecular recognition system (SAMRS) to avoid primer dimer formation, the artificially expanded genetic information system (AEGIS) for amplification specificity, and next-generation sequencing for amplicon identification. Using parallelized multitarget TaqMan Array Cards (TAC) to benchmark performance of the 20-plex assay on wastewater, soil, and human stool samples, we found 90% agreement on positive calls and 89% agreement on negative calls. Additionally, we show how long-read and short-read sequencing information from the 20-plex can be used to further classify allelic variants of genes and distinguish subspecies. The strategy presented offers sensitive, affordable, and robust multiplex detection that can be used to support efforts in wastewater-based epidemiology, environmental monitoring, and human/animal diagnostics.

{"title":"Harnessing Non-standard Nucleic Acids for Highly Sensitive Icosaplex (20-Plex) Detection of Microbial Threats for Environmental Surveillance.","authors":"Hinako Kawabe, Luran Manfio, Sebastian Magana Pena, Nicolette A Zhou, Kevin M Bradley, Cen Chen, Chris McLendon, Steven A Benner, Karen Levy, Zunyi Yang, Jorge A Marchand, Erica R Fuhrmeister","doi":"10.1021/acssynbio.4c00619","DOIUrl":"10.1021/acssynbio.4c00619","url":null,"abstract":"<p><p>Environmental surveillance and clinical diagnostics heavily rely on the polymerase chain reaction (PCR) for target detection. A growing list of microbial threats warrants new PCR-based detection methods that are highly sensitive, specific, and multiplexable. Here, we introduce a PCR-based icosaplex (20-plex) assay for detecting 18 enteropathogen and two antimicrobial resistance genes. This multiplexed PCR assay leverages the self-avoiding molecular recognition system (SAMRS) to avoid primer dimer formation, the artificially expanded genetic information system (AEGIS) for amplification specificity, and next-generation sequencing for amplicon identification. Using parallelized multitarget TaqMan Array Cards (TAC) to benchmark performance of the 20-plex assay on wastewater, soil, and human stool samples, we found 90% agreement on positive calls and 89% agreement on negative calls. Additionally, we show how long-read and short-read sequencing information from the 20-plex can be used to further classify allelic variants of genes and distinguish subspecies. The strategy presented offers sensitive, affordable, and robust multiplex detection that can be used to support efforts in wastewater-based epidemiology, environmental monitoring, and human/animal diagnostics.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143078035","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
De Novo Synthesis of Reticuline and Taxifolin Using Re-engineered Homologous Recombination in Yarrowia lipolytica. 利用 Yarrowia lipolytica 中重新设计的同源重组技术从头合成 Reticuline 和 Taxifolin。
IF 3.7 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-02-03 DOI: 10.1021/acssynbio.4c00853
Changtai Zhang, Mengsu Liu, Xinglong Wang, Junyi Cheng, Jinbo Xiang, Mingyu Yue, Yang Ning, Zhengxuan Shao, Chalak Najat Abdullah, Jingwen Zhou

Yarrowia lipolytica has been widely engineered as a eukaryotic cell factory to produce various important compounds. However, the difficulty of gene editing and the lack of efficient neutral sites make rewiring of Y. lipolytica metabolism challenging. Herein, a Cas9 system was established to redesign the Y. lipolytica homologous recombination system, which caused a more than 56-fold increase in the HR efficiency. The fusion expression of the hBrex27 sequence in the C-terminus of Cas9 recruited more Rad51 protein, and the engineered Cas9 decreased NHEJ, achieving 85% single-gene positive efficiency and 25% multigene editing efficiency. With this system, neutral sites on different chromosomes were characterized, and a deep learning model was developed for gRNA activity prediction, thus providing the corresponding integration efficiency and expression intensity. Subsequently, the tool and platform strains were validated by applying them for the de novo synthesis of (S)-reticuline and (2S)-taxifolin. The developed platform strains and tools helped transform Y. lipolytica into an easy-to-operate model cell factory, similar to Saccharomyces cerevisiae.

脂肪分解酵母已被广泛改造为真核细胞工厂,用于生产各种重要化合物。然而,基因编辑的难度和高效中性位点的缺乏使得脂溶性亚罗维氏菌新陈代谢的重新布线具有挑战性。在此,我们建立了一个Cas9系统,重新设计了Y. lipolytica同源重组系统,使HR效率提高了56倍以上。在Cas9的C端融合表达了hBrex27序列,招募了更多的Rad51蛋白,工程化的Cas9降低了NHEJ,实现了85%的单基因阳性效率和25%的多基因编辑效率。利用该系统,对不同染色体上的中性位点进行了表征,并开发了一个深度学习模型来预测gRNA的活性,从而提供相应的整合效率和表达强度。随后,应用该工具和平台菌株进行了(S)-reticuline 和 (2S)-taxifolin的从头合成验证。所开发的平台菌株和工具有助于将脂溶性酵母转化为类似于酿酒酵母的易于操作的模式细胞工厂。
{"title":"<i>De Novo</i> Synthesis of Reticuline and Taxifolin Using Re-engineered Homologous Recombination in <i>Yarrowia lipolytica</i>.","authors":"Changtai Zhang, Mengsu Liu, Xinglong Wang, Junyi Cheng, Jinbo Xiang, Mingyu Yue, Yang Ning, Zhengxuan Shao, Chalak Najat Abdullah, Jingwen Zhou","doi":"10.1021/acssynbio.4c00853","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00853","url":null,"abstract":"<p><p><i>Yarrowia lipolytica</i> has been widely engineered as a eukaryotic cell factory to produce various important compounds. However, the difficulty of gene editing and the lack of efficient neutral sites make rewiring of <i>Y. lipolytica</i> metabolism challenging. Herein, a Cas9 system was established to redesign the <i>Y. lipolytica</i> homologous recombination system, which caused a more than 56-fold increase in the HR efficiency. The fusion expression of the hBrex27 sequence in the C-terminus of Cas9 recruited more Rad51 protein, and the engineered Cas9 decreased NHEJ, achieving 85% single-gene positive efficiency and 25% multigene editing efficiency. With this system, neutral sites on different chromosomes were characterized, and a deep learning model was developed for gRNA activity prediction, thus providing the corresponding integration efficiency and expression intensity. Subsequently, the tool and platform strains were validated by applying them for the <i>de novo</i> synthesis of (<i>S</i>)-reticuline and (2<i>S</i>)-taxifolin. The developed platform strains and tools helped transform <i>Y. lipolytica</i> into an easy-to-operate model cell factory, similar to <i>Saccharomyces cerevisiae</i>.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-02-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143121666","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Enhanced Integration of Single-Cell Multi-Omics Data Using Graph Attention Networks. 利用图形注意网络增强单细胞多图像数据的整合。
IF 3.7 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-01-31 DOI: 10.1021/acssynbio.4c00864
Xingyu Liao, Yanyan Li, Shuangyi Li, Long Wen, Xingyi Li, Bin Yu

The continuous advancement of single-cell multimodal omics (scMulti-omics) technologies offers unprecedented opportunities to measure various modalities, including RNA expression, protein abundance, gene perturbation, DNA methylation, and chromatin accessibility at single-cell resolution. These advances hold significant potential for breakthroughs by integrating diverse omics modalities. However, the data generated from different omics layers often face challenges due to high dimensionality, heterogeneity, and sparsity, which can adversely impact the accuracy and efficiency of data integration analyses. To address these challenges, we propose a high-precision analysis method called scMGAT (single-cell multiomics data analysis based on multihead graph attention networks). This method effectively coordinates reliable information across multiomics data sets using a multihead attention mechanism, allowing for better management of the heterogeneous characteristics inherent in scMulti-omics data. We evaluated scMGAT's performance on eight sets of real scMulti-omics data, including samples from both human and mouse. The experimental results demonstrate that scMGAT significantly enhances the quality of multiomics data and improves the accuracy of cell-type annotation compared to state-of-the-art methods. scMGAT is now freely accessible at https://github.com/Xingyu-Liao/scMGAT.

{"title":"Enhanced Integration of Single-Cell Multi-Omics Data Using Graph Attention Networks.","authors":"Xingyu Liao, Yanyan Li, Shuangyi Li, Long Wen, Xingyi Li, Bin Yu","doi":"10.1021/acssynbio.4c00864","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00864","url":null,"abstract":"<p><p>The continuous advancement of single-cell multimodal omics (scMulti-omics) technologies offers unprecedented opportunities to measure various modalities, including RNA expression, protein abundance, gene perturbation, DNA methylation, and chromatin accessibility at single-cell resolution. These advances hold significant potential for breakthroughs by integrating diverse omics modalities. However, the data generated from different omics layers often face challenges due to high dimensionality, heterogeneity, and sparsity, which can adversely impact the accuracy and efficiency of data integration analyses. To address these challenges, we propose a high-precision analysis method called scMGAT (single-cell multiomics data analysis based on multihead graph attention networks). This method effectively coordinates reliable information across multiomics data sets using a multihead attention mechanism, allowing for better management of the heterogeneous characteristics inherent in scMulti-omics data. We evaluated scMGAT's performance on eight sets of real scMulti-omics data, including samples from both human and mouse. The experimental results demonstrate that scMGAT significantly enhances the quality of multiomics data and improves the accuracy of cell-type annotation compared to state-of-the-art methods. scMGAT is now freely accessible at https://github.com/Xingyu-Liao/scMGAT.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143070726","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Considerations for Domestication of Novel Strains of Filamentous Fungi.
IF 3.7 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2025-01-30 DOI: 10.1021/acssynbio.4c00672
Randi M Pullen, Stephen R Decker, Venkataramanan Subramanian, Meaghan J Adler, Alexander V Tobias, Matthew Perisin, Christian J Sund, Matthew D Servinsky, Mark T Kozlowski

Fungi, especially filamentous fungi, are a relatively understudied, biotechnologically useful resource with incredible potential for commercial applications. These multicellular eukaryotic organisms have long been exploited for their natural production of useful commodity chemicals and proteins such as enzymes used in starch processing, detergents, food and feed production, pulping and paper making and biofuels production. The ability of filamentous fungi to use a wide range of feedstocks is another key advantage. As chassis organisms, filamentous fungi can express cellular machinery, and metabolic and signal transduction pathways from both prokaryotic and eukaryotic origins. Their genomes abound with novel genetic elements and metabolic processes that can be harnessed for biotechnology applications. Synthetic biology tools are becoming inexpensive, modular, and expansive while systems biology is beginning to provide the level of understanding required to design increasingly complex synthetic systems. This review covers the challenges of working in filamentous fungi and offers a perspective on the approaches needed to exploit fungi as microbial cell factories.

{"title":"Considerations for Domestication of Novel Strains of Filamentous Fungi.","authors":"Randi M Pullen, Stephen R Decker, Venkataramanan Subramanian, Meaghan J Adler, Alexander V Tobias, Matthew Perisin, Christian J Sund, Matthew D Servinsky, Mark T Kozlowski","doi":"10.1021/acssynbio.4c00672","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00672","url":null,"abstract":"<p><p>Fungi, especially filamentous fungi, are a relatively understudied, biotechnologically useful resource with incredible potential for commercial applications. These multicellular eukaryotic organisms have long been exploited for their natural production of useful commodity chemicals and proteins such as enzymes used in starch processing, detergents, food and feed production, pulping and paper making and biofuels production. The ability of filamentous fungi to use a wide range of feedstocks is another key advantage. As chassis organisms, filamentous fungi can express cellular machinery, and metabolic and signal transduction pathways from both prokaryotic and eukaryotic origins. Their genomes abound with novel genetic elements and metabolic processes that can be harnessed for biotechnology applications. Synthetic biology tools are becoming inexpensive, modular, and expansive while systems biology is beginning to provide the level of understanding required to design increasingly complex synthetic systems. This review covers the challenges of working in filamentous fungi and offers a perspective on the approaches needed to exploit fungi as microbial cell factories.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2025-01-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143062250","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
期刊
ACS Synthetic Biology
全部 Acc. Chem. Res. ACS Applied Bio Materials ACS Appl. Electron. Mater. ACS Appl. Energy Mater. ACS Appl. Mater. Interfaces ACS Appl. Nano Mater. ACS Appl. Polym. Mater. ACS BIOMATER-SCI ENG ACS Catal. ACS Cent. Sci. ACS Chem. Biol. ACS Chemical Health & Safety ACS Chem. Neurosci. ACS Comb. Sci. ACS Earth Space Chem. ACS Energy Lett. ACS Infect. Dis. ACS Macro Lett. ACS Mater. Lett. ACS Med. Chem. Lett. ACS Nano ACS Omega ACS Photonics ACS Sens. ACS Sustainable Chem. Eng. ACS Synth. Biol. Anal. Chem. BIOCHEMISTRY-US Bioconjugate Chem. BIOMACROMOLECULES Chem. Res. Toxicol. Chem. Rev. Chem. Mater. CRYST GROWTH DES ENERG FUEL Environ. Sci. Technol. Environ. Sci. Technol. Lett. Eur. J. Inorg. Chem. IND ENG CHEM RES Inorg. Chem. J. Agric. Food. Chem. J. Chem. Eng. Data J. Chem. Educ. J. Chem. Inf. Model. J. Chem. Theory Comput. J. Med. Chem. J. Nat. Prod. J PROTEOME RES J. Am. Chem. Soc. LANGMUIR MACROMOLECULES Mol. Pharmaceutics Nano Lett. Org. Lett. ORG PROCESS RES DEV ORGANOMETALLICS J. Org. Chem. J. Phys. Chem. J. Phys. Chem. A J. Phys. Chem. B J. Phys. Chem. C J. Phys. Chem. Lett. Analyst Anal. Methods Biomater. Sci. Catal. Sci. Technol. Chem. Commun. Chem. Soc. Rev. CHEM EDUC RES PRACT CRYSTENGCOMM Dalton Trans. Energy Environ. Sci. ENVIRON SCI-NANO ENVIRON SCI-PROC IMP ENVIRON SCI-WAT RES Faraday Discuss. Food Funct. Green Chem. Inorg. Chem. Front. Integr. Biol. J. Anal. At. Spectrom. J. Mater. Chem. A J. Mater. Chem. B J. Mater. Chem. C Lab Chip Mater. Chem. Front. Mater. Horiz. MEDCHEMCOMM Metallomics Mol. Biosyst. Mol. Syst. Des. Eng. Nanoscale Nanoscale Horiz. Nat. Prod. Rep. New J. Chem. Org. Biomol. Chem. Org. Chem. Front. PHOTOCH PHOTOBIO SCI PCCP Polym. Chem.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
0
微信
客服QQ
Book学术公众号 扫码关注我们
反馈
×
意见反馈
请填写您的意见或建议
请填写您的手机或邮箱
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
现在去查看 取消
×
提示
确定
Book学术官方微信
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术
文献互助 智能选刊 最新文献 互助须知 联系我们:info@booksci.cn
Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。
Copyright © 2023 Book学术 All rights reserved.
ghs 京公网安备 11010802042870号 京ICP备2023020795号-1