首页 > 最新文献

CRISPR Journal最新文献

英文 中文
Engineering CjCas9 for Efficient Base Editing and Prime Editing. 对 CjCas9 进行工程改造,以实现高效的碱基编辑和基序编辑。
IF 3.7 4区 生物学 Q2 GENETICS & HEREDITY Pub Date : 2024-11-18 DOI: 10.1089/crispr.2024.0018
Siyuan Liu, Yingdi Zhao, Qiqin Mo, Yadong Sun, Hanhui Ma

The CRISPR-Cas9 system has been applied for clinical applications of gene therapy. Most CRISPR-based gene therapies are derived from Streptococcus pyogenes Cas9, which is challenging to package into a single adeno-associated virus vector and limits its clinical applications. Campylobacter jejuni Cas9 (CjCas9) is one of the smallest Cas9 proteins. CjCas9-mediated base editing (CjBE) efficiency varies across genomic sites, while CjCas9-mediated prime editing (CjPE) efficiency is less than 5% on average. Here we developed enhanced cytosine base editors (enCjCBEs) and adenine base editors (enCjABEs) by engineered CjCas9P47K. We demonstrated the robust C-to-T conversion (70% on average) by enCjCBE or A-to-G conversion (76% on average) by enCjABE. Meanwhile, we applied the CjCas9P47K variant to generate enhanced CjPE (enCjPE), which increases the editing efficiency 17-fold at the PRNP site over wild-type CjPE. Fusing nonspecific DNA binding protein Sso7d to enCjCas9 and MS2 stem-loop RNA aptamer to the 3-terminal of cognate pegRNA resulted in 12% editing efficiency on average with a 24-fold increase over wild-type CjPE, and we termed it SsenCjPE. The SsenCjPE can also be combined with hMLH1dn to further increase the editing efficiency and MMLV RTaseΔRnH to reduce size. Finally, we introduced an additional mutation D829R into SsenCjPE and generated SsenCjPE-M2 with a 61-fold increase of PE efficiency over wild-type at the PRNP site. In summary, enCjBEs, SsenCjPEs, or SsenCjPE-M2 are compact Cas9-derived BE or prime editors in biological research or biomedical applications.

CRISPR-Cas9 系统已被应用于基因治疗的临床应用。大多数基于CRISPR的基因疗法都源自化脓性链球菌Cas9,将其包装成单一的腺相关病毒载体具有挑战性,限制了其临床应用。空肠弯曲杆菌 Cas9(CjCas9)是最小的 Cas9 蛋白之一。CjCas9 介导的碱基编辑(CjBE)效率因基因组位点而异,而 CjCas9 介导的质粒编辑(CjPE)效率平均不到 5%。在这里,我们通过工程化 CjCas9P47K 开发了增强型胞嘧啶碱基编辑器(enCjCBEs)和腺嘌呤碱基编辑器(enCjABEs)。我们证明了 enCjCBE 和 enCjABE 可实现强大的 C-T 转换(平均转换率为 70%)或 A-G 转换(平均转换率为 76%)。同时,我们应用 CjCas9P47K 变体生成了增强型 CjPE(enCjPE),它在 PRNP 位点的编辑效率比野生型 CjPE 提高了 17 倍。将非特异性 DNA 结合蛋白 Sso7d 与 enCjCas9 结合,并将 MS2 茎环 RNA 配合物与同源 pegRNA 的 3 端结合,其编辑效率平均为 12%,比野生型 CjPE 提高了 24 倍,我们称之为 SsenCjPE。SsenCjPE 还可以与 hMLH1dn 结合使用,以进一步提高编辑效率,并与 MMLV RTaseΔRnH 结合使用,以缩小体积。最后,我们在 SsenCjPE 中引入了一个额外的突变 D829R,生成的 SsenCjPE-M2 在 PRNP 位点的 PE 效率比野生型提高了 61 倍。总之,enCjBEs、SsenCjPEs 或 SsenCjPE-M2 是用于生物研究或生物医学应用的紧凑型 Cas9 衍生 BE 或素材编辑器。
{"title":"Engineering CjCas9 for Efficient Base Editing and Prime Editing.","authors":"Siyuan Liu, Yingdi Zhao, Qiqin Mo, Yadong Sun, Hanhui Ma","doi":"10.1089/crispr.2024.0018","DOIUrl":"https://doi.org/10.1089/crispr.2024.0018","url":null,"abstract":"<p><p>The CRISPR-Cas9 system has been applied for clinical applications of gene therapy. Most CRISPR-based gene therapies are derived from <i>Streptococcus pyogenes</i> Cas9, which is challenging to package into a single adeno-associated virus vector and limits its clinical applications. <i>Campylobacter jejuni</i> Cas9 (CjCas9) is one of the smallest Cas9 proteins. CjCas9-mediated base editing (CjBE) efficiency varies across genomic sites, while CjCas9-mediated prime editing (CjPE) efficiency is less than 5% on average. Here we developed enhanced cytosine base editors (enCjCBEs) and adenine base editors (enCjABEs) by engineered CjCas9<sup>P47K</sup>. We demonstrated the robust C-to-T conversion (70% on average) by enCjCBE or A-to-G conversion (76% on average) by enCjABE. Meanwhile, we applied the CjCas9<sup>P47K</sup> variant to generate enhanced CjPE (enCjPE), which increases the editing efficiency 17-fold at the <i>PRNP</i> site over wild-type CjPE. Fusing nonspecific DNA binding protein Sso7d to enCjCas9 and MS2 stem-loop RNA aptamer to the 3-terminal of cognate pegRNA resulted in 12% editing efficiency on average with a 24-fold increase over wild-type CjPE, and we termed it SsenCjPE. The SsenCjPE can also be combined with hMLH1dn to further increase the editing efficiency and MMLV RTaseΔRnH to reduce size. Finally, we introduced an additional mutation D829R into SsenCjPE and generated SsenCjPE-M2 with a 61-fold increase of PE efficiency over wild-type at the <i>PRNP</i> site. In summary, enCjBEs, SsenCjPEs, or SsenCjPE-M2 are compact Cas9-derived BE or prime editors in biological research or biomedical applications.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2024-11-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142649684","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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
CRISPR-Cas9-Mediated Targeting of Multidrug Resistance Genes in Methicillin-Resistant Staphylococcus aureus. CRISPR-Cas9 介导的耐甲氧西林金黄色葡萄球菌多药耐药性基因靶向。
IF 3.7 4区 生物学 Q2 GENETICS & HEREDITY Pub Date : 2024-11-08 DOI: 10.1089/crispr.2024.0001
Aysegul Ates, Cihan Tastan, Safak Ermertcan

Antibiotic resistance poses a global health crisis limiting the efficacy of available therapeutic agents. We explored CRISPR-Cas-based antimicrobials to combat multidrug resistance in methicillin-resistant Staphylococcus aureus (MRSA), targeting methicillin (mecA), gentamicin (aacA), and ciprofloxacin (grlA, grlB) resistance genes. Engineered CRISPR plasmids with specific single-guide RNAs were electroporated into MRSA strains. Real-time polymerase chain reaction assessed gene expression changes, while antibiotic susceptibility tests (ASTs) evaluated resistance status. Results showed a 1.5-fold decrease in mecA, a 5.5-fold decrease in grlA, a 6-fold decrease in grlB, and a 4-fold decrease in aacA expression. ASTs demonstrated the reversal of resistance to beta-lactam, quinolone, and aminoglycoside antibiotics. Western blot analysis revealed a 70% decrease in penicillin-binding protein 2a expression. Sanger sequencing confirmed point mutations in the grlB and aacA genes. Our findings highlight the potential of CRISPR-Cas9 technology to restore antibiotic efficacy against multidrug-resistant pathogens.

抗生素耐药性是一个全球性的健康危机,限制了现有治疗药物的疗效。我们针对甲氧西林(mecA)、庆大霉素(aacA)和环丙沙星(grlA、grlB)耐药基因,探索了基于CRISPR-Cas的抗菌药物,以对抗耐甲氧西林金黄色葡萄球菌(MRSA)的多重耐药性。将带有特定单导 RNA 的 CRISPR 质粒电穿孔到 MRSA 菌株中。实时聚合酶链反应评估了基因表达的变化,而抗生素药敏试验(AST)则评估了耐药性状况。结果显示,mecA 表达量减少了 1.5 倍,grlA 减少了 5.5 倍,grlB 减少了 6 倍,aacA 表达量减少了 4 倍。ASTs 表明对 beta-内酰胺类、喹诺酮类和氨基糖苷类抗生素的耐药性发生了逆转。Western 印迹分析显示,青霉素结合蛋白 2a 的表达量减少了 70%。桑格测序证实了 grlB 和 aacA 基因的点突变。我们的研究结果凸显了 CRISPR-Cas9 技术在恢复抗生素对耐多药病原体疗效方面的潜力。
{"title":"CRISPR-Cas9-Mediated Targeting of Multidrug Resistance Genes in Methicillin-Resistant <i>Staphylococcus aureus</i>.","authors":"Aysegul Ates, Cihan Tastan, Safak Ermertcan","doi":"10.1089/crispr.2024.0001","DOIUrl":"https://doi.org/10.1089/crispr.2024.0001","url":null,"abstract":"<p><p>Antibiotic resistance poses a global health crisis limiting the efficacy of available therapeutic agents. We explored CRISPR-Cas-based antimicrobials to combat multidrug resistance in methicillin-resistant <i>Staphylococcus aureus</i> (MRSA), targeting methicillin (<i>mec</i>A), gentamicin (<i>aac</i>A), and ciprofloxacin (<i>grl</i>A, <i>grl</i>B) resistance genes. Engineered CRISPR plasmids with specific single-guide RNAs were electroporated into MRSA strains. Real-time polymerase chain reaction assessed gene expression changes, while antibiotic susceptibility tests (ASTs) evaluated resistance status. Results showed a 1.5-fold decrease in <i>mec</i>A, a 5.5-fold decrease in <i>grl</i>A, a 6-fold decrease in <i>grl</i>B, and a 4-fold decrease in <i>aac</i>A expression. ASTs demonstrated the reversal of resistance to beta-lactam, quinolone, and aminoglycoside antibiotics. Western blot analysis revealed a 70% decrease in penicillin-binding protein 2a expression. Sanger sequencing confirmed point mutations in the <i>grl</i>B and <i>aac</i>A genes. Our findings highlight the potential of CRISPR-Cas9 technology to restore antibiotic efficacy against multidrug-resistant pathogens.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2024-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142607408","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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
Early Detection of Wildlife Disease Pathogens Using CRISPR-Cas System Methods. 利用 CRISPR-Cas 系统方法早期检测野生动物疾病病原体。
IF 3.7 4区 生物学 Q2 GENETICS & HEREDITY Pub Date : 2024-10-31 DOI: 10.1089/crispr.2024.0030
Adam A Pérez, Guelaguetza Vazquez-Meves, Margaret E Hunter

Wildlife diseases are a considerable threat to human health, conservation, and the economy. Surveillance is a critical component to mitigate the impact of animal diseases in these sectors. To monitor human diseases, CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein) biosensors have proven instrumental as diagnostic tools capable of detecting unique DNA and RNA sequences related to their associated pathogens. However, despite the significant advances in the general development of CRISPR-Cas biosensors, their use to support wildlife disease management is lagging. In some cases, wildlife diseases of concern could be rapidly surveyed using these tools with minimal technical, operational, or cost requirements to end users. This review explores the potential to further leverage this technology to advance wildlife disease monitoring and highlights how concerted standardization of protocols can help to ensure data reliability.

野生动物疾病对人类健康、自然保护和经济都构成了巨大威胁。监测是减轻动物疾病对这些领域影响的关键组成部分。为了监测人类疾病,CRISPR-Cas(成簇的有规则间隔短回文重复序列-CRISPR 相关蛋白)生物传感器已被证明是一种诊断工具,能够检测与相关病原体有关的独特 DNA 和 RNA 序列。然而,尽管 CRISPR-Cas 生物传感器的总体发展取得了重大进展,但其在支持野生动物疾病管理方面的应用仍然滞后。在某些情况下,使用这些工具可以快速调查受关注的野生动物疾病,对最终用户的技术、操作或成本要求极低。本综述探讨了进一步利用该技术推进野生动物疾病监测的潜力,并强调了协调一致的标准化协议如何有助于确保数据的可靠性。
{"title":"Early Detection of Wildlife Disease Pathogens Using CRISPR-Cas System Methods.","authors":"Adam A Pérez, Guelaguetza Vazquez-Meves, Margaret E Hunter","doi":"10.1089/crispr.2024.0030","DOIUrl":"10.1089/crispr.2024.0030","url":null,"abstract":"<p><p>Wildlife diseases are a considerable threat to human health, conservation, and the economy. Surveillance is a critical component to mitigate the impact of animal diseases in these sectors. To monitor human diseases, CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein) biosensors have proven instrumental as diagnostic tools capable of detecting unique DNA and RNA sequences related to their associated pathogens. However, despite the significant advances in the general development of CRISPR-Cas biosensors, their use to support wildlife disease management is lagging. In some cases, wildlife diseases of concern could be rapidly surveyed using these tools with minimal technical, operational, or cost requirements to end users. This review explores the potential to further leverage this technology to advance wildlife disease monitoring and highlights how concerted standardization of protocols can help to ensure data reliability.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2024-10-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142548921","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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
CRISPR-GRIT: Guide RNAs with Integrated Repair Templates Enable Precise Multiplexed Genome Editing in the Diploid Fungal Pathogen Candida albicans. CRISPR-GRIT:带有集成修复模板的引导 RNA 可对二倍体真菌病原体白色念珠菌进行精确的多重基因组编辑。
IF 3.7 4区 生物学 Q2 GENETICS & HEREDITY Pub Date : 2024-10-22 DOI: 10.1089/crispr.2024.0052
Christopher J Cotter, Cong T Trinh

Candida albicans, an opportunistic fungal pathogen, causes severe infections in immunocompromised individuals. Limited classes and overuse of current antifungals have led to the rapid emergence of antifungal resistance. Thus, there is an urgent need to understand fungal pathogen genetics to develop new antifungal strategies. Genetic manipulation of C. albicans is encumbered by its diploid chromosomes requiring editing both alleles to elucidate gene function. Although the recent development of CRISPR-Cas systems has facilitated genome editing in C. albicans, large-scale and multiplexed functional genomic studies are still hindered by the necessity of cotransforming repair templates for homozygous knockouts. Here, we present CRISPR-GRIT (Guide RNAs with Integrated Repair Templates), a repair template-integrated guide RNA design for expedited gene knockouts and multiplexed gene editing in C. albicans. We envision that this method can be used for high-throughput library screens and identification of synthetic lethal pairs in both C. albicans and other diploid organisms with strong homologous recombination machinery.

白色念珠菌是一种机会性真菌病原体,会对免疫力低下的人造成严重感染。目前抗真菌药物的种类有限和过度使用导致抗真菌抗药性迅速出现。因此,迫切需要了解真菌病原体的遗传学,以开发新的抗真菌策略。白僵菌的遗传操作受到其二倍体染色体的限制,需要编辑两个等位基因来阐明基因功能。尽管最近 CRISPR-Cas 系统的发展促进了白僵菌的基因组编辑,但大规模和多重功能基因组研究仍然受到同源基因敲除必须共转化修复模板的阻碍。在这里,我们提出了 CRISPR-GRIT(带有整合修复模板的引导 RNA),这是一种整合了修复模板的引导 RNA 设计,用于加速白僵菌的基因敲除和多重基因编辑。我们设想这种方法可用于高通量文库筛选,并在白僵菌和其他具有强大同源重组机制的二倍体生物中鉴定合成致死对。
{"title":"CRISPR-GRIT: Guide RNAs with Integrated Repair Templates Enable Precise Multiplexed Genome Editing in the Diploid Fungal Pathogen <i>Candida albicans</i>.","authors":"Christopher J Cotter, Cong T Trinh","doi":"10.1089/crispr.2024.0052","DOIUrl":"https://doi.org/10.1089/crispr.2024.0052","url":null,"abstract":"<p><p><i>Candida albicans,</i> an opportunistic fungal pathogen, causes severe infections in immunocompromised individuals. Limited classes and overuse of current antifungals have led to the rapid emergence of antifungal resistance. Thus, there is an urgent need to understand fungal pathogen genetics to develop new antifungal strategies. Genetic manipulation of <i>C. albicans</i> is encumbered by its diploid chromosomes requiring editing both alleles to elucidate gene function. Although the recent development of CRISPR-Cas systems has facilitated genome editing in <i>C. albicans</i>, large-scale and multiplexed functional genomic studies are still hindered by the necessity of cotransforming repair templates for homozygous knockouts. Here, we present CRISPR-GRIT (<u>G</u>uide <u>R</u>NAs with <u>I</u>ntegrated Repair <u>T</u>emplates), a repair template-integrated guide RNA design for expedited gene knockouts and multiplexed gene editing in <i>C. albicans</i>. We envision that this method can be used for high-throughput library screens and identification of synthetic lethal pairs in both <i>C. albicans</i> and other diploid organisms with strong homologous recombination machinery.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2024-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142512851","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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
Genome Editing in Apicomplexan Parasites: Current Status, Challenges, and Future Possibilities. 表皮复合寄生虫的基因组编辑:现状、挑战和未来的可能性》。
IF 3.7 4区 生物学 Q2 GENETICS & HEREDITY Pub Date : 2024-10-10 DOI: 10.1089/crispr.2024.0032
Ethel Webi, Hussein M Abkallo, George Obiero, Paul Ndegwa, Shengsong Xie, Shuhong Zhao, Vishvanath Nene, Lucilla Steinaa

Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) technology has revolutionized genome editing across various biological systems, including the Apicomplexa phylum. This review describes the status, challenges, and applications of CRISPR-Cas9 editing technology in apicomplexan parasites, such as Plasmodium, Toxoplasma, Theileria, Babesia, and Cryptosporidium. The discussion encompasses successfully implemented CRISPR-Cas9-based techniques in these parasites, highlighting the achieved milestones, from precise gene modifications to genome-wide screening. In addition, the review addresses the challenges hampering efficient genome editing, including the parasites' complex life cycles, multiple intracellular stages, and the lack of robust genetic tools. It further explores the ethical and policy considerations surrounding genome editing and the future perspectives of CRISPR-Cas applications in apicomplexan parasites.

成簇的有规则间隔短回文重复序列(CRISPR)-CRISPR相关蛋白(Cas)技术已经彻底改变了各种生物系统的基因组编辑,其中包括类囊虫门。本综述介绍了 CRISPR-Cas9 编辑技术的现状、挑战以及在疟原虫、弓形虫、Theileria、巴贝丝菌和隐孢子虫等类群寄生虫中的应用。文章讨论了在这些寄生虫中成功实施的基于CRISPR-Cas9的技术,重点介绍了从精确基因修饰到全基因组筛选所取得的阶段性成果。此外,综述还讨论了阻碍高效基因组编辑的挑战,包括寄生虫复杂的生命周期、多细胞内阶段以及缺乏强大的遗传工具。它还进一步探讨了基因组编辑的伦理和政策考量,以及 CRISPR-Cas 在类凋亡寄生虫中应用的未来前景。
{"title":"Genome Editing in Apicomplexan Parasites: Current Status, Challenges, and Future Possibilities.","authors":"Ethel Webi, Hussein M Abkallo, George Obiero, Paul Ndegwa, Shengsong Xie, Shuhong Zhao, Vishvanath Nene, Lucilla Steinaa","doi":"10.1089/crispr.2024.0032","DOIUrl":"10.1089/crispr.2024.0032","url":null,"abstract":"<p><p>Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) technology has revolutionized genome editing across various biological systems, including the Apicomplexa phylum. This review describes the status, challenges, and applications of CRISPR-Cas9 editing technology in apicomplexan parasites, such as <i>Plasmodium</i>, <i>Toxoplasma</i>, <i>Theileria</i>, <i>Babesia</i>, and <i>Cryptosporidium</i>. The discussion encompasses successfully implemented CRISPR-Cas9-based techniques in these parasites, highlighting the achieved milestones, from precise gene modifications to genome-wide screening. In addition, the review addresses the challenges hampering efficient genome editing, including the parasites' complex life cycles, multiple intracellular stages, and the lack of robust genetic tools. It further explores the ethical and policy considerations surrounding genome editing and the future perspectives of CRISPR-Cas applications in apicomplexan parasites.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2024-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142480623","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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
Mutation-Specific CRISPR Targeting with SaCas9 and AsCas12a Restores Therapeutic Sensitivity in Treatment-Resistant Melanoma. 用 SaCas9 和 AsCas12a 进行突变特异性 CRISPR 靶向可恢复耐药黑色素瘤的治疗敏感性。
IF 3.7 4区 生物学 Q2 GENETICS & HEREDITY Pub Date : 2024-10-10 DOI: 10.1089/crispr.2024.0003
Brett M Sansbury, Sophia B Masciarelli, Salma Kaouser, Olivia M Tharp, Kelly H Banas, Eric B Kmiec

Background: Melanoma remains one of the most challenging cancers to treat effectively with drug resistant remaining a constant concern, primarily with activating BRAF mutations. Mutations in the BRAF gene appear in approximately 50% of patients, 90% of which are V600E. Two frontline BRAF inhibitors (BRAFi), vemurafenib and dabrafenib, are frequently used to treat unresectable or metastatic BRAF V600E melanoma. Initial response rates are high, but soon thereafter, 70-80% of patients develop resistance to treatment within a year. A major mechanism of resistance is the generation of a secondary Q61K mutation in the NRAS gene. Methods: We have developed an approach in which a CRISPR-Cas complex can be designed to distinguish between mutant genes enabling resistance to standard care in tumor cells and normal genomes of healthy cells. For the first time, we demonstrated the utility of two CRISPR-directed mutation-specific editing approaches to restore BRAFi sensitivity in BRAFV600E/NRASQ61K resistant A375 cells. Results: We utilize an AsCas12a protospacer adjacent motif site created by the NRAS Q61K mutation and the Q61K mutation in the critical seed region of an SaCas9 sgRNA for Q61K-selective targeting. We show here that both approaches allow for effective NRAS targeting of only mutated-Q61K and after CRISPR-directed Q61K-targeting, previously resistant A375 cells are re-sensitized to BRAFi treatment. Conclusion: Our data support the feasibility of the development of CRISPR-Cas therapeutic approaches to the treatment of melanoma. Successful therapeutic CRISPR-directed gene editing would enable both specific and efficient editing of a mutation-specific targeting approach eliminate concern for on- and off-target damage to the genomes of healthy cells.

背景:黑色素瘤仍然是最难有效治疗的癌症之一,耐药性始终是一个令人担忧的问题,主要是活化的 BRAF 基因突变。约50%的患者会出现BRAF基因突变,其中90%为V600E。两种一线 BRAF 抑制剂(BRAFi),即维莫非尼(vemurafenib)和达拉菲尼(dabrafenib),常用于治疗不可切除或转移性 BRAF V600E 黑色素瘤。最初的应答率很高,但不久之后,70%-80%的患者会在一年内产生耐药性。耐药的一个主要机制是 NRAS 基因产生了二次 Q61K 突变。方法:我们开发了一种方法,通过设计 CRISPR-Cas 复合物来区分肿瘤细胞中对标准治疗产生耐药性的突变基因和健康细胞的正常基因组。我们首次展示了两种 CRISPR 引导的突变特异性编辑方法在 BRAFV600E/NRASQ61K 耐药的 A375 细胞中恢复 BRAFi 敏感性的实用性。结果:我们利用NRAS Q61K突变产生的AsCas12a原位相邻基序位点和SaCas9 sgRNA关键种子区的Q61K突变进行Q61K选择性靶向。我们在此表明,这两种方法都能有效靶向仅突变 Q61K 的 NRAS,而且在 CRISPR 引导的 Q61K 靶向后,之前耐药的 A375 细胞对 BRAFi 治疗重新敏感。结论我们的数据支持开发 CRISPR-Cas 治疗黑色素瘤方法的可行性。成功的治疗性CRISPR定向基因编辑可实现对突变特异性靶向方法的特异性和高效编辑,消除对健康细胞基因组的靶上和靶下损伤的担忧。
{"title":"Mutation-Specific CRISPR Targeting with SaCas9 and AsCas12a Restores Therapeutic Sensitivity in Treatment-Resistant Melanoma.","authors":"Brett M Sansbury, Sophia B Masciarelli, Salma Kaouser, Olivia M Tharp, Kelly H Banas, Eric B Kmiec","doi":"10.1089/crispr.2024.0003","DOIUrl":"https://doi.org/10.1089/crispr.2024.0003","url":null,"abstract":"<p><p><b>Background:</b> Melanoma remains one of the most challenging cancers to treat effectively with drug resistant remaining a constant concern, primarily with activating <i>BRAF</i> mutations. Mutations in the <i>BRAF</i> gene appear in approximately 50% of patients, 90% of which are V600E. Two frontline <i>BRAF</i> inhibitors (BRAFi), vemurafenib and dabrafenib, are frequently used to treat unresectable or metastatic <i>BRAF</i> V600E melanoma. Initial response rates are high, but soon thereafter, 70-80% of patients develop resistance to treatment within a year. A major mechanism of resistance is the generation of a secondary Q61K mutation in the <i>NRAS</i> gene. <b>Methods:</b> We have developed an approach in which a CRISPR-Cas complex can be designed to distinguish between mutant genes enabling resistance to standard care in tumor cells and normal genomes of healthy cells. For the first time, we demonstrated the utility of two CRISPR-directed mutation-specific editing approaches to restore BRAFi sensitivity in <i>BRAF</i><sup>V600E</sup>/<i>NRAS</i><sup>Q61K</sup> resistant A375 cells. <b>Results:</b> We utilize an AsCas12a protospacer adjacent motif site created by the <i>NRAS</i> Q61K mutation and the Q61K mutation in the critical seed region of an SaCas9 sgRNA for Q61K-selective targeting. We show here that both approaches allow for effective <i>NRAS</i> targeting of only mutated-Q61K and after CRISPR-directed Q61K-targeting, previously resistant A375 cells are re-sensitized to BRAFi treatment. <b>Conclusion:</b> Our data support the feasibility of the development of CRISPR-Cas therapeutic approaches to the treatment of melanoma. Successful therapeutic CRISPR-directed gene editing would enable both specific and efficient editing of a mutation-specific targeting approach eliminate concern for on- and off-target damage to the genomes of healthy cells.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2024-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142480624","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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
Engineering of HEK293T Cell Factory for Lentiviral Production by High-Throughput Selected Genes. 通过高通量选择基因对 HEK293T 细胞工厂进行工程改造,以生产慢病毒。
IF 3.7 4区 生物学 Q2 GENETICS & HEREDITY Pub Date : 2024-10-01 Epub Date: 2024-10-16 DOI: 10.1089/crispr.2024.0016
Zhang Xinyue, Siwei Li, Wang Yujie, Dai Yingcai, Bi Changhao, Zhang Xueli

Lentiviral vectors (LVs) are crucial tools in gene therapy and bioproduction, but high-yield LV production systems are urgently needed. Using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 high-throughput screening, we identified nine critical genes (LDAH, GBP3, BPIFC, NHLRC1, NHLRC3, ZNF425, TTC37, LRRC4B, and SPINK6) from 17,501 genes that limit LV packaging and formation. Knocking out these genes in HEK293T cells significantly increased virus production, with LDAH knockout exhibiting a 6.63-fold increase. Studies on multigene knockouts demonstrated that the cumulative effects of different gene knockouts can significantly enhance lentivirus production in HEK293T cells. Triple knockout of GBP3, BPIFC, and LDAH increased LV titer by ∼8.33-fold, and knockout (or knockdown) of GBP3, NHLRC1, and NHLRC3 increased LV titer by ∼6.53-fold. This study established HEK293T cell lines with multiple genes knockout for efficient LV production, providing reliable technical support for LV production and application and offering new perspectives for studying LV packaging mechanisms and related virus research.

慢病毒载体(LV)是基因治疗和生物生产的重要工具,但目前急需高产的 LV 生产系统。利用聚类规则间隔短回文重复序列(CRISPR)-CRISPR相关蛋白9高通量筛选,我们从17501个基因中发现了9个限制LV包装和形成的关键基因(LDAH、GBP3、BPIFC、NHLRC1、NHLRC3、ZNF425、TTC37、LRRC4B和SPINK6)。在 HEK293T 细胞中敲除这些基因可显著提高病毒产量,其中 LDAH 基因敲除的产量提高了 6.63 倍。对多基因敲除的研究表明,不同基因敲除的累积效应可显著提高 HEK293T 细胞中慢病毒的产量。GBP3、BPIFC和LDAH的三重基因敲除使LV滴度增加了8.33倍,GBP3、NHLRC1和NHLRC3的基因敲除(或敲除)使LV滴度增加了6.53倍。该研究建立了多基因敲除的HEK293T细胞系,实现了LV的高效生产,为LV的生产和应用提供了可靠的技术支持,为研究LV的包装机制和相关病毒研究提供了新的视角。
{"title":"Engineering of HEK293T Cell Factory for Lentiviral Production by High-Throughput Selected Genes.","authors":"Zhang Xinyue, Siwei Li, Wang Yujie, Dai Yingcai, Bi Changhao, Zhang Xueli","doi":"10.1089/crispr.2024.0016","DOIUrl":"10.1089/crispr.2024.0016","url":null,"abstract":"<p><p>Lentiviral vectors (LVs) are crucial tools in gene therapy and bioproduction, but high-yield LV production systems are urgently needed. Using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 high-throughput screening, we identified nine critical genes (<i>LDAH, GBP3, BPIFC, NHLRC1, NHLRC3, ZNF425, TTC37, LRRC4B</i>, and <i>SPINK6</i>) from 17,501 genes that limit LV packaging and formation. Knocking out these genes in HEK293T cells significantly increased virus production, with <i>LDAH</i> knockout exhibiting a 6.63-fold increase. Studies on multigene knockouts demonstrated that the cumulative effects of different gene knockouts can significantly enhance lentivirus production in HEK293T cells. Triple knockout of <i>GBP3, BPIFC</i>, and <i>LDAH</i> increased LV titer by ∼8.33-fold, and knockout (or knockdown) of <i>GBP3, NHLRC1,</i> and <i>NHLRC3</i> increased LV titer by ∼6.53-fold. This study established HEK293T cell lines with multiple genes knockout for efficient LV production, providing reliable technical support for LV production and application and offering new perspectives for studying LV packaging mechanisms and related virus research.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":"7 5","pages":"272-282"},"PeriodicalIF":3.7,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142480626","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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
Five Years of Progress in CRISPR Clinical Trials (2019-2024). CRISPR 临床试验的五年进展(2019-2024 年)。
IF 3.7 4区 生物学 Q2 GENETICS & HEREDITY Pub Date : 2024-10-01 DOI: 10.1089/crispr.2024.0081
Kevin Davies, Alex Philippidis, Rodolphe Barrangou

In July 2019, Victoria Gray became the first patient with sickle cell disease to receive a CRISPR-based cell therapy as a volunteer in the exa-cel clinical trial, sponsored by Vertex Pharmaceuticals and CRISPR Therapeutics. Barely four years later, the ensuing therapy, branded as Casgevy, received approval from regulatory agencies in Europe, the United States, and the Middle East, ushering in a new era of CRISPR-based medicines. During this period, scores of other clinical trials have been launched, including many actively recruiting patients across phase 1, phase 2, and phase 3 clinical trials around the world. In this brief Perspective, we collate the latest information on therapeutic clinical trials featuring CRISPR, base and prime editing, across a range of both in vivo and ex vivo gene and cell therapies.

2019 年 7 月,维多利亚-格雷作为志愿者参加了由 Vertex 制药公司和 CRISPR Therapeutics 赞助的 exa-cel 临床试验,成为第一位接受基于 CRISPR 的细胞疗法的镰状细胞病患者。仅仅四年后,这种被命名为 Casgevy 的疗法就获得了欧洲、美国和中东监管机构的批准,开创了 CRISPR 药物的新时代。在此期间,数十项其他临床试验也相继启动,包括许多正在全球招募患者的一期、二期和三期临床试验。在这篇简短的《透视》中,我们整理了以CRISPR、基质和质粒编辑为特色的治疗性临床试验的最新信息,涉及一系列体内和体外基因和细胞疗法。
{"title":"Five Years of Progress in CRISPR Clinical Trials (2019-2024).","authors":"Kevin Davies, Alex Philippidis, Rodolphe Barrangou","doi":"10.1089/crispr.2024.0081","DOIUrl":"10.1089/crispr.2024.0081","url":null,"abstract":"<p><p>In July 2019, Victoria Gray became the first patient with sickle cell disease to receive a CRISPR-based cell therapy as a volunteer in the exa-cel clinical trial, sponsored by Vertex Pharmaceuticals and CRISPR Therapeutics. Barely four years later, the ensuing therapy, branded as Casgevy, received approval from regulatory agencies in Europe, the United States, and the Middle East, ushering in a new era of CRISPR-based medicines. During this period, scores of other clinical trials have been launched, including many actively recruiting patients across phase 1, phase 2, and phase 3 clinical trials around the world. In this brief Perspective, we collate the latest information on therapeutic clinical trials featuring CRISPR, base and prime editing, across a range of both <i>in vivo</i> and <i>ex vivo</i> gene and cell therapies.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":"7 5","pages":"227-230"},"PeriodicalIF":3.7,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142480628","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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 Research Support of Genome Editing Technologies and Transition to Clinical Trials. 基因组编辑技术研究支持的特点以及向临床试验的过渡。
IF 3.7 4区 生物学 Q2 GENETICS & HEREDITY Pub Date : 2024-10-01 Epub Date: 2024-09-26 DOI: 10.1089/crispr.2024.0011
Riya Mohan, Susanne B Haga

Genome editing technologies have become widely used research tools. To assess the rate of growth with respect to federal funding of gene editing projects, we analyzed publicly available data retrieved from the NIH RePORTER and Clinicaltrials.gov databases. We identified 6,111 awards between 1977 and 2023, the majority being extramural, investigator-driven R (noneducational) awards (66.7%). There was an average growth rate of 40% between 2008 and 2022, and the biggest increase in awards was observed between 2017 and 2018 (doubling from 140 to 280). Five administering institutes/centers accounted for more than 60% of awards with the highest number of awards from the National Cancer Institute (20.0%). The majority of clinical trials involving some type of genome editing (75%) started in or after 2020. This analysis illuminates the rapid and widespread growth of gene editing research across disciplines and the eventual launch of clinical trials using gene editing tools.

基因组编辑技术已成为广泛应用的研究工具。为了评估基因编辑项目的联邦资助增长率,我们分析了从美国国立卫生研究院 RePORTER 和 Clinicaltrials.gov 数据库中检索到的公开数据。我们确定了 1977 年至 2023 年间的 6111 项奖励,其中大部分是校外、研究者驱动的 R(非教育)奖励(66.7%)。2008年至2022年期间的平均增长率为40%,2017年至2018年期间的奖项增幅最大(从140项增加到280项,翻了一番)。五个管理机构/中心的获奖数量占 60% 以上,其中国家癌症研究所的获奖数量最多(20.0%)。大多数涉及某种基因组编辑的临床试验(75%)都是在2020年或之后开始的。这项分析说明了基因编辑研究在各学科中的快速和广泛发展,以及使用基因编辑工具的临床试验的最终启动。
{"title":"Characterization of Research Support of Genome Editing Technologies and Transition to Clinical Trials.","authors":"Riya Mohan, Susanne B Haga","doi":"10.1089/crispr.2024.0011","DOIUrl":"10.1089/crispr.2024.0011","url":null,"abstract":"<p><p>Genome editing technologies have become widely used research tools. To assess the rate of growth with respect to federal funding of gene editing projects, we analyzed publicly available data retrieved from the NIH RePORTER and Clinicaltrials.gov databases. We identified 6,111 awards between 1977 and 2023, the majority being extramural, investigator-driven R (noneducational) awards (66.7%). There was an average growth rate of 40% between 2008 and 2022, and the biggest increase in awards was observed between 2017 and 2018 (doubling from 140 to 280). Five administering institutes/centers accounted for more than 60% of awards with the highest number of awards from the National Cancer Institute (20.0%). The majority of clinical trials involving some type of genome editing (75%) started in or after 2020. This analysis illuminates the rapid and widespread growth of gene editing research across disciplines and the eventual launch of clinical trials using gene editing tools.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"249-257"},"PeriodicalIF":3.7,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142332257","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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
Genome Editing Therapy for the Blood: Ex Vivo Success and In Vivo Prospects. 血液基因组编辑疗法:体内成功与体内前景。
IF 3.7 4区 生物学 Q2 GENETICS & HEREDITY Pub Date : 2024-10-01 Epub Date: 2024-09-26 DOI: 10.1089/crispr.2024.0036
Christy A George, Srishti U Sahu, Lorena de Oñate, Bruno Solano de Freitas Souza, Ross C Wilson

Hematopoietic stem cells (HSCs) provide the body with a continuous supply of healthy, functional blood cells. In patients with hematopoietic malignancies, immunodeficiencies, lysosomal storage disorders, and hemoglobinopathies, therapeutic genome editing offers hope for corrective intervention, with even modest editing efficiencies likely to provide clinical benefit. Engineered white blood cells, such as T cells, can be applied therapeutically to address monogenic disorders of the immune system, HIV infection, or cancer. The versatility of CRISPR-based tools allows countless new medical interventions for diseases of the blood, and rapid ex vivo success has been demonstrated in hemoglobinopathies via transplantation of the patient's HSCs following genome editing in a laboratory setting. Here we review recent advances in therapeutic genome editing of HSCs and T cells, focusing on the progress in ex vivo contexts, the promise of improved access via in vivo delivery, as well as the ongoing preclinical efforts that may enable the transition from ex vivo to in vivo administration. We discuss the challenges, limitations, and future prospects of this rapidly developing field, which may one day establish CRISPR as the standard of care for some diseases affecting the blood.

造血干细胞为人体源源不断地提供健康的功能性血细胞。对于造血恶性肿瘤、免疫缺陷、溶酶体储积症和血红蛋白病患者,治疗性基因组编辑为纠正干预带来了希望,即使编辑效率不高,也可能带来临床益处。经改造的白细胞(如 T 细胞)可用于治疗单基因免疫系统疾病、艾滋病病毒感染或癌症。基于CRISPR技术的工具用途广泛,可为血液疾病提供无数新的医疗干预措施,在实验室环境中进行基因组编辑后,通过移植患者的造血干细胞,已在血红蛋白病方面取得了迅速的体内外成功。在此,我们回顾了造血干细胞和 T 细胞治疗性基因组编辑的最新进展,重点关注体内外治疗的进展、通过体内给药改善治疗的前景,以及为实现从体内外给药到体内给药的过渡而正在进行的临床前工作。我们讨论了这一快速发展领域所面临的挑战、局限性和未来前景,也许有一天 CRISPR 会成为治疗某些血液疾病的标准方法。
{"title":"Genome Editing Therapy for the Blood: <i>Ex Vivo</i> Success and <i>In Vivo</i> Prospects.","authors":"Christy A George, Srishti U Sahu, Lorena de Oñate, Bruno Solano de Freitas Souza, Ross C Wilson","doi":"10.1089/crispr.2024.0036","DOIUrl":"10.1089/crispr.2024.0036","url":null,"abstract":"<p><p>Hematopoietic stem cells (HSCs) provide the body with a continuous supply of healthy, functional blood cells. In patients with hematopoietic malignancies, immunodeficiencies, lysosomal storage disorders, and hemoglobinopathies, therapeutic genome editing offers hope for corrective intervention, with even modest editing efficiencies likely to provide clinical benefit. Engineered white blood cells, such as T cells, can be applied therapeutically to address monogenic disorders of the immune system, HIV infection, or cancer. The versatility of CRISPR-based tools allows countless new medical interventions for diseases of the blood, and rapid <i>ex vivo</i> success has been demonstrated in hemoglobinopathies via transplantation of the patient's HSCs following genome editing in a laboratory setting. Here we review recent advances in therapeutic genome editing of HSCs and T cells, focusing on the progress in <i>ex vivo</i> contexts, the promise of improved access via <i>in vivo</i> delivery, as well as the ongoing preclinical efforts that may enable the transition from <i>ex vivo</i> to <i>in vivo</i> administration. We discuss the challenges, limitations, and future prospects of this rapidly developing field, which may one day establish CRISPR as the standard of care for some diseases affecting the blood.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"231-248"},"PeriodicalIF":3.7,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142332258","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"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
CRISPR Journal
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