Röbbe Wünschiers, Robert Maximilian Leidenfrost, Hauke Holtorf, Bernd Dittrich, Thomas Dürr, Jürgen Braun
{"title":"在课堂上使用质粒 pBR322 进行 CRISPR/Cas9 基因打靶和纳米孔 DNA 测序。","authors":"Röbbe Wünschiers, Robert Maximilian Leidenfrost, Hauke Holtorf, Bernd Dittrich, Thomas Dürr, Jürgen Braun","doi":"10.1128/jmbe.00187-23","DOIUrl":null,"url":null,"abstract":"<p><p>Both nanopore-based DNA sequencing and CRISPR/Cas-based gene editing represent groundbreaking innovations in molecular biology and genomics, offering unprecedented insights into and tools for working with genetic information. For students, reading, editing, and even writing DNA will be part of their everyday life. We have developed a laboratory procedure that includes (i) the biosynthesis of a guide RNA for, (ii) targeting Cas9 to specifically linearize the pBR322 plasmid, and (iii) the identification of the cutting site through nanopore DNA sequencing. The protocol is intentionally kept simple and requires neither living organisms nor biosafety laboratories. We divided the experimental procedures into separate activities to facilitate customization. Assuming access to a well-equipped molecular biology laboratory, an initial investment of approximately $2,700 is necessary. The material costs for each experiment group amount to around $130. Furthermore, we have developed a freely accessible website (https://dnalesen.hs-mittweida.de) for sequence read analysis and visualization, lowering the required computational skills to a minimum. For those with strong computational skills, we provide instructions for terminal-based data processing. With the presented activities, we aim to provide a hands-on experiment that engages students in modern molecular genetics and motivates them to discuss potential implications. The complete experiment can be accomplished within half a day and has been successfully implemented by us at high schools, in teacher training, and at universities. Our tip is to combine CRISPR/Cas gene targeting with nanopore-based DNA sequencing. As a tool, we provide a website that facilitates sequence data analysis and visualization.</p>","PeriodicalId":46416,"journal":{"name":"Journal of Microbiology & Biology Education","volume":null,"pages":null},"PeriodicalIF":1.6000,"publicationDate":"2024-08-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11360410/pdf/","citationCount":"0","resultStr":"{\"title\":\"CRISPR/Cas9 gene targeting plus nanopore DNA sequencing with the plasmid pBR322 in the classroom.\",\"authors\":\"Röbbe Wünschiers, Robert Maximilian Leidenfrost, Hauke Holtorf, Bernd Dittrich, Thomas Dürr, Jürgen Braun\",\"doi\":\"10.1128/jmbe.00187-23\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Both nanopore-based DNA sequencing and CRISPR/Cas-based gene editing represent groundbreaking innovations in molecular biology and genomics, offering unprecedented insights into and tools for working with genetic information. For students, reading, editing, and even writing DNA will be part of their everyday life. We have developed a laboratory procedure that includes (i) the biosynthesis of a guide RNA for, (ii) targeting Cas9 to specifically linearize the pBR322 plasmid, and (iii) the identification of the cutting site through nanopore DNA sequencing. The protocol is intentionally kept simple and requires neither living organisms nor biosafety laboratories. We divided the experimental procedures into separate activities to facilitate customization. Assuming access to a well-equipped molecular biology laboratory, an initial investment of approximately $2,700 is necessary. The material costs for each experiment group amount to around $130. Furthermore, we have developed a freely accessible website (https://dnalesen.hs-mittweida.de) for sequence read analysis and visualization, lowering the required computational skills to a minimum. For those with strong computational skills, we provide instructions for terminal-based data processing. With the presented activities, we aim to provide a hands-on experiment that engages students in modern molecular genetics and motivates them to discuss potential implications. The complete experiment can be accomplished within half a day and has been successfully implemented by us at high schools, in teacher training, and at universities. Our tip is to combine CRISPR/Cas gene targeting with nanopore-based DNA sequencing. As a tool, we provide a website that facilitates sequence data analysis and visualization.</p>\",\"PeriodicalId\":46416,\"journal\":{\"name\":\"Journal of Microbiology & Biology Education\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":1.6000,\"publicationDate\":\"2024-08-29\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11360410/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of Microbiology & Biology Education\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.1128/jmbe.00187-23\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2024/5/10 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q2\",\"JCRName\":\"EDUCATION, SCIENTIFIC DISCIPLINES\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Microbiology & Biology Education","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1128/jmbe.00187-23","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/5/10 0:00:00","PubModel":"Epub","JCR":"Q2","JCRName":"EDUCATION, SCIENTIFIC DISCIPLINES","Score":null,"Total":0}
引用次数: 0
摘要
基于纳米孔的 DNA 测序和基于 CRISPR/Cas 的基因编辑都是分子生物学和基因组学领域的突破性创新,提供了前所未有的洞察力和处理遗传信息的工具。对于学生来说,阅读、编辑甚至书写 DNA 将成为他们日常生活的一部分。我们开发了一套实验室程序,其中包括:(i) 引导 RNA 的生物合成;(ii) 以 Cas9 为靶标对 pBR322 质粒进行特异性线性化;(iii) 通过纳米孔 DNA 测序确定切割位点。该方案有意保持简单,既不需要生物体,也不需要生物安全实验室。我们将实验程序分为不同的活动,以方便定制。假设有一个设备齐全的分子生物学实验室,初始投资大约需要 2,700 美元。每个实验组的材料成本约为 130 美元。此外,我们还开发了一个可免费访问的网站(https://dnalesen.hs-mittweida.de),用于序列读数分析和可视化,从而将所需的计算技能降至最低。对于计算能力较强的人,我们提供了基于终端的数据处理说明。通过所介绍的活动,我们旨在提供一个动手实验,让学生参与现代分子遗传学,并激发他们讨论潜在的影响。整个实验可在半天内完成,我们已在高中、教师培训和大学成功实施了该实验。我们的秘诀是将 CRISPR/Cas 基因打靶与基于纳米孔的 DNA 测序相结合。作为一种工具,我们提供了一个便于序列数据分析和可视化的网站。
CRISPR/Cas9 gene targeting plus nanopore DNA sequencing with the plasmid pBR322 in the classroom.
Both nanopore-based DNA sequencing and CRISPR/Cas-based gene editing represent groundbreaking innovations in molecular biology and genomics, offering unprecedented insights into and tools for working with genetic information. For students, reading, editing, and even writing DNA will be part of their everyday life. We have developed a laboratory procedure that includes (i) the biosynthesis of a guide RNA for, (ii) targeting Cas9 to specifically linearize the pBR322 plasmid, and (iii) the identification of the cutting site through nanopore DNA sequencing. The protocol is intentionally kept simple and requires neither living organisms nor biosafety laboratories. We divided the experimental procedures into separate activities to facilitate customization. Assuming access to a well-equipped molecular biology laboratory, an initial investment of approximately $2,700 is necessary. The material costs for each experiment group amount to around $130. Furthermore, we have developed a freely accessible website (https://dnalesen.hs-mittweida.de) for sequence read analysis and visualization, lowering the required computational skills to a minimum. For those with strong computational skills, we provide instructions for terminal-based data processing. With the presented activities, we aim to provide a hands-on experiment that engages students in modern molecular genetics and motivates them to discuss potential implications. The complete experiment can be accomplished within half a day and has been successfully implemented by us at high schools, in teacher training, and at universities. Our tip is to combine CRISPR/Cas gene targeting with nanopore-based DNA sequencing. As a tool, we provide a website that facilitates sequence data analysis and visualization.