{"title":"基于 Crispr 编辑的人类多能干细胞用于疾病建模。","authors":"Yun Chang, Feng Lan, Yongshuai Zhang, Shuhong Ma","doi":"10.1007/s12015-024-10713-7","DOIUrl":null,"url":null,"abstract":"<p><p>The CRISPR system, as an effective genome editing technology, has been extensively utilized for the construction of disease models in human pluripotent stem cells. Establishment of a gene mutant or knockout stem cell line typically relies on Cas nuclease-generated double-stranded DNA breaks and exogenous templates, which can produce uncontrollable editing byproducts and toxicity. The recently developed adenine base editors (ABE) have greatly facilitated related research by introducing A/T > G/C mutations in the coding regions or splitting sites (AG-GT) of genes, enabling mutant gene knock-in or knock-out without introducing DNA breaks. In this study, we edit the AG bases in exons anterior to achieve gene knockout via the ABE8e-SpRY, which recognizes most expanded protospacer adjacent motif to target the genome. Except for gene-knockout, ABE8e-SpRY can also efficiently establish disease-related A/T-to-G/C variation cell lines by targeting coding sequences. The method we generated is simple and time-saving, and it only takes two weeks to obtain the desired cell line. This protocol provides operating instructions step-by-step for constructing knockout and point mutation cell lines.</p>","PeriodicalId":21955,"journal":{"name":"Stem Cell Reviews and Reports","volume":null,"pages":null},"PeriodicalIF":4.5000,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Crispr-Based Editing of Human Pluripotent Stem Cells for Disease Modeling.\",\"authors\":\"Yun Chang, Feng Lan, Yongshuai Zhang, Shuhong Ma\",\"doi\":\"10.1007/s12015-024-10713-7\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>The CRISPR system, as an effective genome editing technology, has been extensively utilized for the construction of disease models in human pluripotent stem cells. Establishment of a gene mutant or knockout stem cell line typically relies on Cas nuclease-generated double-stranded DNA breaks and exogenous templates, which can produce uncontrollable editing byproducts and toxicity. The recently developed adenine base editors (ABE) have greatly facilitated related research by introducing A/T > G/C mutations in the coding regions or splitting sites (AG-GT) of genes, enabling mutant gene knock-in or knock-out without introducing DNA breaks. In this study, we edit the AG bases in exons anterior to achieve gene knockout via the ABE8e-SpRY, which recognizes most expanded protospacer adjacent motif to target the genome. Except for gene-knockout, ABE8e-SpRY can also efficiently establish disease-related A/T-to-G/C variation cell lines by targeting coding sequences. The method we generated is simple and time-saving, and it only takes two weeks to obtain the desired cell line. This protocol provides operating instructions step-by-step for constructing knockout and point mutation cell lines.</p>\",\"PeriodicalId\":21955,\"journal\":{\"name\":\"Stem Cell Reviews and Reports\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":4.5000,\"publicationDate\":\"2024-07-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Stem Cell Reviews and Reports\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.1007/s12015-024-10713-7\",\"RegionNum\":3,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2024/4/2 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q2\",\"JCRName\":\"CELL & TISSUE ENGINEERING\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Stem Cell Reviews and Reports","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1007/s12015-024-10713-7","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/4/2 0:00:00","PubModel":"Epub","JCR":"Q2","JCRName":"CELL & TISSUE ENGINEERING","Score":null,"Total":0}
引用次数: 0
摘要
CRISPR系统作为一种有效的基因组编辑技术,已被广泛用于在人类多能干细胞中构建疾病模型。基因突变或基因敲除干细胞系的建立通常依赖于Cas核酸酶产生的双链DNA断裂和外源模板,这可能产生不可控的编辑副产品和毒性。最近开发的腺嘌呤碱基编辑器(ABE)在基因的编码区或分裂位点(AG-GT)引入 A/T > G/C 突变,在不引入 DNA 断裂的情况下实现突变基因的敲入或敲出,极大地促进了相关研究。在这项研究中,我们编辑了外显子前部的 AG 碱基,通过 ABE8e-SpRY 实现基因敲除,ABE8e-SpRY 可识别大多数扩展的原位相邻基序,从而靶向基因组。除基因敲除外,ABE8e-SpRY 还能通过靶向编码序列有效建立与疾病相关的 A/T-to-G/C 变异细胞系。我们采用的方法简单省时,只需两周就能获得所需的细胞系。本方案提供了逐步构建基因敲除和点突变细胞系的操作指南。
Crispr-Based Editing of Human Pluripotent Stem Cells for Disease Modeling.
The CRISPR system, as an effective genome editing technology, has been extensively utilized for the construction of disease models in human pluripotent stem cells. Establishment of a gene mutant or knockout stem cell line typically relies on Cas nuclease-generated double-stranded DNA breaks and exogenous templates, which can produce uncontrollable editing byproducts and toxicity. The recently developed adenine base editors (ABE) have greatly facilitated related research by introducing A/T > G/C mutations in the coding regions or splitting sites (AG-GT) of genes, enabling mutant gene knock-in or knock-out without introducing DNA breaks. In this study, we edit the AG bases in exons anterior to achieve gene knockout via the ABE8e-SpRY, which recognizes most expanded protospacer adjacent motif to target the genome. Except for gene-knockout, ABE8e-SpRY can also efficiently establish disease-related A/T-to-G/C variation cell lines by targeting coding sequences. The method we generated is simple and time-saving, and it only takes two weeks to obtain the desired cell line. This protocol provides operating instructions step-by-step for constructing knockout and point mutation cell lines.
期刊介绍:
The purpose of Stem Cell Reviews and Reports is to cover contemporary and emerging areas in stem cell research and regenerative medicine. The journal will consider for publication:
i) solicited or unsolicited reviews of topical areas of stem cell biology that highlight, critique and synthesize recent important findings in the field.
ii) full length and short reports presenting original experimental work.
iii) translational stem cell studies describing results of clinical trials using stem cells as therapeutics.
iv) papers focused on diseases of stem cells.
v) hypothesis and commentary articles as opinion-based pieces in which authors can propose a new theory, interpretation of a controversial area in stem cell biology, or a stem cell biology question or paradigm. These articles contain more speculation than reviews, but they should be based on solid rationale.
vi) protocols as peer-reviewed procedures that provide step-by-step descriptions, outlined in sufficient detail, so that both experts and novices can apply them to their own research.
vii) letters to the editor and correspondence.
In order to facilitate this exchange of scientific information and exciting novel ideas, the journal has created five thematic sections, focusing on:
i) the role of adult stem cells in tissue regeneration;
ii) progress in research on induced pluripotent stem cells, embryonic stem cells and mechanism governing embryogenesis and tissue development;
iii) the role of microenvironment and extracellular microvesicles in directing the fate of stem cells;
iv) mechanisms of stem cell trafficking, stem cell mobilization and homing with special emphasis on hematopoiesis;
v) the role of stem cells in aging processes and cancerogenesis.