转染 CRISPR/Cas9 质粒 DNA 的多肽树枝状聚合物:优化与机制

IF 4.2 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
Susanna Zamolo, Elena Zakharova, Lise Boursinhac, Florian Hollfelder, Tamis Darbre and Jean-Louis Reymond
{"title":"转染 CRISPR/Cas9 质粒 DNA 的多肽树枝状聚合物:优化与机制","authors":"Susanna Zamolo, Elena Zakharova, Lise Boursinhac, Florian Hollfelder, Tamis Darbre and Jean-Louis Reymond","doi":"10.1039/D4CB00116H","DOIUrl":null,"url":null,"abstract":"<p >Gene editing by CRISPR/Cas9 offers great therapeutic opportunities but requires delivering large plasmid DNA (pDNA) into cells, a task for which transfection reagents are better suited than viral vectors. Here we performed a structure–activity relationship study of <strong>Z22</strong>, a <small>D</small>-enantiomeric, arginine containing, lipidated peptide dendrimer developed for pDNA transfection of a CRISPR/Cas9 plasmid co-expressing GFP. While all dendrimer analogs tested bound pDNA strongly and internalized their cargo into cells, <small>D</small>-chirality proved essential for transfection by avoiding proteolysis of the dendrimer structure required for endosome escape and possibly crossing of the nuclear envelope. Furthermore, a cysteine residue at the core of <strong>Z22</strong> proved non-essential and was removed to yield the more active analog <strong>Z34</strong>. This dendrimer shows &gt;83% GFP transfection efficiency in HEK cells with no detrimental effect on cell viability and promotes functional CRISPR/Cas9 mediated gene editing. It is accessible by solid-phase peptide synthesis and therefore attractive for further development.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":null,"pages":null},"PeriodicalIF":4.2000,"publicationDate":"2024-07-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.rsc.org/en/content/articlepdf/2024/cb/d4cb00116h?page=search","citationCount":"0","resultStr":"{\"title\":\"Peptide dendrimers transfecting CRISPR/Cas9 plasmid DNA: optimization and mechanism†\",\"authors\":\"Susanna Zamolo, Elena Zakharova, Lise Boursinhac, Florian Hollfelder, Tamis Darbre and Jean-Louis Reymond\",\"doi\":\"10.1039/D4CB00116H\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p >Gene editing by CRISPR/Cas9 offers great therapeutic opportunities but requires delivering large plasmid DNA (pDNA) into cells, a task for which transfection reagents are better suited than viral vectors. Here we performed a structure–activity relationship study of <strong>Z22</strong>, a <small>D</small>-enantiomeric, arginine containing, lipidated peptide dendrimer developed for pDNA transfection of a CRISPR/Cas9 plasmid co-expressing GFP. While all dendrimer analogs tested bound pDNA strongly and internalized their cargo into cells, <small>D</small>-chirality proved essential for transfection by avoiding proteolysis of the dendrimer structure required for endosome escape and possibly crossing of the nuclear envelope. Furthermore, a cysteine residue at the core of <strong>Z22</strong> proved non-essential and was removed to yield the more active analog <strong>Z34</strong>. This dendrimer shows &gt;83% GFP transfection efficiency in HEK cells with no detrimental effect on cell viability and promotes functional CRISPR/Cas9 mediated gene editing. It is accessible by solid-phase peptide synthesis and therefore attractive for further development.</p>\",\"PeriodicalId\":40691,\"journal\":{\"name\":\"RSC Chemical Biology\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":4.2000,\"publicationDate\":\"2024-07-29\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://pubs.rsc.org/en/content/articlepdf/2024/cb/d4cb00116h?page=search\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"RSC Chemical Biology\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://pubs.rsc.org/en/content/articlelanding/2024/cb/d4cb00116h\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"RSC Chemical Biology","FirstCategoryId":"1085","ListUrlMain":"https://pubs.rsc.org/en/content/articlelanding/2024/cb/d4cb00116h","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0

摘要

CRISPR/Cas9 基因编辑技术提供了很好的治疗机会,但需要将大型质粒 DNA(pDNA)转入细胞,而转染试剂比病毒载体更适合这项任务。在这里,我们对 Z22 进行了结构-活性关系研究,Z22 是一种 D-对映体、含精氨酸、脂化肽树枝状聚合物,开发用于 CRISPR/Cas9 质粒共表达 GFP 的 pDNA 转染。虽然所有测试过的树枝状聚合物类似物都能与 pDNA 紧密结合,并将货物内化到细胞中,但 D 手性被证明是成功转染的关键,因为它能避免树枝状聚合物结构被蛋白酶降解,而这种降解是内质体逃逸和可能穿过核膜所必需的。此外,Z22 核心的一个半胱氨酸残基被证明是非必需的,因此被移除以产生活性更强的类似物 Z34,该类似物已被证明能够实现 CRISPR/Cas9 介导的功能性基因编辑。多肽树枝状聚合物很容易通过固相多肽合成获得,而且其序列可以针对特定应用进行微调,因此是有吸引力进一步开发的试剂。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Peptide dendrimers transfecting CRISPR/Cas9 plasmid DNA: optimization and mechanism†

Peptide dendrimers transfecting CRISPR/Cas9 plasmid DNA: optimization and mechanism†

Gene editing by CRISPR/Cas9 offers great therapeutic opportunities but requires delivering large plasmid DNA (pDNA) into cells, a task for which transfection reagents are better suited than viral vectors. Here we performed a structure–activity relationship study of Z22, a D-enantiomeric, arginine containing, lipidated peptide dendrimer developed for pDNA transfection of a CRISPR/Cas9 plasmid co-expressing GFP. While all dendrimer analogs tested bound pDNA strongly and internalized their cargo into cells, D-chirality proved essential for transfection by avoiding proteolysis of the dendrimer structure required for endosome escape and possibly crossing of the nuclear envelope. Furthermore, a cysteine residue at the core of Z22 proved non-essential and was removed to yield the more active analog Z34. This dendrimer shows >83% GFP transfection efficiency in HEK cells with no detrimental effect on cell viability and promotes functional CRISPR/Cas9 mediated gene editing. It is accessible by solid-phase peptide synthesis and therefore attractive for further development.

求助全文
通过发布文献求助,成功后即可免费获取论文全文。 去求助
来源期刊
CiteScore
6.10
自引率
0.00%
发文量
128
审稿时长
10 weeks
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
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
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
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 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术官方微信