期刊文献

Prime editing in mice with an engineered pegRNA 收藏

带有工程的pegrna的老鼠的主要编辑

原文求助发布源

作者

Joseph M. Miano [a] [1]

作者单位

[a]aVascular Biology Center, Medical College of Georgia at Augusta University, Augusta, GA 30912, United States of AmericaVascular Biology Center and 2Department of Pharmacology and Toxicology, Medical College of Georgia at Augusta University, Augusta, Georgia 30912[b]bDepartment of Pharmacology and Toxicology, Medical College of Georgia at Augusta University, Augusta, GA 30912, United States of AmericaDepartment of Pharmacology and Toxicology, Medical College of Georgia at Augusta University, Augusta, Georgia 30912, United States of America[1]1Given his role as Editor in Chief, Joseph Miano had no involvement in the peer review of this article and has no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to Rosalinda Madonna.

发布日期

1 March 2024

页码

107269

DOI

10.1016/j.vph.2023.107269

来源信息

Vascular Pharmacology ISSN：1537-1891, 2024年, 154卷, 107269页

摘要

CRISPR editing involves double-strand breaks in DNA with attending insertions/deletions (indels) that may result in embryonic lethality in mice. The prime editing (PE) platform uses a prime editing guide RNA (pegRNA) and a Cas9 nickase fused to a modified reverse transcriptase to precisely introduce nucleotide substitutions or small indels without the unintended editing associated with DNA double-strand breaks. Recently, engineered pegRNAs (epegRNAs), with a 3′-extension that shields the primer-binding site of the pegRNA from nucleolytic attack, demonstrated superior activity over conventional pegRNAs in cultured cells. Here, we show the inability of three-component CRISPR or conventional PE to incorporate a nonsynonymous substitution in the Capn2 gene, expected to disrupt a phosphorylation site (S50A) in CAPN2. In contrast, an epegRNA with the same protospacer correctly installed the desired edit in two founder mice, as evidenced by robust genotyping assays for the detection of subtle nucleotide substitutions. Long-read sequencing demonstrated sequence fidelity around the edited site as well as top-ranked distal off-target sites. Western blotting and histological analysis of lipopolysaccharide-treated lung tissue revealed a decrease in phosphorylation of CAPN2 and notable alleviation of inflammation, respectively. These results demonstrate the first successful use of an epegRNA for germline transmission in an animal model and provide a solution to targeting essential developmental genes that otherwise may be challenging to edit.

摘要译文

CRISPR编辑涉及DNA中的双链断裂，并与参加插入/缺失（Indels）可能导致小鼠胚胎致死性。Prime Editing（PE）平台使用Prime编辑指南RNA（PEGRNA）和CAS9 NICKASE融合到改良的逆转录酶中，以精确引入核苷酸取代或小indels，而无需与DNA双链断裂有关的意外编辑。最近，具有3'-延伸的工程性pegrnas（Epegrnas），它屏蔽了pegrna的引物结合位点免受核溶解的攻击，在培养细胞中表现出优于常规PEGRNA的优质活性。在这里，我们表明三成分CRISPR或常规PE无法在CAPN2基因中掺入非同义替代，预计将破坏CAPN2中的磷酸化位点（S50A）。相比之下，具有相同原始探针的Epegrna在两只创始人小鼠中正确安装了所需的编辑，这可以通过可靠的基因分型测定法来检测微妙的核苷酸取代。长阅读的测序表明，在编辑的站点以及排名最高的远端离心位置周围表明了序列保真度。脂多糖治疗的肺组织的蛋白质印迹和组织学分析表明，CAPN2的磷酸化分别降低和显着缓解炎症。这些结果表明，在动物模型中首次成功使用Epegrna进行种系传播，并为靶向基本发展基因的解决方案，否则可能会挑战编辑。

Joseph M. Miano [a] [1]. Prime editing in mice with an engineered pegRNA[J]. Vascular Pharmacology, 2024,154: 107269