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
Siyuan Liu, Yingdi Zhao, Qiqin Mo, Yadong Sun, Hanhui Ma
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引用次数: 0

摘要

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。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Engineering CjCas9 for Efficient Base Editing and Prime Editing.

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.

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来源期刊
CRISPR Journal
CRISPR Journal Biochemistry, Genetics and Molecular Biology-Biotechnology
CiteScore
6.30
自引率
2.70%
发文量
76
期刊介绍: In recognition of this extraordinary scientific and technological era, Mary Ann Liebert, Inc., publishers recently announced the creation of The CRISPR Journal -- an international, multidisciplinary peer-reviewed journal publishing outstanding research on the myriad applications and underlying technology of CRISPR. Debuting in 2018, The CRISPR Journal will be published online and in print with flexible open access options, providing a high-profile venue for groundbreaking research, as well as lively and provocative commentary, analysis, and debate. The CRISPR Journal adds an exciting and dynamic component to the Mary Ann Liebert, Inc. portfolio, which includes GEN (Genetic Engineering & Biotechnology News) and more than 80 leading peer-reviewed journals.
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