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使用真正的 Cas9 切口酶的 Prime 编辑最小化了不必要的插入缺失。

Prime editing with genuine Cas9 nickases minimizes unwanted indels.

机构信息

Center for Genome Engineering, Institute for Basic Science, Daejeon, Republic of Korea.

Department of Chemistry, Seoul National University, Seoul, Republic of Korea.

出版信息

Nat Commun. 2023 Mar 30;14(1):1786. doi: 10.1038/s41467-023-37507-8.


DOI:10.1038/s41467-023-37507-8
PMID:36997524
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10063541/
Abstract

Unlike CRISPR-Cas9 nucleases, which yield DNA double-strand breaks (DSBs), Cas9 nickases (nCas9s), which are created by replacing key catalytic amino-acid residues in one of the two nuclease domains of S. pyogenesis Cas9 (SpCas9), produce nicks or single-strand breaks. Two SpCas9 variants, namely, nCas9 (D10A) and nCas9 (H840A), which cleave target (guide RNA-pairing) and non-target DNA strands, respectively, are widely used for various purposes, including paired nicking, homology-directed repair, base editing, and prime editing. In an effort to define the off-target nicks caused by these nickases, we perform Digenome-seq, a method based on whole genome sequencing of genomic DNA treated with a nuclease or nickase of interest, and find that nCas9 (H840A) but not nCas9 (D10A) can cleave both strands, producing unwanted DSBs, albeit less efficiently than wild-type Cas9. To inactivate the HNH nuclease domain further, we incorporate additional mutations into nCas9 (H840A). Double-mutant nCas9 (H840A + N863A) does not exhibit the DSB-inducing behavior in vitro and, either alone or in fusion with the M-MLV reverse transcriptase (prime editor, PE2 or PE3), induces a lower frequency of unwanted indels, compared to nCas9 (H840A), caused by error-prone repair of DSBs. When incorporated into prime editor and used with engineered pegRNAs (ePE3), we find that the nCas9 variant (H840A + N854A) dramatically increases the frequency of correct edits, but not unwanted indels, yielding the highest purity of editing outcomes compared to nCas9 (H840A).

摘要

与产生 DNA 双链断裂 (DSBs) 的 CRISPR-Cas9 核酸酶不同,酿脓链球菌 Cas9 (SpCas9) 的两个核酸酶结构域之一中的关键催化氨基酸残基被替换后产生的 Cas9 切口酶 (nCas9s) 会产生切口或单链断裂。两种 SpCas9 变体,即分别切割靶标 (向导 RNA 配对) 和非靶标 DNA 链的 nCas9 (D10A) 和 nCas9 (H840A),被广泛用于各种目的,包括配对切口、同源定向修复、碱基编辑和先导编辑。为了确定这些切口酶引起的脱靶切口,我们进行了 Digenome-seq,这是一种基于感兴趣的核酸酶或切口酶处理的基因组 DNA 的全基因组测序的方法,发现 nCas9 (H840A) 但不是 nCas9 (D10A) 可以切割两条链,产生不需要的 DSBs,尽管效率低于野生型 Cas9。为了进一步使 HNH 核酸酶结构域失活,我们将额外的突变引入 nCas9 (H840A)。双突变体 nCas9 (H840A + N863A) 既不在体外表现出 DSB 诱导行为,也没有在与 M-MLV 逆转录酶 (prime editor,PE2 或 PE3) 融合时,与 nCas9 (H840A) 相比,由于 DSB 易错修复而导致不需要的插入缺失的频率更低。当整合到 prime editor 中并与工程化 pegRNA (ePE3) 一起使用时,我们发现 nCas9 变体 (H840A + N854A) 显著增加了正确编辑的频率,但不会增加不需要的插入缺失,与 nCas9 (H840A) 相比,编辑结果的纯度更高。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f88a/10063541/315f93e19186/41467_2023_37507_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f88a/10063541/9c14300b4eee/41467_2023_37507_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f88a/10063541/e542fda904fd/41467_2023_37507_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f88a/10063541/76955f843a9d/41467_2023_37507_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f88a/10063541/27a8480f15f4/41467_2023_37507_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f88a/10063541/315f93e19186/41467_2023_37507_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f88a/10063541/9c14300b4eee/41467_2023_37507_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f88a/10063541/e542fda904fd/41467_2023_37507_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f88a/10063541/76955f843a9d/41467_2023_37507_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f88a/10063541/27a8480f15f4/41467_2023_37507_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f88a/10063541/315f93e19186/41467_2023_37507_Fig5_HTML.jpg

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引用本文的文献

[1]
An integrated enzymatic and computational pipeline for quantifying off-target base-editing.

bioRxiv. 2025-8-26

[2]
Advances in large-scale DNA engineering with the CRISPR system.

Exp Mol Med. 2025-9-1

[3]
The Interface of Gene Editing with Regenerative Medicine.

Engineering (Beijing). 2025-3

[4]
Emerging trends in prime editing for precision genome editing.

Exp Mol Med. 2025-7

[5]
Programmable DNA aptamer logic gates: from structural design to integrated systems for intelligent nanoscale biosensors.

Anal Bioanal Chem. 2025-7-10

[6]
High-Fidelity, One-Pot Nucleic Acid Amplification via OMEGA IsrB Nickase Cycling for Clinical Pathogen Detection.

JACS Au. 2025-6-9

[7]
CRISPR/Cas9-mediated editing of jasmonic acid pathways to enhance biotic & abiotic stress tolerance: An overview & prospects.

Funct Integr Genomics. 2025-6-10

[8]
Prime editor with rational design and AI-driven optimization for reverse editing window and enhanced fidelity.

Nat Commun. 2025-6-3

[9]
Circular RNA-mediated inverse prime editing in human cells.

Nat Commun. 2025-5-31

[10]
Advances in CRISPR/Cas-Based Strategies on Zoonosis.

Transbound Emerg Dis. 2023-8-3

本文引用的文献

[1]
Highly efficient prime editing by introducing same-sense mutations in pegRNA or stabilizing its structure.

Nat Commun. 2022-3-29

[2]
Comprehensive analysis of prime editing outcomes in human embryonic stem cells.

Nucleic Acids Res. 2022-1-25

[3]
Engineered pegRNAs improve prime editing efficiency.

Nat Biotechnol. 2022-3

[4]
Comprehensive deletion landscape of CRISPR-Cas9 identifies minimal RNA-guided DNA-binding modules.

Nat Commun. 2021-9-27

[5]
Chromothripsis as an on-target consequence of CRISPR-Cas9 genome editing.

Nat Genet. 2021-6

[6]
Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice.

Nat Commun. 2021-4-9

[7]
Allele-Specific Chromosome Removal after Cas9 Cleavage in Human Embryos.

Cell. 2020-12-10

[8]
Unbiased investigation of specificities of prime editing systems in human cells.

Nucleic Acids Res. 2020-10-9

[9]
Genome-wide specificity of dCpf1 cytidine base editors.

Nat Commun. 2020-8-13

[10]
Search-and-replace genome editing without double-strand breaks or donor DNA.

Nature. 2019-10-21

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