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CRISPR-Cas9 基因编辑可诱导造血细胞内中靶基因座的大片段拷贝数中性缺失。

CRISPR-Cas9 globin editing can induce megabase-scale copy-neutral losses of heterozygosity in hematopoietic cells.

机构信息

Bordeaux University, Bordeaux, France.

INSERM U1035, Biotherapy of Genetic Diseases, Inflammatory disorders and Cancers, Bordeaux, France.

出版信息

Nat Commun. 2021 Aug 13;12(1):4922. doi: 10.1038/s41467-021-25190-6.

DOI:10.1038/s41467-021-25190-6
PMID:34389729
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC8363739/
Abstract

CRISPR-Cas9 is a promising technology for gene therapy. However, the ON-target genotoxicity of CRISPR-Cas9 nuclease due to DNA double-strand breaks has received little attention and is probably underestimated. Here we report that genome editing targeting globin genes induces megabase-scale losses of heterozygosity (LOH) from the globin CRISPR-Cas9 cut-site to the telomere (5.2 Mb). In established lines, CRISPR-Cas9 nuclease induces frequent terminal chromosome 11p truncations and rare copy-neutral LOH. In primary hematopoietic progenitor/stem cells, we detect 1.1% of clones (7/648) with acquired megabase LOH induced by CRISPR-Cas9. In-depth analysis by SNP-array reveals the presence of copy-neutral LOH. This leads to 11p15.5 partial uniparental disomy, comprising two Chr11p15.5 imprinting centers (H19/IGF2:IG-DMR/IC1 and KCNQ1OT1:TSS-DMR/IC2) and impacting H19 and IGF2 expression. While this genotoxicity is a safety concern for CRISPR clinical trials, it is also an opportunity to model copy-neutral-LOH for genetic diseases and cancers.

摘要

CRISPR-Cas9 是一种很有前途的基因治疗技术。然而，CRISPR-Cas9 核酸酶由于 DNA 双链断裂而导致的靶向 ON 基因的遗传毒性却很少受到关注，而且可能被低估了。在这里，我们报告说，针对珠蛋白基因的基因组编辑会导致从珠蛋白 CRISPR-Cas9 切割位点到端粒（5.2 Mb）的大片段杂合性丢失（LOH）。在已建立的细胞系中，CRISPR-Cas9 核酸酶诱导频繁的 11p 端染色体 11p 截断和罕见的拷贝中性 LOH。在原代造血祖细胞/干细胞中，我们检测到 7/648（1.1%）个克隆通过 CRISPR-Cas9 获得了大片段 LOH。通过 SNP 芯片进行的深入分析揭示了存在拷贝中性 LOH。这导致了 11p15.5 部分单亲二体性，包含两个 Chr11p15.5 印迹中心（H19/IGF2：IG-DMR/IC1 和 KCNQ1OT1：TSS-DMR/IC2），并影响 H19 和 IGF2 的表达。虽然这种遗传毒性是 CRISPR 临床试验的一个安全隐患，但它也是一种针对遗传疾病和癌症的模型拷贝中性 LOH 的机会。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/75da/8363739/cbe9b6f78724/41467_2021_25190_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/75da/8363739/c5dba47cda76/41467_2021_25190_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/75da/8363739/75974c0113ba/41467_2021_25190_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/75da/8363739/fd0e9db98131/41467_2021_25190_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/75da/8363739/5d6c5d7f35ed/41467_2021_25190_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/75da/8363739/cbe9b6f78724/41467_2021_25190_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/75da/8363739/c5dba47cda76/41467_2021_25190_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/75da/8363739/75974c0113ba/41467_2021_25190_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/75da/8363739/fd0e9db98131/41467_2021_25190_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/75da/8363739/5d6c5d7f35ed/41467_2021_25190_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/75da/8363739/cbe9b6f78724/41467_2021_25190_Fig5_HTML.jpg

相似文献

[1]
CRISPR-Cas9 globin editing can induce megabase-scale copy-neutral losses of heterozygosity in hematopoietic cells.

Nat Commun. 2021-8-13

[2]
CRISPR-Cas9 Editing Induces Loss of Heterozygosity in the Pathogenic Yeast Candida parapsilosis.

mSphere. 2022-12-21

[3]
Paternal 132 bp deletion affecting in 11p15.5 is associated with growth retardation but does not affect imprinting.

J Med Genet. 2021-3

[4]
Reactivation of γ-globin in adult β-YAC mice after ex vivo and in vivo hematopoietic stem cell genome editing.

Blood. 2018-5-22

[5]
Probing gene function in wild-type strains by Cas9-facilitated one-step integration of two dominant selection markers: a systematic analysis of recombination events at the target locus.

mSphere. 2024-7-30

[6]
Cell cycle arrest and p53 prevent ON-target megabase-scale rearrangements induced by CRISPR-Cas9.

Nat Commun. 2023-7-10

[7]
Genomic profiling maps loss of heterozygosity and defines the timing and stage dependence of epigenetic and genetic events in Wilms' tumors.

Mol Cancer Res. 2005-9

[8]
ON-Target Adverse Events of CRISPR-Cas9 Nuclease: More Chaotic than Expected.

CRISPR J. 2022-2

[9]
Association of chromosome arm 16q loss with loss of imprinting of insulin-like growth factor-II in Wilms tumor.

Genes Chromosomes Cancer. 2005-6

[10]
CRISPR Gene Editing of Murine Blood Stem and Progenitor Cells Induces MLL-AF9 Chromosomal Translocation and MLL-AF9 Leukaemogenesis.

Int J Mol Sci. 2020-6-15

引用本文的文献

[1]
Precise, predictable genome integrations by deep-learning-assisted design of microhomology-based templates.

Nat Biotechnol. 2025-8-12

[2]
Off-target effects in CRISPR-Cas genome editing for human therapeutics: Progress and challenges.

Mol Ther Nucleic Acids. 2025-7-17

[3]
Targeting of acute myeloid leukemia by five-gene engineered T cells expressing transgenic T-cell receptor specific to WT1, chimeric antigenic receptor specific to GM-CSF receptor, bispecific T-cell engager specific to CD33, and tEGFR suicide gene system.

Immunother Adv. 2025-6-11

[4]
Dissecting the epigenetic regulation of the fetal hemoglobin genes to unravel a novel therapeutic approach for β-hemoglobinopathies.

Nucleic Acids Res. 2025-7-8

[5]
Durable silencing using non-evolved dCas9 epigenome editors in patient-derived cells.

Mol Ther Nucleic Acids. 2025-5-14

[6]
Efficient GBA1 editing via HDR with ssODNs by outcompeting pseudogene-mediated gene conversion upon CRISPR/Cas9 cleavage.

Front Genome Ed. 2025-4-30

[7]
Gene editing without ex vivo culture evades genotoxicity in human hematopoietic stem cells.

Cell Stem Cell. 2025-2-6

[8]
Genome editing with the HDR-enhancing DNA-PKcs inhibitor AZD7648 causes large-scale genomic alterations.

Nat Biotechnol. 2024-11-27

[9]
Adenine base editors induce off-target structure variations in mouse embryos and primary human T cells.

Genome Biol. 2024-11-11

[10]
Pythia: Non-random DNA repair allows predictable CRISPR/Cas9 integration and gene editing.

bioRxiv. 2024-9-23

本文引用的文献

[1]
Frequent loss of heterozygosity in CRISPR-Cas9-edited early human embryos.

Proc Natl Acad Sci U S A. 2021-6-1

[2]
Chromothripsis as an on-target consequence of CRISPR-Cas9 genome editing.

Nat Genet. 2021-6

[3]
Mechanisms restraining break-induced replication at two-ended DNA double-strand breaks.

EMBO J. 2021-5-17

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

Cell. 2020-12-10

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POLQ suppresses interhomolog recombination and loss of heterozygosity at targeted DNA breaks.

Proc Natl Acad Sci U S A. 2020-9-1

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Mutation-Specific Guide RNA for Compound Heterozygous Porphyria On-target Scarless Correction by CRISPR/Cas9 in Stem Cells.

Stem Cell Reports. 2020-9-8

[7]
Author Correction: Cas9 activates the p53 pathway and selects for p53-inactivating mutations.

Nat Genet. 2020-7

[8]
Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors.

Nat Biotechnol. 2020-6-22

[9]
Detection of Deleterious On-Target Effects after HDR-Mediated CRISPR Editing.

Cell Rep. 2020-5-26

[10]
Unintended on-target chromosomal instability following CRISPR/Cas9 single gene targeting.

Ann Oncol. 2020-9

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