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通过瞬时表达 CRISPR/Cas9 DNA 或 RNA 在小麦中进行高效且无转基因的基因组编辑。

Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA.

机构信息

State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.

University of Chinese Academy of Sciences, Beijing 100049, China.

出版信息

Nat Commun. 2016 Aug 25;7:12617. doi: 10.1038/ncomms12617.


DOI:10.1038/ncomms12617
PMID:27558837
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC5007326/
Abstract

Editing plant genomes is technically challenging in hard-to-transform plants and usually involves transgenic intermediates, which causes regulatory concerns. Here we report two simple and efficient genome-editing methods in which plants are regenerated from callus cells transiently expressing CRISPR/Cas9 introduced as DNA or RNA. This transient expression-based genome-editing system is highly efficient and specific for producing transgene-free and homozygous wheat mutants in the T0 generation. We demonstrate our protocol to edit genes in hexaploid bread wheat and tetraploid durum wheat, and show that we are able to generate mutants with no detectable transgenes. Our methods may be applicable to other plant species, thus offering the potential to accelerate basic and applied plant genome-engineering research.

摘要

在难以转化的植物中编辑植物基因组在技术上具有挑战性,并且通常涉及转基因中间体,这引起了监管方面的关注。在这里,我们报告了两种简单有效的基因组编辑方法,其中植物是从瞬时表达 CRISPR/Cas9 的愈伤组织细胞再生的,该 CRISPR/Cas9 是作为 DNA 或 RNA 引入的。这种基于瞬时表达的基因组编辑系统在 T0 代产生无转基因和纯合小麦突变体方面非常高效和特异。我们展示了我们在六倍体面包小麦和四倍体硬粒小麦中编辑基因的方案,并表明我们能够生成无可检测转基因的突变体。我们的方法可能适用于其他植物物种,从而有可能加速基础和应用植物基因组工程研究。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/048c/5007326/c8c3b90ef25c/ncomms12617-f4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/048c/5007326/c9ebe2e6c639/ncomms12617-f1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/048c/5007326/aef9a29c0e99/ncomms12617-f2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/048c/5007326/ff4835779417/ncomms12617-f3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/048c/5007326/c8c3b90ef25c/ncomms12617-f4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/048c/5007326/c9ebe2e6c639/ncomms12617-f1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/048c/5007326/aef9a29c0e99/ncomms12617-f2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/048c/5007326/ff4835779417/ncomms12617-f3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/048c/5007326/c8c3b90ef25c/ncomms12617-f4.jpg

相似文献

[1]
Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA.

Nat Commun. 2016-8-25

[2]
CRISPR-based genome editing in wheat: a comprehensive review and future prospects.

Mol Biol Rep. 2019-4-2

[3]
Development of an Agrobacterium-delivered CRISPR/Cas9 system for wheat genome editing.

Plant Biotechnol J. 2019-3-12

[4]
Biolistic-delivery-based transient CRISPR/Cas9 expression enables in planta genome editing in wheat.

Sci Rep. 2018-9-26

[5]
Efficient generation of stable, heritable gene edits in wheat using CRISPR/Cas9.

BMC Plant Biol. 2018-10-3

[6]
Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes.

Nat Commun. 2017-1-18

[7]
Efficient genome editing of Brassica campestris based on the CRISPR/Cas9 system.

Mol Genet Genomics. 2019-5-25

[8]
CRISPR-Cas9 Based Genome Editing in Wheat.

Curr Protoc. 2021-3

[9]
Multiplex CRISPR/Cas9-mediated genome editing to address drought tolerance in wheat.

GM Crops Food. 2025-12

[10]
Genome Editing to Produce Knockout Mutations of Seed Dormancy Genes in Wheat.

Methods Mol Biol. 2024

引用本文的文献

[1]
From convention to innovation: the role of genetic modification and genome editing in Australian wheat breeding.

AoB Plants. 2025-8-7

[2]
Genome analyses and breeding of polyploid crops.

Nat Plants. 2025-8-28

[3]
Advances in genome editing in plants within an evolving regulatory landscape, with a focus on its application in wheat breeding.

J Plant Biochem Biotechnol. 2025

[4]
CRISPR/Cas system-mediated transgene-free or DNA-free genome editing in plants.

Theor Appl Genet. 2025-8-12

[5]
Large-scale identification and association analysis of wheat grain weight genes using rice orthologs.

Theor Appl Genet. 2025-8-5

[6]
transformation methods to accelerate the domestication of perennial grain crops.

Front Plant Sci. 2025-7-17

[7]
Comprehensive approaches to design efficient gRNA for SDN1-CRISPR/Cas9 genome editing in wheat.

Front Genome Ed. 2025-7-1

[8]
An accelerated transgene-free genome editing system using microparticle bombardment of sorghum immature embryos.

aBIOTECH. 2025-3-4

[9]
Emerging trends in transgene-free crop development: insights into genome editing and its regulatory overview.

Plant Mol Biol. 2025-7-9

[10]
Enhancing biolistic plant transformation and genome editing with a flow guiding barrel.

Nat Commun. 2025-7-1

本文引用的文献

[1]
Regulatory uncertainty over genome editing.

Nat Plants. 2015-1-8

[2]
Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease.

Genome Biol. 2015-11-30

[3]
DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins.

Nat Biotechnol. 2015-10-19

[4]
Generation of inheritable and "transgene clean" targeted genome-modified rice in later generations using the CRISPR/Cas9 system.

Sci Rep. 2015-6-19

[5]
Targeted genome modifications in soybean with CRISPR/Cas9.

BMC Biotechnol. 2015-3-12

[6]
Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo.

Nat Biotechnol. 2015-1

[7]
Genome editing in rice and wheat using the CRISPR/Cas system.

Nat Protoc. 2014-9-18

[8]
Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew.

Nat Biotechnol. 2014-7-20

[9]
Precision genome engineering and agriculture: opportunities and regulatory challenges.

PLoS Biol. 2014-6-10

[10]
Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA.

Genome Res. 2014-4-2

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