Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system

doi:10.1186/1746-4811-9-39

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Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system

Journal Article•DOI•

Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system

Khaoula Belhaj¹, Angela Chaparro-Garcia¹, Sophien Kamoun¹, Vladimir Nekrasov¹•Institutions (1)

Norwich Research Park¹

11 Oct 2013-Plant Methods (BioMed Central)-Vol. 9, Iss: 1, pp 39-39

TL;DR: The CRISPR/Cas system allows targeted cleavage of genomic DNA guided by a customizable small noncoding RNA, resulting in gene modifications by both non-homologous end joining (NHEJ) and homology-directed repair (HDR) mechanisms.

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Abstract: Targeted genome engineering (also known as genome editing) has emerged as an alternative to classical plant breeding and transgenic (GMO) methods to improve crop plants. Until recently, available tools for introducing site-specific double strand DNA breaks were restricted to zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs). However, these technologies have not been widely adopted by the plant research community due to complicated design and laborious assembly of specific DNA binding proteins for each target gene. Recently, an easier method has emerged based on the bacterial type II CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) immune system. The CRISPR/Cas system allows targeted cleavage of genomic DNA guided by a customizable small noncoding RNA, resulting in gene modifications by both non-homologous end joining (NHEJ) and homology-directed repair (HDR) mechanisms. In this review we summarize and discuss recent applications of the CRISPR/Cas technology in plants.

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Journal Article•DOI•

Plant salt-tolerance mechanisms

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Ulrich Deinlein¹, Aaron B. Stephan¹, Tomoaki Horie², Wei Luo¹, Wei Luo³, Guohua Xu³, Julian I. Schroeder¹ - Show less +3 more•Institutions (3)

University of California, San Diego¹, Shinshu University², Nanjing Agricultural University³

01 Jun 2014-Trends in Plant Science

TL;DR: The understanding of the core salt-tolerance mechanisms in plants is reviewed and key Na+ transport and detoxification pathways and the impact of epigenetic chromatin modifications on salinity tolerance are reviewed.

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1,216 citations

Cites background from "Plant genome editing made easy: tar..."

...119 Belhaj, K. et al. (2013) Plant genome editing made easy: targeted...
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Journal Article•DOI•

A CRISPR/Cas9 toolkit for multiplex genome editing in plants

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Hui Li Xing¹, Li Dong¹, Zhi-Ping Wang¹, Hai-Yan Zhang¹, Chun Yan Han¹, Bing Liu¹, Xue Chen Wang¹, Qi-Jun Chen¹ - Show less +4 more•Institutions (1)

University of Minnesota¹

29 Nov 2014-BMC Plant Biology

TL;DR: A toolkit that facilitates transient or stable expression of the CRISPR/Cas9 system in a variety of plant species, which will facilitate plant research, as it enables high efficiency generation of mutants bearing multiple gene mutations.

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Abstract: To accelerate the application of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9) system to a variety of plant species, a toolkit with additional plant selectable markers, more gRNA modules, and easier methods for the assembly of one or more gRNA expression cassettes is required. We developed a CRISPR/Cas9 binary vector set based on the pGreen or pCAMBIA backbone, as well as a gRNA (guide RNA) module vector set, as a toolkit for multiplex genome editing in plants. This toolkit requires no restriction enzymes besides BsaI to generate final constructs harboring maize-codon optimized Cas9 and one or more gRNAs with high efficiency in as little as one cloning step. The toolkit was validated using maize protoplasts, transgenic maize lines, and transgenic Arabidopsis lines and was shown to exhibit high efficiency and specificity. More importantly, using this toolkit, targeted mutations of three Arabidopsis genes were detected in transgenic seedlings of the T1 generation. Moreover, the multiple-gene mutations could be inherited by the next generation. We developed a toolkit that facilitates transient or stable expression of the CRISPR/Cas9 system in a variety of plant species, which will facilitate plant research, as it enables high efficiency generation of mutants bearing multiple gene mutations.

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1,021 citations

Cites methods from "Plant genome editing made easy: tar..."

...Agrobacterium-mediated transformation is a routine method used to generate transgenic plants, and a few binary vectors have been developed to deliver the CRISPR/Cas9 system into plant genomes via this method [15,20,23,24,33-40]....
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Journal Article•DOI•

Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation.

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Zhi-Ping Wang¹, Hui Li Xing¹, Li Dong¹, Hai-Yan Zhang¹, Chun Yan Han¹, Xue Chen Wang¹, Qi-Jun Chen¹ - Show less +3 more•Institutions (1)

University of Minnesota¹

21 Jul 2015-Genome Biology

TL;DR: Comparisons of 12 combinations of eight promoters and two terminators found that the efficiency of the egg cell-specific promoter-controlled CRISPR/Cas9 system depended on the presence of a suitable terminator, and the composite promoter generated by fusing two eggcell-specific promoters resulted in much higher efficiency of mutation in the T1 generation compared with the single promoters.

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Abstract: Arabidopsis mutants produced by constitutive overexpression of the CRISPR/Cas9 genome editing system are usually mosaics in the T1 generation. In this study, we used egg cell-specific promoters to drive the expression of Cas9 and obtained non-mosaic T1 mutants for multiple target genes with high efficiency. Comparisons of 12 combinations of eight promoters and two terminators found that the efficiency of the egg cell-specific promoter-controlled CRISPR/Cas9 system depended on the presence of a suitable terminator, and the composite promoter generated by fusing two egg cell-specific promoters resulted in much higher efficiency of mutation in the T1 generation compared with the single promoters.

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715 citations

Cites background from "Plant genome editing made easy: tar..."

...This highly efficient, easy-to-use system can potentially be used to make highly multiplexed genome modifications, and is supplanting the use of ZFNs and TALENs to become the standard genome-editing technology [3, 4, 6, 7]....
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Journal Article•DOI•

Engineering Quantitative Trait Variation for Crop Improvement by Genome Editing.

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Daniel Rodríguez-Leal¹, Zachary H. Lemmon¹, Jarrett Man², Madelaine E. Bartlett², Zachary B. Lippman¹ - Show less +1 more•Institutions (2)

Cold Spring Harbor Laboratory¹, University of Massachusetts Amherst²

05 Oct 2017-Cell

TL;DR: It is demonstrated that CRISPR/Cas9 genome editing of promoters generates diverse cis-regulatory alleles that provide beneficial quantitative variation for breeding that provide a foundation for dissecting complex relationships between gene-reg regulatory changes and control of quantitative traits.

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673 citations

Additional excerpts

...…for qRT-PCR, see Table S8 This study N/A Recombinant DNA MoClo Toolkit (Werner et al., 2012) Addgene #1000000044 pICH86966::AtU6p::sgRNA_PDS (Belhaj et al., 2013) Addgene #46966 pICH47732::NOSp::NPTII (Belhaj et al., 2013) Addgene #51144 pICH47742::35S::Cas9 (Belhaj et al., 2013) Addgene…...
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...…(Belhaj et al., 2013) Addgene #46966 pICH47732::NOSp::NPTII (Belhaj et al., 2013) Addgene #51144 pICH47742::35S::Cas9 (Belhaj et al., 2013) Addgene #49771 Software and Algorithms Trimmomatic (Bolger et al., 2014) http://www.usadellab.org/cms/?page=trimmomatic BWA-MEM…...
[...]
...…DNA MoClo Toolkit (Werner et al., 2012) Addgene #1000000044 pICH86966::AtU6p::sgRNA_PDS (Belhaj et al., 2013) Addgene #46966 pICH47732::NOSp::NPTII (Belhaj et al., 2013) Addgene #51144 pICH47742::35S::Cas9 (Belhaj et al., 2013) Addgene #49771 Software and Algorithms Trimmomatic (Bolger et al.,…...
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Journal Article•DOI•

Efficient gene editing in tomato in the first generation using the clustered regularly interspaced short palindromic repeats/CRISPR-associated9 system.

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Christopher Brooks¹, Vladimir Nekrasov², Zachary B. Lippman, Joyce Van Eck³•Institutions (3)

Cold Spring Harbor Laboratory¹, Sainsbury Laboratory², Boyce Thompson Institute for Plant Research³

01 Nov 2014-Plant Physiology

TL;DR: The CRISPR/Cas9 system is highly efficient at generating targeted mutations in stable transgenic tomato plants, and homozygous deletions of a desired size can be created in the first generation.

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Abstract: During the past 12 years, there has been rapid development of genome-editing strategies that make it possible to directly target regions of genes in a DNA sequence-specific manner. Two of these strategies, zinc finger nucleases ([Urnov et al., 2010][1]) and transcription activator-like nucleases ([

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640 citations

Cites background or methods from "Plant genome editing made easy: tar..."

...Level 1 constructs carrying sgRNAs placed under the control of the Arabidopsis (Arabidopsis thaliana) U6 promoter were assembled as described (Belhaj et al., 2013)....
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...The CRISPR/Cas9 construct we designed to target SlAGO7 contained two single guide RNAs (sgRNAs) with the intention to create large, defined deletions (Belhaj et al., 2013)....
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References

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Open Access

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Journal Article•DOI•

A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.

[...]

Martin Jinek¹, Krzysztof Chylinski², Krzysztof Chylinski³, Ines Fonfara³, Michael H. Hauer¹, Jennifer A. Doudna, Emmanuelle Charpentier³ - Show less +3 more•Institutions (3)

University of California, Berkeley¹, Max F. Perutz Laboratories², Umeå University³

17 Aug 2012-Science

TL;DR: This study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.

...read moreread less

Abstract: Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.

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12,865 citations

"Plant genome editing made easy: tar..." refers background in this paper

...The ability to reprogram CRISPR/Cas endonuclease specificity using customizable small noncoding RNAs has set the stage for novel genome editing applications [2-8]....
[...]
...The HNH nuclease domain cleaves the complementary DNA strand whereas the RuvC-like domain cleaves the non-complementary strand and, as a result, a blunt cut is introduced in the target DNA [2]....
[...]
...As mentioned above, the sgRNA is a synthetic RNA chimera created by fusing crRNA with tracrRNA [2]....
[...]
...This is consistent with earlier reports [2,5,8]....
[...]
...As a result, the number of components in the CRISPR/Cas system was brought down to two, Cas9 and sgRNA [2]....
[...]

Journal Article•DOI•

Multiplex Genome Engineering Using CRISPR/Cas Systems

[...]

Le Cong¹, Le Cong², F. Ann Ran¹, F. Ann Ran², David M. Cox¹, David M. Cox², Shuailiang Lin³, Shuailiang Lin², Robert P. J. Barretto⁴, Naomi Habib², Patrick D. Hsu², Patrick D. Hsu¹, Xuebing Wu², Wenyan Jiang⁵, Luciano A. Marraffini⁵, Feng Zhang² - Show less +12 more•Institutions (5)

Harvard University¹, Massachusetts Institute of Technology², Tsinghua University³, Columbia University⁴, Rockefeller University⁵

15 Feb 2013-Science

TL;DR: The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage as discussed by the authors.

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Abstract: Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.

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12,265 citations

Multiplex Genome Engineering Using CRISPR/Cas Systems

[...]

Le Cong, F. A. Ran, David Benjamin Turitz Cox, Shuailiang Lin, Robert P. J. Barretto, Naomi Habib, Patrick D. Hsu, Xuebing Wu, Wenyan Jiang, Luciano A. Marraffini, Feng Zhang - Show less +7 more

01 Feb 2013

TL;DR: Two different type II CRISPR/Cas systems are engineered and it is demonstrated that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.

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Abstract: Genome Editing Clustered regularly interspaced short palindromic repeats (CRISPR) function as part of an adaptive immune system in a range of prokaryotes: Invading phage and plasmid DNA is targeted for cleavage by complementary CRISPR RNAs (crRNAs) bound to a CRISPR-associated endonuclease (see the Perspective by van der Oost). Cong et al. (p. 819, published online 3 January) and Mali et al. (p. 823, published online 3 January) adapted this defense system to function as a genome editing tool in eukaryotic cells. A bacterial genome defense system is adapted to function as a genome-editing tool in mammalian cells. [Also see Perspective by van der Oost] Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.

...read moreread less

10,746 citations

"Plant genome editing made easy: tar..." refers background in this paper

...The ability to reprogram CRISPR/Cas endonuclease specificity using customizable small noncoding RNAs has set the stage for novel genome editing applications [2-8]....
[...]
...This is consistent with earlier reports [2,5,8]....
[...]
...As in the case of human cells [4,5], the Cas9 protein was expressed in plants as a fusion to a nuclear localization signal (NLS) to ensure delivery into nuclei....
[...]

Journal Article•DOI•

RNA-Guided Human Genome Engineering via Cas9

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Prashant Mali¹, Luhan Yang¹, Kevin M. Esvelt², John Aach¹, Marc Güell¹, James E. DiCarlo³, Julie E. Norville¹, George M. Church², George M. Church¹ - Show less +5 more•Institutions (3)

Harvard University¹, Wyss Institute for Biologically Inspired Engineering², Boston University³

15 Feb 2013-Science

TL;DR: The type II bacterial CRISPR system is engineer to function with custom guide RNA (gRNA) in human cells to establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.

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Abstract: Bacteria and archaea have evolved adaptive immune defenses, termed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems, that use short RNA to direct degradation of foreign nucleic acids. Here, we engineer the type II bacterial CRISPR system to function with custom guide RNA (gRNA) in human cells. For the endogenous AAVS1 locus, we obtained targeting rates of 10 to 25% in 293T cells, 13 to 8% in K562 cells, and 2 to 4% in induced pluripotent stem cells. We show that this process relies on CRISPR components; is sequence-specific; and, upon simultaneous introduction of multiple gRNAs, can effect multiplex editing of target loci. We also compute a genome-wide resource of ~190 K unique gRNAs targeting ~40.5% of human exons. Our results establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.

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8,197 citations

"Plant genome editing made easy: tar..." refers background or methods in this paper

...sgRNA1 was PCR-amplified with primers PDS_gRNA1_BsaIf and gRNA_AGCG_BsaIr, and sgRNA2 – with primers PDS_gRNA2_BsaIf and gRNA_AGCG_BsaIr using the plasmid gRNA_GFP_T1 [4] as a template....
[...]
...The ability to reprogram CRISPR/Cas endonuclease specificity using customizable small noncoding RNAs has set the stage for novel genome editing applications [2-8]....
[...]
...In order to use the human codon optimised Cas9 [4] in the GG system, all BsaI and BbsI sites had to be removed from its sequence, while preserving the amino acid composition of the protein, in a process called “domestication”....
[...]
...As in the case of human cells [4,5], the Cas9 protein was expressed in plants as a fusion to a nuclear localization signal (NLS) to ensure delivery into nuclei....
[...]

Journal Article•DOI•

DNA targeting specificity of RNA-guided Cas9 nucleases

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Patrick D. Hsu¹, David A. Scott², David A. Scott¹, Joshua A. Weinstein², Joshua A. Weinstein¹, F. Ann Ran², F. Ann Ran³, F. Ann Ran¹, Silvana Konermann², Silvana Konermann¹, Vineeta Agarwala², Vineeta Agarwala⁴, Vineeta Agarwala³, Yinqing Li², Yinqing Li¹, Eli J. Fine⁵, Xuebing Wu⁴, Ophir Shalem¹, Ophir Shalem², Thomas J. Cradick⁵, Luciano A. Marraffini⁶, Gang Bao⁵, Feng Zhang¹, Feng Zhang² - Show less +20 more•Institutions (6)

McGovern Institute for Brain Research¹, Broad Institute², Harvard University³, Massachusetts Institute of Technology⁴, Georgia Institute of Technology⁵, Rockefeller University⁶

01 Sep 2013-Nature Biotechnology

TL;DR: In this article, the Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing.

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Abstract: The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.

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4,113 citations