CRISPR-transient expression in soybean for simplified gRNA screening in planta

The objective of this work was to develop a method to create and validate CRISPR-Cas systems and different gRNAs in soybean (Glycine max) embryos. Two model genes were used for simple mutation with one gRNA or partial gene deletion with two guides. The gRNAs were inserted into the CRISPR transformation vectors by a type IIS restriction enzyme or by subcloning and inserting the promoter + gRNA2 in the final transformation vector using the classic restriction enzyme cloning method. The vectors were successfully constructed for one and two gRNAs. Agrobacterium-mediated transient transformation in soybean was carried out to test the quality of gRNAs and of the system itself (expression cassette). Simple mutation and gene deletion were detected in the embryos transformed after DNA enrichment by enzyme digestion followed by polymerase chain reaction and sequencing, which indicates that the CRISPR-Cas system and guides were working. This protocol can be used to accelerate CRISPR-based genome editing strategies for genetic transformation in soybean.

Index terms
Glycine max; CRISPR vector construction; gRNA validation; mutation detection

Authorship

Alessandra Koltun

Universidade Estadual de Maringá, Centro de Ciências Agrárias, Avenida Colombo, nº 5.790, Bloco J45, CEP 87020-900 Maringá, PR, BrazilUniversidade Estadual de MaringáBrazilMaringá, PR, BrazilUniversidade Estadual de Maringá, Centro de Ciências Agrárias, Avenida Colombo, nº 5.790, Bloco J45, CEP 87020-900 Maringá, PR, Brazil

http://orcid.org/0000-0001-8889-5264

Nathalia Volpi e Silva

Embrapa Soja, Rodovia Carlos João Strass, s/nº , Acesso Orlando Amaral, Distrito de Warta, Caixa Postal 4006, CEP 86085-981 Londrina, PR, BrazilEmbrapa SojaBrazilLondrina, PR, BrazilEmbrapa Soja, Rodovia Carlos João Strass, s/nº , Acesso Orlando Amaral, Distrito de Warta, Caixa Postal 4006, CEP 86085-981 Londrina, PR, Brazil

http://orcid.org/0000-0002-6222-1576

Jéssika Angelotti-Mendonça

http://orcid.org/0000-0002-8147-5547

Silvana Regina Rockenbach Marin

http://orcid.org/0000-0002-6764-1422

Leandro Simões Azeredo Gonçalves

Universidade Estadual de Londrina, Rodovia Celso Garcia Cid, PR 445, Km 380, Campus Universitário, Caixa Postal 10.011, CEP 86057-970 Londrina, PR, BrazilUniversidade Estadual de LondrinaBrazilLondrina, PR, BrazilUniversidade Estadual de Londrina, Rodovia Celso Garcia Cid, PR 445, Km 380, Campus Universitário, Caixa Postal 10.011, CEP 86057-970 Londrina, PR, Brazil

http://orcid.org/0000-0001-9700-9375

Alexandre Lima Nepomuceno

http://orcid.org/0000-0003-2012-6201

Liliane Marcia Mertz-Henning Corresponding author

http://orcid.org/0000-0001-7622-0649

Corresponding author

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Figures

Figures (3)

Thumbnail

Figure 1
gRNA insertion into the C034p7ioR-35sCas CRISPR-Cas9 modular vector, following three steps: digestion of the modular vector with the BsmBI type IIS restriction nuclease (A), phosphorylation and annealing of gRNA oligonucleotides designed with borders complementary to the vector (B), and confirmation of gRNA insertion by polymerase chain reaction using a forward primer that anneals to the U6At promoter and the reverse gRNA oligonucleotide (C). The agarose gel presents expected bands of ~200 bp amplified from plasmids cloned into Escherichia coli. C-, negative control; and CDS, coding DNA sequence.

Thumbnail

Figure 2
Steps of the genetic engineering approach to create a CRISPR-Cas vector bearing two gRNAs. In steps 1 and 2, gRNA1 and gRNA2 are inserted into the CRISPR-Cas modular vector as described in Figure 1 and separately, respectively. In step 3, the clone U6 promoter + gRNA2 is ligated into an entry vector with enzyme restriction borders that correspond to the modular vector using SalI and KpnI, whereas, in step 4, the modular vector + gRNA1 and pENTR+U6::gRNA2 are digested separately with SalI and KpnI, ligating the purified open modular vector + gRNA1 and fragment (U6::gRNA2). Finally, in step 5, the insertion of gRNA2 into the modular vector + gRNA1 is confirmed by polymerase chain reaction using one oligonucleotide of each gRNA as primers. Agar gel shows expected bands of ~600 bp amplified from plasmids cloned into Escherichia coli. C, negative control.

Thumbnail

Figure 3
Strategies for CRISPR-Cas-mediated gene editing and results of transient expression to test gRNA functionality in soybean (Glycine max), using gRNA designed in a restriction enzyme site that, once edited, may be lost (A) and two gRNAs to induce a partial gene deletion of 305 bp (B). In the first strategy, the DNA (target gene) from embryos of the BRS 283 and BRS 537 soybean cultivars transformed with Agrobacterium was previously digested with AflII, visualized in 2.0% agarose gel, and amplified in two polymerase chain reactions (PCRs). C1- and C2- are untransformed negative controls, positive samples were sequenced, and gene editing was confirmed using the ICE software (Synthego, Red Wood City, CA, USA). In the second strategy, the whole gene from PvII-digested embryos of the BMX Potência RR and BRS 537 soybean cultivars transformed with Agrobacterium was amplified by PCR and visualized in 1.5% agarose gel. C1- and C2- are untransformed and undigested negative controls, whereas C3- and C4- are untransformed and not PvuII-digested negative controls. In the protein representation from Protter, amino acids in red and green represent the protein N-terminal and N-glycan motifs, respectively.

Figure 1 gRNA insertion into the C034p7ioR-35sCas CRISPR-Cas9 modular vector, following three steps: digestion of the modular vector with the BsmBI type IIS restriction nuclease (A), phosphorylation and annealing of gRNA oligonucleotides designed with borders complementary to the vector (B), and confirmation of gRNA insertion by polymerase chain reaction using a forward primer that anneals to the U6At promoter and the reverse gRNA oligonucleotide (C). The agarose gel presents expected bands of ~200 bp amplified from plasmids cloned into Escherichia coli. C-, negative control; and CDS, coding DNA sequence.

Figure 2 Steps of the genetic engineering approach to create a CRISPR-Cas vector bearing two gRNAs. In steps 1 and 2, gRNA1 and gRNA2 are inserted into the CRISPR-Cas modular vector as described in Figure 1 and separately, respectively. In step 3, the clone U6 promoter + gRNA2 is ligated into an entry vector with enzyme restriction borders that correspond to the modular vector using SalI and KpnI, whereas, in step 4, the modular vector + gRNA1 and pENTR+U6::gRNA2 are digested separately with SalI and KpnI, ligating the purified open modular vector + gRNA1 and fragment (U6::gRNA2). Finally, in step 5, the insertion of gRNA2 into the modular vector + gRNA1 is confirmed by polymerase chain reaction using one oligonucleotide of each gRNA as primers. Agar gel shows expected bands of ~600 bp amplified from plasmids cloned into Escherichia coli. C, negative control.

Figure 3 Strategies for CRISPR-Cas-mediated gene editing and results of transient expression to test gRNA functionality in soybean (Glycine max), using gRNA designed in a restriction enzyme site that, once edited, may be lost (A) and two gRNAs to induce a partial gene deletion of 305 bp (B). In the first strategy, the DNA (target gene) from embryos of the BRS 283 and BRS 537 soybean cultivars transformed with Agrobacterium was previously digested with AflII, visualized in 2.0% agarose gel, and amplified in two polymerase chain reactions (PCRs). C1- and C2- are untransformed negative controls, positive samples were sequenced, and gene editing was confirmed using the ICE software (Synthego, Red Wood City, CA, USA). In the second strategy, the whole gene from PvII-digested embryos of the BMX Potência RR and BRS 537 soybean cultivars transformed with Agrobacterium was amplified by PCR and visualized in 1.5% agarose gel. C1- and C2- are untransformed and undigested negative controls, whereas C3- and C4- are untransformed and not PvuII-digested negative controls. In the protein representation from Protter, amino acids in red and green represent the protein N-terminal and N-glycan motifs, respectively.

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CRISPR-transient expression in soybean for simplified gRNA screening in planta

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[1] Corresponding author