Single guide RNA (sgRNA) sequences targeting the ZFP36 genomic locus were designed using Benchling online software (https://www.benchling.com ) and cross-validated with Synthego’s guide design verification tool (https://design.synthego.com/#/validate ). sgRNA sequences were synthesized by Integrated DNA Technologies (IDT). After screening three sgRNAs against ZFP36, the top sgRNA was selected for downstream experiments (sgRNA sequence: 5′-CGCTCCACCAGCCTAGTGGA-3′). Gene disruption was performed following an optimized protocol previously described19 (link). Briefly, T cells were washed three times in Opti-MEM™ reduced serum medium (Gibco) and resuspended at 1 × 108 cells/mL in P3 nucleofection solution (Lonza). Ribonucleoprotein (RNP) complexes were generated by incubating each sgRNA (5ug per 10 × 106 cells) individually with Cas9 nuclease (Aldevron, 10ug per 10 × 106 cells) for 10 min at room temperature. For mock groups, no sgRNA was used and for multiple knockout groups, RNPs targeting each gene were incubated separately before combining. Cells were electroporated in batches of 10 × 106 cells (100 µL) with a mixture of RNP complex and 16.8 pmol of electroporation enhancer (IDT) in electroporation cuvettes (electroporation code EH111) in a 4D-Nucleofector X-Unit (Lonza).
Cas9 nuclease
Manufactured by Aldevron
Sourced in United States
About the product
Cas9 nuclease is a protein derived from the CRISPR-Cas9 system. Its core function is to act as a molecular scissors, capable of precisely cutting targeted DNA sequences. This allows for the modification or manipulation of genetic material.
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9 protocols using «cas9 nuclease»
1
CRISPR Knockout of ZFP36 in T Cells
2
CRISPR-Mediated Regnase-1 and Roquin-1 Knockout
Single guide RNA (sgRNA) sequences
targeting Regnase-1 and Roquin-1 from a previous study validating
their efficiency were used to perform knockouts.19 (link) sgRNA sequences were synthesized by Integrated DNA Technologies
(IDT). Gene disruption was performed using a previously described
protocol.26 (link) Briefly, T cells were washed
three times in Opti-MEM and resuspended in P3 nucleofection solution
(Lonza) at 1 × 108 cells/mL. Each sgRNA (5 μg
per 10 × 106 cells) was incubated with Cas9 nuclease
(Aldevron, 10 μg per 10 × 106 cells) for 10
min at room temperature to generate ribonucleoprotein (RNP) complexes.
For no knockout (Mock) groups, no sgRNA was used and for multiple
knockout groups, RNPs targeting each gene were incubated separately
before combining. Cells were electroporated in batches of 10 ×
106 cells (100 μL) with a mix of RNP complex and
16.8pmol of electroporation enhancer (IDT) in nucleofection cuvettes
(pulse code EH111) using a 4D-Nucleofector X-Unit (Lonza). To quantify
indel percentages, genomic DNA was isolated from cells using the DNeasy
Blood and Tissue Kit (Qiagen) per the manufacturer’s protocol.
The target knockout locus was then PCR amplified using previously
reported primers, gel extracted, and then Sanger sequenced. Sequencing
traces were analyzed with Synthego’s browser-based ICE analysis
tool to calculate the discordance in sequences for each sample compared
to an unedited sequencing trace. This metric is used to estimate the
percentage of indels present in the experimental sequencing trace.
targeting Regnase-1 and Roquin-1 from a previous study validating
their efficiency were used to perform knockouts.19 (link) sgRNA sequences were synthesized by Integrated DNA Technologies
(IDT). Gene disruption was performed using a previously described
protocol.26 (link) Briefly, T cells were washed
three times in Opti-MEM and resuspended in P3 nucleofection solution
(Lonza) at 1 × 108 cells/mL. Each sgRNA (5 μg
per 10 × 106 cells) was incubated with Cas9 nuclease
(Aldevron, 10 μg per 10 × 106 cells) for 10
min at room temperature to generate ribonucleoprotein (RNP) complexes.
For no knockout (Mock) groups, no sgRNA was used and for multiple
knockout groups, RNPs targeting each gene were incubated separately
before combining. Cells were electroporated in batches of 10 ×
106 cells (100 μL) with a mix of RNP complex and
16.8pmol of electroporation enhancer (IDT) in nucleofection cuvettes
(pulse code EH111) using a 4D-Nucleofector X-Unit (Lonza). To quantify
indel percentages, genomic DNA was isolated from cells using the DNeasy
Blood and Tissue Kit (Qiagen) per the manufacturer’s protocol.
The target knockout locus was then PCR amplified using previously
reported primers, gel extracted, and then Sanger sequenced. Sequencing
traces were analyzed with Synthego’s browser-based ICE analysis
tool to calculate the discordance in sequences for each sample compared
to an unedited sequencing trace. This metric is used to estimate the
percentage of indels present in the experimental sequencing trace.
3
Efficient CRISPR-Cas9 Knockout of Regnase-1 and Roquin-1 in T Cells
Single-guide RNA (sgRNA) sequences targeting Regnase-1 and Roquin-1 were designed using Benchling online software (https://www.benchling.com ) and cross-validated with Synthego’s guide design verification tool (https://design.synthego.com/#/validate ). sgRNA sequences were synthesized by Integrated DNA Technologies (IDT). After screening several (3+) sgRNAs targeting each gene (SI Appendix, Table S1 ), the top sgRNA was selected for downstream experiments. Gene disruption was performed following an optimized protocol previously described (33 ). Briefly, T cells were washed three times in Opti-MEM™ reduced serum medium (Gibco) and resuspended at 1 × 108 cells/mL in P3 nucleofection solution (Lonza). Ribonucleoprotein (RNP) complexes were generated by incubating each sgRNA (5 μg per 10 × 106 cells) individually with Cas9 nuclease (Aldevron, 10 μg per 10 × 106 cells) for 10 min at room temperature. For no knockout (Mock) groups, no sgRNA was used and for multiple knockout groups, RNPs targeting each gene were incubated separately before combining. Cells were electroporated in batches of 10 × 106 cells (100 µL) with a mixture of RNP complex and 16.8 pmol of electroporation enhancer (IDT) in electroporation cuvettes (electroporation code EH111) in a 4D-Nucleofector X-Unit (Lonza).
4
Genetic Engineering of Capsaicin Production in Tomato
Example 32
This example describes the preparation of reagents for the modification of three genes in tomato. It is predicted that the activation of these three genes will result in a tomato that produces capsaicin, the molecule that gives peppers their spicy taste. Peppers and tomatoes are closely related and share many genes. The genes involved in the biosynthesis of capsaicin in pepper were used to identify the homologous genes in tomato. Based on the pepper genome (see: doi: 10.1038/ng.2877) three genes were selected for modification and enhanced expression: capsaicin synthase (CS), BCAT, and KAS. Putative tomato homologues were identified by BLAST analysis and manual annotation.
A partial genomic sequence of the tomato capsaicin synthase (CS) gene (Solyc02g081740.1.1) is provided as SEQ ID NO:392. CS is the last step in the synthesis of capsaicin. The expression of CS is constitutively increased by inserting an expression enhancing oligonucleotide in the 5′ region of the gene. A maize OCS homologue (see Examples 14 and 23) encoded by a chemically modified single-stranded DNA with the sequence of SEQ ID NO:343 (Integrated DNA Technologies, Coralville, Iowa), phosphorylated on the 5′ end and containing two phosphorothioate linkages at each terminus (i. e., the two linkages between the most distal three bases on either end of the strand) is designed for insertion at a double-strand break effected between nucleotides at positions 46/47, 216/217, and 333/334 of SEQ ID NO:392 in order to provide constitutively increased expression of CS. A CS crRNA is designed to target the nucleotides shown in bold italic in SEQ ID NO: 392 (Table 23) and has the sequences of SEQ ID NO:424, 425, and 426. The crRNA and tracrRNA are purchased from Integrated DNA Technologies, Coralville, Iowa Ribonucleoprotein (RNP) complexes are prepared using gRNA (crRNA:tracrRNA) and Cas9 nuclease (Aldevron, Fargo, N. Dak.). Integration of the OCS homologue sequence in the CS gene is carried out using procedures similar to those described in Examples 5 and 10.
The tomato BCAT gene (Solyc07g021630.2.1) has the partial genomic sequence of SEQ ID NO:393. Expression of BCAT is constitutively increased by inserting an expression enhancing oligonucleotide in the 5′ region of the gene. A maize OCS homologue (see Examples 14 and 23) encoded by a chemically modified single-stranded DNA with the sequence of SEQ ID NO:343 (Integrated DNA Technologies, Coralville, Iowa), phosphorylated on the 5′ end and containing two phosphorothioate linkages at each terminus (i. e., the two linkages between the most distal three bases on either end of the strand) is designed for insertion at a double-strand break effected between nucleotides at positions 103/104, 330/331, or 362/363 of SEQ ID NO:393 in order to provide constitutively increased expression of BCAT. Three BCAT crRNAs are designed to target the nucleotides shown in bold italic in SEQ ID NO: 393 (Table 23) and have the sequences of SEQ ID NO:427, 428, and 429. The crRNAs and tracrRNA are purchased from Integrated DNA Technologies, Coralville, Iowa Ribonucleoprotein (RNP) complexes are prepared using gRNA (crRNA:tracrRNA) and Cas9 nuclease (Aldevron, Fargo, N. Dak.). Integration of the OCS homologue sequence in the BCAT gene is carried out using procedures similar to those described in Examples 5 and 10.
The tomato KAS gene (Solyc08g082620.2.1) has the partial genomic sequence of SEQ ID NO:394. Expression of KAS is constitutively increased by inserting an expression enhancing oligonucleotide in the 5′ region of the gene. A maize OCS homologue (see Examples 14 and 23) encoded by a chemically modified single-stranded DNA with the sequence of SEQ ID NO:343 (Integrated DNA Technologies, Coralville, Iowa), phosphorylated on the 5′ end and containing two phosphorothioate linkages at each terminus (i. e., the two linkages between the most distal three bases on either end of the strand) is designed for insertion at a double-strand break effected between nucleotides at positions 103/104, 168/169 or 259/260 of SEQ ID NO:394 in order to provide constitutively increased expression of KAS. Three BCAT crRNAs are designed to target the nucleotides shown in bold italic in SEQ ID NO: 394 (Table 23) and have the sequences of SEQ ID NO:430, 431, and 432. The crRNAs and tracrRNA are purchased from Integrated DNA Technologies, Coralville, Iowa Ribonucleoprotein (RNP) complexes are prepared using gRNA (crRNA:tracrRNA) and Cas9 nuclease (Aldevron, Fargo, N. Dak.). Integration of the OCS homologue sequence in the KAS gene is carried out using procedures similar to those described in Examples 5 and 10.
In one embodiment, one or more genomic changes are made in the plant's genome during vegetative development; this is useful especially in plants that can be vegetatively propagated (e. g., from cuttings), such as tomato and tobacco. Such plants are conveniently grown in soil or in tissue culture media.
In this example, tomato meristem microinjection and meristem cell preparation are described. The apical or axillary meristem of a young plant is surgically exposed and genome editing reagents are introduced just below the L1 layer, into the L2 layer, of the meristem using a microinjection apparatus. The injected tissue is allowed to recover, and the resulting newly formed tissue is examined for the presence of the intended genomic edits. This cycle can be repeated many times, facilitated by propagating cuttings of the edited plant material from time to time. Modifications to the plant genome can be monitored by one or more molecular assays. Once the intended changes are complete, the plant is permitted to flower, is selfed or crossed, and produces seed. The next generation is examined for the presence and activity of all intended edits.
The genome editing reagents for this work are selected from DNA, RNA, protein, or a combination thereof (such as the ribonucleoproteins described elsewhere in this application). The reagents are delivered using an appropriate microinjection apparatus in a volume of about 2 to about 20 nanoliters per cell. The editing reagents can be delivered alone or as part of a formulation to aid in uptake by the targeted meristematic cells. These reagents can include saponin (e.g., Sigma Cat. No. 47036-50g-F), pectinase, DMSO, Silwet-77, Tween-20, or any other agent that permeabilizes or otherwise makes penetrable the plant cell wall without compromising the cell's activity or interfering with the activity of the editing reagents.
To introduce editing reagents, the newly formed leaf tissue in the target plant is carefully removed to expose the meristem without damaging it. The stem is gently, but firmly supported to counteract the pressure of the microinjection needle. A dissecting or compound microscope with appropriate optics is used to ensure that the microinjection needle accurately contacts the meristematic cells (specifically, the L2 cell layer, which gives rise to the germline). Once the meristem cells are treated, the stem is marked and the plant allowed to recover for several days. It is possible that more than one meristem per plant can be treated with identical or distinct editing reagents. The recovery period is long enough for the plant to grow 3-5 new leaves from the treated meristem. When sufficient new leaf tissue is present, a small piece of a newly formed leaf is excised for molecular analysis.
Molecular analysis encompasses a variety of assays or tests designed to detect the presence of the intended genomic modifications. For example, mRNA from the targeted gene(s) can be amplified by RT-PCR and sequenced to determine if genomic edits are present. Genomic DNA can be examined by targeted sequence analysis for the presence of the intended genomic modifications. Leaf tissue can be examined for visual or physical evidence (i. e., a detectable phenotype) of an intended genomic modification. The time required for evaluation of genomic modifications allows the plant to further recover from the microinjection and makes it competent for further genomic editing, if necessary.
If more genomic edits are required prior to flowering, tissue segments representing edited material can be vegetatively propagated. Excised plantlets are rooted in fresh soil or tissue culture media prior to the next editing step. Care is taken to insure the propagated plant is actively growing (e. g., displays evidence of robust root growth and new leaf formation) before initiating the next microinjection.
When genomic editing activities are complete, the plants are grown to reproductive maturity. If desired further vegetative propagation can be done to produce multiple clones of the edited plant prior to flowering. This may be desirable to ensure adequate seed production. Plants can be selfed or crossed as appropriate to produce seed. The resulting seeds are planted and the germinated seedlings are tested for the presence of the intended edits.
5
Improving Maize Drought Tolerance
Example 40
This example describes the modification of two maize genes to improve water use efficiency and to increase biomass and seed yield in a maize cell or plant.
An increase in expression of NAC111 is predicted to improve water use efficiency. The MITE insertion in the 5′ untranslated region (5′ UTR) of the maize NAC111 gene is modified in order to increase ZmNAC111 expression. The promoter sequence of NAC111 is provided as SEQ ID NO:1002, with the MITE sequence located at nucleotide positions 103-186 of SEQ ID NO:1002 (i. e., 414-498 base-pairs upstream of the NAC111 transcription start site).
In one approach, the MITE sequence is deleted or made non-functional by effecting at least one DSB in the MITE sequence. Two crRNAs are designed to effect DSBs on either side of the MITE sequence, resulting in deletion of the MITE sequence; the first crRNA with the sequence of SEQ ID NO:1003 is designed to effect a DSB 525 base-pairs upstream of the transcription start site (TSS) and the second crRNA with the sequence of SEQ ID NO:1004 is designed to effect a DSB at 396 base-pairs upstream of the TSS. The crRNAs and tracrRNA are purchased from Integrated DNA Technologies, Coralville, Iowa. Ribonucleoprotein (RNP) complexes are prepared using gRNA (crRNA:tracrRNA) complexes and Cas9 nuclease (Aldevron, Fargo, N. Dak.). The two RNPs are delivered together to the maize protoplasts using procedures similar to those described in Examples 13-16. The resulting partial sequence deletion in non-coding sequence of the ZmNAC111 gene is expected to result in upregulation of ZmNAC111 expression. Similar genomic modification is carried out in maize germline cells using procedures as described in Examples 31-34; the resulting maize plants containing such a genomic modification (deletion of the MITE sequence) are predicted to have increased water use efficiency (measured, e. g., by comparison of biomass or of seed yield of plants grown under different water availability regimes) and therefore enhanced drought tolerance.
In another approach, the MITE sequence is modified by targeted demethylation. Constructs encoding a modified dCas9-SunTag and an anti-GCN4 scFv fused to ten-eleven hydroxylase 1 (TET1), as described in detail by Morita et al. (2016) Nature Biotechnol., 34:1060-1065; doi: 10.1038/nbt.3658), are used to demethylate the ZmNAC111 MITE region; these plasmids are publicly available from Addgene (see www[dot]addgene[dot]org/browse/article/22324/). Two crRNAs targeting the MITE region in the promoter of ZmNAC111 were designed to bring TET1 to MITE region and trigger demethylation; the first crRNA with the sequence of SEQ ID NO:1003 is designed to target 508-528 base-pairs upstream of the transcription start site (TSS) and the second crRNA with the sequence of SEQ ID NO:1005 is designed to target 425-445 base-pairs upstream of the TSS. The crRNAs and tracrRNA are purchased from Integrated DNA Technologies, Coralville, Iowa. Ribonucleoprotein (RNP) complexes are prepared using gRNA (crRNA:tracrRNA) complexes and Cas9 nuclease (Aldevron, Fargo, N. Dak.). The two RNPs are delivered together with the dCas9-SunTag and anti-GCN4 scFv/TET1 fusion plasmids to the maize protoplasts using procedures similar to those described in Examples 13-16. The resulting demethylation of the MITE region of the ZmNAC111 gene is expected to result in upregulation of ZmNAC111 expression. Similar genomic modification is carried out in maize germline cells using procedures as described in Examples 31-34; the resulting maize plants containing such a genomic modification (demethylation of the MITE sequence) are predicted to have increased water use efficiency (measured, e. g., by comparison of biomass or of seed yield of plants grown under different water availability regimes) and therefore enhanced drought tolerance.
An increase in expression of NAC111 and an increase in expression of PLA1 simultaneously is predicted to result in improved water use efficiency, increased leaf size, increased biomass, and increased seed yield. A partial genomic sequence of ZmPLA1 is provided as SEQ ID NO:1015. Three crRNAs targeting this region upstream of the ZmPLA1 TSS were designed effect double-strand breaks (DSBs) upstream or slightly downstream of the TSS; the first crRNA with the sequence of SEQ ID NO:1016 is designed to effect a DSB 150 base-pairs upstream of (5′ to) the transcription start site (TSS), the second crRNA with the sequence of SEQ ID NO:1017 is designed to effect a DSB 95 base-pairs upstream of the TSS, and the third crRNA with the sequence of SEQ ID NO:1018 is designed to effect a DSB 19 base-pairs downstream of (3′ to) the TSS. The sequence to be inserted at a DSB was a 2,046-bp promoter sequence of the maize GA2-oxidase with the sequence of SEQ ID NO:1019. Polynucleotide (such as a double-stranded DNA, a single-stranded DNA, a single-stranded DNA/RNA hybrid, or a double-stranded DNA/RNA hybrid) donor molecules are designed including the ZmGA2ox promoter (SEQ ID NO:1019) with the addition of homology arms (generally including about 50 to about 1000 nucleotides having sufficient sequence identity or complementarity to the genomic sequence flanking the site of the DSB) on one or on both sides of the promoter sequence to facilitate HDR-type integration of the donor sequence at the site of the DSB. The crRNAs, tracrRNA, and donor polynucleotides are purchased from Integrated DNA Technologies, Coralville, Iowa. Ribonucleoprotein (RNP) complexes are prepared using gRNA (crRNA:tracrRNA) complexes and Cas9 nuclease (Aldevron, Fargo, N. Dak.). Each individual RNP is delivered together with GA2ox promoter donor molecule to maize protoplasts using procedures similar to those described in Examples 13-16. Integration of the heterologous promoter is expected to result in localized upregulation of ZmPLA1 expression. Similar genomic modification is carried out in maize germline cells using procedures as described in Examples 31-34; the resulting maize plants containing such a genomic modification (operably linking the ZmGA2ox promoter to ZmPLA1) are predicted to have increased leaf area, increased biomass, and/or increased seed yield, compared to plants lacking the genomic modification. Growth chamber and field assays for measuring leaf area, biomass, or seed yield are described in detail in Sun et al. (2017) Nature Communications, 8:14752; DOI: 10.1038/ncomms14752.
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