{ "experiment": { "id": 422, "name": "CRISPR transformations with Agrobacterium tumefaciens", "description": "This template will allow you to do CRISPR transformation of plants with Agrobacterium tumefaciens. The clustered regularly interspaced short palindromic repeats(CRISPR) associated Cas9/sgRNA system is a novel targeted genome-editing technique derived from the bacterial immune system. This technique enables precise genomic modifications in many different organisms and tissues.", "project_id": 223, "created_by_id": 202, "last_modified_by_id": 202, "archived": false, "archived_by_id": null, "archived_on": null, "restored_by_id": null, "restored_on": null, "created_at": "2018-11-08T10:28:00.553Z", "updated_at": "2019-02-07T14:56:54.966Z", "uuid": "1a3d39cf-ea51-48a8-b622-d7a9492fd1e7" }, "my_modules": [ { "my_module": { "id": 2089, "name": "Design and construction of gene-specific sgRNA", "due_date": null, "description": null, "x": 0, "y": 0, "my_module_group_id": 1182, "created_at": "2018-11-08T10:28:01.009Z", "updated_at": "2018-12-19T11:33:27.356Z", "archived": false, "archived_on": null, "created_by_id": 202, "last_modified_by_id": null, "archived_by_id": null, "restored_by_id": null, "restored_on": null, "workflow_order": 0, "experiment_id": 422, "state": "uncompleted", "completed_on": null }, "outputs": [ { "id": 5991, "input_id": 2090, "output_id": 2089 } ], "my_module_tags": [ ], "task_comments": [ ], "my_module_repository_rows": [ ], "user_my_modules": [ ], "protocols": [ { "protocol": { "id": 3391, "name": null, "authors": null, "description": null, "added_by_id": null, "my_module_id": 2089, "team_id": 1, "protocol_type": "unlinked", "parent_id": null, "parent_updated_at": null, "archived_by_id": null, "archived_on": null, "restored_by_id": null, "restored_on": null, "created_at": "2018-11-08T10:28:01.077Z", "updated_at": "2019-02-20T07:40:35.070Z", "published_on": null, "nr_of_linked_children": 0 }, "protocol_protocol_keywords": [ ], "steps": [ { "step": { "id": 4261, "name": "Use the online tools", "position": 1, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-11-08T10:28:01.386Z", "updated_at": "2018-12-21T13:40:38.295Z", "last_modified_by_id": 202, "protocol_id": 3391 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n\n

Paste selected sequence in the selected online tool.

Parameters that should be considered are:
\n

\n\nSelect a genome to query for potential off-target recognition and analyze it. Evaluate and prioritize targets using sequence identity as well as off-target sequence identity." }, "position": 0 } ] }, { "step": { "id": 4571, "name": "Considerations", "position": 2, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-11-15T13:25:17.662Z", "updated_at": "2019-02-20T07:41:30.052Z", "last_modified_by_id": 202, "protocol_id": 3391 }, "step_comments": [ ], "assets": [ { "asset": { "id": 3756, "created_at": "2019-02-20T07:40:34.833Z", "updated_at": "2019-02-20T07:41:30.043Z", "created_by_id": 202, "last_modified_by_id": null, "estimated_size": 15033, "lock": null, "lock_ttl": null, "version": 1, "file_processing": false, "team_id": 1 }, "asset_blob": { "filename": "Design_of_sgRNA.jpg" } } ], "step_orderable_elements": [ { "step_text": { "text": "\n

Consider choosing several different target sites, since not all sgRNAs work equally well. 
Consider using a 20 bp target site that also contains a recognition site of a commercially available restriction enzyme. The target site of the restriction enzyme should span the site of Cas9 cleavage (3 bp in front of the PAM). This will allow you to use the restriction enzyme length polymorphism assay to detect mutations in your gene of interest.

" }, "position": 0 } ] }, { "step": { "id": 4570, "name": "Selecting genomic DNA region", "position": 0, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-11-15T12:25:48.867Z", "updated_at": "2019-02-20T07:40:29.591Z", "last_modified_by_id": 202, "protocol_id": 3391 }, "step_comments": [ ], "assets": [ { "asset": { "id": 3755, "created_at": "2019-02-20T07:40:21.348Z", "updated_at": "2019-02-20T07:40:29.583Z", "created_by_id": 202, "last_modified_by_id": null, "estimated_size": 15690, "lock": null, "lock_ttl": null, "version": 1, "file_processing": false, "team_id": 1 }, "asset_blob": { "filename": "Genomic_target.jpg" } } ], "step_orderable_elements": [ { "step_text": { "text": "\n\n

Genomic DNA region must correspond to the crRNA sequence of the sgRNA:

The 3' end of the DNA target sequence must have a PAM sequence. The 20 nucleotides upstream of the PAM sequence will be your targeting sequence (crRNA), and Cas9 nuclease will cleave approximately three bases upstream of the PAM.

\n
    \n
  1. The PAM sequence itself is absolutely required for cleavage, but it is NOT part of the sgRNA sequence and therefore should not be included in the sgRNA.
  2. \n
  3. The target sequence can be on either DNA strand.
  4. \n
\n" }, "position": 0 } ] } ] } ], "results": [ ] }, { "my_module": { "id": 2090, "name": "Subcloning of the construct in plasmid", "due_date": null, "description": null, "x": 35, "y": 0, "my_module_group_id": 1182, "created_at": "2018-11-08T10:28:01.476Z", "updated_at": "2018-12-19T11:33:27.359Z", "archived": false, "archived_on": null, "created_by_id": 202, "last_modified_by_id": null, "archived_by_id": null, "restored_by_id": null, "restored_on": null, "workflow_order": 1, "experiment_id": 422, "state": "uncompleted", "completed_on": null }, "outputs": [ { "id": 5992, "input_id": 2208, "output_id": 2090 } ], "my_module_tags": [ ], "task_comments": [ ], "my_module_repository_rows": [ ], "user_my_modules": [ ], "protocols": [ { "protocol": { "id": 3393, "name": null, "authors": null, "description": null, "added_by_id": null, "my_module_id": 2090, "team_id": 1, "protocol_type": "unlinked", "parent_id": null, "parent_updated_at": null, "archived_by_id": null, "archived_on": null, "restored_by_id": null, "restored_on": null, "created_at": "2018-11-08T10:28:01.537Z", "updated_at": "2019-02-20T07:53:32.436Z", "published_on": null, "nr_of_linked_children": 0 }, "protocol_protocol_keywords": [ ], "steps": [ { "step": { "id": 4579, "name": "Verification", "position": 3, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-11-15T14:54:15.366Z", "updated_at": "2019-02-20T07:53:32.379Z", "last_modified_by_id": 202, "protocol_id": 3393 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n\n

Verify eight E. coli colonies by colony PCR using primer and your protospacer primer with the following setup.

PCR program:

\n\n

Inoculate positive colony in LB medium supplemented with Amp and grow overnight." }, "position": 0 }, { "table": { "id": 579, "created_at": "2018-11-15T14:54:15.369Z", "updated_at": "2019-02-20T07:53:32.362Z", "created_by_id": 202, "last_modified_by_id": 202, "name": "Mixture", "team_id": 1, "contents": "eyJkYXRhIjpbWyI1eCBSZWFjdGlvbiBidWZmZXIiLCI0IM68TCJdLFsicHJp\nbWVyICgxMM68TSkiLCIxzrxMIl0sWyJwcm90b3NwYWNlciBwcmltZXIgICgx\nMM68TSkiLCIxIM68TCJdLFsiZE5UUHMgKDEwbU0gZWFjaCkiLCIwLjTOvEwi\nXSxbImRkSDIwIiwiMTMuNM68TCJdLFsiVGFxICBwb2x5bWVyYXNlICg1IFUv\nzrxsKSIsIjAuMs68TCJdXX0=\n", "data_vector": "JzAuMic6MzggJzAuNCc6MjUgJzEnOjExLDE5ICcxMCc6OCwxNiAnMTBtbSc6\nMjMgJzEzLjQnOjI5ICcyNzRsJzo2LDEzLDIxLDI3LDMxLDM3LDQwICcyNzRt\nJzoxMCwxOCAnMzE2Jzo1LDksMTIsMTcsMjAsMjYsMzAsMzYsMzkgJzQnOjQg\nJzUnOjM0ICc1eCc6MSAnYnVmZmVyJzozICdkZGgyMCc6MjggJ2RudHBzJzoy\nMiAnZWFjaCc6MjQgJ3BvbHltZXJhc2UnOjMzICdwcmltZXInOjcsMTUgJ3By\nb3Rvc3BhY2VyJzoxNCAncmVhY3Rpb24nOjIgJ3RhcSc6MzIgJ3UnOjM1\n" }, "position": 1 } ] }, { "step": { "id": 4577, "name": "Transformation", "position": 2, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-11-15T14:38:34.247Z", "updated_at": "2019-02-20T07:53:25.630Z", "last_modified_by_id": 202, "protocol_id": 3393 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n

Transform competent E. coli cells with the ligation reaction and spread the transformed cells on LB agar plates supplemented with Amp.


Recipe:
15 g/L Bacto-tryptone 
5 g/L yeast extract 
5 g/L NaCl
with 200 μg/mL ampicillin

" }, "position": 0 } ] }, { "step": { "id": 4575, "name": "Digestion", "position": 1, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-11-15T14:00:37.365Z", "updated_at": "2019-02-07T13:01:07.241Z", "last_modified_by_id": 202, "protocol_id": 3393 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n

Digesting plasmid with a restriction enzyme for 3 hours at 37°C. Run it on an agarose gel and extract the vector backbone from the gel using the DNA Purification Kit.

" }, "position": 0 }, { "table": { "id": 577, "created_at": "2018-11-15T14:16:48.703Z", "updated_at": "2019-02-07T13:01:07.192Z", "created_by_id": 202, "last_modified_by_id": 202, "name": "Restriction digest", "team_id": 1, "contents": "eyJkYXRhIjpbWyJwbGFzbWlkIiwiM868ZyJdLFsiMTB4IGJ1ZmZlciIsIjXO\nvEwiXSxbImRkSDIwIiwidG8gNDnOvEwiXSxbInJlc3RyaWN0aW9uIGVuenlt\nZSAoMjAgVS/OvGwpIiwiMc68TCJdXX0=\n", "data_vector": "JzEnOjIxICcxMHgnOjUgJzIwJzoxNyAnMjc0Zyc6NCAnMjc0bCc6OSwxNCwy\nMCwyMyAnMyc6MiAnMzE2JzozLDgsMTMsMTksMjIgJzQ5JzoxMiAnNSc6NyAn\nYnVmZmVyJzo2ICdkZGgyMCc6MTAgJ2VuenltZSc6MTYgJ3BsYXNtaWQnOjEg\nJ3Jlc3RyaWN0aW9uJzoxNSAndG8nOjExICd1JzoxOA==\n" }, "position": 1 }, { "table": { "id": 677, "created_at": "2018-12-19T10:48:05.316Z", "updated_at": "2019-02-07T13:01:07.218Z", "created_by_id": 202, "last_modified_by_id": 202, "name": "Ligation", "team_id": 1, "contents": "eyJkYXRhIjpbWyJ2ZWN0b3IiLCIzMDAgbmciXSxbIkFubmVhbGVkIHByaW1l\ncnMiLCIxzrxMIl0sWyIxMHggbGlnYXRpb24gYnVmZmVyIiwiMs68TCJdLFsi\nZGRIMjAiLCJ0byAxOc68TCJdLFsiRE5BIGxpZ2FzZSIsIjHOvEwiXV19\n", "data_vector": "JzEnOjYsMjIgJzEweCc6OSAnMTknOjE3ICcyJzoxMiAnMjc0bCc6OCwxNCwx\nOSwyNCAnMzAwJzoyICczMTYnOjcsMTMsMTgsMjMgJ2FubmVhbGVkJzo0ICdi\ndWZmZXInOjExICdkZGgyMCc6MTUgJ2RuYSc6MjAgJ2xpZ2FzZSc6MjEgJ2xp\nZ2F0aW9uJzoxMCAnbmcnOjMgJ3ByaW1lcnMnOjUgJ3RvJzoxNiAndmVjdG9y\nJzox\n" }, "position": 2 } ] }, { "step": { "id": 4574, "name": "Generating double stranded DNA", "position": 0, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-11-15T13:57:43.072Z", "updated_at": "2019-02-19T14:53:00.254Z", "last_modified_by_id": 202, "protocol_id": 3393 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n\n

Order forward and reverse primers for your candidate target sequence N20 with overlaps for cloning and dilute them to a final concentration of 10 μM.
For example:
Forward primer: TTCG-GN19
Reverse primer: AAAC-N19

Pipette 10 μL of each primer dilution into a 1.5 mL microcentrifuge tube and mix.

Heat primer mix: 

\n\n
By doing so, a double-stranded DNA including your protospacer sequence will be generated." }, "position": 0 } ] } ] } ], "results": [ ] }, { "my_module": { "id": 2208, "name": "Cloning of final plant vector", "due_date": null, "description": null, "x": 69, "y": 0, "my_module_group_id": 1182, "created_at": "2018-11-16T10:48:03.813Z", "updated_at": "2018-12-19T11:33:27.361Z", "archived": false, "archived_on": null, "created_by_id": 202, "last_modified_by_id": 202, "archived_by_id": null, "restored_by_id": null, "restored_on": null, "workflow_order": 2, "experiment_id": 422, "state": "uncompleted", "completed_on": null }, "outputs": [ { "id": 5993, "input_id": 2091, "output_id": 2208 } ], "my_module_tags": [ ], "task_comments": [ ], "my_module_repository_rows": [ ], "user_my_modules": [ ], "protocols": [ { "protocol": { "id": 3583, "name": null, "authors": null, "description": null, "added_by_id": null, "my_module_id": 2208, "team_id": 1, "protocol_type": "unlinked", "parent_id": null, "parent_updated_at": null, "archived_by_id": null, "archived_on": null, "restored_by_id": null, "restored_on": null, "created_at": "2018-11-16T10:48:03.821Z", "updated_at": "2019-02-19T14:55:34.883Z", "published_on": null, "nr_of_linked_children": 0 }, "protocol_protocol_keywords": [ ], "steps": [ { "step": { "id": 4621, "name": "Transformation of E.coli cells", "position": 2, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-11-16T12:34:00.600Z", "updated_at": "2019-02-19T14:54:57.484Z", "last_modified_by_id": 202, "protocol_id": 3583 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n\n

Transform competent E. coli cells with the reaction mixture and spread the transformed cells on LB agar plates supplemented with Kan.

LB-medium:

\n\n" }, "position": 0 } ] }, { "step": { "id": 4618, "name": "Amplify the sgRNA cassette", "position": 1, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-11-16T11:50:40.125Z", "updated_at": "2019-02-19T14:54:39.448Z", "last_modified_by_id": 202, "protocol_id": 3583 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n\n

Amplify it from a plasmid with target sequence using primers and polymerase and then extract the sgRNA cassette from the gel. Assemble the plasmid backbone and the sgRNA cassette to generate the final vector using Assembly Master Mix according to the manufacturer’s instruction with an incubation time of 1 h.

PCR program: 

\n\n" }, "position": 0 }, { "table": { "id": 583, "created_at": "2018-11-16T12:13:30.368Z", "updated_at": "2019-02-19T14:54:39.422Z", "created_by_id": 202, "last_modified_by_id": 202, "name": "Mixture", "team_id": 1, "contents": "eyJkYXRhIjpbWyI1eCBQaHVzaW9uIEhGLWJ1ZmZlciIsIjEwzrxMIl0sWyJG\nb3J3YXJkIHByaW1lciAoMTDOvE0pIiwiMi41zrxMIl0sWyJSZXZlcnNlIHBy\naW1lciAoMTDOvE0pIiwiMi41zrxMIl0sWyJkTlRQcyAoMTBtTSBlYWNoKSIs\nIjHOvEwiXSxbImRkSDJPIiwiMzPOvEwiXSxbInBGSDYgd2l0aCB0YXJnZXQg\nc2VxdWVuY2UgKDEwMCBuZy/OvGwpIiwiMC41zrxMIl0sWyJQaHVzaW9uIERO\nQSBwb2x5bWVyYXNlICgyVS/OvGwpIiwiMC41zrxMIl1dfQ==\n", "data_vector": "JzAuNSc6NDMsNTIgJzEnOjI4ICcxMCc6NiwxMSwxOSAnMTAwJzozOSAnMTBt\nbSc6MjYgJzIuNSc6MTQsMjIgJzI3NGwnOjgsMTYsMjQsMzAsMzQsNDIsNDUs\nNTEsNTQgJzI3NG0nOjEzLDIxICcydSc6NDkgJzMxNic6NywxMiwxNSwyMCwy\nMywyOSwzMyw0MSw0NCw1MCw1MyAnMzMnOjMyICc1eCc6MSAnYnVmZmVyJzo1\nICdkZGgybyc6MzEgJ2RuYSc6NDcgJ2RudHBzJzoyNSAnZWFjaCc6MjcgJ2Zv\ncndhcmQnOjkgJ2hmJzo0ICdoZi1idWZmZXInOjMgJ25nJzo0MCAncGZoNic6\nMzUgJ3BodXNpb24nOjIsNDYgJ3BvbHltZXJhc2UnOjQ4ICdwcmltZXInOjEw\nLDE4ICdyZXZlcnNlJzoxNyAnc2VxdWVuY2UnOjM4ICd0YXJnZXQnOjM3ICd3\naXRoJzozNg==\n" }, "position": 1 } ] }, { "step": { "id": 4623, "name": "Inoculation", "position": 4, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-11-16T12:53:26.057Z", "updated_at": "2019-02-19T14:55:34.834Z", "last_modified_by_id": 202, "protocol_id": 3583 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n\n

Inoculate positive colony into LB medium supplemented with Kan and let grow overnight.

LB-medium:

\n\n" }, "position": 0 } ] }, { "step": { "id": 4616, "name": "Digestion", "position": 0, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-11-16T10:52:10.517Z", "updated_at": "2019-02-19T14:53:39.075Z", "last_modified_by_id": 202, "protocol_id": 3583 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n\n

Digestion of a plasmid with restriction enzymes.

Conditions:

\n\n
Run it on an agarose gel and extract the vector backbone from the gel using DNA Purification Kit." }, "position": 0 }, { "table": { "id": 582, "created_at": "2018-11-16T10:52:10.520Z", "updated_at": "2019-02-19T14:53:39.061Z", "created_by_id": 202, "last_modified_by_id": 202, "name": "Digestion mixture", "team_id": 1, "contents": "eyJkYXRhIjpbWyJwbGFzbWlkIiwiM868ZyJdLFsiMTB4IGJ1ZmZlciIsIjXO\nvEwiXSxbImRkSDIwIiwidG8gNDjOvEwiXSxbInJlc3RyaWN0aW9uIGVuenlt\nZSAoMjAgVS/OvEwpIiwiMc68TCJdLFsicmVzdHJpY3Rpb24gZW56eW1lKDIw\nIFUvzrxMKSIsIjHOvEwiXV19\n", "data_vector": "JzEnOjIxLDMwICcxMHgnOjUgJzIwJzoxNywyNiAnMjc0Zyc6NCAnMjc0bCc6\nOSwxNCwyMCwyMywyOSwzMiAnMyc6MiAnMzE2JzozLDgsMTMsMTksMjIsMjgs\nMzEgJzQ4JzoxMiAnNSc6NyAnYnVmZmVyJzo2ICdkZGgyMCc6MTAgJ2Vuenlt\nZSc6MTYsMjUgJ3BsYXNtaWQnOjEgJ3Jlc3RyaWN0aW9uJzoxNSwyNCAndG8n\nOjExICd1JzoxOCwyNw==\n" }, "position": 1 } ] }, { "step": { "id": 4622, "name": "Verification", "position": 3, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-11-16T12:46:27.019Z", "updated_at": "2019-02-19T14:55:10.602Z", "last_modified_by_id": 202, "protocol_id": 3583 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n\n

Verify eight positive colonies by colony PCR.

PCR program:

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With 1 μg of (pUB-Cas9-gene of interest) and spread the transformed cells on YEP agar plates supplemented with Rif/Gent/Kan.

Recipe: 

\n\n
With 150 μg/mL rifampicin, 50 μg/mL gentamycin sulfate, 50 μg/mL kanamycin.

Coculture growth:
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Verify plasmid presence in at least three colonies by colony PCR.

\n
PCR program:
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Transform plants using Agrobacterium-mediated T-DNA transfer with the floral dipping method.
Simple dipping of developing floral tissues into a solution containing Agrobacterium tumefaciens, 5% sucrose and 500 microliters per litre of surfactant Silwet L‐77. Sucrose and surfactant are critical to the success of the floral dip method. Plants inoculated when numerous immature floral buds and few siliques are present produce transformed progeny at the highest rate. 

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PCR/RE Genotyping: Genotyping of clones with targeted mutation
DNA sequencing

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The transgene free homozygous mutants with desired genetic modifications at the targeted loci could be selected by selfing of GE0 generation plants and after segregation of the transgene in the next GE1 generation. The GE plants could be selected by PCR/RE genotyping and DNA sequencing of clones and negatively selecting for the transgene free plants with desired modification in the first generation only.

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