1. Gene Editing Design Demo
-GenomeEditor 2.0
EGFR Gene Editing
Example 1: Create (2369C>T) T790M mutation
Example 2: Knockout/knockin: delete a portion of exon #2
and insert a drug selection cassette
Example 3: Add a HA Tag to the C-terminus of EGFR
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2. Data preparation: starting with the sequence ID: NM_005228
Click on Submit
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3. Retrieved EGFR sequence info
Click on Process Data to record the sequence info
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4. Recorded sequence data
Click on Blast Genome to the blast page
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5. Launch Blast Human Genome Database
Click on Blast and wait for the data to return
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6. Returned Blast data Click on Process Data to record the blast data
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7. Recorded Blast data Step 1. Click on select Exon Group
Step 2. Click on Verify Exons
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8. Data preparation completed
Defined EGFR exons and introns
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9. Export to Gene Editing Design Form
Select Gene editing design
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10. Gene Editing Design Form
Mapped amino acid locations
Mapped exon and intron locations
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11. Select a focal point and download the extended sequence
Example 1: Creation of (2369C>T) T790M mutation
Select a focal point and download the extended sequence
Step 1: Select 790 aa as focal point
Step 2: Click on Download sequence and wait download to complete
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12. Select the sequence to design gRNA oligos for cloning
Example 1: Creation of (2369C>T) T790M mutation
Select the sequence to design gRNA oligos for cloning
Step 1: Select -75/+75 item (75 bp up stream and 75 bp down stream of the focal point)
Step 2: Click on Export to gRNA selection
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13. gRNA selection form Example 1: Creation of (2369C>T) T790M mutation
Select a gRNA sequence near the focal point
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14. Select vector system to design oligos for cloning
Example 1: Creation of (2369C>T) T790M mutation
Select vector system to design oligos for cloning
Step 1: Select a vector system
Step 2: Design oligos
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15. Designed Oligos for selected vector
Example 1: Creation of (2369C>T) T790M mutation
Designed Oligos for selected vector
Forward and reverse complement oligos for gRNA cloning. Copy and save for oligo ordering
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16. Design homologous donor oligo with C to T mutation
Example 1: Creation of (2369C>T) T790M mutation
Design homologous donor oligo with C to T mutation
Select a sequence (-/+75) for editing
+75 nn highlighted
Target aa (T)
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17. Design homologous donor oligo with C to T mutation
Example 1: Creation of (2369C>T) T790M mutation
Design homologous donor oligo with C to T mutation
Make the change ACG (T) to ATG (M) using key board
then highlight +75 nn
C to T
T changed to M
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18. Save or copy the edited sequence as the donor sequence
Example 1: Creation of (2369C>T) T790M mutation
Save or copy the edited sequence as the donor sequence
copy
Reverse complement
Highlight the entire sequence. This is the plus donor sequence. Select Reverse Complement menu to get the reverse complemental strand. Both can be copied and saved for ordering.
Export sequence
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19. Design PCR primers for validating the editing
Example 1: Creation of (2369C>T) T790M mutation
Design PCR primers for validating the editing
Step 1: Select -/+ 300 item for primer design
Step 2: Click on Export to Primer Design
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20. Primer design form Example 1: Creation of (2369C>T) T790M mutation
Step 2: Click on design
Step 1: Set the locations of forward and reverse primer regions by moving the vertical bars
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21. Designed PCR primers for a regular or nested PCR
Example 1: Creation of (2369C>T) T790M mutation
Designed PCR primers for a regular or nested PCR
PCR primer sequences, copy and save for ordering
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22. Summary: Oligos for EGFR T790M editing
Example 1: Creation of (2369C>T) T790M mutation
Summary: Oligos for EGFR T790M editing
-gRNA cloning to Zhang lab- pX330 :
Forward: 5’ caccgTGCAACTCATCACGCAGCTC 3’
Reverse complement: 5’ aaacGAGCTGCGTGATGAGTTGCAc 3’
-Homologous repair donor:
Forward:
5’GTGATGGCCAGCGTGGACAACCCCCACGTGTGCCGCCTGCTGGGCATCTGCCTCACCTCCACCGTGCAACTCATCAtGCAGCTCATGCCCTTCGGCTGCCTCCTGGACTATGTCCGGGAACACAAAGACAATATTGGCTCCCAGTACCTG 3’
Reverse Complement:
5’CAGGTACTGGGAGCCAATATTGTCTTTGTGTTCCCGGACATAGTCCAGGAGGCAGCCGAAGGGCATGAGCTGCATGATGAGTTGCACGGTGGAGGTGAGGCAGATGCCCAGCAGGCGGCACACGTGGGGGTTGTCCACGCTGGCCATCAC 3’
-PCR primers for editing validation:
Num Name Oligo Sequence Direction Location Amplicom Size
Pair 1 NC_ _1_F CTGTGCTAGGTCTTTTGCAGGC Forward 61 -
Pair 1 NC_ _1_R GCAGATGGGACAGGCACTGAT Reverse
Pair 2 NC_ _2_F GGCACAGCTTTTCCTCCATGAG Forward 80 -
Pair 2 NC_ _2_R GGCAAACTCTTGCTATCCCAGG Reverse
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23. Design a knockout/knockin
Example 2: Knockout/knockin within exon 2
Design a knockout/knockin
Step 2: Click download sequence
Step 1: Select exon 2 start as the focal point
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24. gRNA design Example 2: Knockout/knockin Set 200bp sequence
Click on export to gRNA design
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25. Design oligos for gRNA construct
Example 2: Knockout/knockin
Design oligos for gRNA construct
Select gRNA location
Select Vector system
Oligos for cloning
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26. Design homologous Donor construct
Example 2: Knockout/knockin
Design homologous Donor construct
Step 2: Select 5’ Arm
Step 1: Check Assembly
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27. Design donor construct
Example 2: Knockout/knockin
Design donor construct
Select a predesigned CMV-eGFP-IRES-Puro cassette
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28. Design donor construct
Example 2: Knockout/Knockin
Design donor construct
Select 3’-Arm
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29. Donor construct completed
Example 2: Knockout/Knockin
Donor construct completed
Export and save the donor sequence
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30. Export the donor sequence to PCR primer design
Example 2: Knockout/Knockin
Export the donor sequence to PCR primer design
Step 1: Click on Export to Primer Design
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31. Design PCR primers for validating knockout/knockin at 5’ integration
Example 2: Knockout/Knockin within Exon 2
Design PCR primers for validating knockout/knockin at 5’ integration
Outside 3’Arm
Outside 5’ Arm
5’Arm
3’Arm
cassette
Step 1: click
Step 2: Click on Design
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32. Designed PCR primers for validating knockout/knockin at 5’ integration
Example 2: Knockout/Knockin
Designed PCR primers for validating knockout/knockin at 5’ integration
For 3’ integration detection primer design
Designed primers for one step or nested PCR. Copy and save oligos for 5’ integration detection
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33. Summary: Oligos for knockout/knockin editing
Example 2: knockout/knockin
Summary: Oligos for knockout/knockin editing
-gRNA cloning to Zhang lab- PX462:
Forward: caccgAATAACTGTGAGGTGGTCCT
Reverse complement: aaacAGGACCACCTCACAGTTATTc
-Homologous repair donor:
Sequence not shown
-PCR primers for knockout/knockin (5’ integration) validation:
# Name Oligo Sequence Direction Location Amplicom Size
Pair 1 NC_ _1_F TGGTGAGGGAAGTAACTGTGCC Forward 877 -
Pair 1 NC_ _1_R CCTGAGGAGTTAATTTCCGAGAG Reverse
Pair 2 NC_ _2_F ATGGAGTGTGGACGAGCATAGG Forward 923 -
Pair 2 NC_ _2_R GTTCCCAGCACTGCCCCTCT Reverse
* PCR primers for 3’ integration detection can be designed by clicking on 3’ Integration Detection
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34. Design of HA tag insertion at c-terminus
Example 3: add HA Tag to the EGFR C-terminus
Design of HA tag insertion at c-terminus
Step 2: Click on Download Sequence
Step 1. Select stop codon as the focal point
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35. Select sequence for gRNA design and cloning
Example 3: add HA Tag to the EGFR C-terminus
Select sequence for gRNA design and cloning
Step 1: Select -/+75 and highlight all sequence
Step 2: Export to gRNA design
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36. gRNA selection and oligos for gRNA cloning
Example 3: add HA Tag to the EGFR C-terminus
gRNA selection and oligos for gRNA cloning
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37. Design homologous donor with a HA tag
Example 3: Add a HA Tag to the EGFR C-terminus
Design homologous donor with a HA tag
Step 1: Select -/+75 item
Step 3: Select HA-Tag
Step 2: Put the cursor right before the stop codon
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38. Design homologous donor with a HA tag
Example 3: add HA Tag to the EGFR C-terminus
Design homologous donor with a HA tag
HA tag nn sequence
HA-Tag aa sequence
Export and save sequence for homologous donor repair
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39. Design PCR primers for detecting HA tag insertion
Example 3: add HA Tag to the EGFR C-terminus
Design PCR primers for detecting HA tag insertion
1. Select -/+300 item
2. Export to Primer Design to design PCR primers for validating tag insertion as described in example 1
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40. Design PCR primers for detecting HA tag insertion
Example 3: add HA Tag to the EGFR C-terminus
Design PCR primers for detecting HA tag insertion
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41. Summary: Oligos for HA tag insertion at C terminus
Example 3: add HA Tag to the EGFR C-terminus
Summary: Oligos for HA tag insertion at C terminus
-gRNA cloning to Zhang lab- pX330 :
Forward: 5' caccgAATTTATTGGAGCATGACCA 3'
Reverse complement: 5' aaacTGGTCATGCTCCAATAAATTc 3'
-Homologous repair donor:
Forward:
5’ATCTTTAAGGGCTCCACAGCTGAAAATGCAGAATACCTAAGGGTCGCGCCACAAAGCAGTGAATTTATTGGAGCAtacccatacgatgttccagattacgctTGACCACGGAGGATAGTATGAGCCCTAAAAATCCAGACTCTTTCGATACCCAGGACCAAGCCACAGCAGGTCCTC 3’
Reverse Complement:
5’GAGGACCTGCTGTGGCTTGGTCCTGGGTATCGAAAGAGTCTGGATTTTTAGGGCTCATACTATCCTCCGTGGTCAAGCGTAATCTGGAACATCGTATGGGTATGCTCCAATAAATTCACTGCTTTGTGGCGCGACCCTTAGGTATTCTGCATTTTCAGCTGTGGAGCCCTTAAAGAT 3’
Primers for validation:
Num Name Oligo_Sequence Direction Location PCR amplicom
Pair 1 NC_ _1_F CAGCAGAGACCCACACTACCA Forward 27 -
Pair 1 NC_ _1_R GCAACTTCCCAAAATGTGCCCG Reverse
Pair 2 NC_ _2_F CCCGAGTATCTCAACACTGTCC Forward 76 -
Pair 2 NC_ _2_R AATGTGCCCGAGGTGGAAGTAC Reverse
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42. Export any region of a genomic sequence with coding annotation
Other useful function
Export any region of a genomic sequence with coding annotation
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43. Design qPCR primer cross intron-exon
Other useful function
Design qPCR primer cross intron-exon
Intron location and length
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44. Feedback Support@geneharbor.com Toll free 1-800-710-3618
Fax
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