Identify 23bp CRISPR Targeting sequence in the form N20NGG:
5’- NNNNNNNNNNNNNNNNNNNN NGG -3’ ____________________ ___ targetsite PAM
*Note the PAM sequence is not incorporated into the gRNA
Design oligos to insert 20bp target sequence into H1 promoter vector via Gibson Assembly:
5’- NNNNNNNNNNNNNNNNNNNN -3’ |||||||||||||||||||| 3’- nnnnnnnnnnnnnnnnnnnn -5’
*N corresponds to N20 target site (of N20NGG);
*n corresponds to complement sequence
Forward oligo; CTTATAAGTTCTGTATGAGACCACTTTTTCCCNNNNNNNNNNNNNNNNNNNNG
Reverse oligo; CCTTATTTTAACTTGCTATTTCTAGCTCTAAAACnnnnnnnnnnnnnnnnnnnnG
PCR reaction to anneal and extend the two oligos as below:
CTTATAAGTTCTGTATGAGACCACTTTTTCCCNNNNNNNNNNNNNNNNNNNNG |||||||||||||||||||||| GnnnnnnnnnnnnnnnnnnnnCAAAATCTCGATCTTTATCGTTCAATTTTATTCC
CTTATAAGTTCTGTATGAGACCACTTTTTCCCNNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GAATATTCAAGACATACTCTGGTGAAAAAGGGnnnnnnnnnnnnnnnnnnnnCAAAATCTCGATCTTTATCGTTCAATTTTATTCC ------------------------------- ---------------------------------- overlap overlap
Digest pH1v1 with AvrII
Avr II ------ TGGGAATCTTATAAGTTCTGTATGAGACCACTTTTTCCCTAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCC ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ACCCTTAGAATATTCAAGACATACTCTGGTGAAAAAGGGATCCAAAATCTCGATCTTTATCGTTCAATTTTATTCCGATCAGG
TGGGAATCTTATAAGTTCTGTATGAGACCACTTTTTCC CTAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCC |||||||||||||||||||||||||||||||||||||| ||||||||||||||||||||||||||||||||||||||||| ACCCTTAGAATATTCAAGACATACTCTGGTGAAAAAGGGATC CAAAATCTCGATCTTTATCGTTCAATTTTATTCCGATCAGG
Gibson Assembly
5'->3 vector resection:
TTGGGAATCTTATAAGTTCTGTATGAGACCACTTTTTCC CTAGTCCGTTA |||||||| ||||||||||| AACCCTTA CAAAATCTCGATCTTTATCGTTCAATTTTATTCCGATCAGGCAAT
5'->3' oligo resection:
CNNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG |||||||||||||||||||||| GAATATTCAAGACATACTCTGGTGAAAAAGGGnnnnnnnnnnnnnnnnnnnnC
annealing, extension, and ligation:
H1 promoter gRNA Scaffold ------------------------------------- ----------------------------> GGAATCTTATAAGTTCTGTATGAGACCACTTTTTCCCNNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAA |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CCTTAGAATATTCAAGACATACTCTGGTGAAAAAGGGnnnnnnnnnnnnnnnnnnnnCAAAATCTCGATCTTTATCGTTCAATTTT -------------------- target
Sequence verify with M13R:
Final sequence (+ strand)
GGAATTCGAACGCTGACGTCATCAACCCGCTCCAAGGAATCGCGGGCCCAGTGTCACTAG GCGGGAACACCCAGCGCGCGTGCGCCCTGGCAGGAAGATGGCTGTGAGGGACAGGGGAGT GGCGCCCTGCAATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAAT GTCTTTGGATTTGGGAATCTTATAAGTTCTGTATGAGACCACTTTTTCCCNNNNNNNNNN NNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC TTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT
Final sequence (- strand)
AAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAAC TTGCTATTTCTAGCTCTAAAACnnnnnnnnnnnnnnnnnnnnGGGAAAAAGTGGTCTCAT ACAGAACTTATAAGATTCCCAAATCCAAAGACATTTCACGTTTATGGTGATTTCCCAGAA CACATAGCGACATGCAAATATTGCAGGGCGCCACTCCCCTGTCCCTCACAGCCATCTTCC TGCCAGGGCGCACGCGCGCTGGGTGTTCCCGCCTAGTGACACTGGGCCCGCGATTCCTTG GAGCGGGTTGATGACGTCAGCGTTCGAATTCC
truncated T7 promoter gRNA Scaffold ------------- ---------------------------------------------------------------> ATCGATAATACGACTCACCTAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCA ------ AvrII
Identify 23bp CRISPR Targeting sequence in the form GN19NGG:
5’- GNNNNNNNNNNNNNNNNNNN NGG -3’ ____________________ ___ targetsite PAM
*Note the PAM sequence is not incorporated into the gRNA
Design oligos to insert 20bp target sequence into pT7gRNA via Gibson Assembly:
5’- GNNNNNNNNNNNNNNNNNNN -3’ |||||||||||||||||||| 3’- Cnnnnnnnnnnnnnnnnnnn -5’
*N corresponds to N20 target site (of N20NGG);
*n corresponds to complement sequence
Forward oligo; CCATCGATAATACGACTCACTATAGNNNNNNNNNNNNNNNNNNNNG
Reverse oligo; AACTTGCTATTTCTAGCTCTAAAACnnnnnnnnnnnnnnnnnnnnC
PCR reaction to anneal and extend the two oligos as below:
CCATCGATAATACGACTCACTATAGNNNNNNNNNNNNNNNNNNNNG |||||||||||||||||||||| CnnnnnnnnnnnnnnnnnnnnCAAAATCTCGATCTTTATCGTT
CCATCGATAATACGACTCACTATAGNNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAA ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GGTAGCTATTATGCTGAGTGATATCnnnnnnnnnnnnnnnnnnnnCAAAATCTCGATCTTTATCGTT -------------------- ---------------------- overlap overlap
Digest pT7gRNA with AvrII
Avr II ------ AGCAGATTGTACTGAGAGTGCACCATCGATAATACGACTCACCTAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGT ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| TCGTCTAACATGACTCTCACGTGGTAGCTATTATGCTGAGTGGATCCAAAATCTCGATCTTTATCGTTCAATTTTATTCCGATCA
AGCAGATTGTACTGAGAGTGCACCATCGATAATACGACTCAC CTAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC |||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||||||||||||||||||| TCGTCTAACATGACTCTCACGTGGTAGCTATTATGCTGAGTGGATC CAAAATCTCGATCTTTATCGTTCAATTTTATTCCG
Gibson Assembly
5'->3 vector resection:
GAGCAGATTGTACTGAGAGTGCACCATCGATAATACGACTCAC AATAAGGCTAGTCC |||||||| |||||||||||||| AACCCTTA CAAAATCTCGATCTTTATCGTTCAATTTTATTCCGATCAGG
5'->3' oligo resection:
GNNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAA |||||||||||||||||||||| GGTAGCTATTATGCTGAGTGATATCnnnnnnnnnnnnnnnnnnnnC
annealing, extension, and ligation:
T7 promoter gRNA Scaffold ----------------- ----------------------------------------------> ATAATACGACTCACTATAGNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATC ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| TATTATGCTGAGTGATATCnnnnnnnnnnnnnnnnnnnCAAAATCTCGATCTTTATCGTTCAATTTTATTCCGATCAGGCAATAG -------------------- target
Sequence verify with M13R:
Final sequence (+ strand)
CCATCGATAATACGACTCACTATAGNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAA ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGT GCGGCCGC
Final sequence (- strand)
GCGGCCGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAA CTTGCTATTTCTAGCTCTAAAACnnnnnnnnnnnnnnnnnnnCTATAGTGAGTCGTATT ATCGATGG