pNTI647 Expression of the CRISPRi effector protein dCas9-Mxi1, along with the TetR regulator controlling guide expression, is provided by pNTI647. This integrating plasmid is based on the KanR-marked EasyClone 2.0 vector pCfB2225.
pNTI661 The base guide RNA expression vector is an episomal, Leu-marked plasmid. Barcoded guide RNA libraries are constructed in a two-step process.
Guide RNAs are introduced by digesting the pNTI661 vector with BamHI and HindIII, amplifying the guide RNA pool using primers NM636 and NM637, and performing Gibson-style assembly (we like NEBuilder HiFi) to introduce guide RNAs into the plasmid:
pNTI661 …cgcggctgggaacgaaactctgggagctgcgattgg/gatcctcgaagct/ttttagagctagaaatagcaagttaaaataaggctag… digest …cgcggctgggaacgaaactctgggagctgcgattgg gatcctcgaagct ttttagagctagaaatagcaagttaaaataaggctag… ||||||||||||||||||||||||||||||||| |||||||||||||||||||||||||||||||||| PCR 5'-ggctgggaacgaaactctgggagctgcgattggcaCATTTCATTACCCGCAGAGCgttttagagctagaaatagcaagttaaaataaggc-3' NM636 ggctgggaacgaaactctgggagctgcgattggca pool tctgggagctgcgattggcaCATTTCATTACCCGCAGAGCgttttagagctagaaatagc NM637 reverse complement gttttagagctagaaatagcaagttaaaataaggc
After the guide library is cloned and amplified, barcodes are introduced in a second Gibson-style assembly reaction. The library is digested with SphI, leaving 3’ overhangs that are not resected during the assembly reaction. The barcodes are amplified from a degenerate oligo template using primers that introduce the Illumina TruSeq Read 1 primer site and a T7 RNA polymerase promoter.
pNTI661 …AGCTATCAGGCGCGCCACTTCACG/CATG/CTCAAGAGCTCGATCCGCAGGC… digest …AGCTATCAGGCGCGCCACTTCACGCATG CATGCTCAAGAGCTCGATCCGCAGGC… ||||||||||||||||||||||||| ||||||||||||||||||||||| PCR 5'-TATCAGGCGCGCCACTTCACGCATGCNNNNNNNNNNNNNNNNNNNNNNNNNAGATCGGAAGAGCGTCGTGCTATAGTGAGTCGTATTACATGCTCAAGAGCTCGATCCGCA-3' NI-1027 TATCAGGCGCGCCACTTCACGCATGC NI-1026 CGCCACTTCACGCATGCNNNNNNNNNNNNNNNNNNNNNNNNNAGATCGGAAGAGCGTCGT NI-1041 reverse complement AGATCGGAAGAGCGTCGTGCTATAGTGAGTCGTATTACATGCTCAAGAGCTCGATCCGCA