TABLE 1.
Primer sequences for sgRNA cloning and validation.
Step | Primer | Sequence (5′-3′) | Purpose |
---|---|---|---|
5A(iii) | U6-Fwd | GAGGGCCTATTTCCCATGATTCC | Amplify any U6-sgRNA |
5A(iii) | U6-Rev | AAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAcNNNNNNNNNNNNNNNNNNNCCGGTGTTTCGTCCTTTCCACAAG | Amplify specifically designed U6-sgRNA; N is the reverse complement of target; appended cytosine (complementary to appended guanine) in lowercase |
5B(i) | sgRNA-top | CACCgNNNNNNNNNNNNNNNNNNN | Clone sgRNA into pSpCas9(BB); appended guanine in lowercase |
5B(i) | sgRNA-bottom | AAACNNNNNNNNNNNNNNNNNNNc | Clone sgRNA into pSpCas9(BB); appended cytosine (complementary to appended guanine) in lowercase |
117 | pUC-Fwd (M13 -20 primer) | GTAAAACGACGGCCAGT | Sanger sequencing of modified genomic regions cloned into pUC19 |
117 | pUC-Rev (M13 -26 primer) | CAGGAAACAGCTGTAAC | Sanger sequencing of modified genomic regions cloned into pUC19 |