Engineered dual selection for directed evolution of SpCas9 PAM specificity.
Goldberg GW, Spencer JM, Giganti DO, Camellato BR, Agmon N, Ichikawa DM, Boeke JD, Noyes MB
Nat Commun. 2021 Jan 13;12(1):349. doi: 10.1038/s41467-020-20650-x.
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Plasmids from Article
| ID | Plasmid | Purpose |
|---|---|---|
| 165486 | pGG438 | Vector for expression of the SpCas9 VRKG variant in human cells: CMV-T7-humanVRKG(SpCas9, D1135V/S1136R/D1332K/R1333G)-1xNLS(SV40)-3xFLAG |
| 165604 | pGG184 | Reporter plasmid for B1H-dependent expression of the HIS3/GFP operon with a single hEGFP protospacer: PS1(upstream of promoter with 'CAGCG' PAM)-lac-HIS3 and GFP |
| 165605 | pGG198 | Reporter plasmid for B1H-dependent expression of the HIS3/GFP operon with two hEGFP protospacers: PS1(upstream of promoter with 'CAGCG' PAM)-lac-PS2(downstream with 'CGGCG' PAM)-HIS3 and GFP |
| 165606 | pGG369 | Vector for expression of ω-dCas9 and sgRNA in E. coli: lacUV2-ω-1xFLAG-dCas9(Wt PID)-1xNLS-3xHA-1xNLS and UV5-BsaI-sgRNA (BsaI sites in place of the spacer) |
| 165607 | pGG422_UV5 | Vector for expression of the SpCas9 KG variant with sgRNA in E. coli: KG(SpCas9, D1332K/R1333G) and UV5-sgRNA (hEGFP spacer) |
| 165608 | pGG426_UV5 | Vector for expression of the SpCas9 VRKG variant with sgRNA in E. coli: lacUV5-VRKG(SpCas9, D1135V/S1136R/D1332K/R1333G) and UV5-sgRNA (hEGFP spacer) |
| 165612 | pNA0304 | Vector for expression of sgRNAs in yeast: SNR52p-NotI-sgRNA-SUP4t (NotI site in place of the spacer) |
| 165615 | pGG431 | Vector for expression of SpCas9 in yeast after assembly with PID fragments: TEF1p-SpCas9::KanR-1xNLS-CYC1t (KanR cassette in place of the PID) |
| 165616 | pGG442 | Vector for expression of SpCas9-Zif268 in yeast after assembly with PID fragments: TEF1p-SpCas9::KanR-1xNLS-3xHA-1xNLS-Zif268-1xNLS-CYC1t (KanR cassette in place of the PID) |