Scalable characterization of the PAM requirements of CRISPR-Cas enzymes using HT-PAMDA.
Walton RT, Hsu JY, Joung JK, Kleinstiver BP
Nat Protoc. 2021 Feb 5. pii: 10.1038/s41596-020-00465-2. doi: 10.1038/s41596-020-00465-2.
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Plasmids from Article
| ID | Plasmid | Purpose |
|---|---|---|
| 160132 | p11-3’_8xN_PAM-site1 (RTW554) | Plasmid library harboring a randomized 8nt PAM 3’ of target site 1. Can be used to profile CRISPR-Cas PAM requirements of enzymes that recognize 3’ PAMs (e.g. Cas9) |
| 160133 | p11-3’_8xN_PAM-site2 (RTW555) | Plasmid library harboring a randomized 8nt PAM 3’ of target site 2. Can be used to profile CRISPR-Cas PAM requirements of enzymes that recognize 3’ PAMs (e.g. Cas9) |
| 160135 | p11-5’_10xN_PAM-site4 (RTW574) | Plasmid library harboring a randomized 10nt PAM 5’ of target site 4. Can be used to profile CRISPR-Cas PAM requirements of enzymes that recognize 5’ PAMs (e.g. Cas12a) |
| 160136 | pT7-SpCas9_sgRNA-site1 (RTW443) | T7 promoter expression plasmid for in vitro transcription of SpCas9 sgRNA with spacer #1 |
| 160137 | pT7-SpCas9_sgRNA-site2 (RTW448) | T7 promoter expression plasmid for in vitro transcription of SpCas9 sgRNA with spacer #2 |
| 160138 | pT7-AsCas12a_crRNA-site3 (RTW549) | T7 promoter expression plasmid for in vitro transcription of AsCas12a crRNA with spacer #3 |
| 160139 | pT7-AsCas12a_crRNA-site4 (MSP3511) | T7 promoter expression plasmid for in vitro transcription of AsCas12a crRNA with spacer #4 |
| 160140 | pCMV-T7-AsCas12a-P2A-EGFP (RTW2861) | CMV and T7 promoter expression plasmid for human codon optimized AsCas12a with a c-terminal NLS(nucleoplasmin), 3x HA tag, and P2A-EGFP |