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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.
PubMed Article

Plasmids from Article

ID Plasmid Purpose  
160132p11-3’_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)
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160133p11-3’_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)
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160134p11-5’_10xN_PAM-site3 (RTW572)Plasmid library harboring a randomized 10nt PAM 5’ of target site 3. Can be used to profile CRISPR-Cas PAM requirements of enzymes that recognize 5’ PAMs (e.g. Cas12a)
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160135p11-5’_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)
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160136pT7-SpCas9_sgRNA-site1 (RTW443)T7 promoter expression plasmid for in vitro transcription of SpCas9 sgRNA with spacer #1
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160137pT7-SpCas9_sgRNA-site2 (RTW448)T7 promoter expression plasmid for in vitro transcription of SpCas9 sgRNA with spacer #2
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160138pT7-AsCas12a_crRNA-site3 (RTW549)T7 promoter expression plasmid for in vitro transcription of AsCas12a crRNA with spacer #3
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160139pT7-AsCas12a_crRNA-site4 (MSP3511)T7 promoter expression plasmid for in vitro transcription of AsCas12a crRNA with spacer #4
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160140pCMV-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
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