Custom CRISPR-Cas9 PAM variants via scalable engineering and machine learning.
Silverstein RA, Kim N, Kroell AS, Walton RT, Delano J, Butcher RM, Pacesa M, Smith BK, Christie KA, Ha LL, Meis RJ, Clark AB, Spinner AD, Lazzarotto CR, Li Y, Matsubara A, Urbina EO, Dahl GA, Correia BE, Marks DS, Tsai SQ, Pinello L, De Ravin SS, Liu Q, Kleinstiver BP
Nature. 2025 Apr 22. doi: 10.1038/s41586-025-09021-y.
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PubMed
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Article
Plasmids from Article
| ID | Plasmid | Purpose |
|---|---|---|
| 223065 | pACYC-T7-SpCas9(BspMI/SapI/BsaI_cassettes)-T7-gRNA1 (BPK1807) | Dual pT7 entry plasmid for SpCas9(6AA) library; bacterial expression plasmid with type IIS RE cassettes around D1135/S1136, G1218/E1219, R1335/T1337 (precursor to MMW94). Expresses a gRNA. |
| 223067 | pCAG-SpCas9-P2A-EGFP (MSP2582) | pCAG human codon optimized wild-type SpCas9 expression plasmid with C-terminal NLS(SV40)-3xFLAG-P2A-EGFP |
| 223068 | pCMV-T7-SpCas9(MRRWMR)-P2A-EGFP (RAS2816) | pCMV and pT7 Human expression plasmid for SpCas9 enzyme with MRRWMR amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223069 | pCMV-T7-SpCas9(ARGIMR)-P2A-EGFP (AHK147) | pCMV and pT7 Human expression plasmid for SpCas9 enzyme with ARGIMR amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223070 | pCMV-T7-SpCas9(ARTWGR)-P2A-EGFP (RAS2995) | pCMV and pT7 Human expression plasmid for SpCas9 enzyme with ARTWGR amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223071 | pCMV-T7-SpCas9(RRTIMR)-P2A-EGFP (AHK149) | pCMV and pT7 Human expression plasmid for SpCas9 enzyme with RRTIMR amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223072 | pCMV-T7-SpCas9(MRRCQR)-P2A-EGFP (RAS2823) | pCMV and pT7 Human expression plasmid for SpCas9 enzyme with MRRCQR amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223073 | pCMV-T7-SpCas9(IRAVQL)-P2A-EGFP (RTW3820) | pCMV and pT7 Human expression plasmid for SpCas9 enzyme with IRAVQL amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223074 | pCMV-T7-SpCas9(SRSCMV)-P2A-EGFP (RTW3732) | pCMV and pT7 Human expression plasmid for SpCas9 enzyme with SRSCMV amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223075 | pCMV-T7-SpCas9(MKRCMV)-P2A-EGFP (AHK162) | pCMV and pT7 Human expression plasmid for SpCas9 enzyme with MKRCMV amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223076 | pCMV-T7-SpCas9(MWNVML)-P2A-EGFP (AHK156) | pCMV and pT7 Human expression plasmid for SpCas9 enzyme with MWNVML amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223077 | pCMV-T7-SpCas9(MKKCMN)-P2A-EGFP (AHK129) | pCMV and pT7 Human expression plasmid for SpCas9 enzyme with MKKCMN amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223078 | pCMV-T7-SpCas9(KKKCMN)-P2A-EGFP (AHK131) | pCMV and pT7 Human expression plasmid for SpCas9 enzyme with KKKCMN amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223079 | pCMV-T7-SpCas9(LWKYQS)-P2A-EGFP (AHK34) | pCMV and pT7 Human expression plasmid for SpCas9 enzyme with LWKYQS amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223080 | pCMV-T7-SpCas9(LWKYSS)-P2A-EGFP (RAS3024) | pCMV and pT7 Human expression plasmid for SpCas9 enzyme with LWKYSS amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223081 | pCMV-T7-SpCas9(MWKYQA)-P2A-EGFP (RAS2965) | pCMV and pT7 Human expression plasmid for SpCas9 enzyme with MWKYQA amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223082 | pCMV-T7-SpCas9(LWQYQH)-P2A-EGFP (RAS2885) | pCMV and pT7 Human expression plasmid for SpCas9 enzyme with LWQYQH amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223083 | pCMV-T7-SpCas9(RQMYQG)-P2A-EGFP (RAS3009) | pCMV and pT7 Human expression plasmid for SpCas9 enzyme with RQMYQG amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223084 | pCMV-T7-SpCas9(MWKYQS)-P2A-EGFP (RAS2898) | pCMV and pT7 Human expression plasmid for SpCas9 enzyme with MWKYQS amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223085 | pCMV-T7-SpCas9(LWSSLY)-P2A-EGFP (RAS2997) | pCMV and pT7 Human expression plasmid for SpCas9 enzyme with LWSSLY amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223086 | pCMV-T7-SpCas9(KWMQLC)-P2A-EGFP (RAS3025) | pCMV and pT7 Human expression plasmid for SpCas9 enzyme with KWMQLC amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223087 | pCMV-T7-SpCas9(KWRQLC)-P2A-EGFP (RAS2864) | pCMV and pT7 Human expression plasmid for SpCas9 enzyme with KWRQLC amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223088 | pCMV-T7-SpCas9(KWAVIG)-P2A-EGFP (RAS2999) | pCMV and pT7 Human expression plasmid for SpCas9 enzyme with KWAVIG amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223089 | pCMV-T7-SpCas9(MWRQLM)-P2A-EGFP (RAS2868) | pCMV and pT7 Human expression plasmid for SpCas9 enzyme with MWRQLM amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223090 | pCMV-T7-SpCas9(KWHMLM)-P2A-EGFP (RAS2869) | pCMV and pT7 Human expression plasmid for SpCas9 enzyme with KWHMLM amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223091 | pCMV-T7-SpCas9(MRKCLN)-P2A-EGFP (AHK130) | pCMV and pT7 Human expression plasmid for SpCas9 enzyme with MRKCLN amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223092 | pCMV-T7-SpCas9(LWKFEG)-P2A-EGFP (RTW3931) | pCMV and pT7 Human expression plasmid for SpCas9 enzyme with LWKFEG amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223093 | pCMV-T7-SpCas9(CWNWNS)-P2A-EGFP (RAS3011) | pCMV and pT7 Human expression plasmid for SpCas9 enzyme with CWNWNS amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223094 | pCMV-T7-SpCas9(LWRYEK)-P2A-EGFP (RAS2986) | pCMV and pT7 Human expression plasmid for SpCas9 enzyme with LWRYEK amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223095 | pCMV-T7-SpCas9(KWKYES)-P2A-EGFP (RAS2900) | pCMV and pT7 Human expression plasmid for SpCas9 enzyme with KWKYES amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223096 | pCMV-T7-SpCas9(LWKYEY)-P2A-EGFP (RAS3001) | pCMV and pT7 Human expression plasmid for SpCas9 enzyme with LWKYEY amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223097 | pCMV-T7-SpCas9(MKACKV)-P2A-EGFP (AHK136) | pCMV and pT7 Human expression plasmid for SpCas9 enzyme with MKACKV amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223098 | pCMV-T7-SpCas9(MKRCKS)-P2A-EGFP (AHK140) | pCMV and pT7 Human expression plasmid for SpCas9 enzyme with MKRCKS amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223099 | pCMV-T7-SpCas9(KRRCKV)-P2A-EGFP (RAS3014) | pCMV and pT7 Human expression plasmid for SpCas9 enzyme with KRRCKV amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223100 | pCMV-T7-SpCas9(VKMAKG)-P2A-EGFP (RAS3015) | pCMV and pT7 Human expression plasmid for SpCas9 enzyme with VKMAKG amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223101 | pCMV-T7-SpCas9(SKNCKS)-P2A-EGFP (RAS2951) | pCMV and pT7 Human expression plasmid for SpCas9 enzyme with SKNCKS amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223102 | pCMV-T7-SpCas9(MRKCRS)-P2A-EGFP (RAS2821) | pCMV and pT7 Human expression plasmid for SpCas9 enzyme with MRKCRS amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223103 | pCMV-T7-SpCas9(SRQMRG)-P2A-EGFP (RAS3017) | pCMV and pT7 Human expression plasmid for SpCas9 enzyme with SRQMRG amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223104 | pCMV-T7-SpCas9(MRKQKC)-P2A-EGFP (RAS2838) | pCMV and pT7 Human expression plasmid for SpCas9 enzyme with MRKQKC amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223105 | pCMV-T7-SpCas9(MRKSKC)-P2A-EGFP (RAS2840) | pCMV and pT7 Human expression plasmid for SpCas9 enzyme with MRKSKC amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223106 | pCBh-T7-SpCas9(KRTMQR)-P2A-mTagBFP2(RAS3591) | pCBh Human expression plasmid for SpCas9 enzyme with KRTMQR amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-mTagBFP2 |
| 223107 | pCBh-T7-SpCas9(KRHWMR)-P2A-mTagBFP2(RAS3594) | pCBh Human expression plasmid for SpCas9 enzyme with KRHWMR amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-mTagBFP2 |
| 223108 | pCBh-T7-SpCas9(MRRWYR)-P2A-mTagBFP2(RAS3598) | pCBh Human expression plasmid for SpCas9 enzyme with MRRWYR amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-mTagBFP2 |
| 223109 | pCBh-T7-SpCas9(MRRWMR)-P2A-mTagBFP2(RAS3579) | pCBh Human expression plasmid for SpCas9 enzyme with MRRWMR amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-mTagBFP2 |
| 223110 | pCBh-T7-SpG-P2A-mTagBFP2(RAS3583) | pCBh Human expression plasmid for SpG with with C-term BPNLS-P2A-mTagBFP2 |
| 223111 | pCBh-T7-SpCas9-P2A-mTagBFP2(RAS3575) | pCBh Human expression plasmid for SpCas9 with with C-term BPNLS-P2A-mTagBFP2 |
| 223112 | pCMV-T7-ABE8e-SpCas9(KWRQLC)-P2A-EGFP (RAS3467) | pCMV and pT7 Human expression plasmid for ABE8e SpCas9 enzyme with KWRQLC amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223113 | pCMV-T7-ABE8e-SpCas9(LWKYQS)-P2A-EGFP (AHK404) | pCMV and pT7 Human expression plasmid for ABE8e using SpCas9 enzyme with LWKYQS amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223114 | pCMV-T7-ABE8e-SpCas9(LWRYEK)-P2A-EGFP (BKS1028) | pCMV and pT7 Human expression plasmid for ABE8e using SpCas9 enzyme with LWRYEK amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223115 | pCMV-T7-ABE8e-SpCas9(MKRCMV)-P2A-EGFP (BKS1030) | pCMV and pT7 Human expression plasmid for ABE8e using SpCas9 enzyme with MKRCMV amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223116 | pCMV-T7-ABE8e-SpCas9(MKRCKS)-P2A-EGFP (RAS3455) | pCMV and pT7 Human expression plasmid for ABE8e using SpCas9 enzyme with MKRCKS amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223117 | pCMV-T7-ABE8e-SpCas9(MRKCRS)-P2A-EGFP (BKS1034) | pCMV and pT7 Human expression plasmid for ABE8e using SpCas9 enzyme with MRKCRS amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223118 | pCMV-T7-ABE8e-SpCas9(ARGIMR)-P2A-EGFP (AHK507) | pCMV and pT7 Human expression plasmid for ABE8e using SpCas9 enzyme with ARGIMR amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223119 | pCMV-T7-ABE8e-SpCas9(LWQYQH)-P2A-EGFP (AHK504) | pCMV and pT7 Human expression plasmid for ABE8e using SpCas9 enzyme with LWQYQH amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223120 | pCMV-T7-ABE8e-SpCas9(MWKYQS)-P2A-EGFP (AHK505) | pCMV and pT7 Human expression plasmid for ABE8e using SpCas9 enzyme with MWKYQS amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223122 | pCMV-T7-ABE8e-SpCas9(MWKYQA)-P2A-EGFP (AHK503) | pCMV and pT7 Human expression plasmid for ABE8e using SpCas9 enzyme with MWKYQA amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223123 | pCMV-T7-TadCBEd-SpCas9-P2A-EGFP (BKS327) | pCMV and pT7 Human expression plasmid for TadCBEd-SpCas9 with C-term BPNLS-P2A-EGFP |
| 223124 | pCMV-T7-TadCBEd-SpG-P2A-EGFP (BKS777/AHK571) | pCMV and pT7 Human expression plasmid for TadCBEd-SpG with C-term BPNLS-P2A-EGFP |
| 223125 | pCMV-T7-TadCBEd-SpRY-P2A-EGFP (BKS428) | pCMV and pT7 Human expression plasmid for TadCBEd-SpRY with C-term BPNLS-P2A-EGFP |
| 223126 | pCMV-T7-TadCBEd-SpCas9(KWRQLC)-P2A-EGFP (BKS1026) | pCMV and pT7 Human expression plasmid for TadCBEd using SpCas9 CBE enzyme with KWRQLC amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223127 | pCMV-T7-TadCBEd-SpCas9(LWKYQS)-P2A-EGFP (BKS1014) | pCMV and pT7 Human expression plasmid for TadCBEd using SpCas9 CBE enzyme with LWKYQS amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223128 | pCMV-T7-TadCBEd-SpCas9(LWRYEK)-P2A-EGFP (BKS1022) | pCMV and pT7 Human expression plasmid for TadCBEd using SpCas9 CBE enzyme with LWRYEK amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223129 | pCMV-T7-TadCBEd-SpCas9(MKRCMV)-P2A-EGFP (BKS1032) | pCMV and pT7 Human expression plasmid for TadCBEd using SpCas9 CBE enzyme with MKRCMV amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223130 | pCMV-T7-TadCBEd-SpCas9(MKRCKS)-P2A-EGFP (RAS3445) | pCMV and pT7 Human expression plasmid for TadCBEd using SpCas9 CBE enzyme with MKRCKS amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223132 | pCMV-T7-TadCBEd-SpCas9(ARGIMR)-P2A-EGFP (BKS1036) | pCMV and pT7 Human expression plasmid for TadCBEd using SpCas9 CBE enzyme with ARGIMR amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223133 | pCMV-T7-TadCBEd-SpCas9(MRKCRS)-P2A-EGFP (AHK545) | pCMV and pT7 Human expression plasmid for TadCBEd using SpCas9 CBE enzyme with MRKCRS amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223134 | pCMV-T7-TadCBEd-SpCas9(MRKQKC)-P2A-EGFP (AHK574) | pCMV and pT7 Human expression plasmid for TadCBEd using SpCas9 CBE enzyme with MRKQKC amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223135 | pCMV-T7-TadCBEd-SpCas9(MRKSKC)-P2A-EGFP (AHK446) | pCMV and pT7 Human expression plasmid for TadCBEd using SpCas9 CBE enzyme with MRKSKC amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 223136 | pCMV-T7-PEmax-P2A-EGFP (LM1589) | pCMV and pT7 Human expression plasmid for PEmax(nSpCas9(H840A)-M-MMLV_RT**)-P2A-EGFP |
| 223137 | pUC19-U6-[BsmBI_pegRNA_entry]-tevopreQ1-term (LM1138) | pU6 entry plamid for cloning prime editor tevopreQ1 epegRNAs (requires BsmBI or Esp3I digest and ligation of duplexed oligos) |
| 228251 | pCMV-T7-SpCas9(KRHWMR)-P2A-EGFP_(RAS3808) | pCMV human expression plasmid for SpCas9 enzyme with KRHWMR amino acids at positions 1135,1136,1218,1219,1335,T1337 with C-term BPNLS-P2A-EGFP |
| 228252 | pAAV-U6-SpCas9_gRNA(RHO-P23H)-CMV-mTagBFP2_(RAS3613) | pU6 (human U6) expression of SpCas9 sgRNA targeting RHO P23H mutation and pCMV expression of mTagBFP2 |
| 228253 | pUC19-U6-BsmBI_epegRNA_entry-mpknot_(LM1140) | pU6 entry plamid for cloning prime editor mpknot epegRNAs (requires BsmBI or Esp3I digest and ligation of duplexed oligos) |