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Engineering domain-inlaid SaCas9 adenine base editors with reduced RNA off-targets and increased on-target DNA editing.

Nguyen Tran MT, Mohd Khalid MKN, Wang Q, Walker JKR, Lidgerwood GE, Dilworth KL, Lisowski L, Pebay A, Hewitt AW
Nat Commun. 2020 Sep 25;11(1):4871. doi: 10.1038/s41467-020-18715-y. (Link opens in a new window) PubMed (Link opens in a new window) Article

Plasmids from Article

ID Plasmid Purpose
1353401010-AIDx-CPExpression of circular permutant of nSpyCas9 at amino acid position 1010 encoding truncated human cytosine deaminase, hAIDx, at the C-terminus.
1353411029-AIDx-CPExpression of circular permutant nSpyCas9 at amino acid position 1029 encoding truncated human cytosine deaminase, hAIDx, at the C-terminus.
1353421058-AIDx-CPExpression of circular permutant of nSpyCas9 at amino acid position 1058 encoding truncated human cytosine deaminase, hAIDx, at the C-terminus.
135343SpyCas9-AIDxExpression of wildtype nSpyCas9 encoding truncated human cytosine deaminase, hAIDx, at the C-terminus.
135344AIDx-ID-202Expression of intradomain insertion of truncated human cytosine deaminase, hAIDx, at position 202 of nSpyCas9 used for base editing
135345AIDx-ID-208Expression of intradomain insertion of truncated human cytosine deaminase, hAIDx, at position 208 of nSpyCas9
135346AIDx-ID-468Expression of intradomain insertion of truncated human cytosine deaminase, hAIDx, at position 468 of nSpyCas9
135347AIDx-ID-1058Expression of intradomain insertion of truncated human cytosine deaminase, hAIDx, at position 1058 of nSpyCas9 with uracil DNA glycosylase inhibitor (UGI) at the C terminus
135348BE3-NLS-NLS-P2A-BLASTExpression of BE3 (Base editor generation three) with dual C-terminal NLS in a single operon encoding P2A blasticidine resistance
135349S2 1058 AIDx no UGIExpression of intradomain insertion of truncated human cytosine deaminase, hAIDx, at position 1058 of nSpyCas9 lacking uracil DNA glycosylase inhibitor.
135350S2 1058 APOBEC3 no UGIExpression of intradomain insertion of rat cytosine deaminase, rAPOBEC3, at position 1058 of nSpyCas9 lacking uracil DNA glycosylase inhibitor.
135352S1 1029 CP APOBEC3 no UGIExpression of circular permutant of nSpyCas9 lacking uracil DNA glycosylase inhibitor at amino acid position 1029 encoding rat cytosine deaminase, rAPOBEC3 at the N-terminus
135354S1 1029 AIDx with UGIExpression of nSpyCas9 encoding truncated human cytosine deaminase hAIDx at amino acid 1029 and uracil DNA glycosylase inhibitor (UGI) at the C terminus
135355pCMV-ABEMAX-NLS-P2A_BLASTExpression of ABEMax (wildtype Tada monomer coupled with evolved Tada monomer) with dual C-terminal NLS in a single operon encoding P2A blasticidine resistance
135356S2 1058 ABEMaxExpression of intradomain insertion of ABEMax (wildtype Tada monomer coupled with evolved Tada monomer) at position 1058 of nSpyCas9
135357S1 1029 CP ABEMax strategy 1Expression of circular permutant of nSpyCas9 at amino acid position 1029 encoding ABEMax (wildtype Tada monomer coupled with evolved Tada monomer) at the N-terminus
135358S1 1029 CP ABEMax strategy 2Expression of circular permutant of nSpyCas9 at amino acid position 1029 encoding de-coupled ABEMax (wildtype Tada monomer at N terminus, evolved Tada monomer at C terminus)
135359S1 1029 CP ABEMax strategy 3Expression of circular permutant of nSpyCas9 at amino acid position 1029 encoding ABEMax (wildtype Tada monomer coupled with evolved Tada monomer) at the C-terminus
135360S1 1029 miniABEMax V82G strategy1Expression of circular permutant of nSpyCas9 at amino acid position 1029 encoding miniABEMax (evolved Tada monomer with V82G) at the N-terminus
135361S2 1058 V82G ABEMaxExpression of intradomain insertion of miniABEMax (evolved Tada monomer with V82G) at position 1058 of nSpyCas9
135362miniABEMax V82G wtExpression of nSpyCas9 with N-terminal miniABEMax (V82G)
135363S1 1029 V82G ABEMax Strategy 3Expression of circular permutant of nSpyCas9 at amino acid position 1029 encoding miniABEMax (evolved Tada monomer with V82G) at the N-terminus
135364ABEMax nSaCas9Expresion of SaCas9 nickase (D10A) with ABEMax at the N terminus
135365miniABEMax V82G nSaCas9Expression of SaCas9 nickase (D10A) with single evolved TadA monomer (ABEMax) with a V82G substitution
135366nSaCas9 D127 miniABEMax V82GExpression of nSaCas9 intradomain insertion of single evolved TadA monomer (V82G) at the specified position (D127 )
135367nSaCas9 D126 miniABEMax V82GExpression of nSaCas9 intradomain insertion of single evolved TadA monomer (V82G) at the specified position (D126 )
135368nSaCas9 E739 miniABEMax V82GExpression of nSaCas9 intradomain insertion of single evolved TadA monomer (V82G) at the specified position (E739 )
135369nSaCas9 E743 miniABEMax V82GExpression of nSaCas9 intradomain insertion of single evolved TadA monomer (V82G) at the specified position (E743 )
135370nSaCas9 E745 miniABEMax V82GExpression of nSaCas9 intradomain insertion of single evolved TadA monomer (V82G) at the specified position (E745 )
135371nSaCas9 F733 miniABEMax V82GExpression of nSaCas9 intradomain insertion of single evolved TadA monomer (V82G) at the specified position (F733 )
135372nSaCas9 N730 miniABEMax V82GExpression of nSaCas9 intradomain insertion of single evolved TadA monomer (V82G) at the specified position (N730 )
135373nSaCas9 E125 miniABEMax V82GExpression of nSaCas9 intradomain insertion of single evolved TadA monomer (V82G) at the specified position (E125 )
135374nSaCas9 E131 miniABEMax V82GExpression of nSaCas9 intradomain insertion of single evolved TadA monomer (V82G) at the specified position (E131 )
135375nSaCas9 L132 miniABEMax V82GExpression of nSaCas9 intradomain insertion of single evolved TadA monomer (V82G) at the specified position (L132 )
135376nSaCas9 N130 miniABEMax V82GExpression of nSaCas9 intradomain insertion of single evolved TadA monomer (V82G) at the specified position (N130 )
135377nSaCas9 E735 miniABEMax V82GExpression of nSaCas9 intradomain insertion of single evolved TadA monomer (V82G) at the specified position (E735 )
135379nSaCas9 I744 miniABEMax V82GExpression of nSaCas9 intradomain insertion of single evolved TadA monomer (V82G) at the specified position (I744 )
135380nSaCas9 G129 miniABEMax V82GExpression of nSaCas9 intradomain insertion of single evolved TadA monomer (V82G) at the specified position (G129 )
135381nSaCas9 H119 miniABEMax V82GExpression of nSaCas9 intradomain insertion of single evolved TadA monomer (V82G) at the specified position (H119 )
135382nSaCas9 M732 miniABEMax V82GExpression of nSaCas9 intradomain insertion of single evolved TadA monomer (V82G) at the specified position (M732 )
135383nSaCas9 M741 miniABEMax V82GExpression of nSaCas9 intradomain insertion of single evolved TadA monomer (V82G) at the specified position (M741 )
135384nSaCas9 N120 miniABEMax V82GExpression of nSaCas9 intradomain insertion of single evolved TadA monomer (V82G) at the specified position (N120 )
135385nSaCas9 P742 miniABEMax V82GExpression of nSaCas9 intradomain insertion of single evolved TadA monomer (V82G) at the specified position (P742 )
135386nSaCas9 Q731 miniABEMax V82GExpression of nSaCas9 intradomain insertion of single evolved TadA monomer (V82G) at the specified position (Q731 )
135387nSaCas9 Q737 miniABEMax V82GExpression of nSaCas9 intradomain insertion of single evolved TadA monomer (V82G) at the specified position (Q737 )
135388nSaCas9 S740 miniABEMax V82GExpression of nSaCas9 intradomain insertion of single evolved TadA monomer (V82G) at the specified position (S740 )
135389nSaCas9 T128 miniABEMax V82GExpression of nSaCas9 intradomain insertion of single evolved TadA monomer (V82G) at the specified position (T128 )

Antibodies from Article