We deactivated the remaining nuclease domain of Cas9 from nCBE4 (Addgene #100802), nCBE4-gam (Addgene #100806), pCMV-ABE7.10 (Addgene #102919), pCMV-AncBE4max (Addgene #112094) and pCMB_ABEmax (Addgene #112095). Agilent QuikChange XL Site-Directed Mutagenesis Kit (catalogue # 200517) was used with the following primer sequences:
Pcmv ancbe4max
Manufactured by Addgene
Sourced in China
PCMV_AncBE4max is a plasmid that contains a cytomegalovirus (CMV) promoter and the AncBE4max gene. The AncBE4max gene encodes a base editor, which is a type of enzyme used for targeted DNA base editing.
Automatically generated - may contain errors
Lab products found in correlation
Pcmv abemax, by Addgene (6 mentions)
Pcmv abemax p2a gfp, by Addgene (4 mentions)
Pcmv abe7, by Addgene (3 mentions)
T4 ligase, by Enzynomics (2 mentions)
Nebuilder hifi dna assembly master mix, by New England Biolabs (2 mentions)
Pcmv be3, by Addgene (2 mentions)
Quikchange xl site directed mutagenesis kit, by Agilent Technologies (1 mentions)
P3s cas9 hn, by Addgene (1 mentions)
Hifi dna assembly kit, by New England Biolabs (1 mentions)
Phusion polymerase, by Thermo Fisher Scientific (1 mentions)
T4 ligase, by New England Biolabs (1 mentions)
Midi prep kit, by Macherey-Nagel (1 mentions)
Puc57 sgrna, by Addgene (1 mentions)
Paavs1 ndi crispri, by Addgene (1 mentions)
Paavs1 pdi crisprn, by Addgene (1 mentions)
Pcmv pe2, by Addgene (1 mentions)
Abe8e, by Addgene (1 mentions)
Kod multi epi, by Toyobo (1 mentions)
Amaxa 4d nucleofector system, by Lonza (1 mentions)
Miniprep kit, by Qiagen (1 mentions)
14 protocols using pcmv ancbe4max
1
Deactivating Cas9 Nuclease Domains
2
Plasmid Engineering for CRISPR Genome Editing
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The plasmids in this study were provided from Addgene, including p3s-Cas9-HN (addgene no.104171), pCMV_ABEmax (addgene no.112095), pCMV-AncBE4max (addgene no. 112094), pUGI-NLS (addgene no.101091), and pRG2 (Addgene no. 104174). Sequences corresponding to sgRNAs were cloned into BsaI-digested pRG2 vector (Addgene no. 104174). For this step, oligos containing the spacer sequence were annealed to form double-stranded DNA fragments with compatible overhangs and ligated using T4 ligase (Enzynomics). To construct pCMV-AncBE4max without UGI, the c-terminal part of BE4max contacting 2× UGI was digested by Cas9 and AgeI endonuclease (NEB) and Gibson cloned using NEBuilder HiFi DNA Assembly master mix (NEB). All plasmids used for transfection experiments were prepared using a NucleoBond Xtra Midi Plus EF kit (MN).
3
Rapid CRISPR Vector Construction
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Synthesized oligos were used for each of the target sgRNAs. The oligos were extended using Phusion polymerase (Thermo Fisher Scientific) and then ligated with the pRG2-GG vector (Addgene 104174) using T4 ligase (NEB). The cloned vector was transformed into competent DH5a cells (Invitrogen). The plasmids were extracted using a Midi Prep Kit (MACHEREY-NAGEL), and the sequences were confirmed by Sanger sequencing analysis (Bionics). Next, pCMV-BE3 (Addgene 73021), pCMV-AncBE4max (Addgene 112094), pCMV-ABE7.10 (Addgene 102919), and pCMV-ABEmax (Addgene 112095) were obtained from Addgene. The newly designed vectors containing dCas9 or CMPs or TadAmax of ABE8e were structured using the HiFi DNA Assembly Kit (NEB). The target sequences are listed in Supplementary Table 1 .
4
CRISPR Plasmid Construction for Screening
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Primers and plasmids are listed in Supplementary Table 1 . pCMV-nCas9-KanR-AmpR(A118X)-sgRNA, the all-in-one plasmid for insertion screening, was assembled from pCMV-ABEmax (Addgene 112095), pUC57-Kan (Addgene, 51132), and pGL3-U6-sgRNA (Addgene, 51133). The sgRNA expression vector for mammalian cells was constructed using BsaI-digested pGL3-U6-sgRNA-EGFP with annealed DNA oligos (Supplementary Table 1 ). The sgRNA expression vector for GOTI was constructed by cloning annealed DNA oligos (Supplementary Table 1 ) into BbsI-digested pUC57-sgRNA (Addgene, 51132). CE-ABEs and CE-CBEs were derived from pCMV-ABEmax (Addgene 112095) and pCMV-AncBE4max (112094), respectively.
5
Inducible Gene Editing Cell Lines
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The plasmid for inducible PE2 expression used in this study was derived from pAAVS1-NDi-CRISPRi (Addgene, 73497) by replacing the sequences encoding KRAB-dCas9-mCherry with that encoding PE2 (amplified from pCMV-PE2; Addgene, 132775). CBEs and ABEs were amplified form pCMV_AncBE4max (Addgene, 112094) and ABE8e (Addgene, 138489) respectively and cloned into pAAVS1-PDi-CRISPRn (Addgene, 75300) by replacing with the sequence encoding Cas9. IRES2 EGFP was introduced downstream of AncBE4max and T2A mCherry was fused with ABE8e to monitor transgene expression. To generate iPE2, iCas9, iCBE and iABE cell lines, two million H9 hESCs were co-electroporated with the appropriate knockin vector (5 μg) and plasmids encoding AAVS1-targeting TALENs (2 μg; addgene, 59025 and 59026) using an Amaxa 4D Nucleofector system (Lonza). Serial cell dilutions were then seeded in six-well plates in E8 supplemented with Y-27632 (10 μM). After selection with the appropriate antibiotic, clones were picked, expanded, and screened by treating with dox and staining for Cas9. For genotyping, genomic DNA was extracted with a DNeasy Blood & Tissue Kit. KOD -Multi & Epi (Toyobo, KME-101) was used for junction PCR according to the manufacturer's protocol.
6
Base Editing Constructs Preparation
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Plasmids for the following four base editing constructs: pCMV_BE4max (Addgene plasmid # 112093), pCMV_BE4max_P2A_GFP (Addgene plasmid # 112099), pCMV_AncBE4max (Addgene plasmid # 112094), and pCMV_AncBE4max_P2A_GFP (Addgene plasmid # 112100) [40 (link)] were kind gifts from Dr. David Liu. Plasmid DNA was purified from an overnight culture of TOP10 cells (Thermo Fisher Scientific, Waltham, MA, USA) using a miniprep kit (Qiagen, Germantown, MD, USA). All four plasmid DNAs were linearized with SapI (New England BioLabs, Ipswich, MA, USA) and mRNA was synthesized with the T7 mMessage mMachine kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions with the following modifications: addition of 1 µL GTP, an incubation time of 3 h and LiCl precipitation.
7
CRISPR Plasmid Construction and Acquisition
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All the gRNA sequences listed (Table S1 ) were synthesized by GENEWIZ Biotechnology, Ltd. (Suzhou, China) and then inserted into the pBluescriptSKII+ U6-sgRNA(F+E) empty plasmid (Addgene #74707) through a BbsI site. pCMV-BE3, pCMV-ABE7.10, pCMV_AncBE4max and pCMV_ABEmax were obtained from Addgene (Plasmid #73021, #102919, #112094 and #112095). NG-AncBE4max, NG-ABEmax, xCas9-AncBE4max and xCas9-ABEmax were kept in this lab.
pJH372, pJH373, pJH375, and pJH376 (mammalian expression of AcrIIA1, AcrIIA2, AcrIIA3, and AcrIIA4) were obtained from Addgene (Plasmid #86839, #86840, #86841, and #86842). pCMV-T7-hAcrVA1-NLS (sv40) (BPK5050), pCMV-T7-hAcrVA2.1-NLS (sv40) (BPK5059), pCMV-T7-hAcrVA2-NLS (sv40) (AAS2283), pCMV-T7-hAcrVA3.1-NLS (sv40) (RTW2624), and pCMV-T7-hAcrVA3-NLS (sv40) (BPK5077) were obtained from Addgene (Plasmid #115136, #115137, #115138, #115139, and #115140). Human codon-optimized AcrIIA5, AcrIIC1, AcrIIC2, and AcrIIC3 [28 (link)] anti-CRISPR sequences (Table S4 ) were synthesized by GenScriptBiotechnology, Ltd. and then inserted into pcDNA3.1(+) expression vectors through BamHI/EcoRI sites.
pJH372, pJH373, pJH375, and pJH376 (mammalian expression of AcrIIA1, AcrIIA2, AcrIIA3, and AcrIIA4) were obtained from Addgene (Plasmid #86839, #86840, #86841, and #86842). pCMV-T7-hAcrVA1-NLS (sv40) (BPK5050), pCMV-T7-hAcrVA2.1-NLS (sv40) (BPK5059), pCMV-T7-hAcrVA2-NLS (sv40) (AAS2283), pCMV-T7-hAcrVA3.1-NLS (sv40) (RTW2624), and pCMV-T7-hAcrVA3-NLS (sv40) (BPK5077) were obtained from Addgene (Plasmid #115136, #115137, #115138, #115139, and #115140). Human codon-optimized AcrIIA5, AcrIIC1, AcrIIC2, and AcrIIC3 [28 (link)] anti-CRISPR sequences (
8
Constructing Mammalian Expression Plasmids
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To construct pEX-FlagR-ABEmax and pEX-FlagR-BE4max, the ABEmax and BE4max coding sequences (CDSs) were amplified from pCMV_ABEmax (Addgene no.112095) and pCMV_AncBE4max (Addgene no. 112094), respectively, and cloned into the mammalian expression pEX-FlagR vector using the Xho I and Xba I restriction sites. To construct pCMV-NGABEmax, we replaced the Cas9 sequence in pCMV_ABEmax with the SpCas9-NG sequence from pX330-SpCas9-NG (Addgene no. 117919) using the Pml I and Eco RI restriction sites. To construct pEX-FlagR-NGABEmax, the NGABEmax sequence from pCMV-NGABEmax was cloned into pEX-FlagR. Gibson fragments containing the ABEmax or BE4max CDS with matching overlaps were polymerase chain reaction (PCR)–amplified using Phusion High-Fidelity DNA Polymerase [New England Biolabs (NEB)]. Fragments were gel-purified and assembled using NEBuilder HiFi DNA Assembly master mix (NEB) for 1 hour at 50°C and transformed into chemically competent E. coli (DH5α; Enzynomics). Sequences corresponding to sgRNAs were cloned into Bsa I–digested pRG2 vector (Addgene no. 104174). For this step, oligos containing the spacer sequence (table S3) were annealed to form double-stranded DNA fragments with compatible overhangs and ligated using T4 ligase (Enzynomics). All plasmids used for transfection experiments were prepared using a NucleoBond Xtra Midi Plus EF kit [Macherey-Nagel (MN)].
9
Construction of SB transposon vectors
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The sequences of the oligonucleotides are listed in Supplementary Data 13 . The SB transposon (pT2-SV40-BSD-ABEmax and pT2-SV40-BSD-BE4max) was constructed as follows: first, we replaced the NeoR gene (AvrII-KpnI site) on pT2-SV40-NeoR with BSD, resulting in the pT2-SV40-BSD vector; second, the backbone fragment of pT2-SV40-BSD was PCR-amplified with Gibson-SV40-F and Gibson-SV40-R, and the ABEmax fragment was PCR-amplified from pCMV_ABEmax_P2A_GFP (Addgene#112101) with Gibson-ABE/BE4-F and Gibson-ABE/BE4-R, and the BE4max fragment was PCR-amplified from pCMV_AncBE4max (Addgene#112094) with Gibson-ABE/BE4-F and Gibson-ABE/BE4-R; third, the backbone fragments were ligated with ABEmax and BE4max using Gibson Assembly (NEB), resulting in pT2-SV40-BSD-ABEmax and pT2-SV40-BSD-BE4max, respectively.
10
Modular AAV-based base editors
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The CBE plasmids including pCAG-CBE4max-SpG-P2A-EGFP [52 (link)] (Addgene #139,998), pCMV_AncBE4max [23 (link)] (Addgene #112,094) and pAAV-pCMV-dSaCas9-VP64-pU6-sgRNA (Addgene #158,990) were obtained from Addgene (Watertown, MA). All gRNA oligos were annealed and cloned into pLenti-ogRNA via BsmBI site. The hAAT promoter sequence was PCR amplified from the genomic DNA of HEK293 cells. The hAAT promoter, Gp41-1 intein split base editor fragments, and the human U6-driven gRNA fragment, were PCR amplified and inserted into the AAV transfer plasmids pZC0031 and pZC0033 as described previously [22 (link)].
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