Plasmids pCMV-PE2 (Addgene #132775), pU6-pegRNA-GG-acceptor (Addgene #132777) and pU6-Sp-pegRNA-HEK3_CTT_ins (Addgene #132778) were a gift from David Liu. pMD2.G and psPAX2 (second-generation lenti-viral packaging construct, Addgene #12259, #12260) were a gift from Didier Trono. pLenti_PE2_P2A_Puro vector was constructed by assembling amplified PE2 (from Addgene #132775; primers listed in Table) into BamH1 and Nhe1 digested pLenti-ABERA-P2A-Puro vector (Addgene#112675; a gift from Lucas Dow) using NEBuilder HiFi DNA Assembly Master Mix as per the manufacturer’s instructions. Lenti_pegRNA_GFP/RFP vector was constructed by removing the gRNA scaffold from pLKO5.sgRNA.EFS.GFP/RFP (Addgene #57822/57823; Gift from Benjamin Ebert).
Pcmv pe2
Manufactured by Addgene
PCMV-PE2 is a plasmid vector that contains the cytomegalovirus (CMV) promoter and the mCherry fluorescent protein. This plasmid is commonly used for expressing the mCherry protein in various cell types.
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Lab products found in correlation
Pu6 pegrna gg acceptor, by Addgene (7 mentions)
Ng abemax, by Addgene (2 mentions)
Pu6 sp pegrna hek3 ctt ins, by Addgene (1 mentions)
Nebuilder hifi dna assembly master mix, by Addgene (1 mentions)
Paavs1 ndi crispri, by Addgene (1 mentions)
Paavs1 pdi crisprn, by Addgene (1 mentions)
Pcmv ancbe4max, by Addgene (1 mentions)
Abe8e, by Addgene (1 mentions)
Kod multi epi, by Toyobo (1 mentions)
Amaxa 4d nucleofector system, by Lonza (1 mentions)
Pbatc, by Addgene (1 mentions)
Prg2 vector, by Addgene (1 mentions)
Px330 spcas9 ng, by Addgene (1 mentions)
Pspgrna, by Addgene (1 mentions)
Megashortscript kit, by Thermo Fisher Scientific (1 mentions)
T7 ultra kit, by Thermo Fisher Scientific (1 mentions)
Pmei enzyme, by New England Biolabs (1 mentions)
Megaclear kit, by Thermo Fisher Scientific (1 mentions)
Xhoi enzyme, by New England Biolabs (1 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions)
17 protocols using pcmv pe2
1
Lentiviral Vector Construction for Gene Editing
2
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.
3
Generating NG-PAM Targetable PE Vectors
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To construct PE plasmid, PE cassette was amplified from pCMV-PE2 (Addgene no.132775) and amplified product was inserted into pBAtC (Addgene no.78097), generating pBAtC-NGG-PE2 vector. To build NG-PAM targetable PE vector, we introduced same mutations with pX330-SpCas9-NG (Addgene no.117919) in our Cas9 fragment. For introducing pegRNA cassette, oligos representing the target sequences, sgRNA scaffold and 3’ extensions were annealed and cloned into pRG2 vector (Addgene no. 104174) with additional AtU6-26 promoter using BsaI to build AtU6-26p-pegRNA vector. Restriction enzyme-digested fragment encoding AtU6-26p-pegRNA cassette was inserted into pBAtC-NG-PE2 vector digested with same restriction enzyme. To construct nicking sgRNA cassette, oligos representing nicking sequences were annealed and cloned into AarI-digested PE plasmid. Oligos used for preparing plasmid was designed using Cas-designer (22 (link)) and PE-designer (23 (link)).
4
CRISPR Plasmid Construction for Genome Editing
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pSPgRNA (#47108), pCMV-PE2 (#132775), pU6-Sp-pegRNA-GG-acceptor (#132777), and pU6-Sp-pegRNA_HEK3_CTT_ins (#132778) were obtained from Addgene (Watertown, MA). The U6-pegRNA vectors were constructed with pU6-Sp-pegRNA-GG-acceptor and the U6-nicksing sgRNA vectors were constructed with pSPgRNA. pegRNAs were constructed with pU6-Sp-pegRNA-GG-acceptor, as previously reported.3 (link) Nicking sgRNAs were generated by T4 ligation of annealed oligos into the BbsI-digested pSPgRNA plasmid. The pegRNA and nicking sgRNA sequences for different targets in HEK293 cells and HEK293-rd12 cells are listed in Supplementary Tables S1 and S2 . By in-fusion cloning of PCR amplicons to restriction enzyme-digested backbones, the N- and C-terminals of PE2 were produced. Every plasmid was confirmed by sequencing.
5
Plasmid Linearization and In Vitro Transcription
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The pCMV-PE2 was purchased from Addgene (132775) [15 (link)]. The PE2 plasmid was linearized by the PmeI enzyme (NEB, Ipswich, MA, USA), and in vitro transcription was performed using the T7 Ultra Kit (Ambion, Austin, TX, USA). According to the manufacturer’s protocols, RNA was purified by the Mini Kit (Qiagen, Hilden, Germany). The expression plasmids of T7 + pegRNA and T7 + nick sgRNA were linearized by the XhoI enzyme (NEB, Ipswich, MA, USA). According to the manufacturer’s protocols, the pegRNAs and sgRNAs were transcribed in vitro by the MEGA shortscript Kit (Invitrogen, Carlsbad, CA, USA) and purified by the MEGA clear Kit (Invitrogen, Carlsbad, CA, USA).
6
Generating plasmids for RPA70-C and Rad51DBD
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Sequences encoding human RPA70-C and Rad51DBD were synthesized by GeneScript, after which they were amplified by PCR and cloned into the pCMV-PE2 (Addgene, no. 132775) plasmid to generate the ssDBD-PE2-encoding plasmids. These plasmids were named PE2-mid_RPA70, hyPE2, PE2-N_Rad51, and PE2-C_Rad51 (Fig. 1a ). Linker variants were derived from the hyPE2 plasmid and cloned using Gibson assembly16 (link). The sequences of the primers and plasmids used in this study are shown in Supplementary Table 4 and the Supplementary Note, respectively.
7
Engineered Variants of PE2 Plasmids
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The pCMV-PE2 (Addgene plasmid #132775) and pU6-pegRNA-GG-acceptor (Addgene plasmid #132777) plasmids were a gift from David Liu. pegRNA-encoding plasmids were generated by Golden Gate assembly using a custom acceptor plasmid according to methods described in a previous paper. (23 (link)) pCMV-PE2 was modified to generate a set of plasmids encoding the PE2 variants, which included pCMV-PE2-HF (N497A, R661A, Q695A and Q926A), pCMV-ePE2 (K848A, K1003A and R1060A), pCMV-Evo-PE2 (M495V, Y515N, K526E and R661Q), pCMV-Hypa-PE2 (N692A, M694A, Q695A and H698A) and pCMV-Sniper-PE2 (F539S, M763I and K890N), by incorporating mutations via NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs) with gblocks containing mutations (IDT) and site-directed mutagenesis (Q5 Site-Directed Mutagenesis Kit, New England Biolabs).
8
Prime Editing of MFN1 in HEK293T Cells
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We introduced MFN1 editing sites to the genome of HEK293T cells via Prime editing (38 (link)). The spacer and extension sequences were designed according to the guidelines provided previously (38 (link)). The designed oligos were cloned into the pU6-pegRNA-GG-acceptor (Addgene, 132777) to generate both pegRNA and nick guide RNA (gRNA) expressing constructs (oligo sequences were listed in table S4). Plasmids expressing pegRNA (250 ng), nick gRNA (83 ng), and prime editor (750 ng), namely, pCMV-PE2 (Addgene, 132775), were cotransfected into HEK293T (7500 cells per well in 48-well plates) with Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific, catalog no. L3000015) according to the manufacturer’s protocol. After 72 hours, genomic DNA was extracted, amplified by PCR, and sequenced via Sanger sequencing to confirm genome editing events. To optimize the editing efficiency, different combinations of pegRNAs and nick gRNAs (PE2, PE3, and PE3b) were tested. The prime binding site (PBS) and RT template were also optimized to 9 nt for PBS and 19 nt for RT template. The optimal condition (PE3b, 9-nt PBS, and 19-nt RT template) was used in scaled-up experiments to generate single-cell clones with MFN1 genome editing by serial dilution.
9
Plasmid Cloning for Mammalian Transfection
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Editor plasmids used for mammalian cell transfection were generated using Gibson assembly or USER assembly using pCMV-PE2 (Addgene, 132775) and pCMV-PEmax (Addgene, 174820) plasmids. pegRNA and epegRNA constructs were cloned by Golden Gate assembly using custom pU6-pegRNA-GG-acceptor (Addgene,132777) and pU6-tevopreQ1-GG-acceptor (Addgene, 174038) plasmids. All nicking sgRNA plasmids were generated by KLD assembly or Golden Gate assembly using pFYF1320 (Addgene, 47511) as a template plasmid. rAAV vector plasmids were cloned by restriction digestion of v5 AAV CBE (Addgene, 137176) or v5 AAV ABE (Addgene, 137177) followed by Gibson assembly with eBlocks or polymerase chain reaction (PCR) amplicons. All plasmids used for mammalian tissue culture were purified from MACH1, DH5alpha or NEBstable Escherichia coli using Plasmid Plus Maxiprep or Midiprep kits (Qiagen), ZymoPURE II Midiprep Kit (Zymo Research) or PureYield Plasmid Miniprep kit (Promega).
10
Constructing Versatile PE2 Variants
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To construct PE2 variants, we used pCMV-PE2 (Addgene plasmid #132775) plasmid DNA as vector DNA, and the coding sequences of spCas9 variants were obtained by PCR amplification from NG-ABEmax (NG variant, Addgene plasmid #124163), RTW3520 (VQR variant, Addgene plasmid #139990), RTW3160 (VRER variant, Addgene plasmid #139991), RTW3161 (VRQR variant, Addgene plasmid #139992), RTW4177 (SpG variant, Addgene plasmid #139998), and RTW4830 (SpRY variant, Addgene plasmid #139989). All of the PE2 variants used in this study were available from Addgene. The pegRNAs were cloned into the pU6-pegRNA-GG-acceptor (Addgene plasmid #132777), and the spacer sequences and RT template and PBS sequences are listed in Table S2 .
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