Px601

Manufactured by Addgene

Sourced in Canada

The PX601 is a versatile lab equipment product. It is designed for general laboratory use and can perform various tasks. The core function of the PX601 is to assist in standard laboratory procedures. For a more detailed and unbiased description, please consult the manufacturer's specifications.

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10 protocols using px601

CRISPR-Cas9 mRNA and gRNA Synthesis

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The pT7-3 × Flag-hCas9 plasmid was constructed as reported previously37 (link). The transcription template of SaCas9 mRNA was PCR amplified from pX601 (Addgene) using the primer listed in Supplementary information, Table S2. We added the T7 promoter sequence to the 5′ end of the SaCas9 forward primer. The transcription templates were then purified using the PCR Clean Up Kit (Qiagen). SpCas9 and SaCas9 were transcribed using the mMESSAGE mMACHINE T7 ULTRA kit (Life Technologies) following the manufacturer’s instruction. Sequences for SpCas9 gRNAs were cloned into the pDR274 vector (Addgene), using primers listed in Supplementary information Table S2. These recombinant vectors were then linearized with Dra I (NEB) and transcribed using the MEGAshortscript T7 kit (Life Technologies) following the manufacturer’s instruction. The transcription templates of SaCas9 gRNAs were PCR amplified from pX601 (Addgene) using primers listed in Supplementary information, Table S2. We also added the T7 promoter sequence to the 5′ end of the gRNA forward primer. The gRNAs were then transcribed using the MEGAshortscript T7 kit (Life Technologies). Cas9 mRNAs and the gRNAs were subsequently purified using the MEGAclear kit (Life Technologies) and resuspended in RNase-free water.

Zhang X., Liang P., Ding C., Zhang Z., Zhou J., Xie X., Huang R., Sun Y., Sun H., Zhang J., Xu Y., Songyang Z, & Huang J. (2016). Efficient Production of Gene-Modified Mice using Staphylococcus aureus Cas9. Scientific Reports, 6, 32565.

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Codon-optimized SaCas9 and Chimeric gRNA Protocol

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The human codon-optimized SaCas9 and chimeric gRNA expression plasmid pX601 were obtained from Addgene (plasmid 61591). The reproduced rcccDNA system, including plasmid prcccDNA-shB2M (genotype D: GenBank accession no. V01460.1) and pCMV-KRAB-Cre, was a generous gift from Qiang Deng (Fudan University; Li et al., 2018a (link)). pAAV/HBV1.2 (genotype A: GenBank accession no. AF305422.1) was a generous gift from Pei-jer Chen (National Taiwan University). The HBV replicons (genotype B: GenBank accession no. EU570069.1; genotype C: GenBank accession no. FJ899793.1) were generous gifts from Ying Zhu (Wuhan University). Several candidate promoters were inserted into the SacI and HindIII restriction sites of pGL3-Basic (Promega). Three promoters with linked luciferase fragments were inserted between the XhoI and BamHI restriction sites of pHAGE (Addgene). The pSV-β-gal and pRL-TK plasmids were obtained from Promega.

Yan K., Feng J., Liu X., Wang H., Li Q., Li J., Xu T., Sajid M., Ullah H., Zhou L., Zhou L, & Chen Y. (2021). Inhibition of Hepatitis B Virus by AAV8-Derived CRISPR/SaCas9 Expressed From Liver-Specific Promoters. Frontiers in Microbiology, 12, 665184.

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CXCR4 Gene Editing Using SaCas9

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Based on the SaCas9 PAM sequence 5′-NNGRRT-3′, 12 gRNAs targeting CXCR4 exon 2 were designed and synthesized with 5′-CACC and 5′-AAAC overhangs. For lenti-SaCas9-CXCR4-gRNA plasmid, we modified the lentiCRISPR v2 plasmid (Addgene #52961) by replacing the SpCas9 with SaCas9, and cloned the sgRNAs into the vector using the Bsmb1 (Fermentas). For AAV-SaCas9-CXCR4-gRNA plasmid, sgRNAs were inserted into the PX601 plasmid (Addgene #61591) digested with Bsa1 (Fermentas). Oligonucleotides for sgRNAs targeting CXCR4 are shown in Additional file 1: Table S1.

Wang Q., Chen S., Xiao Q., Liu Z., Liu S., Hou P., Zhou L., Hou W., Ho W., Li C., Wu L, & Guo D. (2017). Genome modification of CXCR4 by Staphylococcus aureus Cas9 renders cells resistance to HIV-1 infection. Retrovirology, 14, 51.

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In Vivo CRISPR-Cas9 Editing of RHO Gene

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K912 was packaged with an SaCas9 construct (Addgene; pX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA;U6::BsaI-sgRNA, Plasmid #61591). The gRNA was designed to target 285 bp downstream of the RHO start codon. A cynomolgus macaque and a rhesus macaque were both injected intravitreally, and 9 weeks (cyno) or 6 weeks (rhesus) later, tissue was collected for processing. Genomic DNA was extracted using a Qiagen DNeasy Kit and the target site in the RHO gene was PCR amplified with primers attached to Illumina adapter sequences. Amplicon sequences targeting RHO were sequenced on an Illumina iSeq and ~1,000,000 reads were recovered for each sample. CRISPResso2 (Clement et al., 2019 (link)) (v2.0.34) was used to quantify and visualize the edits, using the amplicon sequence and guide sequence as input. Reads were filtered using an average base quality of 30 and single base quality of 20.

Öztürk B.E., Johnson M.E., Kleyman M., Turunç S., He J., Jabalameli S., Xi Z., Visel M., Dufour V.L., Iwabe S., Pompeo Marinho L.F., Aguirre G.D., Sahel J.A., Schaffer D.V., Pfenning A.R., Flannery J.G., Beltran W.A., Stauffer W.R, & Byrne L.C. (2021). scAAVengr, a transcriptome-based pipeline for quantitative ranking of engineered AAVs with single-cell resolution. eLife, 10, e64175.

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Type I-F CRISPR System Plasmid Construction

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Type I–F Cascade (from Pseudomonas aeruginosa) E. coli expression plasmids were obtained from Addgene (pCsy_complex, 89232). Type I–Fv (from Shewanella putrefaciens) Cas7fv, Cas5fv, Cas6fv cassettes were cloned into the pET28a vector (Sigma-Aldrich, 69864-3CN) as a polycistronic operon and include an N-terminal His-tagged Cas7fv fusion (pET28-type I–Fv). The crRNA sequence was cloned into pACYC184 (NEB, X06403) for bacterial expression. Condon-optimized Cas subunits were sub-cloned into px601 (Addgene, #61591) (replacing the SaCas9 gene) for transfection into mammalian cells. A site for spacer cloning flanked by two Csy4 direct repeats (DR) or Cas6f direct repeats was ligated into lentiGuide-Puro (addgene #52963) between BsmBI and EcoRI restriction sites to generate pLenti-crRNA-IF or pLenti-crRNA-IFv vectors. Oligos containing spacer sequences were annealed and ligated into pLenti-crRNA-IF or pLenti-crRNA-IFv for crRNA expression in mammalian cells. For spacer mutant crRNA cloning, oligos with various of mutant spacer were annealed and ligated into pLenti-crRNA-IF. Sequences are listed in Supplementary Data 1–4. Sequences of plasmids for expression of PaeCascade-VPR, including pCsy1-Csy2, pCsy3-VPR-Csy4, and pCsy-crRNA-EV, are listed in Supplementary Data 5.

Chen Y., Liu J., Zhi S., Zheng Q., Ma W., Huang J., Liu Y., Liu D., Liang P, & Songyang Z. (2020). Repurposing type I–F CRISPR–Cas system as a transcriptional activation tool in human cells. Nature Communications, 11, 3136.

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Optimized Dual-Luciferase Assay Protocol

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24 hr prior to transfection, 10,000 HEK293FT cells were seeded into 96-well plates. Transfections were performed in triplicate, with each mix containing 100 ng DNA measured by Qubit and 0.4 μL Lipofectamine 2000. A typical reaction contained 20 ng Firefly target, 40 ng px601 (Addgene plasmid #61591, a gift from Feng Zhang), 24 ng self-editing plasmid, 4 ng renilla, and 11 ng pUC19 plasmid. Luciferase expression was measured 48 hr post-transfection using the Dual-Glo Luciferase Assay System (Promega, E2920).

Li A., Lee C.M., Hurley A.E., Jarrett K.E., De Giorgi M., Lu W., Balderrama K.S., Doerfler A.M., Deshmukh H., Ray A., Bao G, & Lagor W.R. (2018). A Self-Deleting AAV-CRISPR System for In Vivo Genome Editing. Molecular Therapy. Methods & Clinical Development, 12, 111-122.

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AAV8 Viral Particle Production

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To generate AAV8 viral particles, 293T cells in a 15cm dish (1 million cells, culture media: DMEM supplemented with 10%v/v fetal bovine serum) were transfected with pAAV2/8 (Addgene #112864), pAdDeltaF6 (Addgene #112867), and pX601 (Addgene #61591). Seventy-two hours later, media was removed and AAV particles were purified by Iodixanol gradient ultracentrifugation method (Millipore Sigma) according to the manufacturer’s instructions. The viral particles were further concentrated by Amicon^® Ultra-15 centrifugal filter units (Millipore Sigma) according to the manufacturer’s instructions. Viral titers were determined via a PCR based method utilizing the primers described above. The AAV8 viral particles (2.0×10¹² genome copies/rat diluted in 400μl PBS) were injected into a lateral tail vein.

Sun H., Hodgkinson C.P., Pratt R.E, & Dzau V.J. (2021). Crispr-Cas9 deletion of hepatic Angiotensinogen gene results in sustained control of Hypertension: A Potential for Cure?. Hypertension (Dallas, Tex. : 1979), 77(6), 1990-2000.

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Expression and Purification of CRISPR Proteins

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The gene sequences of SaCas9, SpCas9, AsCas12a, and LbCas12a proteins were derived from pX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA;U6::BsaI-sgRNA (Addgene plasmid # 61591), pSpCas9(BB)-2A-GFP (PX458) (Addgene plasmid # 48138), pY010 (pcDNA3.1-hAsCpf1) (Addgene plasmid # 69982), and pY016 (pcDNA3.1-hLbCpf1) (Addgene plasmid # 69988), respectively, which were gifts from Feng Zhang [33] (link)[34] (link)[35] (link). By comparing the fragments of the expression vector and four target genes, we selected SalI and XbaI as the insertion sites between the multiple clone site of the pET28b prokaryotic expression vector. Then primers were designed for the gene sequences of SaCas9, SpCas9, AsCas12a and LbCas12a. The forward primer 5' was added with SalI digestion site sequence, and the reverse primer

Xiao P., Bai R., Zhang T., Wu R., Chen L., Hou Y., Shen B., Niu Y., Li S., Ji W, & Chen Y. (2021). Extensive humoral immune response to AAVs and Cas proteins in nonhuman primates. Science bulletin, 66(20).

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CRISPR/Cas-Mediated Gene Editing in BETLE Cells

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BETLE reporter cells were edited following transfection with CRISPR/Cas encoding plasmids LentiCRISPRv2 (Addgene #98290), pX601 (Addgene #107055), or pY30 (Addgene #84745) with specific gRNAs. gRNAs were designed using http://crispor.tefor.net/ (accessed on 18 September 2021). The cells were seeded on a 24-well plate and transfected with the plasmids, using Lipofectamine 3000 according to manufacturer’s instructions. For HDR, the respective plasmid was mixed with the 199 bp ssDNA HDR template (synthesized by IDT as an Ultramer) in a DNA mass ratio of 1:1, using Lipofectamine 3000 according to manufacturer’s instructions.

Wettengel J.M., Hansen-Palmus L., Yusova S., Rust L., Biswas S., Carson J., Ryu J., Bimber B.N., Hennebold J.D, & Burwitz B.J. (2023). A Multifunctional and Highly Adaptable Reporter System for CRISPR/Cas Editing. International Journal of Molecular Sciences, 24(9), 8271.

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Engineered Catalytically Inactive SaCas9

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A fragment containing a catalytically inactive SaCas9 coupled to two flanking VP64 transactivator domains was synthesized by BioBasic Canada and cloned into pX601 (Addgene 61591) using AgeI and EcoRI directional cloning to generate 3XFLAG-VP64-SadCas9 (D10A/N580A)-NLS-VP64 plasmid (Figs. 2a, S11, Supplemental Table S6).
Each sgRNA (Supplemental Table S6) was subsequently introduced using BsaI directional cloning. To generate the three guides only construct (Fig. 2a), a fragment containing three repetitive regions of U6 promoter and S. aureus guide scaffold was assembled, with short linkers in between each region (BioBasic Canada). The fragment was cloned into KpnI and NotI sites of a pX601-derivative plasmid.

Kemaladewi D.U., Bassi P.S., Erwood S., Al-Basha D., Gawlik K.I., Lindsay K., Hyatt E., Kember R., Place K.M., Marks R.M., Durbeej M., Prescott S.A., Ivakine E.A, & Cohn R.D. (2019). A mutation-independent approach for muscular dystrophy via upregulation of a modifier gene. Nature, 572(7767).

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