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Cas9m4 vp64

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Cas9m4-VP64 is a modified version of the Cas9 protein, which is a key component of the CRISPR-Cas9 gene editing system. Cas9m4-VP64 contains the Cas9 endonuclease fused to the VP64 transcriptional activator domain. This fusion protein can be used to activate gene expression in targeted genomic locations.

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Lab products found in correlation

M st1n vp64, by Addgene (2 mentions) Sp dcas9 vpr, by Addgene (2 mentions) Pcdna dcas9 p300 core, by Addgene (1 mentions) Geneticin selective antibiotic g418, by Thermo Fisher Scientific (1 mentions) Pspcas9 2a puro px459, by Addgene (1 mentions) Hcas9 d10a, by Addgene (1 mentions) Be4max, by Addgene (1 mentions) Kapa hifi hotstart readymix pcr kit, by Roche (1 mentions) Psmvp cas9n, by Addgene (1 mentions) Psmvp cas9c, by Addgene (1 mentions) Paav cmv cas9c vpr, by Addgene (1 mentions)

5 protocols using cas9m4 vp64

1

Activation Domain Cloning and dCas9 Constructs

Cited 2 times
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Activation domains were cloned using a combination of Gibson and Gateway assembly or Golden Gate assembly methods. For experiments involving multiple activation domains, ADs were separated by short glycine-serine linkers. Activator sequences are listed in the Supplementary Data (vectors to be deposited in Addgene). All SP-dCas9 plasmids were based on Cas9m4-VP64 (Addgene #47319)6 (link), ST1-dCas9 plasmids were based on M-ST1n-VP64 (Addgene #48675)15 (link). Sequences for gRNAs are listed in the supplementary information. gRNAs for endogenous human gene activation were selected to bind between 1 and 1000 bp upstream of the transcriptional start site (TSS). gRNAs for iPSC differentiation to iNeurons, targeting NGN2 and NEUROD1, were selected to bind between 1 and 2000 base pairs upstream of the transcriptional start site. All human gRNAs were expressed from either cloned plasmids (Addgene #41817)5 (link) or integrated into the genome through lentiviral delivery (plasmid SB700). Guide RNA sequences are listed within the Supplementary Data. Reporter targeting gRNAs were previously described (Addgene #48671 and #48672)6 (link).
Chavez A., Scheiman J., Vora S., Pruitt B.W., Tuttle M., Iyer E., Lin S., Kiani S., Guzman C.D., Wiegand D.J., Ter-Ovanesyan D., Braff J.L., Davidsohn N., Housden B.E., Perrimon N., Weiss R., Aach J., Collins J.J, & Church G.M. (2015). Highly-efficient Cas9-mediated transcriptional programming. Nature methods, 12(4), 326-328.
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2

Generating CRISPRa Cell Line using CRISPR/Cas9-NHEJ

Cited 3 times
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To generate the CRISPRa cell line, the CRISPR/Cas9 and nonhomologous end joining (NHEJ) pathway (CRISPR/Cas9-NHEJ)-mediated genome editing strategy was applied [34 (link)]. First, the CRISPR/Cas9 plasmids, pSpCas9 2A-Puro (PX459) (a gift from Feng Zhang, Addgene plasmid #62988) targeting the 3′ region of chicken glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene (GAPDH #1) and targeting the common region of the three CRISPRa vectors (CRISPRa #1), Cas9m4-VP64 (Addgene plasmid #47316) [35 (link)], SP-dCas9-VPR (Addgene plasmid #63798) [13 (link)], or pcDNA-dCas9-p300 Core (Addgene plasmid #61357) were constructed [35 (link),36 (link)]. The 2.5 × 105 DF-1 cells were then transfected with 1 µg of GAPDH #1, 1 µg of CRISPRa #1, and 2 µg of each CRISPRa vector using Lipofectamine 3000 according to the manufacturer’s instructions. The transfected cells were treated with Geneticin Selective Antibiotic (G418, 300 µg/mL) (Thermo Fisher Scientific, Waltham, MA, USA, 24–48 h post transfection, and the drug selection was maintained to establish cell lines for at least 2 weeks. The gRNA and oligo sequences used in all-in-one CRISPR/Cas9 vector construction are listed in Table S1.
Chapman B., Han J.H., Lee H.J., Ruud I, & Kim T.H. (2023). Targeted Modulation of Chicken Genes In Vitro Using CRISPRa and CRISPRi Toolkit. Genes, 14(4), 906.
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3

Modular CRISPR Activation with AID Variants

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MEGA-2 was assembled by introducing AID*Δ-XTEN-Linker at the N-terminus of dCas9-VP64 in the backbone vector Cas9m4VP64 (Addgene #47319) through two-step ligation. AID*Δ was amplified from the pGH335_MS2-AID*Δ-Hygro plasmid (Addgene #85406) with primers including the XTEN-Linker at the C-terminus. For MEGA-1 the same cloning strategy was used but with human full-length wild type AID. MEGA-3 was constructed in a one-step ligation process whereby AID*Δ-XTEN was introduced into the Cas9m2 vector (Addgene #47317). MEGA-4 was cloned in the same way as MEGA-3 but using the hCas9_D10A (Addgene #41816) backbone instead. Cytosine base editors AID-BE3 (Addgene #100803) , BE4max (Addgene #112093) and CP1012 CBEmax (Addgene #119801) were purchased through Addgene.
Weischedel J., Higgins L., Rogers S., Gramalla-Schmitz A., Wyrzykowska P., Borgoni S., MacCarthy T, & Chahwan R. (2023). Modular cytosine base editing promotes epigenomic and genomic modifications. Nucleic Acids Research, 52(2), e8.
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4

Activation Domain Cloning and dCas9 Constructs

Cited 2 times
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Activation domains were cloned using a combination of Gibson and Gateway assembly or Golden Gate assembly methods. For experiments involving multiple activation domains, ADs were separated by short glycine-serine linkers. Activator sequences are listed in the Supplementary Data (vectors to be deposited in Addgene). All SP-dCas9 plasmids were based on Cas9m4-VP64 (Addgene #47319)6 (link), ST1-dCas9 plasmids were based on M-ST1n-VP64 (Addgene #48675)15 (link). Sequences for gRNAs are listed in the supplementary information. gRNAs for endogenous human gene activation were selected to bind between 1 and 1000 bp upstream of the transcriptional start site (TSS). gRNAs for iPSC differentiation to iNeurons, targeting NGN2 and NEUROD1, were selected to bind between 1 and 2000 base pairs upstream of the transcriptional start site. All human gRNAs were expressed from either cloned plasmids (Addgene #41817)5 (link) or integrated into the genome through lentiviral delivery (plasmid SB700). Guide RNA sequences are listed within the Supplementary Data. Reporter targeting gRNAs were previously described (Addgene #48671 and #48672)6 (link).
Chavez A., Scheiman J., Vora S., Pruitt B.W., Tuttle M., Iyer E., Lin S., Kiani S., Guzman C.D., Wiegand D.J., Ter-Ovanesyan D., Braff J.L., Davidsohn N., Housden B.E., Perrimon N., Weiss R., Aach J., Collins J.J, & Church G.M. (2015). Highly-efficient Cas9-mediated transcriptional programming. Nature methods, 12(4), 326-328.
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5

Inducible and Tissue-Specific CRISPR-Cas9 Activation

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The Cas9m4-VP64, SP-dCas9-VPR, pSMVP-Cas9N, pSMVP-Cas9C, and pAAV-CMV-Cas9C-VPR plasmids were obtained from Addgene (#47319, #63798, #80934, #80939, and #80933, respectively). sgRNAs expressed via a U6 promoter were added using standard cloning techniques. Cas9N and Cas9C were rendered catalytically inactive by introducing the D10A and the H840A point mutations via a standard site-directed mutagenesis protocol using the KAPA HiFi HotStart ReadyMix PCR kit (Kapa Biosystems). For the generation of stable cell lines, dCas9-VPR was subcloned into a pb expression vector containing the Tet-On system for DOX-inducible expression of dCas9-VPR. For expression in mouse photoreceptors, the split dCas9-VPR driven by a human rhodopsin promoter and corresponding sgRNAs each driven by a human U6 promoter were subcloned into the pAAV2.1 vector (27 (link)). All transgenes were sequenced before use (Eurofins Genomics).
Böhm S., Splith V., Riedmayr L.M., Rötzer R.D., Gasparoni G., Nordström K.J., Wagner J.E., Hinrichsmeyer K.S., Walter J., Wahl-Schott C., Fenske S., Biel M., Michalakis S, & Becirovic E. (2020). A gene therapy for inherited blindness using dCas9-VPR–mediated transcriptional activation. Science Advances, 6(34), eaba5614.
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