Be4 gam

Manufactured by Addgene

BE4-Gam is a plasmid that encodes a modified Cas9 protein, known as BE4. BE4 is a base editor that can convert cytosine (C) to thymine (T) in the genome. The plasmid is available from Addgene.

Automatically generated - may contain errors

Lab products found in correlation

Pcmv be3, by Addgene (2 mentions) Megaclear transcription clean up kit, by Thermo Fisher Scientific (1 mentions) Px330 mcherry, by Addgene (1 mentions) Mmachine t7 ultra transcription kit, by Thermo Fisher Scientific (1 mentions) Pcmv ha3a be3 y130f, by Addgene (1 mentions) Megashortscript t7 kit, by Thermo Fisher Scientific (1 mentions) Universal dna purification kit, by Tiangen Biotech (1 mentions) Maxiscript t7 kit, by Thermo Fisher Scientific (1 mentions) Hiscribe t7 arca mrna kit, by New England Biolabs (1 mentions) Mirneasy mini kit, by Qiagen (1 mentions) Fast site directed mutagenesis kit, by Tiangen Biotech (1 mentions) Be4max, by Addgene (1 mentions) Megashortscript t7 transcription kit, by Thermo Fisher Scientific (1 mentions) Px459 plasmid, by Addgene (1 mentions) Megaclear kit, by Thermo Fisher Scientific (1 mentions) Premix taq, by Takara Bio (1 mentions) Pcmv abe7, by Addgene (1 mentions) Mmessage mmachine t7 ultra kit, by Thermo Fisher Scientific (1 mentions)

4 protocols using be4 gam

Synthesizing mRNA and sgRNA for Gene Editing

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The mRNA transcriptional templates of different base editors and Cre were amplified by PCR using KOD-Plus-Neo (TOYOBO) from plasmids pCMV-BE3 (Addgene#73021), BE4-Gam (Addgene#100806), pCMV-hA3A-BE3-Y130F (Addgene#113428), pCMV-hA3A-eBE3-Y130F (Addgene#113423), xCas9(3.7)-BE3 (Addgene#108380), xCas9(3.7)-BE4 (Addgene#108381) and pZ4f-Cre, purified by the Universal DNA Purification Kit (TIANGEN, Cat#DP214) and then transcribed using the mMACHINE T7 ULTRA Transcription Kit (Invitrogen, Cat#AM1345) following the manufacturer’s instruction. The transcriptional templates of sgRNAs were amplified from pX330-mCherry (Addgene#98750) and transcribed in vitro using the MEGAshortscript T7 kit (Invitrogen, Cat#AM1354) following the manufacturer’s instruction. mRNAs and sgRNAs were subsequently purified with the MEGAclear Transcription Clean-Up Kit (Invitrogen, Cat# AM1908), resuspended in hot (95 °C) RNase-free water and then stored at −80 °C.

Li Q., Lu J., Yin X., Chang Y., Wang C., Yan M., Feng L., Cheng Y., Gao Y., Xu B., Zhang Y., Wang Y., Cui G., Xu L., Sun Y., Zeng R., Li Y., Jing N., Xu G.L., Wu L., Tang F, & Li J. (2023). Base editing-mediated one-step inactivation of the Dnmt gene family reveals critical roles of DNA methylation during mouse gastrulation. Nature Communications, 14, 2922.

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CRISPR mRNA and sgRNA production

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pCMV-BE3, BE4-Gam, and ABE7.10 plasmids were obtained from Addgene (#73021, #100806 and #102919). The plasmid was linearized with Not I, and mRNA was synthesized using an in vitro RNA transcription kit (HiScribe™ T7 ARCA mRNA kit (with tailing), NEB). sgRNA oligos were annealed into pUC57-sgRNA expression vectors with T7 promoter. Then, sgRNAs were amplified and transcribed in vitro using the MAXIscript T7 kit (Ambion) and purified with miRNeasy Mini Kit (Qiagen) according to manufacturer’s instructions. sgRNA-oligo sequences used in this study were listed in Supplementary Table 1.

Liu Z., Chen M., Chen S., Deng J., Song Y., Lai L, & Li Z. (2018). Highly efficient RNA-guided base editing in rabbit. Nature Communications, 9, 2717.

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Engineered Base Editor Plasmids

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The BE4-Gam and BE4max plasmids were obtained from Addgene (#100806 and #112093). The corresponding mutations were introduced into BE4-Gam or BE4max to obtain YE1 base editors (W90Y + R126E in rA1), YEE base editors (W90Y + R126E + R132E in rA1), Y120F-base editors (Y120F in rA1) and YFE-base editors (W90Y + Y120F + R126E in rA1). Plasmid site-directed mutagenesis was performed using the Fast Site-Directed Mutagenesis Kit (TIANGEN, Beijing). All site-directed mutation primers are listed in Table S1.

Liu Z., Chen S., Shan H., Jia Y., Chen M., Song Y., Lai L, & Li Z. (2020). Efficient base editing with high precision in rabbits using YFE-BE4max. Cell Death & Disease, 11(1), 36.

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Synthesis and Purification of CRISPR Components

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The pCMV-BE3 (#73021), pCMV-ABE7.10 (#102919), saKKH-BE3 (#85170), and BE4-Gam (#100806) plasmids were obtained from Addgene. These plasmids were digested with NotI (NEB, Cat. No. R0189S) and BE3, BE4-Gam, and ABE7.10 mRNAs were synthesized using mMESSAGE mMACHINE T7 Ultra kit (Life Technologies, Cat. No. AM1345). T7-sgRNA cassettes were synthesized by PCR using pX459 plasmid (Addgene, #62988) and primers containing spacer and T7 sequences (Supplementary Table 7) with Premix Taq (Takara, Cat. No. R003Q). The sgRNAs were synthesized using the MEGAshortscript T7 Transcription Kit (Life Technologies, Cat. No. AM1354). The mRNAs and sgRNAs were purified using the MEGAclear Kit (Life Technologies, Cat, No. AM1908).

Zhang W., Aida T., del Rosario R.C., Wilde J.J., Ding C., Zhang X., Baloch Z., Huang Y., Tang Y., Li D., Lu H., Zhou Y., Jiang M., Xu D., Fang Z., Zheng Z., Huang Q., Feng G, & Yang S. (2020). Multiplex precise base editing in cynomolgus monkeys. Nature Communications, 11, 2325.

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