Be4max

Manufactured by Addgene

The BE4max is a specialized laboratory equipment designed for high-throughput gene editing applications. It features a robust and efficient DNA assembly system to facilitate the rapid construction of complex genetic constructs. The core function of the BE4max is to enable efficient and accurate DNA assembly, supporting researchers in their gene editing workflows.

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Lab products found in correlation

Fast site directed mutagenesis kit, by Tiangen Biotech (2 mentions) Cas9m4 vp64, by Addgene (1 mentions) Hcas9 d10a, by Addgene (1 mentions) T4 dna ligase, by New England Biolabs (1 mentions) Be4 gam, by Addgene (1 mentions)

Spelling variants of this product

PCMV-BE4max-P2A-GFP (2 citations) PCMV_BE4max (2 citations)

4 protocols using be4max

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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Engineered APOBEC3G Variants

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The full-length human codon-optimized wild-type A3G with a set of mutations (P200A + N236A + P247K + Q318K + Q322K) was synthesized as gBlock and inserted into the BE4max construct (Addgene #112093) to replace the rAPOBEC1 region, resulting in A3G3.1. To do so, both insertion and vectors were amplified using primers with overhangs containing Esp3I recognition sites, which would generate compatible complementary sticky ends after cutting. Then the one-pot Golden Gate assembly was employed to cut and ligate two amplified pieces by using Esp3I and T4 DNA ligase (New England Biolabs, cat. R0734L and M0202L). Likewise, the Y315F and N244G variants were respectively generated by designing an extra pair of primers containing the indicated mutations and performing a three-piece assembly. The A3G3.8, 3.9, 3.14, and 3.15 constructs were Golden Gate cloned to introduce point mutations T218G, T218I, T218S, and T218N to A3G3.1.

Wang Q., Yang J., Zhong Z., Vanegas J.A., Gao X, & Kolomeisky A.B. (2021). A general theoretical framework to design base editors with reduced bystander effects. Nature Communications, 12, 6529.

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Engineering Ultra High-Fidelity CRISPR Systems

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The BE4max plasmid was obtained from Addgene (#112093). Seven mutations (R1335A/L1111R/D1135V/G1218R/E1219F/A1322R/T1337R) in CRISPR/Cas9 were introduced into BE4max to obtain NG-BE4max. Five high-fidelity CRISPR/Cas9 systems were introduced into NG-BE4max to obtain NG-eSp (K848A/K1003A/R1060A), NG-HF1 (N497A/R661A/Q695A/Q926A), NG-Sniper (F539S/M761I/K890N), NG-HiFi (R691A), and NG-Opti (R661A/K1003H). Site-directed mutagenesis of the plasmid was performed with the Fast Site-Directed Mutagenesis Kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. All primers used for site-directed mutation are listed in Table S6.

Shan H., Liu Z., Jia Y., Chen S., Chen M., Song Y., Sui T., Lai L, & Li Z. (2021). Reduced off-target effect of NG-BE4max by using NG-HiFi system. Molecular Therapy. Nucleic Acids, 25, 168-172.

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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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