Shen Lab DdCBE assembly kit
(Kit #
1000000212
)
Depositing Lab: Bin Shen
The DddA-derived cytosine base editor (DdCBE) assembly kit is a collection of 192 modules harboring repeat variable di-residue (RVD) arrays for any desired target. With the components of this kit, DdCBE expression plasmids for a desired target can be obtained within 36 hours using Golden Gate assembly.
This kit will be sent as bacterial glycerol stocks in 96-well plate format.
Original Publication
Precision modeling of mitochondrial diseases in zebrafish via DdCBE-mediated mtDNA base editing. Guo J, Zhang X, Chen X, Sun H, Dai Y, Wang J, Qian X, Tan L, Lou X, Shen B. Cell Discov. 2021 Sep 3;7(1):78. doi: 10.1038/s41421-021-00307-9. PubMed (Link opens in a new window) Article (Link opens in a new window)
Description
DdCBEs, composed of split interbacterial cytidine deaminase toxin, DddA, transcription activator-like effector (TALE), and uracil glycosylase inhibitor (UGI), targets gene editing either to nuclear genes or mitochondrial DNA (mtDNA) based on the presence of a nuclear localization signal (NLS) or mitochondrial targeting signal (MTS).
Several approaches have been developed to build TALEs using assemble custom repeat variable di-residues (RVD) arrays, most of which are time-consuming, costly, and unfriendly to ordinary laboratories. To establish a cost-effective, robust, and rapid solution suitable for mitochondria base editing, an RVD plasmid library was constructed that includes 192 modules (Figure 1a): 160 dimer RVD, 16 monomer RVD, and 16 half RVD modules.Two sets of DdCBE backbone plasmids, MTS-DdCBE and NLS-DdCBE, with mitochondrial or nuclear localization signal peptide (Figure 1b and 1c) are available as accessory plasmids. DdCBE expression plasmids, containing the desired RVD array, can be obtained within 36 hours by one-step Golden Gate assembly (Figure 1d).
Figure 1: Architecture of RVD library and DdCBE assembly. (a) The RVD plasmid library, in which each RVD module is flanked by BsaI restriction sites and cloned into a vector containing the tetracycline resistance gene. A total of 192 RVD plasmids were constructed: 160 dimer RVD, 16 monomer RVD, and 16 half RVD modules. (b, c) Backbone plasmids MTS-DdCBE and NLS-DdCBE (available as accessory plasmids) with half DddAtox split at G1333 and G1397 with mitochondrial localization signal (b) or nuclear localization signal (c). (d) One-step assembly of DdCBE. Top, plasmids containing intended RVD are cleaved using BsaI to produce sticky ends. Middle, RVD modules with sticky ends are allowed to ligate to the backbones sequentially. Bottom, the assembled DdCBE vectors contain 13.5–20.5 RVDs. Twenty-seven amino acids (aa) of the C-terminal TALE are from the backbone and 14 aa are from a half RVD module, resulting in 41 aa C-terminal TALE after assembly.
How to Cite this Kit
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which they were created, and include Addgene in the Materials and Methods of your future publications.
For your Materials and Methods section:
“The Shen Lab DdCBE assembly kit was a gift from Bin Shen (Addgene kit #1000000212).”
For your Reference section:
Precision modeling of mitochondrial diseases in zebrafish via DdCBE-mediated mtDNA base editing. Guo J, Zhang X, Chen X, Sun H, Dai Y, Wang J, Qian X, Tan L, Lou X, Shen B. Cell Discov. 2021 Sep 3;7(1):78. doi: 10.1038/s41421-021-00307-9. PubMed (Link opens in a new window) Article (Link opens in a new window)
Additional References
Precision modeling of mitochondrial disease in rats via DdCBE-mediated mtDNA editing. Qi X, Chen X, Guo J, Zhang X, Sun H, Wang J, Qian X, Li B, Tan L, Yu L, Chen W, Zhang L, Ma Y, Shen B. Cell Discov. 2021 Oct 19;7(1):95. doi: 10.1038/s41421-021-00325-7. PubMed (Link opens in a new window) Article (Link opens in a new window)
DdCBE mediates efficient and inheritable modifications in mouse mitochondrial genome. Guo J, Chen X, Liu Z, Sun H, Zhou Y, Dai Y, Ma Y, He L, Qian X, Wang J, Zhang J, Zhu Y, Zhang J, Shen B, Zhou F. Mol Ther Nucleic Acids. 2021 Nov 19;27:73-80. doi: 10.1016/j.omtn.2021.11.016. PubMed (Link opens in a new window) Article (Link opens in a new window)
DdCBE-mediated mitochondrial base editing in human 3PN embryos. Chen X, Liang D, Guo J, Zhang J, Sun H, Zhang X, Jin J, Dai Y, Bao Q, Qian X, Tan L, Hu P, Ling X, Shen B, Xu Z. Cell Discov. 2022 Feb 1;8(1):8. doi: 10.1038/s41421-021-00358-y. PubMed (Link opens in a new window) Article (Link opens in a new window)
Shen Lab DdCBE assembly kit - #1000000212
- Resistance Color Key
Each circle corresponds to a specific antibiotic resistance in the kit plate map wells.
- Inventory
Searchable and sortable table of all plasmids in kit. The Well column links to its kit plate map on the right, as well as lists the plasmid well location in its plate. The Plasmid column links to a plasmid's individual web page.
- Kit Plate Map
96-well plate map for plasmid layout. Hovering over a well reveals the plasmid name, while clicking on a well opens the plasmid page.
Resistance Color Key
| Tetracycline |
Inventory
| Well | Plasmid | Resistance |
|---|---|---|
| Plate 1 / A / 1 | RVD-AA1 | Tetracycline |
| Plate 1 / A / 2 | RVD-AA2 | Tetracycline |
| Plate 1 / A / 3 | RVD-AA3 | Tetracycline |
| Plate 1 / A / 4 | RVD-AA4 | Tetracycline |
| Plate 1 / A / 5 | RVD-AA5 | Tetracycline |
| Plate 1 / A / 6 | RVD-AA6 | Tetracycline |
| Plate 1 / A / 7 | RVD-AA7 | Tetracycline |
| Plate 1 / A / 8 | RVD-AA8 | Tetracycline |
| Plate 1 / A / 9 | RVD-AA9 | Tetracycline |
| Plate 1 / A / 10 | RVD-AA10 | Tetracycline |
| Plate 1 / A / 11 | RVD-pA7 | Tetracycline |
| Plate 1 / A / 12 | RVD-pA8 | Tetracycline |
| Plate 1 / B / 1 | RVD-AC1 | Tetracycline |
| Plate 1 / B / 2 | RVD-AC2 | Tetracycline |
| Plate 1 / B / 3 | RVD-AC3 | Tetracycline |
| Plate 1 / B / 4 | RVD-AC4 | Tetracycline |
| Plate 1 / B / 5 | RVD-AC5 | Tetracycline |
| Plate 1 / B / 6 | RVD-AC6 | Tetracycline |
| Plate 1 / B / 7 | RVD-AC7 | Tetracycline |
| Plate 1 / B / 8 | RVD-AC8 | Tetracycline |
| Plate 1 / B / 9 | RVD-AC9 | Tetracycline |
| Plate 1 / B / 10 | RVD-AC10 | Tetracycline |
| Plate 1 / B / 11 | RVD-pA9 | Tetracycline |
| Plate 1 / B / 12 | RVD-pA10 | Tetracycline |
| Plate 1 / C / 1 | RVD-AG1 | Tetracycline |
| Plate 1 / C / 2 | RVD-AG2 | Tetracycline |
| Plate 1 / C / 3 | RVD-AG3 | Tetracycline |
| Plate 1 / C / 4 | RVD-AG4 | Tetracycline |
| Plate 1 / C / 5 | RVD-AG5 | Tetracycline |
| Plate 1 / C / 6 | RVD-AG6 | Tetracycline |
| Plate 1 / C / 7 | RVD-AG7 | Tetracycline |
| Plate 1 / C / 8 | RVD-AG8 | Tetracycline |
| Plate 1 / C / 9 | RVD-AG9 | Tetracycline |
| Plate 1 / C / 10 | RVD-AG10 | Tetracycline |
| Plate 1 / C / 11 | RVD-hA8 | Tetracycline |
| Plate 1 / C / 12 | RVD-hA9 | Tetracycline |
| Plate 1 / D / 1 | RVD-AT1 | Tetracycline |
| Plate 1 / D / 2 | RVD-AT2 | Tetracycline |
| Plate 1 / D / 3 | RVD-AT3 | Tetracycline |
| Plate 1 / D / 4 | RVD-AT4 | Tetracycline |
| Plate 1 / D / 5 | RVD-AT5 | Tetracycline |
| Plate 1 / D / 6 | RVD-AT6 | Tetracycline |
| Plate 1 / D / 7 | RVD-AT7 | Tetracycline |
| Plate 1 / D / 8 | RVD-AT8 | Tetracycline |
| Plate 1 / D / 9 | RVD-AT9 | Tetracycline |
| Plate 1 / D / 10 | RVD-AT10 | Tetracycline |
| Plate 1 / D / 11 | RVD-hA10 | Tetracycline |
| Plate 1 / D / 12 | RVD-hA11 | Tetracycline |
| Plate 1 / E / 1 | RVD-CA1 | Tetracycline |
| Plate 1 / E / 2 | RVD-CA2 | Tetracycline |
| Plate 1 / E / 3 | RVD-CA3 | Tetracycline |
| Plate 1 / E / 4 | RVD-CA4 | Tetracycline |
| Plate 1 / E / 5 | RVD-CA5 | Tetracycline |
| Plate 1 / E / 6 | RVD-CA6 | Tetracycline |
| Plate 1 / E / 7 | RVD-CA7 | Tetracycline |
| Plate 1 / E / 8 | RVD-CA8 | Tetracycline |
| Plate 1 / E / 9 | RVD-CA9 | Tetracycline |
| Plate 1 / E / 10 | RVD-CA10 | Tetracycline |
| Plate 1 / E / 11 | RVD-pC7 | Tetracycline |
| Plate 1 / E / 12 | RVD-pC8 | Tetracycline |
| Plate 1 / F / 1 | RVD-CC1 | Tetracycline |
| Plate 1 / F / 2 | RVD-CC2 | Tetracycline |
| Plate 1 / F / 3 | RVD-CC3 | Tetracycline |
| Plate 1 / F / 4 | RVD-CC4 | Tetracycline |
| Plate 1 / F / 5 | RVD-CC5 | Tetracycline |
| Plate 1 / F / 6 | RVD-CC6 | Tetracycline |
| Plate 1 / F / 7 | RVD-CC7 | Tetracycline |
| Plate 1 / F / 8 | RVD-CC8 | Tetracycline |
| Plate 1 / F / 9 | RVD-CC9 | Tetracycline |
| Plate 1 / F / 10 | RVD-CC10 | Tetracycline |
| Plate 1 / F / 11 | RVD-pC9 | Tetracycline |
| Plate 1 / F / 12 | RVD-pC10 | Tetracycline |
| Plate 1 / G / 1 | RVD-CG1 | Tetracycline |
| Plate 1 / G / 2 | RVD-CG2 | Tetracycline |
| Plate 1 / G / 3 | RVD-CG3 | Tetracycline |
| Plate 1 / G / 4 | RVD-CG4 | Tetracycline |
| Plate 1 / G / 5 | RVD-CG5 | Tetracycline |
| Plate 1 / G / 6 | RVD-CG6 | Tetracycline |
| Plate 1 / G / 7 | RVD-CG7 | Tetracycline |
| Plate 1 / G / 8 | RVD-CG8 | Tetracycline |
| Plate 1 / G / 9 | RVD-CG9 | Tetracycline |
| Plate 1 / G / 10 | RVD-CG10 | Tetracycline |
| Plate 1 / G / 11 | RVD-hC8 | Tetracycline |
| Plate 1 / G / 12 | RVD-hC9 | Tetracycline |
| Plate 1 / H / 1 | RVD-CT1 | Tetracycline |
| Plate 1 / H / 2 | RVD-CT2 | Tetracycline |
| Plate 1 / H / 3 | RVD-CT3 | Tetracycline |
| Plate 1 / H / 4 | RVD-CT4 | Tetracycline |
| Plate 1 / H / 5 | RVD-CT5 | Tetracycline |
| Plate 1 / H / 6 | RVD-CT6 | Tetracycline |
| Plate 1 / H / 7 | RVD-CT7 | Tetracycline |
| Plate 1 / H / 8 | RVD-CT8 | Tetracycline |
| Plate 1 / H / 9 | RVD-CT9 | Tetracycline |
| Plate 1 / H / 10 | RVD-CT10 | Tetracycline |
| Plate 1 / H / 11 | RVD-hC10 | Tetracycline |
| Plate 1 / H / 12 | RVD-hC11 | Tetracycline |
| Plate 2 / A / 1 | RVD-GA1 | Tetracycline |
| Plate 2 / A / 2 | RVD-GA2 | Tetracycline |
| Plate 2 / A / 3 | RVD-GA3 | Tetracycline |
| Plate 2 / A / 4 | RVD-GA4 | Tetracycline |
| Plate 2 / A / 5 | RVD-GA5 | Tetracycline |
| Plate 2 / A / 6 | RVD-GA6 | Tetracycline |
| Plate 2 / A / 7 | RVD-GA7 | Tetracycline |
| Plate 2 / A / 8 | RVD-GA8 | Tetracycline |
| Plate 2 / A / 9 | RVD-GA9 | Tetracycline |
| Plate 2 / A / 10 | RVD-GA10 | Tetracycline |
| Plate 2 / A / 11 | RVD-pG7 | Tetracycline |
| Plate 2 / A / 12 | RVD-pG8 | Tetracycline |
| Plate 2 / B / 1 | RVD-GC1 | Tetracycline |
| Plate 2 / B / 2 | RVD-GC2 | Tetracycline |
| Plate 2 / B / 3 | RVD-GC3 | Tetracycline |
| Plate 2 / B / 4 | RVD-GC4 | Tetracycline |
| Plate 2 / B / 5 | RVD-GC5 | Tetracycline |
| Plate 2 / B / 6 | RVD-GC6 | Tetracycline |
| Plate 2 / B / 7 | RVD-GC7 | Tetracycline |
| Plate 2 / B / 8 | RVD-GC8 | Tetracycline |
| Plate 2 / B / 9 | RVD-GC9 | Tetracycline |
| Plate 2 / B / 10 | RVD-GC10 | Tetracycline |
| Plate 2 / B / 11 | RVD-pG9 | Tetracycline |
| Plate 2 / B / 12 | RVD-pG10 | Tetracycline |
| Plate 2 / C / 1 | RVD-GG1 | Tetracycline |
| Plate 2 / C / 2 | RVD-GG2 | Tetracycline |
| Plate 2 / C / 3 | RVD-GG3 | Tetracycline |
| Plate 2 / C / 4 | RVD-GG4 | Tetracycline |
| Plate 2 / C / 5 | RVD-GG5 | Tetracycline |
| Plate 2 / C / 6 | RVD-GG6 | Tetracycline |
| Plate 2 / C / 7 | RVD-GG7 | Tetracycline |
| Plate 2 / C / 8 | RVD-GG8 | Tetracycline |
| Plate 2 / C / 9 | RVD-GG9 | Tetracycline |
| Plate 2 / C / 10 | RVD-GG10 | Tetracycline |
| Plate 2 / C / 11 | RVD-hG8 | Tetracycline |
| Plate 2 / C / 12 | RVD-hG9 | Tetracycline |
| Plate 2 / D / 1 | RVD-GT1 | Tetracycline |
| Plate 2 / D / 2 | RVD-GT2 | Tetracycline |
| Plate 2 / D / 3 | RVD-GT3 | Tetracycline |
| Plate 2 / D / 4 | RVD-GT4 | Tetracycline |
| Plate 2 / D / 5 | RVD-GT5 | Tetracycline |
| Plate 2 / D / 6 | RVD-GT6 | Tetracycline |
| Plate 2 / D / 7 | RVD-GT7 | Tetracycline |
| Plate 2 / D / 8 | RVD-GT8 | Tetracycline |
| Plate 2 / D / 9 | RVD-GT9 | Tetracycline |
| Plate 2 / D / 10 | RVD-GT10 | Tetracycline |
| Plate 2 / D / 11 | RVD-hG10 | Tetracycline |
| Plate 2 / D / 12 | RVD-hG11 | Tetracycline |
| Plate 2 / E / 1 | RVD-TA1 | Tetracycline |
| Plate 2 / E / 2 | RVD-TA2 | Tetracycline |
| Plate 2 / E / 3 | RVD-TA3 | Tetracycline |
| Plate 2 / E / 4 | RVD-TA4 | Tetracycline |
| Plate 2 / E / 5 | RVD-TA5 | Tetracycline |
| Plate 2 / E / 6 | RVD-TA6 | Tetracycline |
| Plate 2 / E / 7 | RVD-TA7 | Tetracycline |
| Plate 2 / E / 8 | RVD-TA8 | Tetracycline |
| Plate 2 / E / 9 | RVD-TA9 | Tetracycline |
| Plate 2 / E / 10 | RVD-TA10 | Tetracycline |
| Plate 2 / E / 11 | RVD-pT7 | Tetracycline |
| Plate 2 / E / 12 | RVD-pT8 | Tetracycline |
| Plate 2 / F / 1 | RVD-TC1 | Tetracycline |
| Plate 2 / F / 2 | RVD-TC2 | Tetracycline |
| Plate 2 / F / 3 | RVD-TC3 | Tetracycline |
| Plate 2 / F / 4 | RVD-TC4 | Tetracycline |
| Plate 2 / F / 5 | RVD-TC5 | Tetracycline |
| Plate 2 / F / 6 | RVD-TC6 | Tetracycline |
| Plate 2 / F / 7 | RVD-TC7 | Tetracycline |
| Plate 2 / F / 8 | RVD-TC8 | Tetracycline |
| Plate 2 / F / 9 | RVD-TC9 | Tetracycline |
| Plate 2 / F / 10 | RVD-TC10 | Tetracycline |
| Plate 2 / F / 11 | RVD-pT9 | Tetracycline |
| Plate 2 / F / 12 | RVD-pT10 | Tetracycline |
| Plate 2 / G / 1 | RVD-TG1 | Tetracycline |
| Plate 2 / G / 2 | RVD-TG2 | Tetracycline |
| Plate 2 / G / 3 | RVD-TG3 | Tetracycline |
| Plate 2 / G / 4 | RVD-TG4 | Tetracycline |
| Plate 2 / G / 5 | RVD-TG5 | Tetracycline |
| Plate 2 / G / 6 | RVD-TG6 | Tetracycline |
| Plate 2 / G / 7 | RVD-TG7 | Tetracycline |
| Plate 2 / G / 8 | RVD-TG8 | Tetracycline |
| Plate 2 / G / 9 | RVD-TG9 | Tetracycline |
| Plate 2 / G / 10 | RVD-TG10 | Tetracycline |
| Plate 2 / G / 11 | RVD-hT8 | Tetracycline |
| Plate 2 / G / 12 | RVD-hT9 | Tetracycline |
| Plate 2 / H / 1 | RVD-TT1 | Tetracycline |
| Plate 2 / H / 2 | RVD-TT2 | Tetracycline |
| Plate 2 / H / 3 | RVD-TT3 | Tetracycline |
| Plate 2 / H / 4 | RVD-TT4 | Tetracycline |
| Plate 2 / H / 5 | RVD-TT5 | Tetracycline |
| Plate 2 / H / 6 | RVD-TT6 | Tetracycline |
| Plate 2 / H / 7 | RVD-TT7 | Tetracycline |
| Plate 2 / H / 8 | RVD-TT8 | Tetracycline |
| Plate 2 / H / 9 | RVD-TT9 | Tetracycline |
| Plate 2 / H / 10 | RVD-TT10 | Tetracycline |
| Plate 2 / H / 11 | RVD-hT10 | Tetracycline |
| Plate 2 / H / 12 | RVD-hT11 | Tetracycline |
Kit Plate Map - #1000000212
Accessory Plasmids
Eight additional accessory plasmids are also available. These plasmids are not included in the kit, but can be ordered separately: