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Design and Production Protocols of Plasmids for Base-Editing Experiments

Experiment Summary

Base-editing technologies enable the introduction of point mutations at targeted genomic sites in mammalian cells, with higher efficiency and precision than traditional genome-editing methods that use DNA double-strand breaks, and the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (CRISPR-Cas9) system. Base editing can therefore facilitate genotype-to-phenotype studies in a controlled laboratory setting, with applications in both basic research and clinical applications. Base-editing experiments rely on the preparation of two high-quality plasmids: one expressing the gRNA that designates the target genomic location, and the other expressing the BE protein component (Cas9-deaminase fusion). Here, we provide optimized protocols (including experimental design, methods, and analyses) for designing base-editing constructs for subsequent generation of single-nucleotide variant clonal cell lines.

Schematic of adenine base editor (ABE) process.Fig. 1 Schematic of adenine base editor (ABE) process.

Materials and Reagents

  1. Nuclease-free water
  2. Primers
  3. T4 ligase
  4. T4 polynucleotide kinase
  5. High-Fidelity DNA Polymerase and buffer
  6. dNTP solution mix
  7. Plasmids expressing the base-editing machinery, i.e. gRNA expression plasmid with different spacer sequence: S. pyogenes Cas9 gRNA vector, BE4max-NG-GFP for C•G-to-T•A editing, and/or ABEmax-NG-GFP for A•T-to-G•C editing
  8. 1-kb DNA ladder
  9. DpnI restriction endonuclease
  10. PCR purification kit
  11. Quick ligation kit
  12. Competent cells
  13. 100 mg/ml ampicillin or carbenicillin stock solution
  14. Agar plates with 100 μg/ml ampicillin or carbenicillin (final concentration)
  15. Liquid LB or 2× YT medium with 100 μg/ml ampicillin or carbenicillin (final concentration)
  16. Plasmid mini prep kit
  17. Endotoxin-free plasmid midi prep kit
  18. 50% (v/v) glycerol solution
  19. Sequence-viewing software: e.g., Benchling (https://www.benchling.com) or SnapGene (https://www.snapgene.com)
  20. Primer design software: e.g., Primer3
  21. 0.2-ml PCR tubes
  22. Thermocycler
  23. Vacuum manifold
  24. Nanodrop spectrophotometer
  25. 1.5-ml nuclease-free microcentrifuge tubes
  26. 2-ml cryogenic storage vials
  27. Additional reagents and equipment for agarose gel electrophoresis
  28. Introduction of plasmid DNA into bacterial cells

Overview of Design and production of plasmids for base-editing experiments.Fig. 1 Overview of Design and production of plasmids for base-editing experiments.

A. Design gRNA

  1. Import genomic locus of interest into sequence-viewing software of choice. We frequently use Benchling (https://www.benchling.com) or SnapGene (https://www.snapgene.com).
  2. Identify the target nucleotide (C or A) and find an NG PAM exactly 12-16 nt downstream. The target can be on either strand, as long as there is a suitable PAM on the same strand. Annotate the protospacer. Alternately, free software programs such as the Benchling wizard, BE-designer, or BE-Hive can be used for automated gRNA design. As most automated programs lack flexibility in deaminase and Cas variants, we recommend manually designing your protospacers and checking predicted editing efficiencies in BE-Hive.
  3. If the protospacer does not start with guanine, add a 5' G to create a 21-nt spacer in the gRNA. For protospacers that already start with a 5' G (such as the example V270 protospacer), move to the next step.
  4. Design a custom forward primer with the sequence 5'-GTTTTAGAGCTAGAAATAGCA-3', replacing the portion with the protospacer sequence from the previous step. This will be used with the universal reverse primer. Analyze this primer pair for homo- and heterodimers using the IDT oligo analyzer (https://www.idtdna.com/ pages/tools/ oligoanalyzer). Note that amplification may be difficult when using primers with a ΔG of less than −10 kcal/mol.
  5. Order the primers of choice for custom gRNA construction via site-directed mutagenesis of the spacer or "around-the-horn" cloning.

B. Design primers for amplification of genomic locus

C. Clone new spacer into gRNA expression plasmid via site-directed mutagenesis

1. In 0.2-ml PCR tubes, 5' phosphorylate each primer (forward and reverse,) individually in separate 20-μl reactions by combining the following (in the order stated):

15 μl nuclease-free water

2 μl 100 μM primer stock

2 μl T4 DNA ligase buffer

1 μl T4 polynucleotide kinase.

Mix well and quickly spin to collect sample. The 20 μl of phosphorylated primer is enough for eight PCR reactions—scale up if the primer is needed for more than eight gRNAs.

2. Place the reaction tube in a thermocycler and run the following program:

20 min 37°C

5 min 95°C

Hold 12°C.

3. In a PCR tube on ice, combine the following reagents for a 50-μl reaction (in the order stated):

X μl nuclease-free water (fill to a total volume of 50 μl)

10 μl Phusion HF buffer

1 μl dNTP mix

2.5 μl FWD phosphorylated primer (10 μM, from previous step)

2.5 μl REV phosphorylated primer (10 μM, from previous step)

~1 ng DNA template: gRNA expression vector

0.5 μl Phusion polymerase.

4. Mix, quickly spin (~5 sec at ~2000 rcf) at room temperature, and run a thermocycler program with the following cycling conditions:

Overview of Design and production of plasmids for base-editing experiments.

5. Run a 1-μl aliquot out on a 1-1.5% agarose gel with a 1-kb DNA ladder to check for a PCR product that is ∼2.3 kb long.

6. Add 1 μl DpnI to the PCR product, mix, and quickly spin at room temperature. Incubate 1 h at 37°C.

7. Purify PCR products using the PCR kit following the manufacturer's PCR clean-up protocol.

8. Quantify the concentration of the PCR product using a Nanodrop spectrophotometer. Then prepare a 20-μl ligation reaction to circularize the linear PCR product, which contains the new spacer sequence and 5' phosphate groups. In a PCR tube, combine:

~50 ng PCR product after clean-up (from previous step)

X μl nuclease-free water (fill to a total volume of 20 μl)

10 μl 2× Quick Ligase buffer (from Quick Ligation kit)

1 μl Quick Ligase.

9. Mix, quickly spin at room temperature, and incubate 5-10 min at room temperature.

10. Using sterile technique, transform ~5 μl of the ligated product into competent bacterial cells of choice.

11. Pick 1-2 colonies per clone and grow in liquid LB or 2× YT medium with 100 μg/ml ampicillin or carbenicillin until saturated.

12. When cultures reach saturation, purify plasmid DNA by miniprep.

13. Quantify the prep using a Nanodrop, and then run an aliquot on an agarose gel (1.5%) alongside a 1-kb DNA ladder to check the quality and size of the plasmid.

14. Submit preps to a sequencing company for Sanger sequencing to confirm the presence of the new spacer sequence.

Related Products and Services at Creative Biogene

Base Editing by CRISPR
SgRNA Design and Confirmation
* For research use only. Not intended for any clinical use.
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