Plant Gene Knock-In Service

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Plant Gene Knock-In Service

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CD BioSciences has abundant gene editing experience and successful cases. We can provide a full set of plant genome editing solutions, including CRISPR/Cas9-based gene knock-in and various other plant gene editing technologies. Our services can be applied to improve crop characteristics such as high yield, high nutrition, pest and disease resistance, drought resistance, and freezing resistance.

Principle of CRISPR/Cas9-Based Plant Gene Knock-In

The sgRNA (short guide RNA) in the CRISPR/Cas9 system recognizes and binds to the target sequence of the target gene, guiding Cas9 to shear the binding site and produce a DNA double-strand break. Through homologous recombination repair in cells, the exogenous donor DNA is introduced into the target site of the genome, thus realizing gene knock-in.

Figure 1. CRISPR-induced gene knock-in mediated by geminivirus-derived replicons in plant cells. – CD BioSciences Figure 1. CRISPR-induced gene knock-in mediated by geminivirus-derived replicons in plant cells. (Collonnier, C., et al., 2017)

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Additional services Descriptions
Reverse transcription (RT)-PCR We can validate positive cell libraries/clones at the mRNA level.
Flow cytometry analysis We validate transgene expression by FACS analysis.
Western blotting We can validate positive clones by western blotting.
Expression stability testing We validate GOI expression stability by FACS at generation 15/20.

Optimization services of CRISPR/Cas9-based knock-in experiments in plants

  • Choosing the right DNA donor template.

Designing a DNA template is the first step in a knock-in experiment. We use bioinformatics tools to design DNA donor templates for knock-in experiments.

  • Reducing CRISPR/Cas9 off-targets.

We utilize bioinformatics tools to help clients design gRNAs with low off-target probability, such as CRISPR-PLANT, E-CRISP, and CRISPR-P.

  • Optimizing gRNA expression.

Our technology enables multi-site editing for simultaneous expression of multiple gRNA molecules.

  1. Multiple expression units are cloned in plasmid vectors.
  2. Transcripts containing multiple gRNAs are cleaved into individual gRNAs using Csy4 nuclease.
  3. The organism's endogenous transfer RNA (tRNA) is fused to the gRNA.
  4. Reducing PAM limitations.

We modified the amino acid sites related to PAM recognition in the Cas9 protein to obtain Cas9 mutants recognizing different PAM sequences such as 5′-NGAN-3′ and 5′-NGNG-3′ for plant genome editing.

  • Optimizing experimental conditions for CRISPR knock-in.

Given that knock-in is usually less efficient, we can help clients make every effort to optimize experimental conditions, including

  1. Cell number/density.
  2. gRNA: Cas9 ratio.
  3. Recovery media conditions.
Clients need to provide Deliverables
  • Gene sequence and NCBI ID.
  • Host cells.
  • Cell line culture conditions.
  • Western blot/FACS antibody (for validation experiments).
  • One line of knock-in monoclonal cells.
  • Cell cofA file.
  • Sequencing results.
  • Knock-in cell line protein level verification results.
  • Knock-in cell line mRNA level verification.

CD BioSciences is a leading biological company specializing in plant gene editing services. With our cutting-edge expertise and state-of-the-art technology, we offer tailor-made solutions for precise gene knock-in in plants. If you are interested in our services, please feel free to contact us. We prioritize innovation, accuracy, and efficiency to ensure satisfaction of your scientific requirements.

References

  1. Collonnier, C., et al. (2017). Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens. Methods (San Diego, Calif.). 121-122, 103-117.
  2. Rozov, S. M., et al. (2022). Optimization of genome knock-in method: search for the most efficient genome regions for transgene expression in plants. International journal of molecular sciences. 23(8), 4416.

For research use only, not for clinical use.