Mammalian Cell Gene Editing_Synbio Technologies

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The General Introduction of Mammalian Cell Gene Editing

The emergence of CRISPR-Cas9 gene editing technology provides a simple and fast method for constructing mammalian models and offers new approaches for the treatment of genetic diseases.

Synbio Technologies introduces CRISPR RNP-mediated cell gene editing services, which build on advanced technology and an experienced team, offering end-to-end solutions including project design, sgRNA design and chemical synthesis, high-activity recombinant Cas9 protein, RNP complex assembly, and knockout cell line construction.

Highlights

CRISPR sgRNA design for all species models
Chemically synthesized sgRNA with purity ≥ 99%
Recombinant Cas9 protein with purity ≥ 99%
Delivery in as fast as 2 weeks

Service Details

Services	Deliverables	Trunaround Time
Project Design	Professional analysis report Design sequences	Quote
Synthesis & Assembly	Chemical synthesis of modified sgRNA High-activity, high-purity Cas9 protein RNP complex assembly	Quote
Cell Line Construction	Polyclonal cell group or monoclonal cell strain Cell genotype re-examination, amplification and cryopreservation	Quote
Verification	DNA level detection Protein level detection	Quote

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Workflow

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Case Study

1. Using the dual-sgRNA editing strategy with RNP to achieve DNA-level fragment knockout

Case：Knockout of Exon 5-7 fragments of gene A in X cells
(1) 1.Design one highly targeted sgRNA sequence at each end of the required knockout fragment, as shown below:

(2) After chemically modifying and synthesizing gRNA-A1 and gRNA-A2, they are assembled with Cas9 protein into RNPs, which are then transfected into X cells to knock out the A gene fragment sequence.

(3) Knockout result detection is as follows:

At the DNA level - Sanger sequencing shows that the A gene sequence has been knocked out, and the result is as follows:

Cell phenotype detection is shown in the figure below: A represents normal cells, and B represents the mutated cell morphology after the knockout of the A gene, indicating that the A gene has been knocked out.

2. RNP knockout efficiency surpasses traditional plasmid methods

Case: Comparing the knockout efficiency of plasmids (DNA) and RNP using AAVS1 locus knockout as an example.

(1) The AAVS1-sgRNA1 sequence was cloned into a vector containing Cas9, and after transfection of cells, genomic DNA was extracted. Sanger sequencing was performed using genomic DNA as the sample, followed by analysis with the Synthego ICE software. The results showed a knockout efficiency of 14%;

(2) After transfecting the AAVS1-sgRNA1 RNP complex into cells, detection was performed as described in (1), and the results showed a knockout efficiency of 69%;

(3) Comparison of knockout efficiencies (as shown in the figure below) revealed that the RNP knockout efficiency is 4-5 times higher than the plasmid method, significantly outperforming the plasmid method.

RNP Knockout Method

3. Fragment Knock-In via CRISPR/Cas9 Genome Editing

Case: Functional tag knock-in in X cells

(1)A high-specificity gRNA was designed near the insertion site and complexed with Cas9 protein to form RNPs, which were delivered via electroporation.

(2)The donor vector included homologous arms, a functional tag, a selection marker, and removable elements for efficient targeted integration.

(3)Knock-in result detection is as follows:

Sanger sequencing results:

Cellular phenotype analysis:

FAQs

What Issues Should Be Noted in Mammalian Cell Gene Editing?

1. Cell Culture and Selection:

Choose the appropriate mammalian cell type, such as CHO (Chinese hamster ovary) cell lines, NS0 and Sp2/0 mouse myeloma cells, HEK293 (human embryonic kidney 293) cells, etc., depending on the experimental goals and requirements.

2. Ensure Cell Quality and Purity:

Use appropriate culture conditions and media to maintain normal cell growth and division, ensuring the quality and purity of the cells.

Selection of Target Site and sgRNA Design:

3. Carefully Select the Editing Site:

Ensure the edited gene sequence meets the expected outcomes, while avoiding excessive toxicity or lethal effects on the cells.

4. Design Highly Specific sgRNAs:

Ensure that the CRISPR-Cas9 system can accurately recognize and cut the target DNA sequence by designing highly specific sgRNAs.

Construction and Validation of Cas9/sgRNA Vectors:

5. Construct and Validate Cas9/sgRNA Vectors:

Construct the Cas9/sgRNA vectors for mammalian cells and verify their activity and efficiency. The effectiveness of the vector can be evaluated by transfecting cells and assessing the gene editing results.

Cell Transfection and Screening:

6. Cell Transfection and Screening:

Use appropriate transfection methods to introduce Cas9/sgRNA vectors into mammalian cells, ensuring transfection and gene editing efficiency. Enrich gene-edited cells by screening for specific phenotypes, such as drug resistance or fluorescence expression.

Detection of Gene Editing Effects:

7. Detect Gene Editing Effects:

Use technologies such as next-generation sequencing (NGS) to detect the effects of gene editing, including mutations at the target site and off-target effects. Ensure that the editing results meet expectations and avoid mutations at non-target sites.

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