Gene Knock-Out Cell Line Construction Service

Seattle Genova is experienced to deliver a full CRISPR/Cas9-based single gene knockout service utilizing any mammalian cell line and targeting any gene. Gene knock-out (KO) is a critical method for recognizing the functions of coding and non-coding genomic regions. Our scientists are specialists at completing gene knockout with CRISPR/Cas9, from designing gRNA constructs to transfection and single clone generation of a broad range of cells, containing difficult-to-transfect and tumor cell lines.

Process

• Cell preparation:

Based on the cell type assigned, we prepare cells two weeks in advance to maintain their high activity.

• Identification of target knockout gene:

We then observe the coding sequence (CDS) region of the gene and examine the corresponding genomic structure to determine the exon part of CDS. Based on the nature of genes, candidate knockout areas are selected and then identified.

• gRNA design and vector construction:

Three to five (3–5) groups of gRNA will be designed, and corresponding plasmids will be constructed while also incorporating Cas9.

• Cell transfection and obtaining monoclonal cells:

Constructed plasmids are transfected to the host cell to create a cell pool. We will then sequence cells in the cell pool to verify knockout impacts.

• Screening for positive clones:

Utilizing the limited dilution method, transfected cells are plated on 96-well plates for monoclonal screening. Positive monoclonal cells will be amplified, sequenced, identified, and reserved. Ultimately, a cell line is successfully constructed.

• Stability test

• Mycoplasma detection:

It includes Quantitative polymerase chain reaction and Western Blot.

• Cell line freezing and delivery

Quality of our services

• Professional researchers have productive cell culture experience and plentiful CRISPR genome editing experience.

• High achievement rate and low non-specific target rate: multi gRNA design and verification, with non-specific target examination and testing services

• Fast delivery with full transparency: delivery of knockout cell lines in ~3 months, periodic progress report, and transparency during the whole procedure

• One-stop service: We focus on investigations of genes and recombinant proteins. We also deliver one-stop service from gRNA design to protein detection (optional).

Deliverables

• Two full-allelic knockout cell lines verified by sequencing

• One negative control cell line

• Project reports including host cell line characterization report, cell pool editing efficiency report, and clone validation report

• Add-on functional validation services