Membrane Protein Cell Line Development with CRISPR/Cas9

Gene knock-out (KO) is a critical method for identifying the functions of coding and non-coding genomic regions. The clustered regularly interspaced short palindromic repeats, the CRISPR-associated protein 9 (CRISPR/Cas9) system has emerged as one of the most exciting tools for gene knock-out in recent years. The CRISPR/Cas9 system relies simply on base-pairing between the single-guide RNA (sgRNA) and the target DNA. With a minimal requirement in target design and straightforward construction of sgRNAs, it facilitates widespread application in fundamental research. Creative Biolabs has CRISPR/Cas9 system to edit the membrane protein of interest.

CRISPR/Cas9 system (Creative Biolabs) Fig.1. CRISPR/Cas9 system.

Workflow of Membrane Protein Cell Line Development with CRISPR/Cas9

CRISPR Design	Design Deletion Screening Primers	CRISPR Cloning	Transfecting CRISPRs into Cells of Interest
Design sgRNAs to help identify guide sequences that minimize identical genomic matches or near-matches to reduce the risk of off-target effects.	Design primers internal to the sequence to be deleted and primers upstream and downstream of the sgRNA cleavage sites.	Anneal and phosphorylate oligos Ligate annealed oligos into vector Pick 2 - 3 colonies and choose a sequence-verified colony	Delivery of CRISPR/Cas9 plasmids by electroporation. Transfection of CRISPR/Cas9 plasmids may be successfully adapted to numerous cell types.

FACS of Transfected Cells	Primer Validation and Screening for CRISPR/Cas9-Mediated Deletion	Screening CRISPR/Cas9 Clones for Deletions and Clone Selection	Validation of Biallelic Deletion Clones
FACS sort cells to enrich for cells that received high levels of the CRISPR/Cas9 constructs.	Examine samples for the presence/absence of non-deletion and deletion bands. Consider multiplexing the “deletion” and “non-deletion” PCR primer pairs in a single reaction.	Extract the gDNA from clones Screen each clone Select the clones identified with the desired deletion	To characterize obtained clones and validate a successful knockout, evaluate clones at the DNA as well as RNA or protein levels.

Case Study of Membrane Protein Double Knockout Cell Line Development with CRISPR/Cas9 System

Before stable cell line engineering, we evaluate the transfection efficiency of target host cells.

Fig.2. Host cell transfection evaluation.

Workflow of Membrane Protein Cell Line Development with CRISPR/Cas9

According to the target protein sequence of your interest and the gene editing requirements, we can design sgRNA sequences. Also, we can validate the sgRNA transfection efficiency.

Host cell transfection evaluation (Creative Biolabs) Fig.3. sgRNA sequences.

After sgRNA and Cas9 transfection, knockout efficiency is validated with target gene sequencing. Once knockout is successful, single cell clones will be selected and expanded for further sequencing and FACS validation.

Knockout efficiency and FACS validation of target gene knockout efficiency (Creative Biolabs)

Fig.4. Knockout efficiency and FACS validation of target gene knockout efficiency.

Creative Biolabs has extensive experience in the field of membrane protein cell line development. Our scientists have developed several perfect membrane protein knockout cell lines with CRISPR/Cas9 technology. We can offer you the highest quality, accuracy, precision, and a fast turnaround to meet deadlines.

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