CRISPR Cell Line Development

Make Genome Editing Easy

Overview Service-Specifications Case Studies Contact Us

Home » CRISPR Services » CRISPR Cell Lines

Overview

CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. CRISPR sequences were originally identified in the Escherichia coli (E. coli) genome, and were found to function as part of an RNA-based adaptive immune system to target and destroy genetic parasites at the DNA level. CRISPR-associated protein (Cas) is an endonuclease that cuts foreign DNA, allowing integration into the host genome. Cleavage only occurs when there is a protospacer adjacent motif (PAM) around the targeted sequence of the invading DNA, ensuring highly accurate targeting. Researchers studying CRISPR have adapted it for use as a tool for genetic modification of the target host genome. CRISPR/Cas9 has recently become a popular genome editing tool, due to its simplicity and versatility.

Highlight

Workflow

Service Specifications

Service Name	Service Detail	Cell Line Options	Deliverables	Delivery Time
GenCRISPR™ EZ knockout Service	Single gene knock out cell pool/cell line Multiplexed gene knock out cell pool/cell line	Available for 90+ popular transfection-suitable cell lines, including A549, CHO-K1, HEK293, HEK293T, HT-29, MDA-MB-231, 4T1, A20, HCT116, MCF7, MDCK, U937, RPMI 8866, etc.	One knockout cell pool/two full-allelic knockout cell lines validated by sequencing One negative control cell pool transfected with non-targeting gRNA Biweekly project updates	3-6 weeks (cell pool) 9-15 weeks (cell clone)
GenCRISPR™ Customized Knockout Service				8-11 weeks (cell pool) 16-24 weeks (cell clone)
GenCRISPR™ Customized Knock-In Service	Point mutation Seamless point mutation Endogenous locus/gene tagging/reporter Cell model for genetic variation study (including SNV, SV, InDel, etc.) Safe harbor insertion of GOI	Any cell line	1-2 knock in cell lines validated by sequencing One negative control cell pool transfected with non-targeting gRNA Biweekly project updates	12-25 weeks (Cell clone)
GenCRISPR™ Whole Gene Deletion Service	Whole gene deletion	Any cell line	One deletion cell pool/full-allelic deletion cell line validated by sequencing One negative control cell pool transfected with non-targeting gRNA Biweekly project updates	13-23 weeks (cell clone)

It is preferred that customers provide their own cell lines, despite available cell lines can be purchased by GenScript for an additional fee. Please note that we currently do not provide genome editing service for primary cells, stem cells or iPS cells.
Validation services for CRISPR cell lines can be found under the "Add On Services" tab.

Add-On Service

Add-On Service	Description
Reverse transcription (RT)-PCR	Validate the positive cell pool/clones at mRNA level
FACS analysis	Validate the transgene expression by FACS analysis. A validated antibody with specific binding to target protein or reporter tag must be provided.
Western Blot	Validate the positive clones by western blot. A validated antibody in the host cells with specific binding to target protein must be provided.
Expression stability test	Validate the GOI expression stability by WB or FACS in 15/20 generation.
One additional clone	QC and deliver one additional positive clone.
Off-target analysis	Characterize one knockout clone by sequencing the top 5 of potential off-target sites.

Case Studies

Case Study I
- K-ras locus in human colon cancer cell line, HCT116, was knocked-out by creating indels in exon 4, as shown in the targeting strategy (Fig. 1).
- Viral encapsulation of Cas9 and gRNA delivery particles were carried out, viral titer was optimized for HCT116 cells.
- Individual clones were Sanger sequenced to select for homozygous knock-out indels of K-ras at exon 4. Sanger sequencing data (Fig. 2).
- Absence of K-ras expression in the selected clone was confirmed via Western Blot (Fig. 3).
Case Study II
- A sequence optimized gRNA was designed and synthesized to target a specific region on the GS allele. DG44 cells were transfected with the construct and the cell pool was analyzed by Sanger sequencing.
- Several single clones were obtained and Sanger sequence analyzed. A single clone containing a frame shift mutation was carried forward (Fig. 1).
- The GS knockout clone was also analyzed for GS protein expression (Fig. 2).
- To assess loss of function, GS knockouts were grown in the presence or absence of increasing concentrations of glutamine (Fig. 3). GS knockouts were unable to grow in the absence of glutamine. However growth improved in the presence of increasing concentrations of glutamine, thereby indicating a functional loss in the GS knockout cell line.
- In conclusion, successful targeting of GS generated a single clone that was fully validated for lack of expression and GS function.
Case Study III

A

Validation of A gene KO by RT-PCR

B

Validation of B gene KO by WB

Double gene Knockout