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CRISPR Services
CRISPR Services
Investigate gene function, shut down the production of a particular protein, introduce specific-site mutations, or even introduce a reporter gene or regulatory DNA region by leveraging our high-quality knock-in and knock-out gene editing services and expertise that consistently delivers on challenging projects.
Our Knock-in & Knock-out Services
Knock Out Gene Editing Services
- Single Gene Knockout
- Multi-Gene Knockout
- Large Deletion
- Knockout Pools
Knock In Gene Editing Services
- Point Mutation Insertional Mutation (i.e. epitope tagging)
- Large Insertional Mutation (i.e. fluorescent protein fusion, transcriptional termination, etc.)
- Multi-Gene Knock-in
Whether you need homozygous knockouts, site-specific point mutations, or epitope tagging, partner with Rockland to get a reliable source of gene editing reagents and data:
- Two independent clonal cell lines (2 vials of 1MM cells)
- Parental cells (1MM cells)
- Sequence analysis
- Raw sequence files
- Certificate of analysis
Custom Project Workflow
Rockland's scientific expertise enables highly customized CRISPR services to meet your basic, applied and clinical research demands to drive the next wave of discoveries.
1
Project Design & Assessment
- Custom cell line sterility
- gRNA and HDR template design and synthesis
2
Reagent Synthesis
- PCR design
- Primer synthesis
- Reaction optimization
3
Transfection & Bulk Analysis
- Cell transfections
- Bulk population PCR and Sanger sequencing (for efficiency analysis)
4
Clonal Sorting & Expansion
- Single-cell sorting
- Clonal preparation
5
Clone Verification* & Delivery
- Clonal genomic analysis by Sanger sequencing (for on-target verification)*
- Cell banking
- Delivery of identified engineered clones
*Upon request, we can provide additional services to further evaluate the target gene editing at the protein level using Western Blot, ELISA, or related functional assays.
The CRISPR/Cas System
CRISPR-Cas9 targeting system. In the CRISPR/Cas9 system, a guide RNA hybridizes a 20-nt DNA sequence immediately preceding an NGG DNA motif (protospacer-associated motif or PAM), resulting in a double-strand break (DSB) 3 bp upstream of the NGG. The double-stranded DNA breaks become substrates for endogenous cellular DNA repair machinery that catalyze nonhomologous end joining (NHEJ) or homology-directed repair (HDR). [Adopted from Charpentier & Doudna, Nature, 2013,495:50–1]
Case Studies
1 Homozygous Knockout
Project Goal: Knockout with effective gRNA targeting all 7 coding variants.
Strategy: Target all known splicing variants of ITGβ6 by choosing appropriate gRNA to ensure effective knockout (A). TIDE analysis of the transfected cells (bulk population) shows high knockout efficiency (B). TIDE analysis of a clonally expanded cell shows that all three copies of ITGβ6 are knocked-out via frame-shifting indels (C).
2 Multiplex Knock Out
Project Goal: Knockout using multiple gRNAs targeting all 6 varients by multiplex.
Strategy: Target all known splicing variants of KEAP1 (red text) to ensure effective knockout using two gRNAs simultaneously (A). TIDE analyses of a clone shows frame-shifting indels at both CRISPR targeted loci, generating a knockout of all variants (B, C).
3 Point Mutation via HDR
Project Goal: Introduction of pathological mutations using CRISPR and HDR template DNA.
Strategy: Induce two amino acid changes using CRISPR and HDR template DNA (A). Clonal analysis shows successful homozygous mutations were created at the intended sites (B).
4 Epitope Tagging Project
Project Goal: Knock in of C-terminal epitope tag using CRISPR and HDR template DNA.
Strategy: Knock in 24bp epitope tag sequence at C-terminus using CRISPR and HDR template DNA (A). Clonal analysis shows homozygous in-frame insertion of the 24bp epitope tag sequence at the C-terminus (B).
Interested in CRISPR Services?
We can help move your program forward—contact us to find out how.