CRISPR/Cas9 Safe Harbor Transgene Knockin

Targeted gene insertion at AAVS1 and ROSA26 loci

Transgene expression via random integration into the genome is subjected to position effects and silencing. In addition, random gene insertion might interrupt or activate the neighboring genes. Genomic safe harbor sites are transcriptionally active, therefore allowing robust and stable gene expression. Furthermore, a transgene insertion at genomic safe harbors does not have adverse effect on the host cell genome. CRISPR technology can be utilized to do targeted gene insertion at these genomic loci. For Human cells, AAVS1 has been accepted as a high gene expression and a safe genomic location; for Mouse cells, ROSA26 is proved to be a genomic safe harbor locus.

Transgene insertion at AAVS1 or ROSA26 locus via CRISPR/Cas9

Why is Safe Harbor transgene insertion needed?

• Targeted transgene insertion
• Robust and stable gene expression
• No random genetic interruption and insertional mutagenesis
• Critical for therapeutic cell engineering and many functional screening/assays
• Genome-wide TrueORF collection serves as the source for transgenes
• Easy shuttling from TrueORF clones into AAVS1 and Rosa26 donor vector (PrecisionShuttle System)

AAVS1 Human Safe Harbor knockin via CRISPR

This system needs two components:
AAVS1 gRNA Vector: All-in-one CRISPR vector containing Cas9 expression cassette and specific gRNA targeting AAVS1 locus
AAVS1 Donor Vector: Donor vector for transgene integration, containing the AAVS1 homologous arms (Gene of interest needs to be cloned)
A prevalidated AAVS1 gRNA Vector and many AAVS1 Donor Vectors which contain different combination of drug selection marker and promoter are offered. In addition to the individual gRNA and donor vector, pre-assembled AAVS1 Vector Kits are also available, including not only AAVS1 specific gRNA and donor vector, but also a non-specific scramble control. You can also get our predesigned donor controls for your experiment or request a custom AAVS1 donor plasmid.

ROSA26 Mouse Safe Harbor knockin via CRISPR

The ROSA26 site is the most popular genomic safe harbor site for mouse cell. It has been used to create more than a hundred mouse knock-in cell lines. The transgene inserted in this locus showed a constitutive and strong gene expression in mice. A series of ROSA26 CRISPR products to help researchers insert their gene of interest in the ROSA26 genomic locus through homologous recombination is offered.

This system needs two components:
pCas-Guide-ROSA26: All-in-one CRISPR gRNA vector for ROSA26 targeting
pAAVS1-Puro-DNR Donor Vector:  Donor vector for transgene integration, containing ROSA26 homologous arms (Gene of interest needs to be cloned)

A ROSA26 CRISPR Knockin Kit and a ROSA26 donor control are also provided.

Scheme of transgene knockin AAVS1/ROSA26 via CRISPR