Transgene Knockin via CRISPR 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.

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 ( Precision Shuttling System)
ROSA26 or AAVS1 diagram

gRNA/CRISPR cuts the genome, then the gene of interest (GOI) cassette will be knocked in via homologous recombination.

AAVS1 – Human Targeted Gene Insertion via CRISPR

ROSA26 – Mouse Targeted Gene Insertion via CRISPR


AAVS1 Safe Harbor knockin system via CRISPR

This system needs two components:

  1. AAVS1 gRNA vector,All-in-one CRISPR vector containing Cas9 expression cassette and specific gRNA targeting AAVS1 locus
  2. AAVS1 Donor vector,donor vector for transgene integration, containing the AAVS1 homologous arms (Gene of interest needs to be cloned)

OriGene offers a prevalidated AAVS1 gRNA and many AAVS1 donor vectors, which contains different combination of drug selection marker and promoter. In addition to the individual gRNA and donor vector, the pre-assembled AAVS1 vector kit is also available, including not only AAVS1 specific gRNA and donor vector, but also a non-specific scramble control. You can also get our predesigned positive donor control for your experiment or request a custom AAVS1 donor plasmid.

AAVS1 gRNA Vector

Cat # Product Name Description Manual
GE100023 pCas-Guide-AAVS1 AAVS1 gRNA vector, validated AAVS1 targeting sequence cloned in pCas-Guide vector

AAVS1 Donor Vectors

Cat # Product Name Description Manual
GE100024 pAAVS1-Puro-DNR Donor vector with AAVS1 homologous arms cloned, ready for transgene cloning
GE100035 pAAVS1-BSD-DNR Donor vector with AAVS1 homologous arms cloned (blasticidin selection), ready for exogenous gene cloning (10 ug)
GE100046 pAAVS1-EF1a-Puro-DNR AAVS1 donor vector with AAVS1 homologous arms cloned (Puro selection), EF1a promoter, ready for exogenous gene cloning (10 ug)
GE100048 pAAVS1-EF1a-BSD-DNR AAVS1 donor vector with AAVS1 homologous arms cloned (blasticidin selection), EF1a promoter, ready for exogenous gene cloning (10 ug)

Transgene Knockin Vector Kits

Cat # Product Name Description Manual
GE100027 AAVS1 Transgene knockin vector kit Vector kit for targeted transgene insertion into AAVS1 locus, including GE100023, GE100024 and GE100003
GE100036 AAVS1 Transgene knockin kit (BSD) Vector kit for targeted transgene insertion into AAVS1 locus (blasticidin selection), including GE100023, GE100035 and GE100003
GE100047 AAVS1 Transgene Knockin kit (EF1a, Puro) Vector kit for targeted transgene insertion into AAVS1 locus (EF1a, Puro), including GE100023, GE100046 and GE100003
GE100049 AAVS1 Transgene Knockin kit (EF1a, BSD) Vector kit for targeted transgene insertion into AAVS1 locus (EF1a, blasticidin), including GE100023, GE100048 and GE100003

AAVS1 Donor Controls

Cat # Product Name Promoter Transgene/Selection Marker
GE100037 pAAVS1-Cas9-Puro-DNR CMV Cas9/Puromycin
GE100039 pAAVS1-Cas9-BSD-DNR CMV Cas9/Blasticidin
GE100026 pAAVS1-RFP-DNR CMV tRFP/Puromycin
GE100063 pAAVS1-mGFP-Puro-DNR CMV mGFP/Puromycin
GE100064 pAAVS1-mRFP-Puro-DNR CMV mRFP/Puromycin
GE100065 pAAVS1-mGFP-BSD-DNR CMV mGFP/Blasticidin
GE100066 pAAVS1-mRFP-BSD-DNR CMV mRFP/Blasticidin
GE100068 pAAVS1-EF1a-tGFP-puro-DNR EF1a tGFP/Puromycin
GE100069 pAAVS1-EF1a-tGFP-BSD-DNR EF1a tGFP/Blasticidin
GE100070 pAAVS1-EF1a-tRFP-puro-DNR EF1a tRFP/Puromycin
GE100071 pAAVS1-EF1a-tRFP-BSD-DNR EF1a tRFP/Blasticidin
GE100072 pAAVS1-EF1a-Luc-puro-DNR EF1a Luciferase/Puromycin
GE100073 pAAVS1-EF1a-Luc-BSD-DNR EF1a Luciferase/ Blasticidin

More AAVS1 Products


ROSA26 Mouse Safe Harbor Knockin System 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.

OriGene has developed a series of ROSA26 CRISPR products to help researchers insert their gene of interest in the ROSA26 genomic locus through homologous recombination

This system needs two components:

  1. pCas-Guide-ROSA26, All-in-one CRISPR gRNA vector for ROSA26 targeting
  2. pAAVS1-Puro-DNR, donor vector for transgene integration, containing ROSA26 homologous arms (Gene of interest needs to be cloned)

ROSA26 gRNA Vector

Cat # Product Name Description Manual
GE100050 pCas-Guide-ROSA26 ROSA26 safe harbor gRNA vector, validated ROSA26 gRNA targeting sequence cloned in pCas-Guide vector

ROSA26 Donor Vector

Cat # Product Name Description Manual
GE100051 pROSA26-Puro-DNR ROSA26 donor vector with ROSA26 homologous arms cloned (Puro selection), ready for exogenous gene cloning (10 ug)

ROSA26 donor control

Cat # Product Name Description Manual
GE100067 pRosa26-tGFP-Puro-DNR ROSA26 donor vector with ROSA26 homologous arms, tGFP expression, and Puro selection

ROSA26 CRISPR Knockin Kit

Cat # Product Name Description Manual
GE100052 ROSA26 CRISPR transgene knockin kit (Puro) Vector kit for CRISPR targeted transgene insertion into ROSA26 locus in mice (Puro), including GE100050, GE100051 and GE100003

Other CRISPR Products


Scheme of transgene knockin AAVS1/ROSA26 via CRISPR


AAVS1 transgene knockin diagram
ROSA26 transgene knockin diagram