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)

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)

AAVS1 Knockin Validation Data

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
GE100023	pCas-Guide-AAVS1	AAVS1 gRNA vector, validated AAVS1 targeting sequence cloned in pCas-Guide vector

AAVS1 Donor Vectors

Cat #	Product Name	Description
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
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

Cas9 insertion at AAVS1 locus

RFP AAVS1 donors
(AAVS1 positive control)

AAVS1-int5 PCR Primer Pairs

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 Knockin validation data

ROSA26 gRNA Vector

Cat #	Product Name	Description
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
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
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
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

CRISPR Vectors

CRISPR Knockout Kits

Cas9 Nuclease

Cas9 Ab

Scheme of transgene knockin AAVS1/ROSA26 via CRISPR