CompoZr^® Targeted Integration Kit - AAVS1

Rapid Gene Insertion into the Human Cell Line of Your Choice

The CompoZr Targeted Integration Kit – AAVS1 is designed to rapidly integrate a user-specified gene of interest (GOI) into the preferred site of integration of the adeno-associated virus (AAVS1) on human chromosome 19. The Zinc Finger Nuclease (ZFN) in this kit targets the AAVS1 site with high specificity, creating a double strand break. The cell then uses the pZDonor plasmid, with homology arms to the AAVS1 genomic region, in homology-directed repair to replace the natural AAVS1 locus. The pZDonor plasmid can be engineered to contain your GOI and will direct targeted integration of your GOI into the AAVS1 locus (see figure 1).

Features and Benefits:

• Effective integration efficiency without antibiotic selection
  The ZFNs engineered to target AAVS1 are a well-validated pair that cut the DNA with high efficiency. ZFN-aided targeted integration into the AAVS1 site can achieve high success rates in human cell types up to three to four orders of magnitude higher than spontaneous homologous recombination events.¹
• Rapid biallelic insertions through a single transfection
  ZFN mediated homology-directed repair leads to gene addition within 48 hours of transfection of ZFN mRNA/plasmid donor, with both monoallelic and biallelic insertions.¹
• Controlled transgene effects through use of single genomic locus
  In the past, most transgenic cell lines have been constructed via random integration of a plasmid. This results in a collection of clones with greatly varied expression levels and expression stability. Targeted integration into the AAVS1 locus will provide a common integration site with stable, uniform expression levels.¹
• Eliminates need to engineer cell lines with transgene landing pads
  Transgene integration into the genome has previously been achieved via recombination through recombinases such as Cre and FLP. This requires prior insertion of a transgene landing pad into the genome through random integration followed by transgene addition, making it a lengthy two step process. Targeted integration into AAVS1 eliminates the first step, making transgene expression a simple one-step process.¹

Why is AAVS1 considered an ideal safe harbor locus?

1. It is the only adeno-associated virus type 1 (AAVS1) integration site in the human genome¹
2. This site expresses the p84 protein with no known function and disruption of this site does not have any major effect on the cells¹
3. This site is constitutively expressed, demonstrating transcriptional competence to maintain stable, uniform expression of your gene of interest¹

Figure 1. Schematic of CompoZr Targeted Integration Kit – AAVS1 A. Your Gene of Interest (GOI) is subcloned into the supplied pZDonor plasmid at the multiple cloning site (MCS). The MCS is flanked by left and right homology arms to the AAVS1 genomic integration site. B. The modified pZDonor now contains your GOI between the AAVS1 homology arms. C. The modified pZDonor and supplied mRNA, encoding ZFNs that target the genomic integration site of AAVS1, are co-transfected into the human cell line of your choice. D. The ZFNs bind and cut the genomic AAVS1 site in the nucleus, creating a double strand break. E. The cell uses the modified pZDonor plasmid as a repair template. ZFN stimulated homology-directed repair leads to targeted integration of your gene into the AAVS1 locus in the genome of the human cell line of your choice. Your gene can be transcribed either throught the endogenous promoter at the AAVS1 site or by inserting a promoter of your choice upstream of the GOI.

CompoZr Targeted Integration Kit Components

• 10 Aliquots of Ready-to-Deliver ZFN Pair in mRNA form
• pZDonor plasmid – AAVS1
• Forward MCS primer
• Reverse AAVS1 primer
• Integration Control DNA – AAVS1
  • Positive control for PCR reaction
  • Used to determine integration efficiency

References for Targeted Gene Integration

1. Hockemeyer, D. et al., “Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases.” Nat Biotechnol, 27(4):851-7, 2009.

For other references regarding CompoZr Zinc Finger Nucleases please go to sigma.com/zfnreference

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