What is the AAVS1 safe harbor locus?
The AAVS1 locus, in intron 1 of PPP1R12C (protein phosphatase 1 regulatory subunit 12C), is known as a “safe harbor” because its disruption does not have adverse effects on the cell, and robust transcription can be used to maintain the expression of an exogenously inserted gene5,6,7.
What is the AAVS1 locus?
The adeno-associated virus site 1 (AAVS1) locus in the human genome is a strong candidate for gene therapy by insertion of an exogenous gene into the locus. The AAVS1 locus includes the coding region for myosin binding subunit 85 (MBS85).
What is a safe harbor gene?
Abstract. Genomic safe harbors (GSHs) are sites in the genome able to accommodate the integration of new genetic material in a manner that ensures that the newly inserted genetic elements: (i) function predictably and (ii) do not cause alterations of the host genome posing a risk to the host cell or organism.
What is crispr Cas9 used for?
CRISPR-Cas9 is a unique technology that enables geneticists and medical researchers to edit parts of the genome? by removing, adding or altering sections of the DNA? sequence. It is currently the simplest, most versatile and precise method of genetic manipulation and is therefore causing a buzz in the science world.
What is ROSA26 locus?
ROSA26 is a locus used for constitutive, ubiquitous gene expression in mice. It was first isolated in 1991 in a gene-trap mutagenesis screen of embryonic stem cells (ESCs). Over 130 knock-in lines have been created based on the ROSA26 locus. The human homolog of the ROSA26 locus has been identified.
Where is AAVS1?
Adeno-associated virus integration site 1 is a viral integration site that in humans is encoded by the AAVS1 gene located on chromosome 19.
What is the ROSA26 locus?
What is targeted integration?
Targeted integration in mammalian cells is generally achieved through a double crossover event between the genome and a linear donor containing two homology arms flanking the gene of interest. However, this strategy is generally less efficient at introducing larger DNA fragments.
What is the Crispr CAS system?
The CRISPR-cas system is an adaptive immune system of bacteria and archaea, which protects the bacteria from invaders, including bacteriophages or phages and mobile genetic elements (MGEs) . The CRISPR-cas system degrades foreign genetic elements in three steps (Fig. 1).
What is CRE ERT2?
The Cre-ERT2 fusion protein (Cre-ERT2) consists of Cre recombinase fused to a triple mutant form of the human estrogen receptor; which does not bind its natural ligand (17β-estradiol) at physiological concentrations but will bind the synthetic estrogen receptor ligands 4-hydroxytamoxifen (OHT or tamoxifen) and, with …
What is the ROSA26 gene?
Which is the gene targeting vector for AAVS1?
Two TALEN vectors are designed to target the AAVS1 locus based on gene targeting studies by Jaenisch and colleagues ( Hockemeyer et al., 2011 ): the AAVS1 -TALEN-L vector is designed to target CCCCTCCACCCCACAGT, and the AAVS1 -TALEN-R vector is designed to target TTTCTGTCACCAATCCT (Addgene 59025 and 59026) ( Fig. 11.2 B).
Which is the first intron of the AAVS1?
First, AAVS1-TALEN along with two donor constructs were electroporated to the first intron of the AAVS1 in hESCs and hiPSCs cell lines. A high biallelic mutation was observed in > 50% of clones which was confirmed by Southern blot and qRT-PCR revealed the Cas9 expression after doxycycline induction to all iCas9 clones.
What kind of cells can AAVS1 be found in?
AAVS1 targeting was observed in up to 84% of stable transduced human cell lines (Gli36) (Wang et al., 2002) and up to 50% of stable transduced transgenic mouse fibroblasts carrying the AAVS1 site (Bakowska et al., 2003).
How is the AAVS1 ectopic expression system used?
The AAVS1 ectopic expression system is useful to rapidly and reliably generate panels of isogenic cell lines expressing protein variants (eg, splice variants) ( Dalvai et al., 2015 ).