The ability to insert, or knock-in a gene at a specific locus is a tremendously useful
method for dissecting disease mechanisms.
However, in previous years, the insertion of a foreign gene has been far too tedious
and expensive for many labs to perform.
Thanks to the recent development of CRISPR/Cas9 technology, gene knock-in experiments have
now become a very popular technique in modern molecular biology.
In contrast to the random integration events by the lentiviral system, CRISPR utilizes
a guide RNA that directs gene insertion with extraordinary specificity.
In particular, the AAVS1 safe harbour site is often a preferred target site for gene knock-ins.
This is because it enables consistent and robust transgene expression with minimal toxic effects.
At this safe harbour site, the surrounding DNA maintains an open conformation enabling
stable expression of the newly inserted transgene.
This makes safe harbour sites such as the AAVS1 locus on human chromosome 19, and the
ROSA26 locus on mouse chromosome 6, very popular sites for targeted knock-in experiments.
In this case study, we will walk you through how a red fluorescent protein gene was knocked
into human embryonic kidney cells at the AAVS1 safe harbour site.
We will also demonstrate how the knock-in was verified using a PCR-based method.
To begin, an sgRNA was designed to target the human AAVS1 locus.
Software analysis was performed to ensure maximum efficacy of on-target cleavage, while
avoiding any off-target effects.
The resulting guide sequence was used to construct abm's AAVS1-targeting All-in-One Vector.
This vector expresses the Cas9 endonuclease under a CMV promoter and
the sgRNA under a U6 promoter.
To achieve the knock-in, a donor plasmid containing the gene of interest is required to provide
a template for Homology Directed Repair.
The HDR pathway is a mechanism in cells that repairs double stranded breaks by using a DNA
repair template homologous to the sites flanking upstream and downstream of the break site.
To enable HDR mediated gene insertion, the gene of interest must be flanked by such
homology arms.
Therefore, a second plasmid was constructed by cloning an RFP-puromycin expression cassette
into abm's repair template plasmid for the AAVS1 locus.
The resulting pAAVS1-Donor-RFP plasmid had the RFP gene under a CMV promoter and a puromycin
resistance marker under a PGK promoter.
Once both plasmids were ready, they were introduced into HEK293 cells using abm's transfection
reagent DNAfectin.
Following transfection, the cells were passaged ten times to dilute out the episomal donor vector.
Following, the resulting HEK293 cells were given 3 to 4 weeks to grow in the presence
of puromycin to select for cells with successful knock-in of the RFP-puromycin resistance cassette.
Cells transfected with both AAVS1-targeting All-in-One vector and pAAVS1-Donor-RFP plasmid
were healthy after 4 weeks, and greater than 95% of HEK293 cells were
successfully expressing RFP!
Control cells that were not transfected with the vectors died after puromycin treatment.
PCR genotyping was carried out to confirm that RFP
was successfully knocked-in at the AAVS1 site.
In this method, two primers were designed: primer 1 targeting the 5' homology arm upstream
of RFP, and Primer 2 targeting a site within the RFP-Puromycin resistance cassette.
Gene knock-in was confirmed when a PCR product of 1.1 kb was observed indicating that Primers
1 and 2 were both able to bind to the edited genome to enable successful PCR amplification.
In the case of a non-successful knock-in or in the presence of wild type control cells,
no PCR products were observed as Primer 2 could not anneal to the target site
and enable amplification.
We hope this case study has demonstrated how the CRISPR-based gene knock-in technique can
make your exploration of gene function amazingly easy and efficient.
CRISPR/Cas9 mediated gene knock-ins allows for biallelic modifications of some genes
to be generated with ease, with phenotypic changes in mice observable in a single generation
an unprecedented timeframe!
Let abm's team of CRISPR scientists help you knock-in any gene into any cell line you
want all with minimal toxic effects at the AAVS1 or ROSA26 Safe Harbor Sites.
Explore abm's extensive portfolio of customizable CRISPR services and products today and start
knocking-in genes, tags, markers, and more!
To make it even easier for you, simply describe
your desired knock-in and we'll take care of the rest!