Streamline genome-wide surveys and screens
With cell lines that express Cas9, you can more easily and efficiently conduct high-throughput gRNA validation and genome-wide surveys and screens. SBI’s DIY Cas9-expressing Cells Complete provides everything you need to knock-in the CAS9 gene at the AAVS1 safe harbor site and stably express Cas9 in the cell line of your choice.
The Kit includes:
- A junction PCR primer mix to confirm the Cas9 integration site
- The Cas9 RT-PCR Primer Set to confirm expression of Cas9 mRNA
We’ve chosen the AAVS1 safe harbor site for placement of the CAS9 gene as the site consistently delivers robust gene expression. Insertion at the site has been shown to be safe with no phenotypic effects reported, and the surrounding DNA appears to be kept in an open confirmation, enabling stable expression of a variety of transgenes.
A note about our DIY Cas9-expressing Cells HR Donor
As with all of our AAVS1 HR Targeting Vectors, the DIY Cas9-expressing Cells HR Donor is designed to limit off-target integration. Taking advantage of the AAVS1’s location within an intron, these vectors come with a puromycin marker that has no promoter, only a splice acceptor site—expression of puromycin can only occur when the construct integrates within an intron, reducing the probability of recovering off-target integrants in the presence of puromycin selection.
How It Works
Make your own Cas9-expressing cells
Making your own Cas9-expressing cells follows the same workflow as a gene knock-in (Figure 1)—co-transfect the HR Donor vector with a Cas9 delivery method and a gRNA targeting AAVS1 (we recommend our All-in-one Cas9 SmartNuclease & AAVS1 gRNA Plasmid). The gRNA targeting AAVS1 will direct Cas9 activity to the AAVS1 site resulting in a double-strand break (DSB). Because the donor arms on the DIY Cas9-expressing Cells HR Donor are complementary to the AAVS1 site, they will mediate repair of the DSB through homologous recombination of the donor arms with the AAVS1 site. Repair with the HR Donor will result in the hspCas9 gene being inserted into the AAVS1 site.
Figure 1. Schematic showing CRISPR/Cas9-mediated gene knock-in of Cas9 at the AAVS1 safe harbor site.
Through careful selection of the target sequence and design of a donor plasmid for homologous
recombination, you can achieve efficient and highly targeted genomic modification with CRISPR/Cas9.
Cas9 protein—uses guide RNA (gRNA) to direct site-specific, double-strand DNA cleavage adjacent to a protospacer adapter motif (PAM) in the target DNA.
gRNA—RNA sequence that guides Cas9 to cleave a homologous region in the target genome. Efficient cleavage only where the gRNA homology is adjacent to a PAM.
PAM—protospacer adapter motif, NGG, is a target DNA sequence that spCas9 will cut upstream from if directed to by the gRNA.
The workflow at-a-glance
DESIGN: Select gRNA and HR donor plasmids. Choice of gRNA site and design of donor
plasmid determines whether the homologous recombination event results in a knock-out,
knock-in, edit, or tagging.
CONSTRUCT: Clone gRNA into all-in-one Cas9 vector. Clone 5’ and 3’ homology arms into HR
donor plasmid. If creating a knock-in, clone desired gene into HR donor.
CO-TRANSFECT or CO-INJECT: Introduce Cas9, gRNA, and HR Donors into the target cells
using co-transfection for plasmids, co-transduction for lentivirus, or co-injection for mRNAs.
SELECT/SCREEN: Select or screen for mutants and verify.
VALIDATE: Genotype or sequence putative mutants to verify single or biallelic conversion.
Get good Cas9 expression from the AAVS1 site
Figure 2. Use of the DIY Cas9-expressing Cells HR Donor leads to good expression of Cas9 from the AAVS1 site. We knocked the CAS9 gene into the AAVS1 site using the DIY Cas9-expressing Cells HR Donor and the All-in-one Cas9 SmartNuclease & AAVS1 gRNA Plasmid. We then tested the resulting positive clones for relative Cas9 mRNA expression using qPCR (left panel) and analyzed the two highest-expressing clones (#8 and 17) for Cas9-mediated cleavage activity using a Surveyor Assay (right panel, arrow indicated band showing Cas9-mediated cleavage). In the Surveyor Assay, only the two lanes with the Cas9 clones showed Cas9 activity (arrow), with the non-transfected cells showing no Cas9 activity.