hspCas9 AAVS1 Safe Harbor Knock-in Donor (AAVS1-SA-puro-EF1-hspCas9), Complete Kit with CAS601A-1 (Cas9 SmartNuclease AAVS1-gRNA Targeting Vector), CAS640PR-1 (Junction PCR Primer Mix to confirm Cas9 integration site), and CAS9-PR-1

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.

CRISPR/Cas9 Basics

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.

The system

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.