Confidence from the right controls
With SBI’s Cas9 SmartNucleaseTM Genome Editing Positive Control Kit, you get everything you need to verify your genome editing techniques.
The kit includes:
- The All-in-one Cas9 SmartNuclease & AAVS1 gRNA Plasmid to target Cas9 activity to the AAVS1 sequences used in the included positive control reporter cell line
- The Genome Editing Positive Control EGIP 293T Reporter Cell Line which has a non-fluorescent eGFP gene integrated into the genome—the eGFP mutant (EGIP) is nonfuctional due to the insertion of a premature stop codon embedded in a 53 bp AAVS1 sequence
- An AAVS1-eGFP HR rescue vector designed to restore the full eGFP open reading frame, resulting in the production of fluorescent eGFP.
- T7 Endonuclease, buffer, and primers for a Surveyor assay to assess site-specific genome cleavage
Successful genome engineering will result in the restoration of GFP fluorescence. Note that because the control reporter cell line takes advantage of AAVS1 sequences, this kit can also be used to verify appropriate AAVS1 targeting constructs.
How It Works
Using the Cas9 SmartNuclease Genome Editing Positive Control Kit
The Cas9 SmartNuclease Genome Editing Positive Control Kit is a great way for those new to CRISPR/Cas9 methods to verify their techniques. The experiment starts with the Cas9 Positive Control EGIP 293T Reporter Cell Line (kindly provided by Dr. Jizhong Zou of the NIH Center for Regenerative Medicine, a Common Fund initiative of the U.S. National Institutes of Health) which contains a genomic copy of the EGFP gene that is non-fluorescent due to the insertion of a premature stop codon embedded in 53 bp of AAVS1 safe harbor site sequence (Figure 1).
Figure 1. Schematic of the All-in-one Cas9 SmartNuclease & AAVS1 gRNA Plasmid and Cas9 Positive Control EGIP 293T Reporter Cell Line.
Co-transfection of the included All-in-one Cas9 SmartNuclease & AAVS1 gRNA Plasmid and the AAVS1-eGFP HR rescue vector results in repair of the eGFP gene and restoration of fluorescence (Figure 2B). The All-in-one Cas9 & AAVS1 gRNA plasmid delivers Cas9 activity that is targeted to AAVS1 sequences by the gRNA (Figure 2A).
Using the kit to test AAVS1 targeting constructs
You can use this kit to test your own AAVS1-targeting constructs—simply include the appropriate eGFP-restoring mutations in your AAVS1-targeting construct and use it in place of the included AAVS1-eGFP HR rescue vector.
Figure 2. Restoring GFP fluorescence with the Cas9 SmartNuclease Genome Editing Positive Control Kit. (A) Surveyor Assay data shows ~25% Cas9-mediated cleavage activity. (B) Fluorescence imaging shows restoration of GFP fluorescence only when the Cas9 SmartNuclease & AAVS1 gRNA Plasmid is included.
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.
See the above “How it works” section