Cas9 Nickase: MSCV-hspCas9(D10A)-T2A-Puro-H1-gRNA linearized all-in-one SmartNuclease Lentivector Plasmid

Stay on-target with your genome editing in transfection-resistant cells When you’re genome editing in a transfection-resistant cell line and need to keep off-target events to a minimum, turn to one of SBI’s Cas9 SmartNickase™ Lentivector Systems. Unlike the wildtype Cas9 protein which introduces double-strand breaks (DSBs), the MSCV-hspCas9(D10A)-T2A-Puro-H1-gRNA All-in-one Cas9 SmartNickase introduces paired nicks at the gRNA-directed site. Creating nicks favors the higher-fidelity homologous recombination process over non-homologous end joining (NHEJ), with paired nicking shown to reduce off-target activity by 50- to 1,500-fold in cell lines, and to facilitate gene knockout in mice without losing on-target cleavage efficiency¹.

 

Available as pre-linearized, ready-to-clone lentivector plasmids, the MSCV-hspCas9(D10A)-T2A-Puro-H1-gRNA All-in-one Cas9 SmartNuclease Lentivector expresses human codon-optimized Cas9 wild-type nuclease from the strong MSCV promoter, includes the puromycin selection marker, and delivers gRNA from an H1 promoter.

• Conduct genome editing and engineering in difficult-to-transfect cell lines
• Simultaneously deliver Cas9 and gRNA from a single lentivector
• Stay more on-target with Cas9 nickase activity
• Drive Cas9 expression from the MSCV promoter, for high expression in hematopoietic and stem cells
• Perform in vivo engineering of model organisms
• Supports synthetic biology applications, gene- and cell-based therapy development, and genome-wide functional screening

Why an HR targeting vector is a recommended Even though gene knock-outs can result from DSBs caused by Cas9 alone, SBI recommends the use of HR targeting vectors (also called HR donor vectors) for more efficient and precise mutation. HR donors can supply elements for positive or negative selection ensuring easier identification of successful mutation events. In addition, HR donors can include up to 6-8 kb of open reading frame for gene knock-ins or tagging, and, when small mutations are included in either 5’ or 3’ homology arms, can make specific, targeted gene edits.

 

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.