GBI – Genera Biosystems Ltd.

Advanced Molecular Diagnostic Systems – Multiplex qPCR

hspCas9-RT-PCR Primer Set

Overview

Confirm Cas9 mRNA expression With our ready-to-use Cas9 RT-PCR primer set, you can quickly and easily confirm Cas9 expression in cells or in vitro transcription assays. The amplicon for Game 1 is 219 bp and the amplicon for Game 2 2 is 122 bp. These primer sets allow the detection of nuclease Cas9, Nickase Cas9 and double mutant Cas9 at the messenger level. The primer sets are compatible with the expression of Cas9 mRNA from any SBI Cas9 SmartNuclease construct and vectors from Dr Feng Zhang’s laboratory.

How does it work?

CRISPR / Cas9 basics

By careful selection of the target sequence and design of a donor plasmid for homologous recombination, you can achieve efficient and highly specific genomic modification with CRISPR / Cas9.

Cas9 protein – Uses guide RNA (gRNA) to direct cleavage of site-specific double-stranded DNA adjacent to a protospacer adapter motif (PAM) in target DNA.

GRNA: RNA sequence that guides Cas9 to cleave a homologous region in the target genome. Cleavage is efficient only when gRNA homology is adjacent to a PAM.

PAM: The protospacer adapter motif, NGG, is a target DNA sequence that spCas9 will cut upstream if it is directed toward the gRNA.

Workflow at a glance

DESIGN: Select plasmids from donor gRNA and HR. Choice of gRNA site and donor design plasmid determines if the homologous recombination event results in a knock-out, knock-in, edit or tag.

BUILD: Clone gRNA into an all-in-one Cas9 vector. Clone 5 ‘and 3’ homology arms in HR donor plasmid. If you create a knock-in, clone the desired gene into the HR donor.

CO-TRANSFECT or CO-INJECT: Introduce Cas9, gRNA and HR donors into target cells using cotransfection for plasmids, cotransduction for lentivirus or coinjection for mRNA.

SELECT / SCREEN: Select or search for mutants and verify.

VALIDATE: Genotype or sequence of putative mutants to verify simple or biallelic conversion.

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