in vivo validated Cas9 mRNAs

CRISPR/Cas systems are employed by bacteria and archaea as a defense against invading viruses and plasmids. The type II CRISPR/Cas system from the bacterium Streptococcus pyogenes has been engineered to function in eukaryotic systems using two molecular components: a single Cas9 protein and a non-coding guide RNA (gRNA). Since 2013, the CRISPR/Cas system has been used for gene editing (adding, disrupting or changing the sequence of specific genes) and gene regulation in species throughout the tree of life. By delivering the Cas9 protein and appropriate guide RNAs into a cell, the organism's genome can be cut at desired location. Both wild type Cas9 and Cas9-D10A nickase are widely used in gene editing research. The Cas9-D10A nickase with paired gRNAs dramatically reduces off-target activity.

Applied StemCell’s Cas9 mRNA and Cas9-D10A nickase mRNA are transcribed from mammalian codon- optimized cDNA plasmid. The mRNAs are capped and polyA-tailed. Sequences of Nuclear Localization Signal were added to both 5’ and 3’ of the genes to generate proteins that has high efficiency of nuclear localization in mammalian cells. These mRNAs should be used in conjunction with purified guideRNAs.

Standard -cas9-WT


H840A-Cas9-nickase


D10A-nickase-Cas9


Examples of using ASC’s Cas9 mRNA

Example #1: Site-specific knock-in of a 27bp tag in mice using Applied StemCell’s Cas9 mRNA

A.

B.

C.

Figure 1. Site-specific knock-in of a 27bp tag in mice using Applied StemCell’s Cas9 mRNA. A. gRNA validation: gRNAs were cloned into the gRNA expressing vector pBT-U6-Cas9-2A-GFP. Each construct was then transfected into mouse N2A cells. SURVEYOR assay was performed on the PCR products. g4a was selected for the generation of donor DNA and gRNA transcripts for microinjection. B. Genotyping by PCR: Initial PCR/cut identified 4 out of 14 pups were modified. C. PCR products were sequenced, 2 pups had the tag (yellow) inserted at the right position.

Example #2: gRNA guided gene knockout in rat using Applied StemCell’s Cas9 mRNA.
A.

B.

Figure 2. gRNA guided gene knockout in rat using Applied StemCell’s Cas9 mRNA. A. gRNA validation: gRNAs were cloned into the gRNA expressing vector pBT-U6-Cas9-2A-GFP. Each construct was then transfected into Rat6C cells. SURVEYOR assay was performed on the PCR products. g25 and g28 were used for generation of donor DNA and gRNA transcripts for microinjection. B. Genotyping by PCR: Initial PCR identified 4 out of 16 pups were modified with potential homozygous (#3, 6, 16) or heterozygous (#13) knockout deletions.

References

 

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