Custom Mouse Injection-Ready Kit (Validated)

Injection-Ready Validated CRISPR Kits for Mouse Model Generation

CRISPRCRITTERS Injection-Ready Kit is a CRISPR kit for the generation of transgenic mice and rats. This CRISPR mouse model kit contains in vivo validated gRNAs designed to target your gene of interest, donor DNAs and microinjection-validated Cas9 elements. For customers who prefer a more stringent off-target profile, we also provide mRNAs of Cas9 D10A or H840A nickase.

  • Injection-Ready
  • In vivo validated gRNAs New!
  • Cas9 Nickase Available
  • Full Mouse Generation Service Available

Cas9 Variants

Rat Model Generation

CRISPR Kit

 

 

Technical Details

An Example of gRNA Validation Data (mRosa26 locus)

Two gRNA sequences were cloned into gRNA-expressing vectors individually. Each construct was transfected into mouse N2A cells, individually. gRNA activity was determined by SURVEYOR assay on the PCR products, using the SURVEYOR Mutation Detection Kit, according to the manufacturer’s instructions. The results showed indel frequency of 26% and 12% for the two gRNA constructs, respectively.

An Example of Generating a Point Mutation Mouse Model Using Cas9 D10A nickase CRISPRCRITTERS™ Mouse Point mutation kit

The mixture of two gRNAs, a single-stranded DNA oligo containing desired mutation, and Cas9 D10A nickase mRNA was injected into cytoplasm of mouse embryos. PCR products from genomic DNA were sequenced. Results from a positive pup with desired T>A mutation are shown below.

 

An Example of Generating a Conditional Knock-out Mouse Model Using Cas9 CRISPRCRITTERS™ Knock-in kit

A mixture of active guide RNA (gRNA), a single strand oligo donor containing a HA tag sequence and Cas-9 mRNA was injected into the cytoplasm of C57BL/6 embryos. Mice born from the microinjected embryos were screened using PCR-restriction digest analysis. Pups #2, 6, 13 and 17 showed heterozygous insertion. PCR products were topo cloned and sequenced to confirm the HA tag was inserted at right location.  Positive animals were further verified by sequencing the region adjacent to the targeted location.

 
FAQ

1. Do you provide validated gRNAs in the CRISPRCRITTERS™ kit?

 

Yes, the gRNAs included in the mouse kits are validated in vivo in mouse embryos, and by in vitro validation in cultured rat cells for rat CRISPRCRITTER™ Kits.

 

2. What are the advantages of in vivo gRNA validation for mouse CRISPRCRITTERS™ Kits?

 

In vivo validation of gRNAs is more relevant than in vitro validation, and shows 80-100% correlation with gRNA activity in the final mouse model.

 

3. How many mouse embryos do you microinject for in vivo validation of gRNAs?

 

To test indel activity, 30-50 mouse embryos are injected with each sgRNA and allowed to develop to blastocyst stage. We then confirm indel activity by sequencing DNA extracted from the blastocysts using Sanger sequencing.

 

4. How do you calculate indel rate?

 

We use the formula: indel rate = # blastocysts with indels/ # total blastocysts tested.

 

5. What are the contents of the CRISPRCRITTERS™ Kits?

 

The kit contains individually aliquoted reagents sufficient for 3 rounds of microinjection: (1) in vivo validated total sgRNA (1 µg x 3); (2) total donor ssODN (5 µg x 3) or donor plasmids (0.25 µg x 3); (3) optimized Cas9 protein (2.5 µg x 3). We do not recommend freeze-thawing of reagents after the 1st use for microinjections.

 

6. Do you also validate gRNAs in CRISPCRITTERS™ Kits for Rats using in vivo validation in rat embryos?

 

Due to limitations in culturing rat embryos, we include only in vitro validated gRNAs in the rat kits.

 

7. What are the criteria to be considered for designing a conditional knockout (CKO) CRISPRCRITTERS™ Kit?

 

We use two criteria for designing the CKO CRISPRCRITTERS™ Kits: (1) Retain the 1st exon if possible and target the remaining early exons (provided the length of the targeted exon(s) is not a multiple of 3) for inserting flanking LoxP sites; (2) The distance between the two LoxP sites should be > 6kb.

References

 

 

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