RNA Format – Transgenic

 

RNA-only CRISPR Formats for Embryo Microinjection

It is well-established in the literature that knockout animals can be generated via microinjection of ZFN mRNA and donor constructs14-16, and recently these methods have been extended using CRISPR systems.17 Sigma offers RNA-only CRISPR formats for both single-site and paired nickase formats (Figure 2). The use of RNA vs. plasmid constructs avoids complications with promoter-embryo compatibility as well as the possibility of random integration of nuclease and gRNA-expressing plasmids into the host animal genome.

Sigma CRISPR RNA is supplied at concentrations of 500 ng/μl (50 μl Cas9 mRNA and Cas9-D10A mRNA, capped and polyA-tailed) and 200 ng/μl (20 μl gRNA). Typical microinjection concentrations used in the literature are in the ranges of 20-200 ng/μl for Cas9 mRNA and 10-50 ng/μl for gRNA.

While Sigma CRISPR RNA was not developed for robust application to cell culture, we have seen some success in detecting CEL-I activity using RNA-only formats for both CRISPR nucleases and paired nickases. RNA-only delivery formats maybe favorable for cell types which are sensitive to double stranded DNA (such a dendritic cells18 ) or when promoter-cell incompatibilities exist.

If you suspect that the promoter used to drive Cas9 in your plasmid is not functional in your cell type, a quick possible solution maybe to substitute Cas9-mRNA. Since the human U6 promoter used to drive gRNA has robust performance across different cell types (based on experience with shRNA), try co-transfection Cas9-mRNA or Cas9-D10A-mRNA along with U6-gRNA plasmids. If Cas9-mRNA does not work, then try to modifying your plasmid with an alternate promoter to increase Cas9 expression (see below for plasmid maps, restriction sites, etc.).

Note: while Sigma implements the same high quality RNA synthesis procedures that have been successful in many mRNA-based ZFN transgenic projects, we highly recommend centrifuging your final, concentration-adjusted CRISPR RNA samples immediately prior to microinjection to ensure all particulate material has been removed which may result in clogging of microinjection needles.

This tab is primarily for transgenic applications as it allows the researcher to microinject the gRNA and Cas9 in RNA format directly into the embryo. Packages have been assembled with different levels of support (controls, validation, plasmid counterparts) to allow for different levels of user expertise.

Project Package #1: Reagents Only

2 gRNA in RNA format for the same gene target

Cas9 in mRNA format

no donor design, no free matching plasmids, no validation

turn around time : 3-4 weeks

Project Package #2: Reagents plus Control

2 gRNAs in RNA format for the same gene target (4 ug each)

Cas9 in mRNA format (25 ug)

Free matching plasmids of gRNAs (U6 vector)

Free Rosa26 positive control in plasmid format (U6 vector)

no donor design included

turn around time : 3-4 weeks

Project Package #3: Validated CRISPR

1 validated gRNA RNA format (4 ug)

Cas9 mRNA format (25 ug)

Free ss donor design*

Free ss oligo donor

turn around time : 5-6 weeks

*donor plasmid design service available for $500

Project Package #4: Paired Nickase (2nd paired nickase needed only for conditional KO projects)
4A: Non-validated paired nickase

One pair of gRNAs (for nickases) in RNA format for the same gene target (4 ug each)

Cas9D10A in mRNA format (25 ug)

Matching plasmids of gRNAs (U6 vector)

Rosa26 positive control in plasmid format (U6 vector)

Cas9D10A plasmid (1ug)

Cas9D10A plasmid (1ug)

Cas9D10A plasmid (1ug)

Add another $1,500 for the 2nd target (or loxp site)

One pair of gRNAs (for nickases) in RNA format for the same gene target (4 ug each)

One pair of gRNAs (for nickases) in RNA format for the same gene target (4 ug each)

Free matching plasmids of gRNAs (U6 vector)

No donor design included, no validation

TAT 3-4 weeks

4B: Validated paired nickase

One pair of gRNAs (for nickases) in RNA format for the same gene target (4 ug each)

Cas9D10A in mRNA format (25 ug)

Free oligo donor design for this site

TAT 8-10 weeks

Add another $4,000 for the 2nd target (or loxp site)

One pair of gRNAs (for nickases) in RNA format for the same gene target (4 ug each)

Free oligo donor design for this site

TAT 8-10 weeks

·         no donor design, no free matching plasmids, no validation

·         TAT 3-4 weeks