Protocols

RNA purification for CRISPR injection

(Courtesy of Dr. Jorge Henao-Mejia)

Production of Cas9 mRNA

T7 promoter is added upstream of the Cas9-HA-2NLS open reading frame. A linearized plasmid is used as a template for in vitro transcription of the Cas9 mRNA. The plasmid is linearized with XbaI, which has a single cutting site downstream of the stop codon of the Cas9-HA-2NLS open reading frame. In vitro transcription is performed using the mMESSAGE mMACHINE T7 ULTRA kit (Life Technologies) following the manufacturer’s instructions. The Cas9-HA-2NLS mRNA is purified using MEGAclear kit (Life Technologies) and eluted in elution buffer. Cas9-HA-2NLS mRNA is then diluted to a concentration of 400 ng/ul using injection buffer (10 mM Tris / 0.1 mM EDTA, pH 7.5 prepared with sterile water).

EXTREMELY IMPORTANT: After the Cas9-HA-2NLS mRNA is eluted, it is essential to centrifuge at top speed for 20 minutes and then transfer suspension to a new RNAse-free tube (leaving 10-15 ul in the original tube). If this is not performed the needle used for microinjection will be blocked.

Production of gRNAs

The T7 promoter is added to the gRNAs template by PCR amplification using specific primers. The T7-sgRNA PCR product is purified with a PCR product purification column (QIAGEN) and used as the template for in vitro transcription. In vitro transcription is performed with the MEGAshortscript T7 kit (Life Technologies) following the manufacturer’s instructions. The gRNAs are purified using MEGAclear kit (Life Technologies) and eluted in elution buffer. The purified gRNA is then diluted to a concentration of 500 ng/ul using injection buffer (10 mM Tris / 0.1 mM EDTA, pH 7.5 prepared with sterile water).

EXTREMELY IMPORTANT: After the gRNA is eluted, it is essential to centrifuge at top speed for 20 minutes and then transfer suspension to a new RNAse-free tube (leaving 10-15 ul in the original tube). If this is not performed the needle used for microinjection will be blocked.

For oocyte Injection

Provide Cas9 mRNAs (100 ng/ul) and gRNAs (50-100 ng/ul) to be injected into the cytoplasm of fertilized eggs. If oligo DNAs are also to be injected to promote homologous directed repair (generation of knockin mice), include these at a concentration of 100 ng/ul.  All should be combined at their final concentration and submitted to TCMF on the scheduled injection day.