Hi, Ondrej,

Thank you very much for the answer, it's really helpful. Yes, we synthesized sgRNA by ourselves. More precisely, it was synthesized by one of the collaborator. I don't know the exact procedure he used, but it wasn't commercial Kit for in vitro transcription for sure. I thought about RNA quality as the reason, but tried to take into account another reasons.

Thank you once again,

Liza

For those of you, who are still interested. I've ordered synthetic sgRNAs from Synthego (which are made not through in vitro transcription but direct chemical synthesis) and the system worked for both HEK293 and iPSCs with efficiency about 70-90%

Hi Liza and readers!

We and some of our customers have tested IDT and Synthego RNAs, both are very good. And no doubt that it makes a big difference compared to self-made IVT RNAs. Maybe of interest for you all, we have in hands a very safe and efficient reagent for RNP delivery, based on our Viromer technology (polymer-based). In-house tests and beta-testing (including from IDT staff) has proven outperformance and more consistency compared to lipofection.

For more details follow this link or contact me.

https://viromer-transfection.com/crispr

BR

Sandra

Updated information and some users' data to complete my previous message!

https://viromer-transfection.com/crispr

Hi Liza,

I have the similar problem of low in vivo editing efficiency by using RNP complexes. I ordered the gRNA and Cas9 from IDT company, with RNAimax as transfection reagent, and just followed the protocol. The in vitro cleavage worked. However, the in vivo editing on HEK with positive control HPRT ordered from the same company showed low cleavage band. I don't know which step can go wrong. Could anyone give some suggestions?

BR,

Le Guo

Hi Liza and Le Guo,

Please, could you give us some information about how you transfer the protocol from in vitro to in vivo? animal model? route of injection? etc

Hi Sandra,

Please see below for protocols for in vivo and in vitro assay, and I use HEK293 and Lipofectamine RNAiMAX Transfection Reagent.

http://sfvideo.blob.core.windows.net/sitefinity/docs/default-source/user-guide-manual/alt-r-crispr-cas9-user-guide-ribonucleoprotein-transfections-recommended.pdf?sfvrsn=1c43407_24

http://sfvideo.blob.core.windows.net/sitefinity/docs/default-source/protocol/alt-r-crispr-cas9-protocol-in-vitro-cleavage-of-target-dna-with-rnp-complex.pdf?sfvrsn=88c43107_18

Le Guo

I prefer to use an electroporation. It has high efficiency, and relatively low death rate (compare to a plasmid delivery).

Hi All.

I hate to reactivate a dormant thread, but I am hoping someone here can help me understand what cell types the Cas9 RNP delivery system will work in. Will this work in all cell types? What about prokaryotes?

hey can anyone suggest me RNP use in bacterial system..and its feasibility could not find literature

CRISPR/Cas9 ribonucleoprotein (RNP) is a form of CRISPR genome editing where the Cas9 protein is complexed with a synthetic guide RNA (sgRNA) to form an RNP complex. This complex is then delivered directly into target cells to induce site-specific DNA cleavage and subsequent genome modifications. Here's an overview of CRISPR/Cas9 RNP technology:

CRISPR/Cas9 RNP technology offers a versatile and efficient approach for precise genome editing in a wide range of cell types and organisms, with applications spanning basic research, biotechnology, and therapeutic development.

l Take a look at this protocol list; it could assist in understanding and solving the problem.