CLICK Method
Zygote EP – CLICK Method- Tomoji Mashimo – HR with RNP and short oligo by NEPA21 – can also do HR with long single stranded DNA even for the Rosa locus. Can knock-in less than 1.2 kb cassette with NEPA21
With respect to the recent article: Re-Evaluating One-step Generation of Mice Carrying Conditional Alleles by CRISPR-Cas9-Mediated Genome Editing Technology
| doi: https://doi.org/10.1101/393231
(the Node 03 Oct., 2018): The article cites a previous article that references that reports up to 16% efficiency in generating conditional knockout alleles in mice using 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides (ssODN) (2sgRNA–2ssODN).
This article re-evaluates the reported method from a large data set generated from a consortium consisting of 17 transgenic core facilities or laboratories or programs across the world. The dataset constituted 17,887 microinjected or electroporated zygotes and 1,718 live born mice, of which only 15 (0.87%) mice harbored 2 correct LoxP insertions in cis configuration indicating a very low efficiency of the method.
The reported analysis indicates that the 2sgRNA–2ssODN method generates a large number of undesired alleles (>99%), and a very small number of desired alleles (<1%) requiring, on average 1,192 zygotes.
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For your information, another recent article has been published by your client Prof. Tomoji Mashimo.
Click: one-step generation of conditional knockout mice
| https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5930688/
This article has advanced the art even further and demonstrates successful conditional knockout alleles in mice using 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides (ssODN) (2sgRNA–2ssODN).
Prof Mashimo is performing HR with RNP and short oligo by NEPA21 with high efficiency. He confirms that his lab is also successfully doing HR with long single stranded DNA even for the Rosa locus. He can knock-in less than 1.2 kb cassette with NEPA21. |
Cas9 Consumables
| Please note this link to a recent third-party web-site referencing the Kaneko ‘TAKE method’ (and our NEPA21 system) and outlining the full ‘TAKE Method’ protocol and a NEPA21 protocol for human induced iPS Cells.
Here are the specific links to the NEPA21 protocols on the IDT website: “Here you can access protocols for mouse zygote electroporation and human-induced iPS cells using the NEPA21 Electroporator”. The ‘human-induced iPS cells link is a step-by-step protocol elucidation. (I have copied a pdf version of h=the protocol as: ‘IDT – iPS – crispr-cas9-rnp-delivery-ips-cell-electroporation-nepagene’
For your information, Prof. Kaneko has communicated to us that the quality of Cas9 products is quite important for replicating successful results. Unfortunately, not all companies that supply these products supply products of the same quality. This is why Prof. Kaneko only uses IDT products for his experiments. Also, the concentration is important. Prof. Kaneko uses 100 ug/ml of cas protein for his zygote protocol.. If one uses protein, carefully handling is also required (as protein is fragile).
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