LM Easy KO Knockout Kit (RNP Method)
1. What is RNP gene knockout method?
RNP refers to a complex formed by direct complexation of sgRNA and Cas9 protein. Unlike traditional gene transfection, Cas9 gene does not need to be introduced into cells, but Cas9 protein and sgRNA are directly provided to cells for use.
Using RNP has the following advantages over other methods of gene editing:
(1) High editing efficiency: especially suitable for host cells with low plasmid transfection efficiency, such as stem cells, immune cells, and primary cells that are difficult to transfect.
(2) No immune response interference: the factors that may affect the host cells are completely excluded, and the accuracy of the experiment is improved.
(3) No DNA interference: It can avoid the insertion of unexpected gene fragments and ensure the stability of the gene.
(4) Simple and fast: This technology does not require transcription, expression and degradation of plasmids, which greatly shortens the experimental cycle and is 1-2 months faster than the traditional method.
(5) Reduce off-target effects: Cas9 protein and sgRNA are not continuously expressed and can be degraded, thereby reducing off-target effects.
(6) Avoiding rabbit genetic variation: In the process of RNP transfection, Cas9 protein only exists in the cell for a short time, and is degraded after editing, avoiding potential genetic variation.
2. Why choose Elem LM Easy Knockout Kit?
(1) From editing to verifying all in one box, gene knockout "zero" basis;
(2) chemical synthesis of sgRNA,RNP efficient delivery, optimization of sgRNA and Cas9 ratio;
(3) A novel strategy for sgRNA design targeting early exons;
(4) By simple Sanger sequencing, the knockout efficiency can be determined in 3 days;
(5) Elem has accumulated a large amount of data to prove that the kit can achieve efficient editing of cell lines, primary cells, iPS cells, T cells, etc.
3.Elem LM Easy Knockout KitGene Knockout Analysis PlatformOperation process
(1) according to the Elem LM EasyKO Kit(RNP method) instructions, gene editing efficiency analysis SOP, PCR experiments;
(2) The PCR product (only for column purification, not for gel recovery and purification) is handed over to a generation sequencing company to obtain the sequencing files of wild-type cells and KO cells (.ab1 format);
(3) Consult the technical support of Elem to obtain the account information of the gene editing efficiency analysis platform and log on to the following websites:http://8.138.1.26:8819/dna/page/home/login ;
(4) Click "Report List"-"Add Report" to upload the sequencing files (.ab1 format) of wild type cells and KO cells to the designated locations respectively, and click "Submit"; It is estimated that the gene knockout efficiency results will be obtained in 15 min. The results were used to evaluate the editing efficiency of the gene knockout cell pool (KO Cell Pool) and to evaluate the success rate of subsequent monoclonal acquisition.