1. What is the RNP gene knockout method?
RNP refers to the complex formed by the direct combination of sgRNA and Cas9 protein. Unlike traditional gene transfection methods, it does not require the introduction of the Cas9 gene into the cell, but directly provides Cas9 protein and sgRNA to the cell for use.

 

Compared with other gene editing methods, using RNP has the following advantages:
(1) High editing efficiency: Especially suitable for host cells with low plasmid transfection efficiency, such as difficult-to-transfect stem cells, immune cells, and primary cells.
(2) No immune response interference: Completely eliminates factors that may affect host cells, improving the accuracy of the experiment.
(3) No DNA interference: Avoids unexpected gene fragment insertion and ensures gene stability.
(4) Simple and fast: This technology does not require transcription, expression, and degradation of plasmids, greatly shortening the experimental cycle and being 1-2 months faster than traditional methods.
(5) Reduce off-target effects: Cas9 protein and sgRNA are not continuously expressed and are degradable, thus reducing off-target effects.
(6) Avoid genetic variations: During RNP transfection, the Cas9 protein only exists briefly in the cell and is degraded after editing, avoiding potential genetic variations.

 

2. Why choose the Liman LM Easy Knockout Kit?
(1) From editing to verification, all in one box, zero foundation for gene knockout;
(2) Chemically synthesized sgRNA, efficient RNP delivery, optimized the ratio of sgRNA and Cas9;
(3) A new sgRNA design strategy targets early exons;
(4) Through simple Sanger sequencing, knockout efficiency can be determined in 3 days;
(5) Liman has accumulated a large amount of data to prove that this kit can achieve highly efficient editing for cell lines, primary cells, iPS cells, and T cells.

 

3. Liman LM Easy Knockout Kit Gene knockout analysis platform Operation process

(1) Perform PCR experiments according to the gene editing efficiency analysis SOP in the Liman LM EasyKO Kit (RNP method) instructions;
(2) Send the PCR product (only column purification, no gel recovery purification) to a first-generation sequencing company to obtain sequencing files (.ab1 format) for wild-type cells and KO cells;
(3) Consult Liman technical support to obtain the account information for the gene editing efficiency analysis platform and log in to the following website: http://8.138.1.26:8819/dna/page/home/login ；
(4) Click "Report List" - "New Report", upload the sequencing files (.ab1 format) of wild-type cells and KO cells to the specified location, and click "Submit"; the gene knockout efficiency results will be obtained in approximately 15 minutes. This result is used to evaluate the editing efficiency of the gene knockout cell pool (KO Cell Pool) and to assess the success rate of subsequent single clones.

 

Detailed introduction: Why choose Liman CRISPR EasyKO kit

1. From editing to verification, all in one box, zero foundation for gene knockout

• The Liman LM EasyKO kit contains all the reagents needed to knock out the target gene and sequencing primers for verifying knockout efficiency. In just 3 days, editing efficiency exceeding 70% can be achieved in cell lines and primary cells.
• For difficult-to-transfect cell types such as primary cells, iPS cells, T cells, and B cells, Liman provides an exclusive optimized electroporation system ( Electroporator demo + electroporation reagents/consumables). It accelerates the popularization of CRISPR/Cas9 RNP technology and improves the efficiency of experimental personnel, reducing costs.
• Liman Biology has accumulated a large amount of data to prove that the LM EasyKO kit can achieve highly efficient editing for cell lines, primary cells, iPS cells, and T cells. Commonly used HEK293, Vero, etc. tool cells, A549, Hela, HCT116, etc. common tumor cells, and difficult-to-transfect neuronal cells SH-SY5Y, U87MG, U251, immune cells THP-1, K562, Raji, Raw264.7, embryonic stem cells iPSC, hESC, etc., all have high editing efficiency.

2. Chemically synthesized sgRNA, efficient RNP delivery

• Does not activate the immune system: Whether it is a viral vector, DNA plasmid, or in vitro transcribed tracrRNA, traditional gene editing reagents inevitably activate the DNA/RNA sensing pathway in cells to some extent, leading to cell apoptosis. This is particularly important for the success or failure of gene editing of immune cells (antigen-presenting APC, T cells). Liman chooses chemically modified synthesized sgRNA to reduce the activation of the RIG-I and type I interferon pathways, thereby maximizing cell survival and ensuring that the phenotype of edited cells is not affected by the activation of the type I interferon pathway.
• Efficient RNP delivery: Liman Biology has optimized the optimal mixing ratio of Cas9 protein and Cas9 with sgRNA. Simply mixing Cas9 protein and sgRNA in a certain proportion can deliver RNP into cells through simple liposome transfection or electroporation. Targeted gene editing can be completed in 3 days, effectively avoiding the tedious experiments of viral packaging, plasmid transfection, and waiting for protein expression, optimizing the editing process.
• Low toxicity, low off-target effect: Compared with viral and plasmid expression systems, the RNP complex delivery method adopted by Liman Biology only stably exists in cells for about 72 hours, greatly reducing the off-target effect caused by the continuous expression of Cas9 and sgRNA.

3. All human whole-genome gRNAs have been designed and experimentally verified, no need for de novo design

• Liman Biology's whole-genome array library has been fully verified by high-content screening in human primary cells, and has achieved very high stability in repeated experiments (Z’>0.7).
• More than 8700 target points have been confirmed by PCR sequencing, and the 3-day KO effect has reached >80%.

4. Simple Sanger sequencing can determine editing efficiency within 3 days.

• Utilizing LiMan Bio's unique sgRNA design strategy, gene editing efficiency can be quickly determined through simple PCR Sanger sequencing. Even without suitable antibody validation, it is possible to know if the target gene has been successfully knocked out.

• The LiMan LM EasyKO kit has designed a complete set of sequencing primers for the whole genome and corresponding bioinformatics analysis services. After 3 days of gene editing experiments, simply use the primers and procedures provided in the kit to complete a simple PCR experiment, and the editing efficiency (gene frameshift + fragment deletion mutation) of the target cells can be determined through sequence analysis.

 

LiMan CRISPR EasyKO Kit Operation Procedure

The following briefly describes the usage procedure of the LM CRISPR EasyKO kit:

• Day 1: After ordering the kit, you can start preparing for cell resuscitation and other preliminary work; the kit will be received in about a week.

• Day 8: Mix the sgRNA and Cas9 protein from the kit in vitro according to the ratio in the instructions to form an RNP complex.

• Day 8: Transfer the RNP complex into cells via nuclear electroporation or lipid transfection.

• Day 11: After 3 days of cell culture, use the cell lysis solution provided in the kit to lyse both electroporated cells and WT cells simultaneously. The treated cell lysate is used as a PCR template. Use the primers in the kit and the standard SOP procedure to complete a simple PCR experiment, and perform Sanger sequencing on the PCR product.

• Day 12: The ab1 sequencing files (WT Cell & KO Cell Pool) obtained from the sequencing company can be uploaded to LiMan's designated email address (specified in the kit instructions) to determine the editing efficiency (gene frameshift + fragment deletion mutation) of the target cells (KO Cell Pool) using sequence analysis software. The following is a diagram of the kit operation procedure.

• Day 20: After obtaining a stable genetically knocked-out cell pool, obtain single-clone cell lines through single-clone plating or microfluidic printing, and screen for homozygous gene knockout single-clone cell lines (validated using Sanger sequencing with primers provided in the kit).

 

Gene knockout experiments, like PCR experiments, Western Blot, and flow cytometry analysis, seem simple, but involve a wide variety of cell types and extensive gene background information, and still encounter many difficult problems.

LiMan's team's long-term accumulation and systematic optimization in bioinformatics, gene editing, antibodies, and mass spectrometry analysis provide complete and systematic solutions for our scientific researchers and corporate R&D personnel, comprehensively assisting drug development and basic research. LiMan Bio focuses on creating simpler and easier-to-use CRISPR gene knockout tools, freeing you from tedious virus packaging, plasmid preparation, Cas9 protein concentration testing, and other optimization experiments, allowing you to focus on biological discovery.

 

Detailed introduction: Follow the LiMan official account

User Guide | LiMan CRISPR EasyKO Kit (RNP method) [Collection]