A Guide on RNP System for CRISPR Editing (2)

If you missed A Guide on RNP System for CRISPR Editing(1), we will attach it here. You can find the information about how to prepare RNA oligos and RNP complex. Today, this issue covers topics on：

l  How to test RNP efficacy in cells, through either cationic lipid-mediated transfection or electroporation?

l  How to test RNP efficacy in embryos?

l  How to test RNP in vitro cutting efficiency?

Test RNP Efficacy in Cells

RNP testing is best performed in easy-to-handle cell lines, such as HEK293 cells, to confirm gene editing efficiency. For such cells, Lipofectamine™ CRISPRMAX™ are recommended for transfection. The following protocol is recommended for Lipofectamine™ CRISPRMAX™.

Ø  CRISPRMAXTM Procedure

1.     Seed well-dissociated cells one day (16–24 hours) prior transfection in D10 medium without antibiotics.

TIP: 30–70% confluency at the time of transfection is recommended. If confluency is too high, this can negatively impact transfection efficiency.

2.     Prepare RNP complex and transfect cells following Lipofectamine™ CRISPRMAX™ protocol.

3.     Harvest the cells approximately 48 hours after transfection. Extract the genomic DNA for further analysis.

4.     PCR amplify the fragment containing the target (for best results, design the primers to target >200bp away from the target) and test the genome editing efficiency by T7E1 or by Sanger Sequencing.

Ø  How to Use the HPRT Positive Control?

It is recommended to use GenScript’s human HPRT positive control and a non-coding negative control gRNA to optimize transfection conditions and find the condition that gives the highest gene editing efficiency and cell viability.

1.     Seed well-dissociated cells one day (16-24 hours) prior to transfection in media lacking antibiotics.

2.     Determine different transfection conditions that need to be tested, including cell density at the time of transfection, different gRNA to Cas9 ratio, transfection reagent quantity, incubation time period, etc.

3.     Prepare RNP complex and transfect cells following Lipofectamine™ CRISPRMAX™ protocol. Harvest cells approximately 48 hours to 72 hours after transfection and extract genomic DNA for analysis.

4.     PCR amplify genomic fragments using GenScript Human HPRT Primers and verify genome editing efficiency via sequencing or T7E1 digestion assay.

Ø  Electroporation Procedure for Suspension Cell Line (THP-1, U937)

The following procedure is for electroporation of suspension cells with an electroporator, Celetrix. It is highly suggested to optimize the electroporation condition for each cell line by electroporating a positive control (e.g. HPRT). The RNP can also be electroporated by other electroporators following manufacturer’s manuals.

1.     Collect 3*10⁶ cells and spin cells at 800rpm for 5 minutes. Decant supernatant and wash cells with PBS. Spin cells again and decant PBS.

2.     Resuspend cells with 65μl electroporation buffer.

3.     Mix 1.6μl Cas9 nuclease (10mg/ml) and 200pmol sgRNA in a 1.5ml EP tube. Add

electroporation buffer in the tube to a final volume 65μl and mix gently. Incubate at room temperature for 10 minutes.

4.     Mix the solution of step 3 and the resuspended cells of step 2 gently. Incubate for 10 minutes.

5.     Transfer the mixture of step 4 to the electroporation tube.

6.     Electroporate following Celetrix operation procedure using an appropriate voltage (700V suggested to THP-1, 660V suggested to U937).

7.     Harvest the cells approximately 48-72 hours after transfection. Extract the genomic DNA for further analysis.

8.     PCR amplify the fragment containing the target (for best results, design the primers to target >200bp away from the target) and test the genome editing efficiency by T7E1 or by Sanger Sequencing.

Note: The electroporation can be used for iPS or ES cells following manufacturer’s manuals.

Test RNP Efficacy in Embryos

RNP testing can also be performed in embryos, such as mouse or zebrafish, to confirm gene editing efficiency. The following zebrafish protocol can be used as an example:

1.     Prepare 10μl RNP complex in a nuclease-free tube:

2.     Incubate the tube at 37°C for 10 minutes to allow RNP complexing.

3.     Microinject ~1nl RNP Mix into embryos at the 1-cell stage.

4.     When embryos reach 24hpf, collect at least 5 of the injected embryos and extract their genomic DNA.

5.     PCR amplify the fragment containing the target (for best results, design the primers to target >200bp away from the target) and test the genome editing efficiency by T7E1 or by Sanger Sequencing.

Test RNP in vitro Cutting Efficiency

Depending on the target site, it may be necessary to perform in vitro testing of the CRISPR/Cas9 system prior to introducing RNP into cells.

1.     Prepare the PCR amplicon as the substrate of CRISPR/Cas9 RNP digestion. When designing the amplicon, add at least 200bp on either side of the guide RNA target.

TIP: A longer amplicon will give a clearer band when the sample is run on a gel to verify successful cutting. We typically use amplicons around 1kb.

2.     Prepare 16 μl RNP complex in a nuclease-free tube:

3.     Incubate the tube at 37°C for 10 minutes to allow RNP complexes to assemble.

4.     Add 160ng of PCR amplicon into 16μl RNP Mix. Bring the final volume to 20μl with nuclease-free water and mix gently.

5.     Incubate the reaction for at least 30 minutes at 37°C.

6.     Assess the reaction by gel electrophoresis. If the reaction works correctly, two distinct bands will appear on the gel.

CRISPRRibonucleoprotein (RNP) User Manual

A guide on how to use CRISPR RNP for targeted genome editing.

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