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sgRNA and Cas9 Delivery


 
 

Welcome to our training series on performing your own CRISPR Cas9 experiment for gene knockout. Each week we’ll send you new instructional material including decision-making tools, protocols, and troubleshooting advice on how to design and carry out your gene knockout experiment.

This week we’ll talk about how to decide on a delivery method for your sgRNA and Cas9, and how to proceed with transfection/infection once you’ve decided.

 
 

1. Common Delivery Methods


 
 

So, you’ve decided on a Cas9 and designed your sgRNAs. Next you need to decide how to express those components in your target cells. Asking yourself the following questions can help you make your decision:

  • Do you need high level expression?
  • Do you need long term expression, or is transient expression enough?
  • How concerned are you about off-target effects of your editing?
  • Will you be performing repeated experiments on the same cell line?
  • Are you carrying out the experiment in an animal model?
  • Would you prefer a simplified experimental approach?
  • Are your cells difficult to transfect?
  • Are you using spCas9 (larger) or saCas9 (smaller)?

 

Luckily, no matter your circumstances, there is a delivery method that will suit your needs. abm offers a wide array of expression systems for CRISPR components.

 
 

2. Expression System Comparison


 
 

Expression System

Advantages

Disadvantages

abm Products

Non-Viral Vector / Plasmid

  • Low risk of off-target effects
  • Can express Cas9 and sgRNA from one construct
  • Simple
  • Transient expression, which may be low level
  • Difficult to transfect some cells

sgRNA Non-Viral Vectors and spCas9 Non-Viral Vectors

Cas9-sgRNA Ribonucleo-protein (RNP)

  • Low risk of off-target effects
  • Rapid editing
  • Transient
  • Difficult to transfect some cells

spCas9 and saCas9 proteins, and sgRNA synthesis kit

Lentivirus

  • Stable expression
  • Can express Cas9 and sgRNA from one construct
  • Popular for genome-wide screens
  • Higher chance of off-target effects

sgRNA Lentivirus, All-In-One Lentivirus, and spCas9 Lentivirus

AAV

  • Popular for in vivo delivery
  • Tissue-specific delivery, depending on the serotype
  • Transient expression
  • Small virus: cannot package spCas9

sgRNA AAV, All-In-One AAV, and saCas9 AAV

Adenovirus

  • High transduction efficiency and level of expression
  • Transient expression
  • Immunogenic

sgRNA Adenovirus and spCas9 Adenovirus

Cas9-Expressing Cell Line

  • Ideal for repeated CRISPR experiments, such as to knockout multiple genes
  • Cas9 expression is pre-validated
  • Simple
  • Higher chance of off-target effects
  • More expensive

spCas9 Expressing Stable Cell Lines

 

 
 

3. One-Component vs. Two-Component Systems


 
 

If you decide to proceed with a vector or virus-based delivery method, you have an additional choice between using a one-component (or, All-In-One) or two-component system.

An All-In-One System is one in which sgRNA and Cas9 are expressed from the same vector. This makes it easy to use and less expensive if only one experiment will be done. However, you can’t reuse the vector between different experiments or trying different sgRNAs.

Two-component systems will split expression of the sgRNA and the Cas9 into two separate vectors, or a vector and a Cas9-expressing cell line. The Cas9 vector or cell line can be re-used between experiments, and allows for more flexibility in trying different sgRNAs. However, this can be more expensive and complicated to use than an All-In-One vector.

Additionally, there is the option of using a multiplexed sgRNA vector. Multiplex vectors allow for the co-expression of multiple sgRNAs from one vector. This can be useful for the following applications:

  • Targeting multiple genes simultaneously for multi-gene knockouts.
  • Ensuring efficient knockout, activation, or repression by targeting multiple sites within the same gene.
  • Convenient for use with Cas9 nickase, which requires two sgRNAs for double-stranded DNA cleavage.

 

CRISPR-One-and-Two-Component-2

The All-In-One System expresses Cas9 and sgRNA from a single vector. Multiplex gRNA vectors allow for the co-expression of multiple sgRNAs from one vector. Two-component systems split the expression of Cas9 and sgRNA between two vectors, or between a vector and a stable cell line.

 

 
 

4. Expression Vector Selection Tools


 
 

Overwhelmed by the choices? abm has made a few tools to help you out.

Our specialized CRISPR Experimental Design Tool will give tailored recommendations for your CRISPR experiment, including suggestions for Cas9 and sgRNA delivery, controls, and methods for validation.

We also have a general Vector Selection Tool to help you decide between expression systems.

 
 

5. Protocols for Delivery 


 
 

Depending on the application, proceed with a transfection or a transduction of the vector or virus into the cell line of interest. The following transfection and transduction procedures may serve as general guidelines for each delivery method. 

 

a. Transfection Protocol

Note: To monitor the success of transfection, it is highly recommended to perform transfection with the appropriate GFP control vector in parallel.

  • Approximately 18-24 hours prior to performing the transfection, plate 1 - 3x105 adherent cells (in 2 ml appropriate culture medium complete with serum and antibiotics if they are normally used) into each well of a 6-well plate. Incubate the cells at 37°C in a CO2 incubator until the cells are 50-70% confluent.
  • The next day, set up the transfection reaction. For each transfection sample, prepare the Transfection Reagent (such as DNAfectin 2100) and DNA complexes as follows (per well of a 6-well plate):
    1. Add 2 μg each of vector (sgRNA and Cas9 vectors or All-in-One vector) into 100 μl of serum-free, antibiotic-free media.
    2. Vortex DNAfectin 2100 thoroughly prior to use. Then, add 12.0 μl of DNAfectin 2100 into serum-free, antibiotic-free media.
    3. Mix the DNA solution from step a) and the DNAfectin 2100 solution from step b), and mix gently to ensure uniform distribution.
    4. Incubate for 20 minutes at room temperature to form the DNAfectin 2100-DNA complexes. Complexes are stable at room temperature for 3 - 5 hr.
  • Add 800 μl of serum-free and antibiotic-free media to the DNAfectin 2100-DNA complexes.
  • Aspirate the growth media from the cells to be transfected.
  • Dropwise, add 1 ml of DNAfectin 2100-DNA complexes per well of cells. Incubate the cells for an additional 4-6 hr at 37°C, 5% CO2.
  • After incubation, add 100 μl of 10% FBS to directly into each well. Incubate the cells at 37°C in a CO2 incubator for a total of 18-24 hr.
  • Passage cells at 1:10 (or higher dilution) into fresh growth medium 24 hours post transfection and monitor the cells for the next 1-2 days before adding selection drug.

 

b. Packaging and Transduction Protocol

The transduction efficiency of mammalian cells varies significantly under different experimental conditions. This includes virus concentration, exposure time to the virus, and growth area of the well or plate used for the infection.

Note: If you plan on using sequencing as the primary method of validation and have multiple sgRNAs for 1 gene target, it is recommended to infect each sgRNA separately in different wells.

Note: Include one transduction well with a positive GFP control virus. Leave one well of cells uninfected as an additional drug selection control.

Day 1: Seed cells one day before viral infection to achieve 20-30% density on day of infection. Incubate the cells at 37°C, 5% CO2 overnight.
Day 2:

You may need to optimize the Multiplicity of Infection or MOI (the number of virus particles/cell) of the virus you are using to achieve the highest transduction efficiency. MOI can be optimized by infecting your target cells with a reporter control virus then assessing reporter strength and cell health.

e.g. On the day of infection, cells should be at 20% density. If the virus titer is 107 IU/ml, the following volumes of virus can be added to 105 cells to achieve the below target MOIs.

MOI-example

Therefore, to infect at an MOI of 1, use 10 μl of 107 IU/ml of virus to infect cells that are at 20% density. If the transduction efficiency of the target cells is low, add Transduction Enhancer at a 1:100 ratio (or at the optimized dilution ratio determined for the transduction). Keep the infected cells at 37°C, 5% CO2 for incubation overnight until ready for the drug selection step.

Day 3:

Incubate the cells for another 24-48 hr at 37°C, 5% CO2 to allow cells to recover.

If a vector with fluorescent reporter is being used, observe cells for infection signal at this time. If your vector confers resistance to an antibiotic, you can treat cells with that antibiotic. Only cells which have taken up the vector/virus will survive.

 

c. Supporting Documents 

See our Drug-Selection Killing Curve guidelines for stable cell line generation using lentivirus. We also have a list of suggested MOIs for commonly used cancer cell lines. 

Typically, lentivirus can be used at an MOI of 1, 5, 10, and 50. Usual AAV MOI ranges from 10 000 to 500 000, depending on serotype and cell type. Adenovirus MOIs can range from 1 to 50; for adenoviruses, abm has had good success by simply overlaying cells with viral culture supernatant (at ~106 GC/ml).

 

 

So, now you know how to perform your knockout experiment. But how do you isolate and verify correctly edited cells? Next week we’ll focus on methods of screening and validation of your CRISPR knockout.

 
 

Test Your Knowledge!


 
 

Take our Week 3 Quiz, and graduate to becoming a Gene Expression Journeyman!

 

How did you do? Share your results with #CRISPRCrashCourse: