CRISPR/Cas9 Transfection

* This product is for research use only. Not intended for use in the treatment or diagnosis of disease.

Background of CRISPR/Cas9

CRISPR is a powerful tool that can be used to edit the genome relatively quickly and easily and is at the forefront of gene-editing technology. The CRISPR/Cas9 system contains two important components: programmable guide RNA (gRNA), and nuclease Cas9. gRNA can bring Cas9 nuclease to the target region of the genome, and then Cas9 makes a double-strand break in the DNA. The cell's natural DNA repair mechanism can then be used to make specific genome changes (point mutations, deletions, insertions, etc.). CRISPR has been used for a variety of purposes, including single-base editing, whole-genome screening, and the development of transgenic animals.

CRISPR Handles Multiple Types of Genome Modification. Figure 1: CRISPR Handles Multiple Types of Genome Modification.

CRISPR Transfection Method

  • CRISPR gene editing workflow
  • CRISPR gene editing workflow. Figure 1: CRISPR gene editing workflow.

    The key step of any CRISPR workflow is to deliver gRNA and Cas9 to the cytoplasm or nucleus of the target cell. Transfection methods can be roughly divided into physical, chemical, and virus-mediated categories. Each method has different advantages and disadvantages in terms of efficiency, throughput, equipment, skills, and cost. In this article, we discussed some tips to consider before choosing a transfection protocol, as well as the advantages and disadvantages of each method.

  • Format of CRISPR components
  • The format of the CRISPR component may influence your decision on which transfection method to use. Guide RNA and Cas9 can be delivered to cells in the form of DNA, RNA, or pre-formed Cas9/guide RNA ribonucleoprotein (RNP) complex. Cas9 DNA and RNA formats need to be transcribed and/or translated after they are introduced into cells, while nucleases need to pass through the nuclear envelope after their preparation. The Cas9 sequence contains a nuclear localization sequence (NLS) to ensure that it can enter the nucleus for genome editing. In addition, Cas9 can be directly transferred into the cytoplasm in the form of protein, but care needs to be taken to avoid the loss of Cas9 function caused by the endosomal pathway.

Table 1: Comparison of Cas9 Formats: DNA, RNA and Protein

Plasmid DNAmRNAProtein

CRISPR/Cas9 Transfection

CRISPR/Cas9 Transfection2

CRISPR/Cas9 Transfection3

High efficiency+ + + ++ + + ++ + + +
Low cost+ + + ++ + + ++ + + +
Specificity+ + + ++ + + ++ + + +
FeaturesSimple, low-cost.
  • Achieve rapid gene expression and eliminate the risk of insertional mutagenesis.
  • Can be effectively delivered to cell types that are resistant to plasmid DNA transfection.
  • RNP exhibits the fastest genome editing pulse and reduces the possibility of off-target cleavage events.
  • Can be effectively delivered to cell types that are resistant to plasmid DNA transfection.

Physical transfections of CRISPR/Cas9 system

Temporary holes are created in the plasma membrane through physical methods, and gRNA/Cas9 passes through these holes and then enters the cell. Three popular physical methods of introducing CRISPR components into cells are electroporation, nuclear transfection, and microinjection. Electroporation and nuclear transfection use electrical pulses to create holes in the plasma membrane, while microinjection uses a needle to force the holes through the membrane.

AdvantagesDisadvantages
Electroporation & Nucleofection
  • Quick and easy
  • High efficiency
  • A large number of cells can be transfected in a short time (a few minutes)
  • Need special equipment
  • High voltage pulses and incomplete membrane repair may cause cell death
Cell TypesImmortalized cells, Primary cells, Stem cells, Oocytes/zygotes/embryos
Microinjection
  • High efficiency (near 100%)
  • Lar-intensive & requires skill
  • Damage to the plasma and nuclear membranes can lead to cell death
  • Low-throughput
  • Requires special equipment
Cell TypesSingle cells, Oocytes/zygotes/embryos

Chemical transfections of CRISPR/Cas9 system

Several chemical-based methods can be used to transport molecules into cells, including calcium phosphate, cationic polymers, and cationic amino acids. Lipid transfection is one of the most common methods for introducing CRISPR components into cells.

Lipid transfection uses cationic lipid reagents to deliver CRISPR components into cells. The method first involves building fat-soluble structures around CRISPR components. These components are then transported into the cell through endocytosis, during which the plasma membrane surrounds these components on the outside and germinates inside the cell. The CRISPR components then escape the endosomal pathway and diffuse into the cytoplasm. Unlike nuclear transfection and microinjection, lipofection does not deliver CRISPR components to the nucleus.

AdvantagesDisadvantages
Lipofection
  • Economical & easy
  • No specialized equipment is required
  • Low cytotoxicity
  • Adaptable to high-throughput systems
  • Moderately efficient
  • Limited cell types
  • Can not be delivered directly to the nucleus
Cell TypesStandard cell lines and immortalized cells. Not suitable for transfection in Primary cells or stem cells.
  • Viral transfection methods
  • The process first involves packaging the gRNA/Cas9 sequence into viral particles and then introducing the particles into target cells. To prepare virus particles, plasmids containing gRNA or Cas9 sequences and plasmids containing viral genes are introduced into a packaging cell line (for example, 293T cells). Once the viral particles are produced, they can be harvested from packaging cells and introduced into target cells to be transfected with gRNA or Cas9.

AdvantagesDisadvantages
Viral Transfection
  • High efficiency
  • Can be used in vivo and in vitro
  • May require extra safety measures
  • Laborious & time-consuming
  • Some types may have immunogenicity & cytotoxicity
Cell Types
  • Compatible with many cell types

There is no single “one-size-fits-all” answer to the protocol that everyone should use in CRISPR experiments. The sample type and experiment goal will first be used to determine the best plan for each experiment, and in each case, the selected plan needs to be optimized to achieve the highest editing efficiency.

CRISPR-Cas9 Delivery Solution Selection Guide
Transfection reagentsCRISPR delivery methodCell lines
CRISPR DNACRISPR gRNACRISPR mRNACRISPR proteinCRISPR RNPLentivirus293 cellsHepG2 cellsHela cellsPrimary cells
RNA Transfection Reagent      
Plasmid DNA Transfection Reagent     
HTT mRNA Transfection Reagent      
Protein Transfection Reagent       
Lentiviral Transfection Reagent     
CRISPR RNP Transfection Reagent     
Hela CRISPR Transfection Reagent 
HepG2 CRISPR Transfection Reagent
293 CRISPR Transfection Reagent