CRISPR

CRISPR Cas9 technology is an innovative gene editing technology that allows precise modification of the DNA of living organisms. It consists of a guide RNA (gRNA) that accurately recognizes and binds to the target DNA and a Cas9 nuclease that cuts the target DNA, allowing genes to be manipulated through the process of damaging and repairing DNA double strands.

gRNA consists of crRNA that binds complementary to the target sequence and tracrRNA for Cas9-binding. gRNA serves to guide Cas9 to the target region and binds complementary to the target DNA sequence. There must be a PAM (Protospacer adjacent motif) sequence at the 3' end of the target DNA sequence.

When the gRNA forms a complex with the Cas9 nuclease and specifically binds to the target sequence, the Cas9 nuclease recognizes the PAM sequence and causes a double strand break (DSB) in the 3 bp upstream part of the PAM.

To repair DNA damaged by DSBs, cells generally go through two pathways: Non-homologous end-joining (NHEJ) or Homology-directed repair (HDR) pathway.

• NHEJ pathway: In the absence of a donor template, DSBs are reassembled through the NHEJ pathway, and gene knock-outs are created due to frameshifts and premature stop codons caused by the creation of indels (insertions and deletions) within the gene coding region.
• HDR pathway: The HDR pathway allows precise and accurate modifications and insertion of new genes by donor templates.
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CRISPR-Cas9 technology is a powerful third-generation genome editing tool that is more economical and efficient than the first-generation ZFN (Zinc Finger Nuclease) and the second-generation TALEN (Transcription Activator-Like Effector Nuclease).

The CRISPR workflow involves the following steps:

• 1. gRNA design: To proceed with knock out and knock in, gRNA and donor design must be performed.
• 2. gRNA synthesis: The designed target sequence is synthesized in plasmid or ssRNA oligo form.
• 3. Introducing and editing cells: Transfect the produced Cas9 and gRNA into target cells.
• 4. Validation: After gDNA prep in a cell, you can check the editing efficiency through a mutation detection kit or In/del analysis service.
• 5. Analysis: We conduct follow-up experiments.

It is recommended that the design be such that the GC content is 40-80%, the length is 18-20bp, and the PAM sequence is not included in the sgRNA sequence. Find the PAM sequence of the Cas9 you want to use in the desired gene and design it as 20nt 5’ upstream of the PAM site.

If you have difficulty designing sgRNA, you can use the AccuTool™ Custom Design Service. This service designs and provides 3-4 sequences with high on-target efficiency and low off-target effects. For high editing efficiency, it is recommended to design at least two sgRNAs and use them in experiments.

Off-target effects mean that Cas9 acts at a location other than the target site and cuts DNA unintended. To reduce off-target effects, sgRNA design is most important. Off-target effects can be predicted using in silico tools and are calculated based on the sgRNA sequence.

You can use sgRNA (dRGEN) and Cas9 (pRGEN) by transfection at a 1:1 ratio. If you use a fluorescently tagged plasmid, transfection efficiency and screening are primarily possible.

dRGEN Manual Link

CMV promoter and EF1a promoter are used as promoters to express Cas9 protein.

CMV Promoter:

• The CMV promoter is a powerful promoter that drives high levels of Cas9 protein expression in a variety of mammalian cells.
• Therefore, if high Cas9 expression levels are required, the use of the CMV promoter is recommended.
• However, strong expression of the CMV promoter may cause off-target editing at unintended locations or cause cytotoxicity.

EF1a Promoter:

• The EF1a promoter is a powerful promoter that provides good levels of Cas9 expression in multiple cell types.
• Unlike the CMV promoter, this promoter is suitable for stable expression because it is resistant to gene silencing by methylation.
• Compared to the CMV promoter, there is less possibility of off-target editing or causing cytotoxicity.
• Therefore, when minimizing off-target effects and cytotoxicity is a priority, the use of the EF1a promoter is recommended.

The effectiveness of the promoter may vary depending on the specific cell line used. Consult the literature or technical information on Cas9 plasmids to determine if there are recommendations for your specific cell line.

sgRNA (single guide RNA) is a single RNA molecule that is a complex of a custom-designed crRNA sequence and a scaffold tracrRNA sequence. 2-part gRNA refers to each crRNA and tracrRNA. To apply 2-part gRNA to an experiment, crRNA and tracrRNA must be hybridized to form a complex.

Both are synthesized through RNA oligonucleotides and are suitable for experiments in RNP form. Because sgRNA (aRGEN) is long, 2-part gRNA is more economical, but sgRNA is more stable.

There are two main forms in which sgRNA and Cas9 are delivered to cells for CRISPR-Cas9 gene editing: RNP (ribonucleoprotein complex) and Plasmid DNA.

RNP (ribonucleoprotein complex):

• Composition: Cas9 protein + sgRNA (aRGEN)
• Delivery: RNP complex -> Direct delivery to the nucleus without transcription or translation -> genome editing
• Advantages:
  • Fast gene editing: No transcription process is required, so experiments can be performed faster than plasmid DNA.
  • Low cytotoxicity: RNP is composed of protein and sgRNA, so external DNA delivery is not required, and RNP decomposes quickly, showing low cytotoxicity.
  • Low off-target effect: It has a short remaining time in the cell and is degraded by proteases and RNases, showing low off-target.
  • Can be used both in vitro and in vivo.

Plasmid DNA:

• Composition: Cas9 plasmid + plasmid sgRNA (dRGEN)
• Delivery: Move to nucleus -> sgRNA, Cas9 transcription -> Cas9 translation -> RNP formation -> Move to nucleus -> genome editing
• Advantages:
  • Low cost: Low cost of production and operation compared to RNP.
  • Long shelf life: Plasmid DNA is more stable and has a longer shelf life than RNP.
  • Easy delivery: Because it uses general transformation methods, experiments can be performed easily.
  • Easy screening: A selection marker can be expressed, allowing you to check whether it has been delivered into the cell after transformation.
• Disadvantages:
  • Slow and less efficient: Cas9 expression and sgRNA processing take time, making the editing process slower and potentially less efficient compared to RNP.
  • High off-target effect: Compared to RNP, Cas9 and sgRNA remain in the cell for a longer period of time, making them more likely to be off-target.
  • High cytotoxicity: Foreign DNA is delivered, showing high cytotoxicity.

The average molecular weight of sgRNA is 32,212 g/mol.

If you want to use it in ng, use the formula below to calculate the molecular weight listed on the sheet:

Formula: Molecular weight M.W. (g) X mole number (nmole) = amount of sgRNA (ng)

The length of the homology arm varies depending on the length of the insert:

• Short size inserts (e.g., point mutations): Homology arm length of 40nt-50nt is recommended.
• Large size inserts: Homology arm length of 300-800nt is recommended.

Mutation detection Kit (T7E1):

This kit allows you to quickly and conveniently check KO efficiency using the T7 Endonuclease I (T7E1) enzyme. The T7E1 enzyme identifies mismatches and cleaves them. Although this kit allows simple quantification, accurate analysis of single nucleotides is not possible.

Mutation Detection Kit Manual Link

In/del analysis service:

AccuCRISPR™ In/del analysis service uses a targeted resequencing method to analyze specific parts of the genome through NGS. The targeted sequencing method allows for quick and accurate quantification of gene editing efficiency through CRISPR-Cas9.

Crispr efficiency is determined by factors such as:

sgRNA: sgRNA sequence design is important because efficiency varies depending on the sequence. It is recommended to use 2-3 sgRNAs.

Delivery Method: Use a delivery method appropriate for the host cell or organism.

• Delivery methods: Transfection reagent, viral transduction, electroporation, microinjection

Crispr component format: The forms in which sgRNA and Cas9 are delivered to cells include DNA, RNA, and RNP (pre-formed ribonucleoprotein complex). An appropriate delivery method must be selected depending on each type.

• DNA: Move to nucleus -> sgRNA, Cas9 transcription -> Cas9 translation -> RNP formation -> Move to nucleus -> genome editing
• RNA: Cas9 mRNA is translated in cytoplasm -> Cas9 protein is formed, then gRNA and RNP complex is formed -> moves to the nucleus -> genome editing
• RNP: RNP complex -> Directly delivered to the nucleus without transcription or translation -> genome editing

Use Cas9 variant: Bioneer sells Sniper Cas9, a variant of SpCas9. Sniper Cas9 has lower off-target effects and higher specificity than SpCas9 WT. Lee, J.K., Jeong, E., Lee, J. et al. Directed evolution of CRISPR-Cas9 to increase its specificity. Nat Commun 9, 3048 (2018).