Genome Editing with mRNA Encoding Cas9
Targeted changes in the genome offer the ability to address fundamentally important questions. The CRISPR/Cas system, also known as the third-generation genomic editing tool, is now the most recent platform in the field of genome editing. Genome editing tools based on the CRISPR-Cas9 system are a breakthrough technology, which can precisely modify a genome in a relatively simple manner. The Creative Biogene team is experienced in custom mRNA synthesis, including zinc finger nuclease (ZFN)-mRNA, TALEN-mRNA, Cas9-mRNA, but not limited to them. Moreover, we are able to provide the best delivery strategy of Cas9-mRNA according to the specific applications.
Fig1. Genome editing mediated by site-specific nucleases. (Zhang, H. X., et al, 2019)
Comparison of CRISPR/Cas9 with the ZFNs and TALENs.
The recognition of the DNA site of the two previous technologies (ZFNs and TALENs) is based on sequence recognition by artificial proteins requiring interaction between protein and DNA. While CRISPR/Cas system is based on RNA-DNA interactions. Therefore, CRISPR/Cas system has several advantages over ZFNs and TALENs, including easy prediction regarding off-target sites, easy design for any genomic targets, and the potential of simultaneous multiple loci editing.
Table 1. Comparison of CRISPR/Cas9 with the ZFNs and TALENs.
| The aspect of comparison | ZFN | TALEN | CRISPR/Cas9 |
| Origin | Eukaryotes | Bacteria | Bacteria/archaea |
| Mechanism of action and modification pattern | Introduction of double-strand breaks (DSBs) in target DNA; FokI nuclease | Introduction of double-strand breaks (DSBs) in target DNA, FokI nuclease | Introduction of DSBs in target DNA by wtCas9 or single-strand nicks by Cas9 nickase; Cas9 nuclease |
| Cleavage efficacy and off-target effects | Efficient; Highly possible off-target activities | Efficient; Low possible off-target activities | Highly efficient; Variable; limited off-target activities, not fully studied in plants |
| Delivery vehicle | Easy via electroporation and viral vectors transduction | Easy in vitro delivery; difficult in vivo due to the large size of TALEN DNA and their high probability of recombination | Easy in vitro; the moderate difficulty of delivery in vivo due to poor packaging of the large Cas9 by viral vectors. |
| Cytotoxicity | Variable to high | Low | Low |
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)
The CRISPR-Cas9 system is an RNA-based bacterial defense mechanism designed to recognize and eliminate foreign DNA from invading bacteriophage and plasmids. They consist of a Cas endonuclease that is directed to cleave a target sequence by a single guide RNA (sgRNA). Therefore, the system can be co-opted to cleave any target sequence of choice by modifying the sequence of the gRNA, which is encoded by a "CRISPR/array" in the bacterial genome.
There are three common strategies developed for genome editing with the CRISPR/Cas9 platform, including:(1) the plasmid-based CRISPR/Cas9 strategy, (2) mRNA for Cas9 translation alongside the sgRNA, and (3) directly delivery of Cas9 protein and sgRNA. Among them, the second strategy that genome editing by using modified RNA has unique advantages. Since RNA is an intermediate product of DNA transcription, which is responsible for guiding the generation of downstream proteins. Genome editing with mRNA encoding Cas9 can avoid the irreversible modification of the genome, off-target cleavage as well as unwanted genomic integration of plasmid DNA. Additionally, it can repair protein function in almost all cells to treat a series of diseases.
The vehicle and strategy used to deliver mRNA encoding Cas9 and the sgRNA can be classified into two general groups: physical delivery (such as electroporation and microinjection) and non-viral vectors (such as lipid nanoparticles). Here, we make a brief summary of these delivery vehicles and their common features.
Table 2. CRISPR-Cas9 mRNA delivery vehicles and their common features.
| Delivery vehicle | Composition | Most common cargo | Capacity | Advantages | Limitations |
| Microinjection | Needle | mRNA (Cas9 + sgRNA); | nM levels of Cas9 and sgRNA | Guaranteed delivery into cell of interest | Time-consuming; difficult; generally in vitro only |
| Electroporation; nucleofection | Electric current | mRNA (Cas9 + sgRNA) | nM levels of Cas9 and sgRNA | Delivery to cell population; well-known technique | Generally in vitro only; some cells not amenable |
| Lipid nanoparticles/ liposomes/lipoplexes | Natural or synthetic lipids or polymers | mRNA (Cas9 + sgRNA); Protein (RNP) | nM levels of Cas9 and sgRNA | Virus-free; simple manipulation; low cost | Endosomal degradation of cargo; specific cell tropism |
Benefits of Cas9 mRNA for genome editing
- No need for transcription, avoiding incompatibility between the promoter and cell line.
- DNA free, no risk of DNA integration into the host genome.
- Less off-targeting.
The applications of Cas9 mRNA
- Precise gene knockout
- Gene knockout via indels
- Knockin mutations
- Gene tagging
Creative Biogene is offering a wide series of mRNA research services, which can accelerate the progress of mRNA-based genome editing for customers worldwide. We can support our customers with the most affordable, high-quality custom mRNAs according to the desired applications. If you are interested in this area, please feel free to contact us. We look forward to providing services for your next project.
References
- Lino, C. A.,et al. (2018). "Delivering CRISPR: a review of the challenges and approaches." Drug delivery, 25(1), 1234-1257.
- Khalil, A. M. (2020). "The genome editing revolution.” Journal of Genetic Engineering and Biotechnology, 18(1), 1-16.
- Zhang, H. X., et al. (2019). "Genome editing with mRNA encoding ZFN, TALEN, and Cas9." Molecular Therapy, 27(4), 735-746.
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