CRISPR IVT RNA

CRISPR-Cas9 can be delivered through a variety of methods including plasmid, recombinant virus, gRNA-Cas9 RNP complex, and as a mixture of gRNA and Cas9 IVT mRNA. Each of these methods has its own advantages and limitations which allows researchers to select the method tailored to their application for maximum efficiency with minimal off-target and undesired effects, with IVT RNA rapidly emerging as one of the most promising approaches.

Plasmids are generally the easiest to generate in large quantities cheaply and can be delivered through chemical transfection and electroporation which also tends to be simple. However, due to their method of delivery, they are largely limited to in vitro applications. In addition, the efficiency of plasmid transfection can vary widely between cell types and expression is dependent on cell-type specific promoter activity, which can hinder their efficiency in certain systems. As a result, plasmids tend to use highly active promoters which can lead to high-expression levels of Cas9 which can increase off-target editing and random integration of plasmid DNA into the host genome.

Recombinant viruses tend to be another popular method for delivery of CRISPR-Cas9 as they can be used in difficult to transfect cells and they can be used to create stably expressing Cas9 cell lines for multiple gRNA experiments. However, this system has many of the same limitations of plasmid delivery including cell-type dependent promoter specificity and elevated risk of off-target effects due to prolonged Cas9 expression. In addition, recombinant viruses pose an elevated risk of insertional mutagenesis, especially in retroviral and lentiviral systems where genomic integration of the Cas9 transgene occurs.

Delivery of a gRNA-Cas9 RNP complex circumvents many of the limitations of the plasmid and recombinant viral systems allowing for rapid editing as neither transcription nor translation needs to occur. However, this method is largely limited to in vitro applications as it requires electroporation for efficient delivery. Editing by this method occurs rapidly and quickly drops off as Cas9 is degraded in host cells and is therefore widely limited by the availability of Cas9 protein.

In contrast to the above methods, delivery of a mixture of gRNA and Cas9 IVT mRNA has rapidly emerged as one of the most efficient methods for genome editing with minimal off-target effects. gRNA and Cas9 IVT mRNA can be delivered together co-encapsulated in lipid nanoparticles or chemical transfection reagents making the system suitable for both in vitro and in vivo applications without risk of genomic integration. Further, no transcription is required, making expression independent of cell-type specific promoter activity and translation occurs relatively quickly, allowing for rapid and efficient editing. Compared to gRNA-Cas9 RNP complex, mRNA promotes prolonged expression of Cas9 allowing for sustained levels of editing, however, the mRNA is eventually degraded and editing only occurs transiently, limiting off-target effects.

In summary, delivery of CRISPR-Cas9 components through plasmids, recombinant virus, and gRNA-Cas9 RNP complex methods have limitations that limit their use to a number of applications and in addition, have downsides including limited efficiency, increased risk of off-target effects, and potential for insertional mutagenesis. Further, delivery of a mixture of gRNA and Cas9 IVT mRNA has emerged as one of the most efficient methods of genome editing due to its numerous advantages and fewer limitations and should be considered for genome editing applications.