Dr. Brittany Enzmann from Synthego has recently published an article discussing about why ribonucleoprotein (RNP) is a better alternative to CRISPR plasmids for CRISPR genome editing experiments. Below are highlights from the article:
1. RNPs Reduce the Total Experimental Time
Plasmid-based CRISPR transfection requires multiple steps for the guide preparation and is more labor-intensive compared to RNP workflows. In a comparative analysis performed with immortalized cell lines using Synthego’s synthetic sgRNA RNPs versus plasmids, their scientists found that transfection with RNPs reduced the overall experimental duration by 50%. This included the time involved in sorting cells tagged with fluorescent protein as a positive selection marker.
Figure 1. Plasmid vs RNP CRISPR timeline: The total number of days to complete a CRISPR workflow (guide preparation, transfection, and analysis) using a plasmid delivery system versus synthetic sgRNA and Cas9 protein.
2. CRISPR Plasmid Transfection Increases Off-target Effects
One of the most concerning aspects of any CRISPR experiment is the risk of non-specific editing of the genome. Although well-designed guides will target the correct DNA location most of the time, they may also bind to sequences that differ by only a few nucleotides. These off-target effects are problematic because unintended editing of the genome can alter phenotypes or have harmful effects on the target cell or organism.
The introduction of CRISPR components as plasmids is often associated with high levels of unintended edits. This editing is due to the relatively long time that plasmids persist inside cells, which may last up to several weeks. Because plasmids hang around for so long, a lot of gRNA and Cas9 are expressed, consequently increasing the opportunities for erroneous editing.
Several studies have demonstrated that the frequency of off-target mutations is lower when CRISPR components are delivered as pre-complexed RNPs rather than plasmid DNA (Liang et al. 2015, Kim et al. 2014). For instance, Liang et al. (2015) found that for the gene OT3-18, the ratio of off-target to on-target mutations was 28-fold lower when using RNPs relative to plasmid DNA. The relatively lower incidence of off-targets with RNP use is likely linked to the limited time that RNPs are active inside the cell. Unlike plasmids, RNPs cleave genomic DNA rapidly after transfection and persist in cells for only about a day before being degraded (Kim et al. 2014). This shorter shelf-life ensures fewer unintended cuts to the genome and minimizes off-target effects.
3. Plasmid DNA May Integrate into Host Genome
Another potential problem with plasmid transfection is the random integration of all or part of the plasmid DNA into the genome of target cells. Researchers found a prevalence of plasmid-derived insertions at on- and off-target sites. Unlike insertions at on-target sites that can be readily detected via sequencing, insertions at off-target sites are difficult to ascertain. Transfection using RNPs avoids the risk of DNA integration altogether.
4. RNPs Are Less Toxic to Cells Than CRISPR Plasmids
One of the limitations of using plasmids as the CRISPR cargo is that DNA transfection is often stressful to cells. The cytotoxicity could be both due to transfection reagents and the plasmids. Many chemical transfection methods are detrimental to cell viability and may not be optimal for all cell types. More recently there have been immense improvements in the transfection reagents and delivery vehicles to ensure better cell viability, as well as, efficacy and safety. (Yu et al. 2015, Givens et al. 2019).
In Synthego’s experiments using their synthetic sgRNA RNP versus plasmid-based transient transfection, they found that plasmids reduced cell viability significantly. Their data suggest that plasmid dosage correlates inversely with cell viability, meaning more severe cytotoxicity with higher plasmid concentrations (Figure 2).
Figure 2. Comparison of cell viability between plasmid and RNP delivery of sgRNA and Cas9. Cell viability was assessed using the CellTiter-Glo® Luminescent Cell Viability Assay. For the cell viability assay, cells were nucleofected without RNP or plasmid as a mock control. RNPs were constituted with different ratios of sgRNA and Cas9, 50 pmols sgRNA:10 pmols Cas9, or 90 pmols sgRNA:10 pmols Cas9.
It is important to note that low plasmid concentrations also offer relatively low editing efficiencies. RNPs, on the other hand, are notably less cytotoxic while providing highly efficient edits.
Furthermore, the presence of foreign DNA may trigger cyclic GMP-AMP synthase activation in primary human cells and pluripotent cells. For such specialized cell types, transfection using an RNP format rather than plasmids is likely to have much lower toxicity. According to (Kim et al. 2014), transfection with RNPs has resulted in at least 2x more viable colonies relative to plasmid transfection in embryonic stem cells.
5. Plasmids Demonstrate Variable CRISPR Editing Efficiencies
Plasmids may also have a quality problem. When CRISPR components are transfected as plasmid DNA, transcription (of gRNA and Cas9) and translation (of Cas9) need to occur within the cell before any genome editing occurs. Not only does this extend experimental timelines, but it also risks discrepancies in gRNA expression or the assembly and folding of Cas9. Working with RNPs, on the other hand, avoids these issues because the sgRNA and protein components are made before transfection. The quality of the gRNA and Cas9 can be verified before they are introduced to cells.
Another benefit of the RNP format is that the gRNA is protected by Cas9 such that it is less prone to degradation. Furthermore, synthetically-made gRNA can be chemically modified to prevent endonuclease degradation and immune responses. Both of these factors enable the gRNA to target genomic DNA more efficiently during the short time it is active within the cell (Hendel & Bak et al. 2015).
Given the many benefits of RNPs, it is not surprising that they have consistently yielded high editing efficiencies across a variety of immortalized cells, primary cells, and stem cells (e.g., Hendel & Bak et al. 2015, Kim et al. 2014, Liang et al. 2015, Lin et al. 2014). “Knocking-in” a genetic sequence via homology-directed repair, a notoriously inefficient process, also appears to be enhanced by the use of RNPs (Gaj et al. 2017, Lin et al. 2014, Schumann et al. 2015).