VIROMER® CRISPR for RNP delivery

Genome-editing using Cas9/gRNA Ribonucleoprotein

Easily accessible genome editing by RNA-guided nucleases has transformed all disciplines of molecular biology, and especially the potential for development of therapeutics is tremendous. At first glance, the only requirement is to have in hands a purified Cas9 protein and a guide RNA targeting the desired genome sequence. The endonuclease will then cleave double strand chromosomal DNA with a very high precision making possible all types of genome modifications through subsequent NEHJ and HDR repair pathways (insertions, deletions, directed mutations).

Fig 2 – Viromer® CRISPR for RNP delivery
Fig 1 – CRISPR/Cas9-mediated DNA cleavage and simplified repair mechanisms.
The Cas9 endonuclease is activated by first binding to two different single-stranded RNAs (crRNA and tracrRNA), which are partially complementary in bacteria but have been combined as one-piece gRNA for research purposes. Matching between the gRNA and the target genomic sequence (immediately preceding a 3-nucleotide protospacer adjacent motif, i.e. PAM sequence) enables the Cas9 enzyme to create a double-strand break. In mammalian cells, natural repair pathways will then occur: (i) non-homologous end-joining (NHEJ) with perfect repair (relegation) and insertions/deletions of variable lengths (InDels mutations), or (ii) homology-directed repair (HDR) in presence of a dsDNA or ssDNA donor, resulting in an edited gene sequence.

Despite the theoretical simplicity of the CRISPR methodology, major technical problems can occur at the bench. First, due to the fidelity of the system (off-targets), and second, due to the efficiency of transfecting and editing some specific cell types. Hence, the necessity to choose an appropriate format of CRISPR components and the right method to deliver them into the cells.

Use of plasmids or viral particles as vehicles are the most common and easy approaches. They are perfectly adapted for cell lines transfectable at high efficiency, and for stable or long-term Cas9 expression. The main drawbacks are then the off-target cleavage of DNA due to the kinetics of Cas9 expression (from 12h post-transfection to next cell division in case of plasmids) and the limited efficiency for hard-to-transfect cells.

On the contrary, pre-formed RNP complex enables a direct action of the CRISPR system upon delivery. There is no need to pass through the transcription/translation cell machinery for expressing the Cas9 protein, and it skips the assembly step with the gRNA into the cytosol. It is then faster and safer! Second, the cell rapidly clears Cas9/gRNA RNPs through protein degradation pathways. Obviously, this transient action provides a certain degree of control as it greatly increases the fidelity of the endonuclease (less off-target cleavage) and there is no risk of integration into the cell genome.

Fig 2 – Schematic comparison of plasmid versus RNP approaches for delivery of CRISPR components mediating DNA editing.
Direct delivery of pre-formed RNP complex is obviously advantageous due to a better control of the Cas9 activity (e.g. adjustable amounts of Cas9 and gRNA, transient action limiting off-target cleavage).

How was the new Viromer® CRISPR developed?

While viral transduction, electroporation, microinjection and lipofection or are gold standards in most of CRISPR-Cas9 protocols, there is little consideration for alternative chemical tools. However, polymer-based nanoparticles like the Viromer® reagents have the potential for high performance transfection among various cell types, including some hard-to-transfect cells, with low impact on cell physiology or cell viability.

At Lipocalyx, we have listened to researchers looking for other solutions. While our bestseller reagent, the Viromer® RED, has proven great efficiency for transfecting CRISPR plasmids or Cas9-mRNA, it gave a low output when tested for Cas9/gRNA RNP complex delivery. We therefore screened again over the Viromer® library and worked on formulation to select the best of our technology for that specific purpose.

Based on a collaborative work with OriGene (USA) who provided all CRISPR components for knocking-out RFP expression in stable HEK cells, we first identified two candidates with the required efficiency and outperforming the main leading competitor (lipofection-based reagent).

Fig 4 – CRISPR/Cas9 mediated knock-out of RFP expression in stable HEK293T
Up to 60% reduction of RFP expression was observed 48h post-transfection of RNP complex of Cas9 and gRNA (source: OriGene, US).

We compared the two selected leads through a battery of in-house tests targeting the housekeeping gene HPRT1 in A549, HEK and THP-1 cells with Cas9 and gRNA of different sources and by using variable amounts and ratios of both.

We also accumulated data from independent researchers (beta-testing) confirming success of genome editing efficiency in other cell lines like e.g. HeLa, HUVECs, C2C12 or PDACs. We then optimized the final formulation to have one final product and we adjusted the protocol. As a result, the new Viromer® CRISPR is now available to offer the comfort of an easy-to-use and scalable chemical reagent.

A549 lung adenocarcinoma

C2C12 mouse myoblasts

HUVEC endothelial cells

Pancreatic ductal adenocarcinoma cells, primary mouse

How getting ready?

As aforementioned, the only need is to pre-form in vitro complex of Cas9 and gRNA as ribonucleoproteins!

Cas9 can be easily produced and purified from bacteria by using expression plasmids. Alternatively, it is available from many commercial sources at very affordable prices, under liquid or lyophilized formats, and with different variants: wild-type Cas9 from Streptococcus pyogenes (SpCas9) or Streptococcus aureus (SaCas9), high-fidelity Cas9 with additional Nucleus Localization Sequences (NLS), nickases for single strand DNA breaks, dCas9 etc.

More attention is needed for gRNA as it is crucial to have the optimal design to target the genome upstream of a PAM sequence, at a location that is unique and accessible to the Cas9. While it is possible to work with gRNAs generated by IVT, we highly recommend using synthetic modified gRNA as offered by leading oligo suppliers (as either two-piece tracrRNA + crRNA or one-piece gRNA).

Standards protocols provided with commercial Cas9 and gRNAs recommend mixing Cas9 and gRNA with an equimolar ratio (1:1). We also advise to use this ratio as starting conditions but depending on cell types higher ratios giving an excess of gRNA can improve transfection and editing efficiencies. Some authors also report working with higher ratios (e.g. 1:2 to 1:5) to avoid solubility problems. In addition, they recommend adding Cas9 to the gRNA slowly with manual stirring.

Something special for the transfection protocol?

No! Once you have your stock of RNP complex, simply mix it with the appropriate amount of Viromer® CRISPR, wait for 15 min and go onto your cells.

  • Forward or reverse transfections are both possible
  • Labeled gRNA can be used to monitor transfection efficiency and for FACS sorting
  • Make simply some optimization efforts once to find the best compromise between transfection rate, editing efficiency, amounts of used reagents and toxicity for your special cell type and use our easy manual.