Although the CRISPR system can be delivered to cells via plasmids, direct introduction of Cas9 RNP strengthens and expands the applications of CRISPR genome modification technology by eliminating the possibility of plasmid DNA integration into the host genome. RNP complexes offer faster gene editing because the functional nuclease is immediately available in the cell and the complexes are quickly degraded and cleared making the editing activity short-lived. The reduced time of available RNP in the cells leads to reduced off-target effects.
Figure 1.Schematic diagram of the CRISPR one-part and two-part systems with Cas9 protein, demonstrating the composition of the Ribonucleoprotein (RNP). After a double-strand break is created two possible repair pathways are taken by the cellular DNA machinery; Non-Homologous End Joining (NHEJ), Indel formation or Homology-Directed Repair (HDR) for targeted insertions or knock-ins.
Figure 2.Editing with RNP Nickase systems, with only a single active cutting domain, and then delivering with proximal guide RNAs in a pair allows precise double stranded breaks, and provides hyper-specific and efficient genome editing in cell types that are more difficult to edit.
Ribonucleoprotein (RNP)-based genome editing experiments have never been easier than with SygRNA® synthetic guide RNAs and Sigma-Aldrich® Cas9 proteins, however some cells can be harder to edit than others. We have extensively validated our reagents and protocols to work in the most challenging of applications, including in primary T cells which present a number of significant challenges that need to be overcome when attempting to knock-in or knockout a construct or gene. However, with SygRNA® sgRNAs, editing in primary human T cells at efficiencies and specificities that exceed two-part guide systems can be achieved (Figure 2). In both primary T cells and PBMCs, SygRNA® sgRNAs can be introduced as highly specific paired nickases and Cas9 nickase, or as single site SpCas9 protein to get complete knockouts of the PD-1 gene, as measured by PD-1 expression and NGS (Figure 2). sgRNAs can be rapidly designed for any gene as either a single target RNA or as part of the paired nickase system, ideal for a variety of human cell types, including primary T-cells. Cas nucleases when delivered in protein format, pre-complexed with a single, modified guide RNA (gRNA), rather than encoded on a plasmid, allow researchers to bypass the historically limiting factors of using CRISPR in the therapeutic realm of off-targeting effects, low efficiency, and undesired cellular responses to foreign DNA. When you need to be certain, SygRNA® sgRNAs are the only guide RNAs that are fully backed by a 100% performance guarantee – predesigned and custom sequences.
Figure 3.Gene editing in mouse embryos is traditionally a long process, however, RNP-based editing with CRISPR reduces the complexity. Mouse models can be generated rapidly, for nearly any target. SygRNA® is suitable for both microinjection and electroporation of embryos.
If no cutting is observed and there is reason to suspect an experimental flaw is at fault, the following considerations may aid in troubleshooting the experiment.