From Research to Clinical with a Single Reagent
Aldevron now offers RNP services to help you turn your CRISPR reagents into therapies. Working with guide RNAs from the world’s leading providers, we’ve defined the optimal conditions for complexing, characterizing, and storing CRISPR RNPs created with Aldevron research and GMP-grade CRISPR/Cas9 proteins.
Complexing and Cutting
Aldevron has developed gel- and plate-based in vitro cutting assays that can be used to assess the activity of any CRISPR RNP. We’ll complex your unique gRNA with Aldevron Cas9 nuclease to assess a series of gRNA-to-protein ratios. This allows us to create an activity profile and determine the ideal conditions for creating your RNP.
The CRISPR RNP
The Cas9 protein and single guide RNA (gRNA) combine to make the CRISPR ribonucleoprotein, or RNP (Fig. 1). The RNP represents the functional unit of CRISPR cutting activity for genome editing.
Quantifying RNP Activity
During RNP development, Aldevron will use our proprietary in vitro cutting assays to measure the relative activity of each RNP under uniform conditions. In our gel-based assay (Fig. 2a), a linearized plasmid containing a single copy of the RNP target is used to assess cutting. In this example, CRISPR RNPs made at a 1:1 ratio of Cas9:gRNA were titrated to determine the linear range of activity and the limit of detection for in vitro cutting (Fig. 2b). Aldevron has also developed a faster and more quantitative plate-based assay to determine relative activity for CRISPR RNPs.
Optimizing Complexing Conditions
If making a new RNP for the first time, Aldevron can work with you to identify the optimal complexing ratios, conditions, and formulation for your unique RNP. In this example (Fig. 3a & 3b), an RNP was made with increasing ratios of gRNA:Cas9 protein that were complexed under specific conditions and then assessed for cutting against the same linearized DNA target. For this RNP, increasing the ratio of gRNA to Cas9 protein increased RNP activity, with in vitro cutting maxing out at a 1.5X ratio. After identifying the ideal conditions for complexing your RNP, Aldevron will send you research-grade RNP material for confirmation of the in vitro results in your clinically-relevant cell model. Conducting RNP development at research-grade is faster, less costly, and allows you to save precious GMP reagents for your clinical application. However, you must make sure that you are using identical formulations and purity levels for your CRISPR materials when moving from research-to-GMP in order to maintain comparability. This consideration is one of the primary reasons that Aldevron uses the same formulations at all quality grades (Research, GMP-Source, and GMP) for our purified CRISPR/Cas proteins.
Characterization of RNP, Uncomplexed Guide and Protein
To be delivered as a therapeutic, the composition of the CRISPR RNP needs to be highly defined. Aldevron uses a variety of in-house methods to quantify the amount of complexed RNP, relative to (uncomplexed) free-Cas9 and free-gRNA, in the final product. After assessing several alternatives, analytical cation exchange (CEX-HPLC) was found to be the best method for distinguishing and quantifying fully-formed RNP, relative to free-Cas9.