CRISPR Cargo Formulation

Cargo Type Overview

The CRISPR toolbox has expanded well beyond the original Cas9 + sgRNA paradigm. Each payload type presents distinct physicochemical challenges for LNP encapsulation:

Cas9 RNP (Ribonucleoprotein)

Pre-formed Cas9:sgRNA ribonucleoprotein complex. Advantages: no in-cell transcription required, reduced off-target activity due to transient expression. Challenges: RNP is a protein-nucleic acid complex (~160 kDa), requires gentle encapsulation at physiological pH. Biopathio's protocol uses low-voltage lipid exchange (LVLE) at pH 7.2 to maintain RNP integrity during encapsulation. Encapsulation efficiency for RNP is lower than nucleic acid-only cargo (see table below) but sufficient for in vitro CNS cell editing.

mRNA + sgRNA Dual-Cargo

Co-encapsulation of Cas9 mRNA and single guide RNA. This is the primary cargo type for in vivo delivery, as mRNA is more stable than RNP in systemic circulation. Challenge: mRNA and sgRNA have different charge densities and optimal encapsulation conditions. Sequential loading or co-encapsulation with adjusted N/P ratio (4–6) achieves adequate EE for both components. pKa 6.0–6.2 is optimal for dual-cargo endosomal escape.

Base Editors (ABE, CBE)

Base editor mRNAs are 4.5–5.5 kb in length — significantly larger than Cas9 mRNA (4.5 kb) alone, and substantially larger when the deaminase fusion is added (~5.5 kb for ABE8e). This requires adjusted lipid:RNA mass ratios and may benefit from lower PEG-lipid density to accommodate larger nucleic acid payload. Biopathio has achieved satisfactory EE for ABE8e mRNA + sgRNA formulation with ionizable lipid titration.

Prime Editors (PE2, PE3)

Prime editing requires co-delivery of PE2/PE3 mRNA and pegRNA (prime editing guide RNA). This is the most demanding dual-cargo format: pegRNA is larger than a standard sgRNA (~120 nt vs ~100 nt), and PE mRNA is > 6 kb. Active development area in Biopathio's formulation pipeline.

Comparison Table: Cargo × Encapsulation × Editing Efficiency

In vitro editing efficiency data from primary cortical neurons (DIV7–10) and SH-SY5Y differentiated neuron-like cells. All values are synthetic but plausible preclinical benchmarks.