Poly(Beta-Amino Ester) Nanoparticles Enable Nonviral Delivery of CRISPR-Cas9 Plasmids for Gene Knockout and Gene Deletion

Purpose: CRISPR-Cas9 gene editing requires efficient intracellular delivery of large cargo. Viral vectors have packaging and production limitations, and nonviral CRISPR plasmid delivery had mainly been studied for single-site knockout, not for gene deletion requiring two cleavage events. The transfection requirements for 1-cut vs. 2-cut edits were unclear.

Hypothesis: Biodegradable poly(beta-amino ester) (PBAE) nanoparticles can codeliver plasmid DNA encoding Cas9 and sgRNA to enable both 1-cut gene knockout and 2-cut gene deletion. 2-cut deletion edits will require higher CRISPR component expression than 1-cut knockout edits.

Aims: Develop PBAE nanoparticles for codelivery of Cas9 and sgRNA plasmids. - Design a reporter system to evaluate 1-cut knockout and 2-cut deletion edits. - Compare editing efficiency in easy-to-transfect HEK293T vs. hard-to-transfect B16-F10 cells. - Determine expression thresholds and transfection requirements for 1-cut vs. 2-cut edits. - Test a tRNA-gRNA multiplex sgRNA expression system for 2-cut editing.

Delivery system: Polymers: PBAEs. - 446: linear PBAE from B4 + S4 at 1.1:1, end-capped with E6. - 7,8-4-J11: branched PBAE from B7, B8, and S4 (overall vinyl:amine 2.2:1; 80% B7, 20% B8 by mole fraction), end-capped with J11. - Nanoparticles: PBAE/DNA polyplexes, 100–200 nm diameter, positive zeta potential 12–25 mV. 446 used at 60 w/w; 7,8-4-J11 used at 30 w/w. N/P ratios: 34.5 and 14.5, respectively. - Payload: Plasmid DNA encoding Cas9 and sgRNA, mixed at 1:1 weight ratio. Standard dose 600 ng DNA/well in 96-well plates unless otherwise noted; 2 h incubation. - Targeting ligand: None. - Reporter systems: GFPd2 for 1-cut knockout (loss of GFP); iRFP-STOP-ReNL for 2-cut deletion (gain of ReNL fluorescence/luminescence after stop-cassette removal).

Approach: In vitro only. HEK293T (easy-to-transfect) and B16-F10 murine melanoma (hard-to-transfect) cells. - Stable reporter cell lines generated using PiggyBac transposon/transposase: GFPd2 knockout reporter; iRFP-STOP-ReNL deletion reporter. - Cells plated at 15,000 (HEK293T) or 10,000 (B16-F10) cells/well in 96-well plates. - Nanoparticles incubated 2 h; editing assessed ~day 3 post-transfection. - Controls: untreated cells, Cas9-only plasmid, and commercial reagents jetPrime and Lipofectamine 3000. - Dose titration: 600 ng vs. 300 ng total DNA. Cold shock: transfected cells maintained at 30 °C for 3 days, then returned to 37 °C. - n = 4 replicates; mean + SEM; statistical tests including Holm–Sidak, ANOVA with Tukey.

Key methods: DLS and TEM for nanoparticle size, zeta potential, and morphology. - Flow cytometry for GFP transfection, iRFP knockout, and ReNL gain-of-function. - qRT-PCR for Cas9 mRNA and sgRNA expression. - Western blot for Cas9 protein persistence. - Surveyor assay and TIDE analysis for 1-cut editing. - Gel electrophoresis and band quantification for 2-cut deletion editing. - Sanger sequencing for editing confirmation. - Nano-Glo luciferase assay for ReNL luminescence. - MTS assay for viability.

Key results: Transfection: >80% of cells were transfected in both HEK293T and B16-F10 using GFP reporter. However, geometric mean expression in HEK293T was nearly 1 order of magnitude higher than B16-F10. - 1-cut knockout: Up to 70% gene knockout due to small indels. - 2-cut deletion: Up to 45% gain-of-function ReNL expression after a 600-bp deletion edit. - Expression threshold: 1-cut editing correlated logarithmically with Cas9 mRNA expression (R² = 0.9550); 2-cut editing correlated linearly (R² = 0.9195) in HEK293T. - Dose effect: Reducing DNA dose from 600 ng to 300 ng significantly decreased 2-cut edits but did not significantly change 1-cut edits. - Cold shock: Increased Cas9 mRNA expression and 2-cut editing in B16-F10 cells, but did not significantly change 1-cut editing. - Cell line difference: B16-F10 showed minimal 2-cut edits; 1-cut knockout was lower than HEK293T (12% vs. 33%) but the difference was much smaller than for 2-cut edits. - Multiplex sgRNA: tRNA-gRNA tandem expression under a single U6 promoter achieved similar 2-cut editing to separate U6-driven sgRNAs. - Cas9 persistence: Cas9 mRNA remained high 4.5–48 h; sgRNA peaked at 48 h; Cas9 protein was undetectable by 11 days post-transfection.

Interpretation: PBAE nanoparticles can codeliver Cas9 and sgRNA plasmids for both 1-cut knockout and 2-cut deletion edits. 2-cut deletion edits have a higher expression threshold and are more sensitive to transfection efficiency than 1-cut edits. These findings provide design criteria for nonviral CRISPR-Cas9 delivery systems and support further development of PBAE-based gene-editing nanoparticles.

Limitations: In vitro only: no in vivo animal or clinical validation. - Plasmid DNA format: prolonged Cas9/sgRNA persistence may increase off-target editing risk; plasmid integration risk also exists, though authors note it is lower than viral vectors. - Hard-to-transfect cells: B16-F10 achieved very low 2-cut editing; 2-cut edits remain limited by DNA transfection efficiency. - Limited cell lines: only HEK293T and B16-F10 were tested. - No targeting ligand: delivery specificity was not addressed. - No in vivo safety, biodistribution, or immune response data.