CPP-CRISPR/Cas9 mediated GBA1 Knockout: development of a Gaucher’s disease model

다국어 초록 (Multilingual Abstract)

Genome editing originated from early insights into cellular DNA repair pathways, which revealed that programmed double-strand breaks (DSBs) could be harnessed for targeted genetic modification. Although initial platforms such as triplex-forming oligonucleotides, peptide nucleic acids, and engineered nucleases provided proof of concept, the emergence of CRISPR- Cas systems transformed genome engineering through their simplicity, programmability, and efficiency. The Type II CRISPR system from Streptococcus pyogenes (SpCas9) became the standard tool due to its single effector nuclease and a customizable single-guide RNA (sgRNA). Once directed to a genomic locus via a 20-nt targeting sequence and PAM recognition, Cas9 induces DSBs that are repaired by either error-prone non- homologous end joining (NHEJ) or homology-directed repair (HDR), enabling disruptive or precise modifications. A continuing barrier to effective CRISPR application is intracellular delivery of Cas9 and sgRNA. DNA and viral vectors risk insertional mutagenesis and prolonged nuclease expression, while mRNA delivery depends on lipid nanoparticles that perform poorly for large ribonucleoprotein complexes (RNPs). Electroporation is efficient but unsuitable for in vivo use. Direct RNP delivery offers the safest and most controllable editing profile, yet achieving efficient uptake—particularly in neuronal cells—remains difficult. Cell-penetrating peptides (CPPs) provide an attractive strategy for protein and RNP delivery. These short peptides traverse cellular membranes and can transport macromolecular cargo, but endosomal escape remains a major limitation. Rational CPP design—incorporating pH-responsive, histidine-rich, hydrophobic, or membrane-disruptive elements—can enhance cytosolic release and improve editing outcomes. Gaucher disease, caused by loss-of-function mutations in GBA1, represents a relevant biomedical context where improved delivery technologies are urgently needed. SH-SY5Y neuroblastoma cells serve as a neuronal model but are notoriously difficult to transfect, making them an ideal system to evaluate peptide-mediated CRISPR delivery. This study develops and characterizes two recombinant CPP-fused Cas9 proteins, PolyE-TAT-NLS-Cas9 (PECas9) and TAT-NLS-Cas9 (TATCas9), engineered for efficient RNP assembly, membrane penetration, and nuclear targeting. The poly-glutamate tag in PECas9 improves solubility, balances TAT’s positive charge, and reduces nonspecific sgRNA binding. Both constructs were cloned, expressed, purified, assembled into RNPs, and tested for genome-editing performance in SH-SY5Y cells. The optimized CPP-Cas9 platform was then used to generate GBA1 knockout SH-SY5Y lines, enabling rapid modeling of Gaucher pathology. Overall, this work establishes a scalable, transient, non-integrating CPP-based RNP delivery system for CRISPR editing in hard-to-transfect neuronal cells, addressing a key translational bottleneck in genome engineering.

목차 (Table of Contents)

• Abstract 1
• Introduction 4
• Materials and Methods 31
• Results 49
• Discussion 114
• Conclusion 120
• References 122
• Acknowledgment 134