· Methods in molecular biology· Vol 3075, pp.
311-324
· 0 citations
Medicine
TL;DR
A protocol for generating VSV-G-pseudotyped VLPs that deliver CRISPR/Cas9 RNPs and their functional validation in surrogate cell lines is described and their functional validation in surrogate cell lines is validated.
Abstract
Virus-like particles (VLPs) are self-assembling viral protein complexes that mimic native virions while lacking replicative and infectious capacity. By retaining efficient cellular entry and cargo protection, they overcome key limitations of viral vectors, including insertional mutagenesis and strong immunogenicity. Their ease of production, flexible cargo capacity, and tunable tropism make them highly versatile tools. Here, we describe a protocol for generating VSV-G-pseudotyped VLPs that deliver CRISPR/Cas9 RNPs and their functional validation in surrogate cell lines.
It is revealed that a wide variety of mutation types are possible with the base editors in this VLP collection, revealing that a wide variety of mutation types are possible with the base editors in this collection.
Asfar Lathif Salaudeen, Trevor Shyiak, Carl G. de Boer· bioRxiv· 0 citations
This chapter detail a methodology using a rAAV vector carrying Staphylococcus aureus Cas9 (SaCas9) to target the murine Hao1 gene in liver parenchymal cells as a curative treatment for primary hyperoxaluria type 1 (PH1).
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Cell-penetrating peptides (CPPs) provide a modular nonviral strategy for intracellular delivery of CRISPR/Cas9 components, particularly preassembled Cas9 ribonucleoprotein (RNP) complexes. This chapter summarizes key principles of CPP-mediated Cas9 RNP delivery, including noncovalent complexation and covalent CPP-Cas9...
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This chapter emphasizes that the strategic selection of a delivery vehicle must be harmonized with the specific cargo and the unique biological requirements of the target tissue to ensure therapeutic success.
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Extracellular vesicles (EVs), particles released from cells, have the potential to become an important in vivo delivery vehicle for CRISPR machinery. Unlike viral vectors such as Adeno-associated virus (AAV), EVs reduce risks associated with immunogenicity, long-term expression, and potential off-target effects. EVs ca...
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This protocol enables quantification of INDELs and DNA-SVs, allowing for validation of gene disruption, and describes guide RNA (gRNA) design, plasmid cloning, delivery of AAV-CRISPR to the liver, and execution of liver DNA-editing analysis.
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