# An improved CRISPR-Cas9 protein-based method for knocking out insect Sf9 cell genes

## TL;DR

Researchers developed a more efficient CRISPR-Cas9 method to edit genes in insect Sf9 cells, improving their use in biopharmaceutical production.

## Contribution

A novel CRISPR-Cas9 RNP delivery pipeline for insect cells was implemented, achieving higher knockout efficiency than previous methods.

## Key findings

- A 68% knockout rate was achieved using the new CRISPR-Cas9 pipeline in insect Sf9 cells.
- Sf-Dronc deletion increased apoptosis resistance and delayed cell viability loss during baculovirus infection.
- Sf-Dronc deletion led to over a twofold increase in influenza VLP production compared to wild-type cells.

## Abstract

Insect cells are one of the uprising expression systems in the biopharmaceutical industry to produce vaccines and gene therapy vectors, but cell line development has been limited by the lack of established genetic engineering tools and genomic characterization. CRISPR-Cas9 has arisen as a powerful tool for gene editing but has seen little application in insect cells. In this work, a gene editing pipeline for the delivery of a ribonucleoprotein (RNP) complex comprised of a guide RNA and the enzyme Cas9 to insect Sf9 cells was implemented and then applied to knockout caspase initiator Sf-Dronc, aiming at alleviating cell apoptosis during an infection process. The resulting engineered cell lines were characterized as per their phenotype and production of three different product modalities. Utilizing the established workflow, a knockout rate of 68% was achieved with the implemented protocol (vs. the 12% presumed efficiency of a previously reported system) when targeting the fdl gene. When applied to Sf-Dronc, mutants containing deletions in several alleles of the host genome were identified and confirmed by next-generation sequencing. Generated clones exhibited higher apoptosis resistance and delayed onset of cell viability drop following infection with baculovirus. While Sf-Dronc deletion was shown to have negligible impact on the production of rAAV and PfRipr5, production of iVLPS showed an > twofold increase over wild-type Sf9. Overall, this study showcases the successful implementation of an efficient CRISPR-Cas9 pipeline, further leveraging the usage of genetic engineering in insect Sf9 cells towards the development of enhanced cell hosts for biopharmaceutical production.

• Implementation of an efficient CRISPR-Cas9 RNP complex delivery strategy to insect cells.

• Establishment of the genome editing pipeline demonstrated through Sf-Dronc knockout, resulting in increased apoptosis resistance and delayed loss of viability upon baculovirus infection.

• Sf-Dronc deletion led to over a twofold increase in the production of influenza VLPs compared to wild-type Sf9 cells.

The online version contains supplementary material available at 10.1007/s00253-026-13722-3.