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Cas-CLOVER-mediated knockout of STAT1: A novel approach to engineer packaging HEK-293 cell lines used for rAAV production
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  • Peter Andorfer,
  • Carolin-Isabel Kahlig,
  • Doris Pakusic,
  • Robert Pachlinger,
  • Christiane John,
  • Irene Schrenk,
  • Peter Eisenhut,
  • Johannes Lengler,
  • Bernd Innthaler,
  • Lucia Micutkova,
  • Barbara Kraus,
  • Corey Brizzee,
  • Jack Crawford,
  • Juan Antonio Hernandez Bort
Peter Andorfer
Takeda Orth an der Donau
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Carolin-Isabel Kahlig
Takeda Orth an der Donau
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Doris Pakusic
Takeda Orth an der Donau
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Robert Pachlinger
Takeda Orth an der Donau
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Christiane John
Takeda Orth an der Donau
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Irene Schrenk
Takeda Orth an der Donau
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Peter Eisenhut
Takeda Orth an der Donau
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Johannes Lengler
Takeda Orth an der Donau
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Bernd Innthaler
Takeda Orth an der Donau
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Lucia Micutkova
Takeda Orth an der Donau
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Barbara Kraus
Takeda Orth an der Donau
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Corey Brizzee
Demeetra AgBio
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Jack Crawford
Demeetra AgBio
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Juan Antonio Hernandez Bort
Takeda Orth an der Donau

Corresponding Author:[email protected]

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Abstract

In addressing the limitations of CRISPR-Cas9, including off-target effects and high licensing fees for commercial use, Cas-CLOVER, a dimeric gene editing tool activated by two guide RNAs, was recently developed. This study focused on implementing and evaluating Cas-CLOVER in HEK-293 cells used for rAAV production by targeting the STAT1 locus, which is crucial for cell growth regulation and might influence rAAV production yields. Cas-CLOVER demonstrated impressive efficiency in gene editing, achieving over 90% knockout success. Selected HEK-293 STAT1 KO sub-clones were subjected to extensive analytical characterization to assess their genomic stability, crucial for maintaining cell integrity and functionality. Additionally, rAAV9 productivity, Rep protein pattern profile and potency, among others, were assessed. Our study also established a comprehensive analytical workflow to detect and evaluate the gene knockouts generated by this innovative tool, providing a solid groundwork for future research in precise gene editing technologies.
Submitted to Biotechnology Journal
Submission Checks Completed
Assigned to Editor
Reviewer(s) Assigned
08 Jul 2024Review(s) Completed, Editorial Evaluation Pending
09 Jul 2024Reviewer(s) Assigned
30 Jul 2024Editorial Decision: Revise Major
14 Aug 20241st Revision Received
14 Aug 2024Submission Checks Completed
14 Aug 2024Assigned to Editor
14 Aug 2024Review(s) Completed, Editorial Evaluation Pending
14 Aug 2024Reviewer(s) Assigned
20 Aug 2024Editorial Decision: Accept