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Conquering gene editing off-targets with Gibco™ CTS™ HiFi Cas9 Protein

D Papp(1) E De Meulenaere(1) J Potter(1) T Tang(1) I Pringle(1)

1:Thermo Fisher Scientific

Gibco™ CTS™ HiFi Cas9 Protein is the latest addition to Thermo Scientific's CRISPR/Cas9 portfolio. With this new product, we are now offering researchers working in a clinical environment a low off-target solution for their genome editing projects. It is designed and manufactured in compliance with GMP principles and to meet requirements of standards for Ancillary Materials for Cell, Gene, and Tissue-Based Products, including USP <1043>, Ph.Eur. 5.2.12, and ISO 20399.

A panel of sgRNAs targeting therapeutically relevant genes (TRAC, CD52, TRBC and B2M) was used to compare genome-wide off-target and on-target activities of CTS™ HiFi Cas9, wild-type CTS™ Cas9, and Supplier A GMP hi-fidelity Cas9. Cas9/gRNA RNP complexes were delivered to T cells or iPSC by electroporation (EP). Cells were harvested on day 3 or 11 post EP and analyzed using Attune flow cytometer for protein function or next generation sequencing (NGS) for Indel, and HDR. TEG-seq (PMID 30114933 & 37161298) was used for genome wide off-target discovery. TAV-seq was used for off-target validation, on-target editing efficiency.

At tested loci, CTS™ HiFi Cas9 achieved equivalent on target activity compared to wild-type CTS™ Cas9. TCR KO and CAR KI efficiency is as high as wild-type CTS™ Cas9. However, with CTS™ HiFi Cas9 over 90% reduction in off-target effects in primary immune cells was observed, compared to CTS TrueCut Cas9 (wild type) at tested loci. CTS™ HiFi Cas9 is supported by extensive traceability documentation—including COA, COO, Drug Master Files or Regulatory Support Files when required.

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