CLUSTER guide RNAs enable precise and efficient RNA editing with endogenous ADAR enzymes in vivo

Philipp Reautschnig1, Nicolai Wahn2, Jacqueline Wettengel1, Annika E Schulz1, Ngadhnjim Latifi1, Paul Vogel3, Tae-Won Kang4,5, Laura S Pfeiffer1, Christine Zarges1, Ulrike Naumann6, Lars Zender4,5,7, Jin Billy Li3, Thorsten Stafforst8

  1. Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany.
  2. , Tübingen, Germany.
  3. Department of Genetics, Stanford University, Stanford, CA, USA.
  4. Department of Medical Oncology and Pneumology (Internal Medicine VIII), University Hospital Tübingen, Tübingen, Germany.
  5. German Cancer Research Consortium, Partner Site Tübingen, German Cancer Research Center, Heidelberg, Germany.
  6. Hertie Institute for Clinical Brain Research, Center of Neurology, University Hospital Tübingen, University of Tübingen, Tübingen, Germany.
  7. DFG Cluster of Excellence 2180 'Image-guided and Functional Instructed Tumor Therapy', University of Tübingen, Tübingen, Germany.
  8. Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany. thorsten.stafforst@uni-tuebingen.de.

Abstract

RNA base editing represents a promising alternative to genome editing. Recent approaches harness the endogenous RNA-editing enzyme adenosine deaminase acting on RNA (ADAR) to circumvent problems caused by ectopic expression of engineered editing enzymes, but suffer from sequence restriction, lack of efficiency and bystander editing. Here we present in silico-optimized CLUSTER guide RNAs that bind their target messenger RNAs in a multivalent fashion, achieve editing with high precision and efficiency and enable targeting of sequences that were not accessible using previous gRNA designs. CLUSTER gRNAs can be genetically encoded and delivered using viruses, and are active in a wide range of cell lines. In cell culture, CLUSTER gRNAs achieve on-target editing of endogenous transcripts with yields of up to 45% without bystander editing. In vivo, CLUSTER gRNAs delivered to mouse liver by hydrodynamic tail vein injection edited reporter constructs at rates of up to 10%. The CLUSTER approach opens avenues for drug development in the field of RNA base editing.

Presented By Philipp Reautschnig | ORCID iD

Biotechnology, Biochemistry, Genetics & Genome BiologyNicholas Liao2022-01 (Jan)