Biology subjects

Villiger, L.

Publications and source records attributed to Villiger, L..

3 recordsLinked to original sources

bioRxiv2021

Treatment of a metabolic liver disease by in vivo prime editing in mice

Prime editing is a highly versatile CRISPR-based genome editing technology with the potential to correct the vast majority of pathogenic mutations (1). However, correction of a disease phenotype in vivo in somatic tissues has not been demonstrated thus far. Here, we establish proof-of-concept for in vivo prime editing and repair the metabolic liver disease phenylketonuria (PKU) in mice. We first developed a size-reduced SpCas9 prime editor (PE) lacking the RNaseH domain of the reverse transcriptase (PE2{Delta}RnH), and a linker- and NLS-optimized intein-split PE construct (PE2 p.1153) for delivery by adeno-associated virus (AAV) vectors. Systemic dual AAV-mediated delivery of this variant into the liver of neonatal mice enabled installation of a transversion mutation at the Dnmt1 locus with an average efficiency of 15%, and delivery of unsplit PE2{Delta}RnH using human adenoviral vector 5 (AdV5) further increased editing rates to 58%. PE2{Delta}RnH-encoding AdV5 was also used to correct the disease-causing mutation of the phenylalanine hydroxylase (Pah)enu2 allele in phenylketonuria (PKU) mice with an average efficiency of 8% (up to 17.3%), leading to therapeutic reduction of blood phenylalanine (L-Phe) levels. Our study demonstrates in vivo prime editing in the liver with high precision and editing rates sufficient to treat a number of metabolic liver diseases, emphasizing the potential of prime editing for future therapeutic applications. One Sentence SummaryIn vivo prime editing corrects phenylketonuria in mice.

genetics↗

bioRxiv2020

Replacing the SpCas9 HNH domain by deaminases generates compact base editors with an alternative targeting scope

Base editors are RNA-guided deaminases that enable site-specific nucleotide transitions. The targeting scope of these Cas-deaminase fusion proteins critically depends on the availability of a protospacer adjacent motif (PAM) at the target locus and is limited to a window within the CRISPR-Cas R-loop, where single stranded (ss)DNA is accessible to the deaminase. Here, we reason that the Cas9-HNH nuclease domain sterically constrains ssDNA accessibility, and demonstrate that omission of this domain expands the editing window. By exchanging the HNH nuclease domain with an adenosine deaminase we furthermore engineer adenine base editor variants (HNHx-ABE) with PAM-proximally shifted editing windows. This work expands the targeting scope of base editors, and provides base editor variants that are substantially smaller. It moreover informs of potential future directions in Cas9 protein engineering, where the HNH domain could be replaced by other enzymes that act on ssDNA.

bioengineering↗

bioRxiv2020

Predicting base editing outcomes with an attention-based deep learning algorithm trained on high-throughput target library screens

Base editors are chimeric ribonucleoprotein complexes consisting of a DNA-targeting CRISPR-Cas module and a single-stranded DNA deaminase. They enable conversion of C•G into T•A base pairs and vice versa on genomic DNA. While base editors have vast potential as genome editing tools for basic research and gene therapy, their application has been hampered by a broad variation in editing efficiencies on different genomic loci. Here we perform an extensive analysis of adenine- and cytosine base editors on thousands of lentivirally integrated genetic sequences and establish BE-DICT, an attention-based deep learning algorithm capable of predicting base editing outcomes with high accuracy. BE-DICT is a versatile tool that in principle can be trained on any novel base editor variant, facilitating the application of base editing for research and therapy.Competing Interest StatementThe authors have declared no competing interest.View Full Text

genomics↗