RNA-guided programmable cytosine and adenine base editors are a powerful class of genome editing tool for the introduction of localized base transitions without generating a double-stranded DNA break. Base editors (BE) have an optimal window of activity relative to the PAM recognized by the Cas9 enzyme and these constructs are strand selective. Here we demonstrate that fusion of a programmable DNA-binding domain (pDBD) or another Cas9 orthologue to spCas9-BE, we can produce an RNA-programmable Cas9-BE-pDBD chimera or Cas9-BE-Cas9 chimeras with dramatically improved activities and increased targeting range. Cas9-pDBD or Cas9-Cas9 fusion base editors display an expanded targeting repertoire and achieve highly specific genome editing, which can be tailored to achieve extremely precise genome editing at nearly any genomic locus.

Disclosed herein are methods and compositions for identification of specific DNA binding domains for constructing highly specific nucleases, which allows for pristine genome editing.

The present disclosure provides zinc finger fusion proteins that inhibit expression of the prion gene in the nervous system, and methods of using the proteins to treat prion disease.

The present disclosure relates to base-editing systems including a fusion protein including a DNA-binding domain and a cytidine deaminase domain and a non-protein uracil-DNA glycosylase inhibitor, and methods of using the same. The DNA-binding domains of base-editing systems of the present disclosure include domains with a variety of target region possibilities, which increase the number and type of sequences that can be edited. The npUGIs of the base-editing systems of the present disclosure improve UDG inhibition (e.g., UDG inhibition is more complete) and are suitable for use in a wide range of organisms.

Compositions and methods comprising novel deaminase polypeptides for targeted editing of nucleic acids are provided. Compositions comprise deaminase polypeptides. Also provided are fusion proteins comprising a DNA-binding polypeptide and a deaminase of the invention. The fusion proteins include RNA-guided nucleases fused to deaminases, optionally in complex with guide RNAs. Compositions also include nucleic acid molecules encoding the deaminases or the fusion proteins. Vectors and host cells comprising the nucleic acid molecules encoding the deaminases or the fusion proteins are also provided.

The present invention relates to the identification of a genomic integration site for heterologous polynucleotides in Chinese Hamster Ovary (CHO) cells resulting in high RNA and/or protein production. More specifically it relates to CHO cells comprising at least one heterologous polynucleotide stably integrated into the S100A gene cluster of the CHO genome and to methods for the production of said CHO cells. Further, the invention relates to a method for the production of a protein of interest using said CHO cell and to the use of said CHO cell for producing a protein of interest at high yield. Integration within these specific target regions leads to reliable, stable and high yielding production of an RNA and/or protein of interest, encoded by the heterologous polynucleotide.

Provided are various embodiments relating to compositions and methods for treating vascular disease, including core NOX1 promoters and variants thereof for regulating expression of transgenes in response to vascular pathology and allowing for increased transgene loading capacity. Also provided are variant FOXP polypeptides having a zinc finger and leucine zipper region of a different FOXP polypeptide. Further provided are vectors comprising the core NOX1 promoters and/or a coding sequence for variant FOXP polypeptides described herein and optionally coding sequence(s) for one or more additional therapeutic polypeptide(s), such as IL10, for treating inflammation-associated diseases, such as vascular disease. Also provided is a screening model for testing therapeutic agents capable of treating established and ongoing atherosclerotic pathology.

Described herein are lipid nanoparticles comprising cationic lipids and other lipids and also comprising engineered nucleases facilitate transfer of nucleic acids to cells.

Provided herein are systems, compositions, and methods for the incorporation of unnatural amino acids into proteins via nonsense suppression or rare codon suppression. Nonsense codons and rare codons may be introduced into the coding sequence of a protein of interest using a CRISPR/Cas9-based nucleobase editor described herein. The nucleobase editors are able to be programmed by guide nucleotide sequences to edit the target codons in the coding sequence of the protein of interest. Also provided are application enabled by the technology described herein.

Described herein are methods and compositions for genomic editing. Endonucleases for genomic editing involve inducing breaks in double stranded DNA, for which knock-ins are notoriously inefficient for relying on random integration of homologous DNA sequences into the break site by repair proteins. To address these issues, described herein are novel recombinant fusion proteins that actively recruit linear DNA inserts in closer proximity to the genomic cleavage site, increasing integration efficiency of large DNA fragments into the genome. Such improvements to genomic editing technology allow one to use lower linear DNA concentrations without sacrificing efficiency and can be further combined with other features, such as fluorescent protein reporting systems.