Nucleic acid constructs comprising gene editing multi-sites and uses thereof

Inventors

POPMA, Sicco Hans • Zhang, Di

Assignees

Io Biosciences Inc

Abstract

Disclosed herein is a polynucleotide construct comprising one or more primary endonuclease recognition sequences upstream and downstream of a multiple gene editing site that comprises a plurality of secondary endonuclease recognition sequences. The primary endonuclease recognition sequences facilitate insertion of the multiple gene editing site into a host cell genome. The secondary endonuclease recognition sequences facilitate insertion of one or more exogenous donor genes into the host cell.

Core Innovation

The document describes engineered gene editing multi-site (GEMS) polynucleotide constructs for insertion into a genome at an insertion site. Each GEMS polynucleotide sequence comprises a plurality of nuclease recognition sequences, and at least one nuclease recognition sequence comprises the nucleotide sequence of SEQ ID NO: 14. The GEMS polynucleotide sequence is configured to create a multi-edit region, and guide polynucleotide biology guides a nuclease to the nuclease recognition sequences during insertion.

The disclosure further describes secondary nuclease recognition sites linked to guide target sequences and protospacer adjacent motifs (PAMs), with polynucleotide spacers separating adjacent nuclease recognition sequences. Upstream and downstream flanking insertion sequences homologous to genome sequences are included relative to the insertion site. The design supports multiple nuclease options including Cas9, Cpf1, meganucleases such as I-Scel, ZFN, TALEN, Argonaute, and PfAgo.

The disclosed GEMS approach is implemented in an isolated host cell in which a GEMS polynucleotide sequence is present in the host cell genome, and donor nucleic acid sequences can be inserted within the GEMS polynucleotide sequence. The document further describes engineered GEMS polynucleotide sequences defined by at least 80% sequence identity with SEQ ID NO: 81 or SEQ ID NO: 83, and host cells using an engineered GEMS polynucleotide construct with a nucleic acid vector and a nuclease configured to recognize one or more nuclease recognition sequences via binding to a guide polynucleotide.

Claims Coverage

The independent claims cover four inventive features centered on engineered GEMS constructs and isolated host cells defined by nuclease recognition sequence content or sequence identity. The claims cover constructs and cells with at least one nuclease recognition sequence comprising SEQ ID NO: 14, as well as constructs and cells with at least 80% sequence identity to SEQ ID NO: 81 or SEQ ID NO: 83.

Overall, the claims cover engineered GEMS polynucleotide constructs and isolated host cells defined by a plurality of nuclease recognition sequences with at least one recognition sequence comprising SEQ ID NO: 14, or by a GEMS polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 81 or SEQ ID NO: 83.

Stated Advantages

Documented Applications