Compositions and methods for genomic integration of nucleic acids into exogenous landing pads

Inventors

George, Kevin • Main, Andrew • Tsai, Chia-Hong

Assignees

Amyris Inc

Abstract

Provided herein are compositions and methods of integrating one or more exogenous donor nucleic acids into one or more exogenous landing pads engineered into a host cell's genome. In certain embodiments, the exogenous landing pads and exogenous donor nucleic acids comprise standardized, compatible homology regions so that exogenous donor nucleic acids can integrate into any of the landing pads, independent of the genomic sequences surrounding the landing pads. In certain embodiments, the methods comprise contacting the host cell comprising landing pads with one or more exogenous donor nucleic acids, and a nuclease capable of causing a double-strand break within the landing pads, and recovering a host cell comprising one or more exogenous donor nucleic acids integrated in any of the landing pads.

Core Innovation

The invention relates to a method for integrating one or more exogenous donor nucleic acids into a yeast host cell genome using one or more engineered exogenous landing pads. Each exogenous landing pad comprises a nuclease target sequence (NTS) positioned between an upstream landing pad homology sequence (ULP) and a downstream landing pad homology sequence (DLP), and donor integration is independent of genomic sequences surrounding each landing pad.

The exogenous donor nucleic acids include a nucleic acid of interest positioned between an upstream library sequence (UL) and a downstream library sequence (DL). Each UL is capable of homologously recombining at any ULP, and each DL is capable of homologously recombining at any DLP of any of the one or more exogenous landing pads, so any exogenous donor nucleic acid is integrated at any exogenous landing pad in an arrangement independent of genomic sequences surrounding each landing pad.

The system supports rapid combinatorial integration, pooled libraries, markerless integration with enrichment and barcode-based genotype-phenotype tracking, and optional secondary landing pads for targeted swapping of promoter, terminator, and degron components at native loci. In the disclosed examples, the platform is used for isoprenoid (MEV) pathway engineering and optimized biosynthetic pathway components.

Claims Coverage

One independent claim is identified (clm-00001), and it defines the main landing-pad and donor-library recombination architecture for yeast genome integration. Dependent claims refine the system by specifying locus types, homology constraints, host cell composition, and secondary landing pad architectures.

Claim clm-00001 covers a yeast integration method based on standardized exogenous landing pads defined by NTS between ULP and DLP, together with donor nucleic acids defined by UL and DL that recombine with any ULP and any DLP across the landing pads. It further requires that donor integration at any landing pad is independent of genomic sequences surrounding each landing pad, and that the landing pads are selected from SEQ ID NOs: 8, 9, and 10.

Stated Advantages

Documented Applications