Multiplex production and barcoding of genetically engineered cells
Inventors
Roy, Kevin • Smith, Justin D. • St. Onge, Robert P. • Steinmetz, Lars M. • Haber, James E.
Assignees
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Publication Number
US-12416015-B2
Publication Date
2025-09-16
Expiration Date
Abstract
The present disclosure relates to multiplex production and phenotyping of genetically engineered cells using RNA-guided nucleases and genomic barcoding. In particular, high-throughput multiplex genome editing is achieved utilizing a system that facilitates precise genome editing at desired target chromosomal loci by homology directed repair. Integration of guide RNA and donor DNA sequences as a genomic barcode at a separate chromosomal locus allows identification, isolation, and massively-parallel validation of individual variants from a pool of transformants. Strains can be arrayed according to their precise genetic modifications, as specified by donor DNA incorporation in heterologous or native genes. The present disclosure further relates to a method of editing codons outside of canonical guide RNA recognition regions, which enables complete saturation mutagenesis of protein-coding genes, a marker-based internal cloning method, which removes background due to oligonucleotide synthesis errors and incomplete vector backbone cleavage, and a method of enhancing homology directed repair by active donor recruitment.
Core Innovation
The invention relates to localizing a donor polynucleotide to a genomic target locus in a target cell for genome editing. A genome editing cassette is delivered that includes a promoter operably linked to a nucleic acid sequence encoding a guide RNA capable of hybridizing at the genomic target locus, and a donor polynucleotide comprising a sequence to be integrated into the genomic target locus.
In addition to the genome editing cassette, a second recombinant polynucleotide is delivered that encodes an RNA-guided nuclease and a donor recruitment protein. The RNA-guided nuclease, when complexed with the guide RNA, recognizes and creates a DNA break at the genomic target locus, while the donor recruitment protein binds both the donor polynucleotide via a DNA binding domain and a region near the DNA break via a DNA break site localizing domain.
The donor recruitment protein selectively recruits the donor polynucleotide to the DNA break site, thereby localizing the donor polynucleotide to the genomic target locus. The recruitment protein architecture includes a LexA DNA binding domain with a forkhead-associated (FHA) phosphothreonine-binding domain, or includes a TP53BP1 domain, and a unique polynucleotide barcode is incorporated either in the genome editing cassette or within the donor polynucleotide.
The disclosure further incorporates unique polynucleotide barcodes and a chromosomal barcode locus to enable identification, isolation, and massively-parallel validation/phenotyping of individual variants from pooled transformants. It also describes ordered array workflows using recombinase-directed indexing and chromosomal barcode integration locus approaches, along with sequencing-based linkage between barcodes and guide-donor cassettes.
Claims Coverage
The independent claims identified in the provided material are clm-00001, clm-00014, and clm-00015. Across these claims, the inventive coverage centers on donor localization to a genomic target locus using an RNA-guided nuclease together with a donor recruitment protein that binds both the donor polynucleotide and a region near a DNA break site, with additional barcode and donor-recruitment constraints.
Selective donor recruitment to the dna break site for genomic integration
A method for localizing a donor polynucleotide to a genomic target locus in a target cell by transfecting a target cell with a genome editing cassette comprising a promoter operably linked to a nucleic acid sequence encoding a guide RNA and a donor polynucleotide to be integrated, and transfecting with an RNA-guided nuclease that creates a DNA break at the genomic target locus and a donor recruitment protein that binds the donor polynucleotide via a DNA binding domain and binds a region near the DNA break via a DNA break site localizing domain, so that the donor recruitment protein selectively recruits the donor polynucleotide to the DNA break site to localize it to the genomic target locus.
LexA dna binding with FHA phosphothreonine-binding or TP53BP1 break-site localization architecture
The method of localizing the donor polynucleotide further requires that the DNA binding domain comprises a LexA DNA binding domain and the DNA break site localizing domain comprises a forkhead-associated (FHA) phosphothreonine-binding domain or comprises a TP53BP1 domain.
Unique polynucleotide barcode for donor localization
The method further requires that the genome editing cassette comprises a unique polynucleotide barcode or the donor polynucleotide comprises a unique polynucleotide barcode.
Circular genome editing cassette with second gRNA (guide X)
The method further requires that the first recombinant polynucleotide is circular and comprises a second nucleic acid sequence encoding a second gRNA, wherein the guide X forms a complex with a nuclease in each cell such that the guide X-nuclease complex cleaves the first recombinant polynucleotide.
Across the independent claims, the coverage is directed to donor localization for genomic integration using an RNA-guided nuclease that creates a DNA break together with a donor recruitment protein having donor-binding and break-site localizing domains. The claims further specify a LexA DNA binding domain with an FHA phosphothreonine-binding domain or a TP53BP1 domain, a unique polynucleotide barcode in either the genome editing cassette or the donor polynucleotide, and a circular donor-containing polynucleotide with a second gRNA that cleaves the circular polynucleotide in each cell.
Stated Advantages
Identification, isolation, and massively-parallel validation/phenotyping of individual variants from pooled transformants.
Improving homology-directed repair via active donor recruitment.
Optional inhibition of non-homologous end joining via chemical or CRISPRi/RNAi.
Documented Applications
Identification, isolation, and massively-parallel validation/phenotyping of individual variants from pooled transformants.
Ordered array workflows using recombinase-directed indexing.
Chromosomal barcode integration locus approaches.
Sequencing-based linkage between barcodes and guide-donor cassettes.
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