MULTIPLEX PRODUCTION AND BARCODING OF GENETICALLY ENGINEERED CELLS

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United States of America Patent

APP PUB NO 20200270632A1
SERIAL NO

16646999

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Abstract

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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.

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Patent Owner(s)

Patent OwnerAddress
BRANDEIS UNIVERSITY415 SOUTH STREET WALTHAM MA 02454

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Inventor(s)

Inventor Name Address # of filed Patents Total Citations
Haber, James E Waltham, US 2 31
Roy, Kevin Mountain View, US 3 31
Smith, Justin D Redwood City, US 8 85
St, Onge Robert P San Francisco, US 3 32
Steinmetz, Lars M Stanford, US 4 31

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