The present disclosure relates to the
multiplex production and phenotyping of
genetically engineered cells by using
RNA-guided nucleases and genomic barcoding. In particular, the high-
throughput multiplex genome editing is achieved by utilizing a
system that facilitates the precise
genome editing at desired target chromosomal loci by
homology directed repair. The integration of
guide RNA and donorDNA 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 canbe 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 the 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 the active donor recruitment.