US 2023/0340539 A1Published application

Methods of transient protein and gene expression in cells

OwnerColin Scott MAXWELL, Solomon Henry STONEBLOOM +1inventors

InventorsColin Scott MAXWELL, Solomon Henry STONEBLOOM, Shawn SZYJKA

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Abstract

The present disclosure provides methods for producing gene-edited cells free of gene-editing system molecules through the manipulation of prototrophy. Exemplary system molecules include those required for CRISPR editing techniques, such as plasmids and genes encoding Cas nucleases. The methods may employ constructs that temporarily disrupt prototrophy, the removal of which restores prototrophy. Also disclosed are gene-edited cells and populations of gene-edited cells comprising these constructs. The present methods and compositions may be used to achieve desired gene editing of a host cell in the absence of extraneous genetic material remaining from the genetic engineering technique itself.

A method for producing a population of gene-edited cells free of gene-editing system molecules, comprising:

(a) introducing an integrating nucleic acid construct into a population of cells that comprise a target gene of interest and that are prototrophic for a nutrient,
wherein the integrating nucleic acid construct integrates into a gene that is required for prototrophy for the nutrient;
and
wherein the integrating nucleic acid construct comprises:
a first nucleotide sequence encoding a gene-editing protein;
a second nucleotide sequence encoding a dominant selectable marker;
and
a pair of repeat nucleotide sequences flanking the first nucleotide sequence and the second nucleotide sequence;
(b) selecting for expression of the dominant selectable marker to produce a population of cells that are auxotrophic for the nutrient;
(c) introducing a non-integrating nucleic acid construct into the population of cells produced in step (b);
wherein the non-integrating nucleic acid construct comprises:
a third nucleotide sequence encoding a gene-editing nucleic acid that introduces an edit into the gene of interest;
and
a fourth nucleotide sequence encoding a protein that complements the auxotrophy for the nutrient, wherein the fourth nucleotide sequence cannot recombine with the cellular genome;
(d) simultaneously selecting for expression of the dominant selectable marker and for prototrophy for the nutrient to produce a population of cells that comprise the edited gene of interest;
(e) removing the non-integrating nucleic acid nucleic acid construct from the population of cells produced in step (d) by growing the cells on media that selects against expression of the protein that complements the auxotrophs for the nutrient to produce a population of cells that comprise the edited gene of interest and are free of the non-integrating nucleic acid construct;
and
(f) removing the integrating nucleic acid construct from the population of cells produced in step (e) by growing the cells on media that selects for prototrophy for the nutrient to produce a population of cells that comprise the edited gene of interest and that are free of the integrating nucleic acid construct.

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Abstract

Claims21