Back to results
US 2020/0370203 A1Published application
Construction and methods of use of a barcoded and gene edited dna library
AssigneeThe Regents of the University of Colorado, a body corporate
InventorsKenneth Felsenstein, Dan Theodorescu, Ryan T. Gill
Life of this patent
Estimating the term…
Abstract
Provided herein, in some embodiments, are methods and compositions for gene editing using a trackable gene library, for example, a barcoded and gene edited library.
Claims22
A bacterial cell composition comprising:
- (a) a recipient plasmid comprising:
- (i) a selection marker;
- (ii) a barcode landing sequence flanked by a region that is complementary to a portion of a barcode sequence in donor plasmid of (b);
- and
- (iii) a target nucleic acid sequence that encodes a gene of interest and is flanked by a region that is complementary to a portion of a modified target nucleic acid sequence;
- and
- (b) a donor plasmid comprising:
- (i) a selection marker and a counter selection marker, wherein the selection marker and the counter selection marker are not the same;
- (ii) a first guide nucleic acid sequence comprising a region that is complementary to a portion of the barcode landing sequence of (a)(ii), wherein the first guide nucleic acid sequence is operably linked to a constitutively active promoter;
- (iii) the barcode sequence;
- and
- (iv) an editing cassette comprising the modified target nucleic acid sequence linked to a mutated protospacer adjacent motif (mutated PAM) and a second guide nucleic acid sequence comprising a region that is complementary to a portion of the editing cassette, wherein the second guide nucleic acid sequence is operably linked to an inducible promoter;
- (c) a nucleic acid-guided DNA binding protein;
- and
- (d) a recombination system.
12 more elements
A composition comprising:
- (a) a recipient plasmid comprising:
- (i) a selection marker;
- (ii) a barcode landing sequence flanked by a region that is complementary to a portion of a barcode sequence;
- and
- (iii) a target nucleic acid sequence that encodes a gene of interest and is flanked by a region that is complementary to a portion of a modified target nucleic acid sequence;
- and
- (b) a donor plasmid comprising:
- (i) a selection marker and a counter selection marker wherein the selection marker and the counter selection marker are not the same;
- (ii) a first guide nucleic acid sequence comprising a region that is complementary to a portion of the barcode landing sequence, wherein the first guide nucleic acid sequence is operably linked to a constitutively active promoter;
- (iii) the barcode sequence;
- and
- (iv) an editing cassette comprising the modified target nucleic acid sequence linked to a mutated protospacer adjacent motif (mutated PAM) and a second guide nucleic acid sequence comprising a region that is complementary to a portion of the editing cassette, wherein the second guide nucleic acid sequence is operably linked to an inducible promoter.
9 more elements
A method of gene editing, the method comprising:
- combining a population of cells comprising a nucleic acid-guided DNA binding protein and a recombination system with:
- (a) a recipient plasmid comprising:
- (i) a selection marker;
- (ii) a barcode landing sequence flanked by a region that is complementary to a portion of a barcode sequence;
- and
- (iii) a target nucleic acid sequence that encodes a gene of interest and is flanked by a region that is complementary to a portion of a modified target nucleic acid sequence;
- and
- (b) a donor plasmid comprising:
- (i) a selection marker and a counter selection marker, wherein the selection marker and the counter selection marker are not the same;
- (ii) a first guide nucleic acid sequence comprising a region that is complementary to a portion of the barcode landing sequence, wherein the first guide nucleic acid sequence is operably linked to a constitutively active promoter;
- (iii) the barcode sequence;
- and
- (iv) an editing cassette comprising the modified target nucleic acid sequence linked to a mutated protospacer adjacent motif (mutated PAM) and a second guide nucleic acid sequence comprising a region that is complementary to a portion of the editing cassette, wherein the second guide nucleic acid sequence is operably linked to an inducible promoter, thereby producing a combination;
- maintaining the combination under conditions under which the first guide nucleic acid sequence and the nucleic acid-guided DNA binding protein create a barcode edit in the barcode landing sequence;
- selecting for a population of cells comprising the recipient plasmid and the donor plasmid, thereby producing a selected population of cells;
- maintaining the selected population of cells under conditions under which expression of the second nucleic acid sequence is induced and the expressed second guide nucleic acid sequence and the nucleic acid-guided DNA binding protein create a gene edit in the target nucleic acid sequence;
- and
- counter selecting for a population of cells comprising the barcode edit in the barcode landing sequence and the gene edit in the target nucleic acid sequence.
15 more elements
A bacterial cell composition comprising:
- (a) a recipient plasmid comprising:
- (i) a selection marker;
- (ii) a barcode landing sequence flanked by a region that is complementary to a portion of a barcode sequence in donor plasmid of (b);
- and
- (iii) a target nucleic acid sequence that encodes a gene of interest and is flanked by a region that is complementary to a portion of a modified target nucleic acid sequence;
- and
- (b) a donor plasmid comprising:
- (i) a selection marker and a counter selection marker, wherein the selection marker and the counter selection marker are not the same;
- (ii) a first guide nucleic acid sequence comprising a region that is complementary to a portion of the barcode landing sequence of (au), wherein the first guide nucleic acid sequence is operably linked to a constitutively active promoter;
- (iii) the barcode sequence;
- and
- (iv) an editing cassette comprising the modified target nucleic acid sequence linked to a mutated protospacer adjacent motif (mutated PAM) and a second guide nucleic acid sequence comprising a region that is complementary to a portion of the editing cassette, wherein the second guide nucleic acid sequence is operably linked to an inducible promoter.
9 more elements
Dependencies are inferred from the claim text.