Systems for in vivo site-directed mutagenesis using oligonucleotides
AssigneeThe United States of America, as represented by the Secretary, Department of Health and Human Services
InventorsFrancesca Storici, Lysle Kevin Lewis, Michael A. Resnick
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Abstract
This disclosure provides several methods to generate nucleic acid mutations in vivo, for instance in such a way that no heterologous sequence is retained after the mutagenesis is complete. The methods employ integrative recombinant oligonucleotides (IROs). Specific examples of the described mutagenesis methods enable site-specific point mutations, deletions, and insertions. Also provided are methods that enable multiple rounds of mutation and random mutagenesis in a localized region. The described methods are applicable to any organism that has a homologous recombination system.
Claims91
A method for introducing a mutation into a target double stranded nucleic acid sequence in a cell, wherein the double stranded nucleic acid sequence comprises a first and a second strand, the method comprising:
- introducing a double-stranded nucleic acid cassette into a target nucleic acid sequence at an insertion point, wherein the cassette is a RE-cassette and comprises
- a first portion homologous to a nucleic acid sequence on a first side of the insertion point;
- a second portion homologous to a second nucleic acid sequence on a second side of the insertion point;
- and
- a nucleic acid sequence encoding a reporter located between the first portion and the second portion;
- transforming the cell with a first oligonucleotide comprising:
- a nucleic acid sequence homologous to one strand (the chosen strand) of the target nucleic acid sequence at a position on the first side of the insertion point;
- and
- a nucleic acid sequence homologous to the same strand of the target nucleic acid sequence at a position on the second side of the insertion point, and comprising at least one nucleotide that differs from the chosen strand of the target nucleic acid sequence;
- and
- selecting for loss of the nucleic acid sequence encoding the reporter gene, wherein loss of the nucleic acid sequence encoding the reporter gene indicates integration of the oligonucleotide sequence comprising the at least one nucleotide that differs from the target nucleic acid sequence.
A method of deleting a target double stranded nucleic acid sequence from within a cell, wherein the target double stranded nucleic acid sequence comprises a first and a second strand, the method comprising
- introducing a double-stranded nucleic acid cassette into a target nucleic acid sequence at an insertion point, wherein the cassette is a RE-cassette and comprises
- a nucleic acid sequence encoding a reporter located between a first portion and a second portion of the RE-cassette;
- transforming the cell with a first oligonucleotide comprising:
- a nucleic acid sequence homologous to the first strand of a nucleic acid 5′ of the nucleic acid of interest;
- and
- a sequence homologous to the first strand of a nucleic acid 3′ of the nucleic acid sequence of interest;
- and
- selecting for loss of the nucleic acid sequence encoding the reporter gene, wherein loss of the nucleic acid sequence encoding the reporter gene from the target double stranded nucleic acid indicates deletion of the target double stranded nucleic acid.
A CORE-cassette construct, a RE-DSB-cassette construct, or a CORE-DSB-cassette construct for use in delitto perfetto mutagenesis.
An integrative recombinant oligonucleotide (IRO) for use in delitto perfetto mutagenesis.
A kit for carrying out in vivo mutagenesis or deletion of a nucleic acid sequence, comprising an amount of a CORE-cassette construct, a RE-DSB-cassette construct, or a CORE-DSB-cassette construct, and an amount of an integrative recombinant oligonucleotide.
Dependencies are inferred from the claim text.