Systems and methods for modulating chromosomal rearrangements
AssigneeEditas Medicine, Inc.
InventorsCecilia Cotta-Ramusino, Anne Helen Bothmer
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Abstract
The present disclosure provides systems and methods for modulating the occurrence of chromosomal rearrangements, e.g., translocations, in a cell during genome editing. Embodiments are provided for reducing the occurrence of unwanted chromosomal rearrangements, and for increasing the occurrence of desired chromosomal rearrangements.
Claims18
A method of altering a cell at two target nucleic acids in the cell, the method comprising the step of delivering to the cell two ribonucleoprotein (RNP) complexes, wherein a first RNP complex comprises a SpCas9 RNA-guided nuclease, and wherein a second RNP complex comprises an Acidaminococcus sp. Cpf1 nuclease, thereby altering the cell at the two target nucleic acids.
A method of reducing the risk of translocations in a cell when the cell is altered at two target nucleic acids, the method comprising delivering to the cell two RNP complexes, wherein a first RNP complex comprises a SpCas9 RNA-guided nuclease, and wherein a second RNP complex comprises an Acidaminococcus sp. Cpf1 nuclease, thereby reducing the risk of translocations in the cell.
A method of altering a cell at a first target nucleic acid and a second target nucleic acid, comprising the steps of:
- forming at least one single- or double-stranded break at a first cleavage site in the first target nucleic acid by delivering to the cell a ribonucleoprotein (RNP) complex comprising a first RNA-guided nuclease and a first guide RNA (gRNA) capable of directing the first RNA-guided nuclease to the first target nucleic acid, wherein the first cleavage site is repaired by at least one DNA repair pathway to produce an altered first target nucleic acid;
- and
- forming at least one single- or double-stranded break at a second cleavage site in the second target nucleic acid by delivering to the cell a second RNA-guided nuclease expressed in the cell from an exogenous nucleic acid encoding the second RNA-guided nuclease, wherein the second cleavage site is repaired by at least one DNA repair pathway to produce an altered second target nucleic acid,
- wherein the first RNA-guided nuclease is SpCas9 nuclease and the second RNA-guided nuclease is an Acidaminococcus sp. Cpf1 nuclease, and wherein the first and the second RNA complexes may be delivered simultaneously or sequentially in any order.
A method of altering a cell at a first target nucleic acid and a second target nucleic acid, comprising the steps of:
- forming at least one single- or double-stranded break at a first cleavage site in the first target nucleic acid by delivering to the cell a first ribonucleoprotein (RNP) complex comprising a first RNA-guided nuclease and a first guide RNA (gRNA) capable of directing the first RNA-guided nuclease to the first cleavage site in the first target nucleic acid, wherein the first RNA-guided nuclease is an SpCas9 nuclease, and wherein the first cleavage site is repaired by at least one DNA repair pathway to produce an altered first target nucleic acid;
- and
- after a period of time sufficient for repair of the first cleavage site, forming at least one single- or double-stranded break at a second cleavage site by delivering to the cell a second ribonucleoprotein (RNP) complex comprising a second RNA-guided nuclease and a second guide RNA (gRNA) capable of directing the second RNA-guided nuclease to the second cleavage site in the second target nucleic acid, wherein the second RNA-guided nuclease is an Acidaminococcus sp. Cpf1 nuclease, and wherein the second cleavage site is repaired by at least one DNA repair pathway to produce an altered second target nucleic acid, thereby altering the cell.
Dependencies are inferred from the claim text.