RAPID REMOVAL OF A SELF-REPLICATING FUNGAL PLASMID FOR EFFICIENT MARKER CYCLING

The present disclosure provides compositions and methods for gene editing. The disclosure also provides methods for removing extra-chromosomally replicating plasmids from competent cells.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (ZYMR_074_01 US_SeqList_ST26.xml; Size: 13,565 bytes; and Date of Creation: Jan. 17, 2023) are herein incorporated by reference in its entirety.

FIELD

The present disclosure generally describes methods of removing extra-chromosomally replicating plasmids from competent cells. The disclosure further provides methods and compositions for gene editing.

BACKGROUND

Filamentous fungi are capable of expressing native and heterologous proteins to high levels, making them well-suited for the large-scale production of enzymes, proteins, small molecules, and natural products for industrial, pharmaceutical, animal health, and food and beverage applications. The use of filamentous fungi for large-scale production of products of interest requires genetic manipulation of the selected fungi for improving strain performance in industrial applications.

Extra-chromosomally replicating plasmids are a valuable tool for genome editing of filamentous fungi, because they enable genome editing without integration of a selectable marker gene, so called ‘marker-free’ genome editing. However, the difficulty of removing extra-chromosomally replicating plasmids from the filamentous fungi prevents these plasmids from being used or recycled for successive rounds of genome editing. Known methods for plasmid removal include culturing the fungi in counter-selective conditions or without selective pressure or subjecting the fungus to asexual sporulation to achieve clonality. These methods are time-consuming, may require chemicals that are mutagenic to the microbial host or toxic to humans, and are especially challenging for fungal strains that do not sporulate. Thus, there is a need in the art for new methods of recycling extra-chromosomally replicating plasmids.

SUMMARY

The present disclosure solves the problems in the art by providing compositions and methods for efficient genome editing in filamentous fungi, which are markerless. The present disclosure provides novel methods and compositions for gene editing. The compositions of the disclosure comprise an extra-chromosomally replicating plasmid comprising a selectable marker gene, a gene-editing complex that recognizes a genomic target in a competent cell, and a reagent for the removal of the extra-chromosomally replicating plasmid. The compositions optionally comprise a genetic element of interest. The disclosure further provides methods for using these compositions to recycle an extra-chromosomally replicating plasmid and for making multiple edits to the genome of a filamentous fungi.

In some embodiments, provided herein are compositions for gene editing, comprising: competent cells; an extra-chromosomally replicating plasmid comprising a selectable marker gene; and a gene-editing complex that recognizes a genomic target of a competent cell.

In some embodiments, the compositions comprise a genetic element of interest.

In some embodiments, the compositions do not comprise a genetic element of interest.

In some embodiments, the genetic element of interest is selected from the group consisting of: a nucleic acid sequence, a gene of interest, a gene variant, a genetic edit, a single nucleotide polymorphism, a genetic regulatory sequence, a promoter, a non-coding nucleic acid sequence, a terminator, or any combination thereof.

In some embodiments, the genetic element of interest is a promoter.

In some embodiments, the genetic element of interest is a gene or fragment thereof.

In some embodiments, the gene-editing complex comprises a ribonucleoprotein (RNP).

In some embodiments, the RNP comprises Cas9 and a guide RNA (gRNA) that recognizes the genomic target.

In some embodiments, the gene-editing complex comprises a transcription activator-like effector nuclease (TALEN).

In some embodiments, the gene-editing complex comprises a zinc-finger nuclease (ZFN).

In some embodiments, the competent cells are eukaryotic cells.

In some embodiments, the competent cells are prokaryotic cells.

In some embodiments, the competent cells are fungal cells.

In some embodiments, the competent cells are filamentous fungal cells.

In some embodiments, the competent cells are protoplasts.

In some embodiments, the extra-chromosomally replicating plasmid comprises a plasmid replicator.

In some embodiments, the plasmid replicator is AMA1.

In some embodiments, the selectable marker gene is selected from pvrG, hph, nat, amdS, nptII, niaD, and argB.

In some embodiments, the extra-chromosomally replicating plasmid comprises an endonuclease site.

In some embodiments, the extra-chromosomally replicating plasmid comprises a recombinatorial site.

In some embodiments, the recombinatorial site is a loxP site or a Frt site.

In some embodiments, the compositions comprise a RNP that recognizes the selectable marker gene.

In some embodiments, the RNP comprises Cas9 and a gRNA.

In some embodiments, the compositions comprise an endonuclease, which recognizes an endonuclease site.

In some embodiments, the compositions comprise a recombinase, which recognizes a recombinatorial site.

In some embodiments, the extra-chromosomally replicating plasmid comprises a suicide gene, wherein the suicide gene is under control of an inducible promoter.

In some embodiments, the inducible promoter is an alcohol-regulated promoter, a tetracycline-regulated promoter, a steroid regulated promoter, a metal-regulated promoter, a pathogenesis regulated promoter, a carbon-regulated promoter, a xylose-regulated promoter, a heat shock promoter, a synthetic-transcription factor-dependent promoter, or a light-regulated promoter.

In some embodiments, provided herein is a method for gene editing, comprising: transforming a competent cell with a first composition comprising: an extra-chromosomally replicating plasmid comprising a selectable marker gene and a gene-editing complex that recognizes a genomic target of a competent cell.

In some embodiments, the first composition comprises a genetic element of interest.

In some embodiments, the first composition does not comprise a genetic element of interest.

In some embodiments, the genetic element of interest of the first composition is selected from the group consisting of: a nucleic acid sequence, a gene of interest, a gene variant, a genetic edit, a single nucleotide polymorphism, a genetic regulatory sequence, a promoter, a non-coding nucleic acid sequence, a terminator, or any combination thereof.

In some embodiments, the genetic element of interest of the first composition is a promoter.

In some embodiments, the genetic element of interest of the first composition is a gene or fragment thereof.

In some embodiments, the gene-editing complex of the first composition comprises a ribonucleoprotein (RNP).

In some embodiments, the gene-editing complex of the first composition comprises a ribonucleoprotein (RNP), wherein the RNP comprises Cas9 and a guide RNA (gRNA) that recognizes the genomic target.

In some embodiments, the gene-editing complex of the first composition comprises a transcription activator-like effector nuclease (TALEN).

In some embodiments, the gene-editing complex of the first composition comprises a zinc-finger nuclease (ZFN).

In some embodiments, the methods of the disclosure comprise selecting for competent cells that comprise the extra-chromosomally replicating plasmid.

In some embodiments, the extra-chromosomally replicating plasmid of the first composition comprises a plasmid replicator.

In some embodiments, the extra-chromosomally replicating plasmid of the first composition comprises a plasmid replicator, wherein the plasmid replicator is AMA1.

In some embodiments, the selectable marker gene of the extra-chromosomally replicating plasmid of the first composition is selected from pyrG, hph, nat, amdS, nptII, niaD, and argB.

In some embodiments, the extra-chromosomally replicating plasmid of the first composition comprises a endonuclease site.

In some embodiments, the extra-chromosomally replicating plasmid of the first composition comprises a recombinatorial site.

In some embodiments, the extra-chromosomally replicating plasmid of the first composition comprises a recombinatorial site, wherein the recombinatorial site is a loxP site or a Frt site.

In some embodiments, the methods of the disclosure comprise removing the extra-chromosomally replicating plasmid.

In some embodiments, the methods of the disclosure comprise removing the extra-chromosomally replicating plasmid by applying a RNP to the competent cells comprising the extra-chromosomally replicating plasmid.

In some embodiments, the methods comprise removing the extra-chromosomally replicating plasmid by applying a RNP to the competent cells comprising the extra-chromosomally replicating plasmid, wherein the RNP comprises Cas9 and a gRNA that recognizes the selectable marker gene of the extra-chromosomally replicating plasmid.

In some embodiments, the methods comprise removing the extra-chromosomally replicating plasmid by applying a recombinase to the competent cells comprising the extra-chromosomally replicating plasmid, wherein the recombinase recognizes a recombinatorial site on the extra-chromosomally replicating plasmid.

In some embodiments, the methods comprise removing the extra-chromosomally replicating plasmid by applying an endonuclease to the competent cells comprising the extra-chromosomally replicating plasmid, wherein the endonuclease recognizes an endonuclease site on the extra-chromosomally replicating plasmid.

In some embodiments, the methods comprise introducing a genetic element of interest at a genomic target site.

In some embodiments, the extra-chromosomally replicating plasmid of the first composition comprises a suicide gene, wherein the suicide gene is under control of an inducible promoter.

In some embodiments, the extra-chromosomally replicating plasmid of the first composition comprises a suicide gene, wherein the suicide gene is under control of an inducible promoter, wherein the inducible promoter is an alcohol-regulated promoter, a tetracycline-regulated promoter, a steroid regulated promoter, a metal-regulated promoter, a pathogenesis regulated promoter, a heat shock promoter, a carbon-regulated promoter, a xylose-regulated promoter, a synthetic-transcription factor-dependent promoter or a light-regulated promoter.

In some embodiments, the methods comprise removing the extra-chromosomally replicating plasmid by inducing the promoter to express the suicide gene.

In some embodiments, the methods comprise introducing a gene-editing complex of the first composition that recognizes a genomic target of a competent cell, wherein the competent cell is a eukaryotic cell.

In some embodiments, the methods comprise introducing a gene-editing complex of the first composition that recognizes a genomic target of a competent cell, wherein the competent cell is a prokaryotic cell.

In some embodiments, the methods comprise introducing a gene-editing complex of the first composition that recognizes a genomic target of a competent cell, wherein the competent cell is a fungal cell.

In some embodiments, the methods comprise introducing a gene-editing complex of the first composition that recognizes a genomic target of a competent cell, wherein the competent cell is a filamentous fungal cell.

In some embodiments, the methods comprise introducing a gene-editing complex of the first composition that recognizes a genomic target of a competent cell, wherein the competent cell is a protoplast.

In some embodiments, the methods comprise introducing a second composition, comprising: a second extra-chromosomally replicating plasmid, wherein the extra-chromosomally replicating plasmid comprises a second selectable marker gene and a gene-editing complex that recognizes a genomic target of a competent cell.

In some embodiments, the second composition comprises a genetic element of interest.

In some embodiments, the second composition does not comprise a genetic element of interest.

In some embodiments, the genetic element of interest of the second composition is selected from the group consisting of: a nucleic acid sequence, a gene of interest, a gene variant, a genetic edit, a single nucleotide polymorphism, a genetic regulatory sequence, a promoter, a non-coding nucleic acid sequence, a terminator, or any combination thereof.

In some embodiments, the genetic element of interest of the second composition is a promoter.

In some embodiments, the genetic element of interest of the second composition is a gene or fragment thereof.

In some embodiments, the gene-editing complex of the second composition comprises a ribonucleoprotein (RNP).

In some embodiments, the gene-editing complex of the second composition comprises a ribonucleoprotein (RNP), wherein the RNP comprises Cas9 and a guide RNA (gRNA) that recognizes the genomic target.

In some embodiments, the gene-editing complex of the second composition comprises a transcription activator-like effector nuclease (TALEN).

In some embodiments, the gene-editing complex of the second composition comprises a zinc-finger nuclease (ZFN).

In some embodiments, the methods comprise selecting for competent cells that comprise the second extra-chromosomally replicating plasmid.

In some embodiments, the second extra-chromosomally replicating plasmid comprises a plasmid replicator.

In some embodiments, the second extra-chromosomally replicating plasmid comprises a plasmid replicator, wherein the plasmid replicator is AMA1.

In some embodiments, the second selectable marker gene is selected from pyrG, hph, nat, amdS, nptII, niaD, and argB.

In some embodiments, the second extra-chromosomally replicating plasmid comprises an endonuclease site.

In some embodiments, the second extra-chromosomally replicating plasmid comprises a recombinatorial site.

In some embodiments, the second extra-chromosomally replicating plasmid comprises a recombinatorial site, wherein the recombinatorial site is a loxP site or a Frt site.

In some embodiments, the methods comprise removing the second extra-chromosomally replicating plasmid.

In some embodiments, the methods comprise removing the second extra-chromosomally replicating plasmid by applying a ribonucleoprotein (RNP) to the competent cells comprising the second extra-chromosomally replicating plasmid.

In some embodiments, the methods comprise removing the second extra-chromosomally replicating plasmid by applying a ribonucleoprotein (RNP) to the competent cells comprising the second extra-chromosomally replicating plasmid, wherein the RNP comprises Cas9 and a gRNA that recognizes the selectable marker gene of the second extra-chromosomally replicating plasmid.

In some embodiments, the methods comprise removing the second extra-chromosomally replicating plasmid by applying a recombinase to the competent cells comprising the second extra-chromosomally replicating plasmid, wherein the recombinase recognizes a recombinatorial site on the second extra-chromosomally replicating plasmid.

In some embodiments, the methods comprise removing the second extra-chromosomally replicating plasmid by applying an endonuclease to the competent cells comprising the extra-chromosomally replicating plasmid, wherein the endonuclease recognizes an endonuclease site on the extra-chromosomally replicating plasmid.

In some embodiments, the genetic element of interest of the second composition is introduced at a genomic target site of the second composition.

In some embodiments, the extra-chromosomally replicating plasmid of the second composition comprises a suicide gene, wherein the suicide gene is under control of an inducible promoter.

In some embodiments, the extra-chromosomally replicating plasmid of the second composition comprises a suicide gene, wherein the suicide gene is under control of an inducible promoter, wherein the inducible promoter is an alcohol-regulated promoter, a tetracycline-regulated promoter, a steroid regulated promoter, a metal-regulated promoter, a pathogenesis regulated promoter, a heat shock promoter, a carbon-regulated promoter, a xylose-regulated promoter, a synthetic-transcription factor-dependent promoter, or a light-regulated promoter.

In some embodiments, the methods comprise removing the extra-chromosomally replicating plasmid of the second composition by inducing the promoter which controls the suicide gene to express the suicide gene.

In some embodiments, the methods comprise removing the extra-chromosomally replicating plasmid of the second composition by inducing the promoter which controls the suicide gene to express the suicide gene, wherein inducing comprises introducing an alcohol, a transcription factor, tetracycline, a steroid, a metal, heat, light, an antibiotic, a sugar, xylose, glucose, sucrose, maltose, ethanol, glycerol, methanol, oleic acid, acetate, hexose, lactose, or galactose to the competent cells comprising the second extra-chromosomally replicating plasmid.

In some embodiments, the methods comprise a gene-editing complex of the second composition that recognizes a genomic target of a competent cell, wherein the competent cell is a eukaryotic cell.

In some embodiments, the methods comprise introducing a gene-editing complex of the second composition that recognizes a genomic target of a competent cell, wherein the competent cell is a prokaryotic cell.

In some embodiments, the methods comprise introducing a gene-editing complex of the second composition that recognizes a genomic target of a competent cell, wherein the competent cell is a fungal cell.

In some embodiments, the methods comprise introducing a gene-editing complex of the second composition that recognizes a genomic target of a competent cell, wherein the competent cell is a filamentous fungal cell.

In some embodiments, the methods comprise introducing a gene-editing complex of the second composition that recognizes a genomic target of a competent cell, wherein the competent cell is a protoplast.

In some embodiments, the disclosure provides a method of removing an extra-chromosomally replicating plasmid comprising a selectable marker gene from a competent cell, comprising: administering a reagent to remove the extra-chromosomally replicating plasmid.

In some embodiments, the disclosure provides a method of removing an extra-chromosomally replicating plasmid comprising a selectable marker gene from a competent cell, comprising: administering a reagent to remove the extra-chromosomally replicating plasmid, wherein the extra-chromosomally replicating plasmid comprises a plasmid replicator.

In some embodiments, the disclosure provides a method of removing an extra-chromosomally replicating plasmid comprising a selectable marker gene from a competent cell, comprising: administering a reagent to remove the extra-chromosomally replicating plasmid, wherein the extra-chromosomally replicating plasmid comprises a plasmid replicator, wherein the plasmid replicator is AMA1.

In some embodiments, the disclosure provides a method of removing an extra-chromosomally replicating plasmid comprising a selectable marker gene from a competent cell, wherein the selectable marker gene is selected from pyrG, hph, nat, amdS, nptII, niaD, and argB.

In some embodiments, the disclosure provides a method of removing an extra-chromosomally replicating plasmid comprising a selectable marker gene from a competent cell, wherein the competent cell is a eukaryotic cell.

In some embodiments, the disclosure provides a method of removing an extra-chromosomally replicating plasmid comprising a selectable marker gene from a competent cell, wherein the competent cell is a prokaryotic cell.

In some embodiments, the disclosure provides a method of removing an extra-chromosomally replicating plasmid comprising a selectable marker gene from a competent cell, wherein the competent cell is a fungal cell.

In some embodiments, the disclosure provides a method of removing an extra-chromosomally replicating plasmid comprising a selectable marker gene from a competent cell, wherein the competent cell is a filamentous fungal cell.

In some embodiments, the disclosure provides a method of removing an extra-chromosomally replicating plasmid comprising a selectable marker gene from a competent cell, wherein the competent cell is a protoplast.

In some embodiments, the disclosure provides a method of removing an extra-chromosomally replicating plasmid comprising a selectable marker gene from a competent cell, comprising: administering a reagent to remove the extra-chromosomally replicating plasmid, wherein the reagent comprises a ribonucleoprotein (RNP), an endonuclease, or a recombinase.

In some embodiments, the disclosure provides a method of removing an extra-chromosomally replicating plasmid comprising a selectable marker gene from a competent cell, comprising: administering a reagent to remove the extra-chromosomally replicating plasmid, wherein the reagent comprises a ribonucleoprotein (RNP) that recognizes the selectable marker gene.

In some embodiments, the disclosure provides a method of removing an extra-chromosomally replicating plasmid comprising a selectable marker gene from a competent cell, comprising: administering a reagent to remove the extra-chromosomally replicating plasmid, wherein the reagent comprises a ribonucleoprotein (RNP) that recognizes the selectable marker gene, wherein the RNP comprises Cas9 and a gRNA.

In some embodiments, the disclosure provides a method of removing an extra-chromosomally replicating plasmid, wherein the extra-chromosomally replicating plasmid comprises an endonuclease site.

In some embodiments, the disclosure provides a method of removing an extra-chromosomally replicating plasmid comprising a selectable marker gene from a competent cell, comprising: administering a reagent to remove the extra-chromosomally replicating plasmid, wherein the reagent is an endonuclease that recognizes an endonuclease site.

In some embodiments, the disclosure provides a method of removing an extra-chromosomally replicating plasmid comprising a selectable marker gene from a competent cell, wherein the extra-chromosomally replicating plasmid comprises a recombinatorial site.

In some embodiments, the disclosure provides a method of removing an extra-chromosomally replicating plasmid comprising a selectable marker gene from a competent cell, wherein the extra-chromosomally replicating plasmid comprises a recombinatorial site, wherein the recombinatorial site is a loxP site or a Frt site.

In some embodiments, the disclosure provides a method of removing an extra-chromosomally replicating plasmid comprising a selectable marker gene from a competent cell, comprising administering a reagent to remove the extra-chromosomally replicating plasmid, wherein the reagent comprises a recombinase that recognizes a recombinatorial site.

In some embodiments, the disclosure provides a method of removing an extra-chromosomally replicating plasmid, wherein the extra-chromosomally replicating plasmid comprises a suicide gene, wherein the suicide gene is under control of an inducible promoter.

In some embodiments, the disclosure provides a method of removing an extra-chromosomally replicating plasmid, wherein the extra-chromosomally replicating plasmid comprises a suicide gene, wherein the suicide gene is under control of an inducible promoter, wherein the inducible promoter is an alcohol-regulated promoter, a tetracycline-regulated promoter, a steroid regulated promoter, a metal-regulated promoter, a pathogenesis regulated promoter, a heat shock promoter, a carbon-regulated promoter, a xylose-regulated promoter, a synthetic-transcription factor-dependent promoter, or a light-regulated promoter.

In some embodiments, the disclosure provides a method of removing an extra-chromosomally replicating plasmid, wherein the extra-chromosomally replicating plasmid comprises a suicide gene, wherein the suicide gene is under control of an inducible promoter, wherein the inducible promoter is an alcohol-regulated promoter, a tetracycline-regulated promoter, a steroid regulated promoter, a metal-regulated promoter, a pathogenesis regulated promoter, a heat shock promoter, a carbon-regulated promoter, a xylose-regulated promoter, a synthetic-transcription factor-dependent promoter, or a light-regulated promoter, comprising introducing a reagent to induce expression of the suicide gene, wherein the reagent is selected from the group consisting of a metal, a transcription factor, heat, light, an antibiotic, a sugar, xylose, glucose, sucrose, maltose, ethanol, glycerol, methanol, oleic acid, acetate, hexose, lactose, and galactose.

In some embodiments, the disclosure provides a method for making markerless multiple genomic edits, comprising:

(a) transforming a competent cell with a first composition comprising:(i) an extra-chromosomally replicating plasmid comprising a selectable marker gene; and(ii) a gene-editing complex that recognizes a genomic target of a competent cell;

(b) selecting for competent cells that comprise the extra-chromosomally replicating plasmid of the first composition;

(c) removing the extra-chromosomally replicating plasmid of the first composition by administering a reagent;

(d) transforming a competent cell with a second composition comprising:(i) an extra-chromosomally replicating plasmid comprising a selectable marker gene; and(ii) a gene-editing complex that recognizes a genomic target of a competent cell;

(e) selecting for competent cells that comprise the extra-chromosomally replicating plasmid of the second composition; and

(f) removing the extra-chromosomally replicating plasmid of the second composition by administering a reagent.

In some embodiments, the first composition comprises a genetic element of interest.

In some embodiments, the second composition comprises a genetic element of interest.

In some embodiments, the disclosure provides a method for making markerless multiple genomic edits, comprising:

(a) transforming a competent cell with a first composition comprising:(i) an extra-chromosomally replicating plasmid comprising a selectable marker gene; and(ii) a gene-editing complex that recognizes a genomic target of a competent cell;

(b) selecting for competent cells that comprise the extra-chromosomally replicating plasmid of the first composition;

(c) removing the extra-chromosomally replicating plasmid of the first composition by administering a reagent;

(d) transforming a competent cell with a second composition comprising:(i) an extra-chromosomally replicating plasmid comprising a selectable marker gene; and(ii) a gene-editing complex that recognizes a genomic target of a competent cell;

(e) selecting for competent cells that comprise the extra-chromosomally replicating plasmid of the second composition; and

(f) removing the extra-chromosomally replicating plasmid of the second composition by administering a reagent

(g) transforming the competent cell with a third composition comprising:(i) an extra-chromosomally replicating plasmid comprising a selectable marker gene; and(ii) a gene-editing complex that recognizes a genomic target of a competent cell.

(h) selecting for competent cells that comprise the extra-chromosomally replicating plasmid of the third composition; and(i) removing the extra-chromosomally replicating plasmid of the third composition by administering a reagent.

In some embodiments, the first extra-chromosomally replicating plasmid is removed by administering a recombinase that recognizes a recombinatorial site on the first extra-chromosomally replicating plasmid.

In some embodiments, the second extra-chromosomally replicating plasmid is removed by administering a recombinase that recognizes a recombinatorial site on the second extra-chromosomally replicating plasmid.

In some embodiments, the first extra-chromosomally replicating plasmid is removed by administering an endonuclease that recognizes an endonuclease site on the first extra-chromosomally replicating plasmid.

In some embodiments, the second extra-chromosomally replicating plasmid is removed by administering an endonuclease that recognizes an endonuclease site on the second extra-chromosomally replicating plasmid.

In some embodiments, the first extra-chromosomally replicating plasmid is removed by administering a RNP that recognizes a selectable marker gene on the first extra-chromosomally replicating plasmid.

In some embodiments, the second extra-chromosomally replicating plasmid is removed by administering a RNP that recognizes a selectable marker gene on the second extra-chromosomally replicating plasmid.

In some embodiments, the RNP comprises a gRNA and Cas9.

In some embodiments, the RNP comprises a gRNA and Cas9.

In some embodiments, the first extra-chromosomally replicating plasmid is removed by administering an inducer of a suicide gene on the first extra-chromosomally replicating plasmid.

In some embodiments, the second extra-chromosomally replicating plasmid is removed by administering an inducer of a suicide gene on the second extra-chromosomally replicating plasmid.

DETAILED DESCRIPTION

The term “a” or “an” refers to one or more of that entity. i.e., can refer to a plural referent. As such, the terms “a” or “an”, “one or more” and “at least one” are used interchangeably herein. In addition, reference to “an element” by the indefinite article “a” or “an” does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there is one and only one of the elements.

Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device or the method being employed to determine the value, or the variation that exists among the samples being measured. Unless otherwise stated or otherwise evident from the context, the term “about” means within 10% (i.e., within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less) above or below the reported numerical value (except where such number would exceed 100% of a possible value or go below 0%). When used in conjunction with a range or series of values, the term “about” applies to the endpoints of the range or each of the values enumerated in the series, unless otherwise indicated. As used in this application, the terms “about” and “approximately” are used as equivalents.

Herein, the terms “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

A “eukaryote” is any organism whose cells contain a nucleus and other organelles enclosed within membranes. Eukaryotes belong to the taxon Eukarya or Eukaryota. The defining feature that sets eukaryotic cells apart from prokaryotic cells (the aforementioned Bacteria and Archaea) is that they have membrane-bound organelles, especially the nucleus, which contains the genetic material, and is enclosed by the nuclear envelope.

As used herein, the term “fungus” or “fungi” refers in general to any organism from Kingdom Fungi. Historical taxonomic classification of fungi has been according to morphological presentation. Beginning in the mid-1800's, it was recognized that some fungi have a pleomorphic life cycle, and that different nomenclature designations were being used for different forms of the same fungus. In 1981, the Sydney Congress of the International Mycological Association laid out rules for the naming of fungi according to their status as anamorph, teleomorph, or holomorph (Taylor, 2011). With the development of genomic sequencing, it became evident that taxonomic classification based on molecular phylogenetics did not align with morphological-based nomenclature (Shenoy, 2007). As a result, in 2011 the International Botanical Congress adopted a resolution approving the International Code of Nomenclature for Algae, Fungi, and Plants (Melbourne Code) (2012), with the stated outcome of designating “One Fungus=One Name” (Hawksworth, 2012). However, systematics experts have not aligned on common nomenclature for all fungi, nor are all existing databases and information resources inclusive of updated taxonomies. As such, many fungi referenced herein may be described by their anamorph form, but it is understood that based on identical genomic sequencing, any pleomorphic state of that fungus may be considered to be the same organism. For example, the genusAlternariais the anamorph form of the teleomorph genusLewia(Kwasna 2003), ergo both would be understood to be the same organism with the same DNA sequence. For example, it is understood that the genusAcremoniumis also reported in the literature as genusSarocladiumas well as genusTilachilidium(Summerbell, 2011). For example, the genusCladosporiumis an anamorph of the teleomorph genusDavidiella(Bensch, 2012), and is understood to describe the same organism. In some cases, fungal genera have been reassigned due to various reasons, and it is understood that such nomenclature reassignments are within the scope of any claimed genus. For example, certain species of the genusMierodiplodiahave been described in the literature as belonging to genusParaconiothyrium(Crous and Groenveld, 2006).

As used herein, “selectable marker” is a nucleic acid segment that allows one to select for a molecule (e.g., a replicon) or a cell that contains it, often under particular conditions. These markers can encode an activity, such as, but not limited to, production of RNA, peptide, or protein, or can provide a binding site for RNA, peptides, proteins, inorganic and organic compounds or compositions and the like. Examples of selectable markers include but are not limited to: (1) nucleic acid segments that encode products which provide resistance against otherwise toxic compounds (e.g., antibiotics); (2) nucleic acid segments that encode products which are otherwise lacking in the recipient cell (e.g., tRNA genes, auxotrophic markers); (3) nucleic acid segments that encode products which suppress the activity of a gene product; (4) nucleic acid segments that encode products which can be readily identified (e.g., phenotypic markers such as β-galactosidase, green fluorescent protein (GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), and cell surface proteins); (5) nucleic acid segments that encode products that bind other products which are otherwise detrimental to cell survival and/or function; (6) nucleic acid segments that encode nucleic acids that otherwise inhibit the activity of any of the nucleic acid segments resulting in a visible or selectable phenotype (e.g., antisense oligonucleotides); (7) nucleic acid segments that encode products that bind other products that modify a substrate (e.g. restriction endonucleases); (8) nucleic acid segments that can be used to isolate or identify a desired molecule (e.g. specific protein binding sites); (9) nucleic acid segments that encode a specific nucleotide sequence which can be otherwise non-functional (e.g., for PCR amplification of subpopulations of molecules); and (10) nucleic acid segments, which when absent, directly or indirectly confer resistance or sensitivity to particular compounds.

As used herein, “counterselectable marker” or a “counterselection marker” is a nucleic acid segment that eliminates or inhibits growth of a host organism upon selection. In some embodiments, the counterselectable markers of the present disclosure render the cells sensitive to one or more chemicals/growth conditions/genetic backgrounds. In some embodiments, the counterselectable markers of the present disclosure are toxic genes. In some embodiments, the counterselectable markers are expressed by inducible promoters.

As used herein, the term “nucleic acid” refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, or analogs thereof. This term refers to the primary structure of the molecule, and thus includes double- and single-stranded DNA, as well as double- and single-stranded RNA. It also includes modified nucleic acids such as methylated and/or capped nucleic acids, nucleic acids containing modified bases, backbone modifications, and the like. The terms “nucleic acid” and “nucleotide sequence” are used interchangeably.

As used herein, the term “gene” refers to any segment of DNA associated with a biological function. Thus, genes include, but are not limited to, coding sequences and/or the regulatory sequences required for their expression. Genes can also include non-expressed DNA segments that, for example, form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters.

As used herein, the term “promoter” refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. The promoter sequence may consist of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an “enhancer” is a DNA sequence that can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter.

The term “competent cell” refers to a cell which has the ability to take up and replicate an exogenous nucleic acid.

As used herein, an “extra-chromosomally replicating plasmid” is an autonomously replicating vector that exists as an extra-chromosomal entity. The replication of an extra-chromosomally replicating plasmid is independent of chromosomal replication.

The term “ribonucleoprotein” as used herein refers to a RNA sequence associated with a protein. The association of RNA and protein may be affected by any suitable means, including, for example, protein-nucleic acid interactions. In other words, the term “ribonucleoprotein” as used herein may refer to a RNA-protein complex.

The term “endonuclease” or “nuclease” refers to any wild-type or mutant enzyme that has the ability to catalyze the hydrolysis (cleavage) of bonds between nucleic acids within a DNA or RNA molecule.

The term “recombinase” generally refers to an enzyme that catalyzes recombination.

The term “transform” refers to the introduction of a molecule, such as a polynucleotide, into a competent cell.

The term “percent identity” in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared. Percentage identity can be calculated using the tools CLUSTALW2 or Basic Local Alignment Search Tool (BLAST), which are available online. The following default parameters may be used for CLUSTALW2 Pairwise alignment: Protein Weight Matrix=Gonnet; Gap Open=10; Gap Extension=0.1.

The term “gene edit” refers to the introduction of a genetic element of interest (e.g., a nucleic acid sequence, a gene of interest, a gene variant, a genetic edit, a single nucleotide polymorphism, a genetic regulatory sequence, a promoter, a non-coding nucleic acid sequence, a terminator, or a combination thereof) at a genomic target site. In embodiments, a gene edit comprises an insertion of a genetic element of interest into the genome of a competent cell, substitution of a genomic target of a competent cell with a genetic element of interest, or generation of a single-nucleotide polymorphism within a competent cell.

The term “gene-editing complex” refers to an enzyme and/or nucleic acid that cleaves a genomic target. Non-limiting examples of gene-editing complexes include ribonucleoproteins (RNPs, e.g., a Cas9 nuclease and a guide RNA), a zinc-finger nuclease (ZFN), and a transcription activator-like effector nuclease (TALEN).

II. Compositions for Gene Editing

Provided herein are compositions for gene editing comprising:

(a) an extra-chromosomally replicating plasmid comprising a selectable marker gene;

(c) a gene-editing complex that recognizes a genomic target of a competent cell.

In some embodiments, the compositions provided herein may be used to silence a gene. In some embodiments, the compositions provided herein may be used to upregulate a gene. In some embodiments, the compositions provided herein may be used to mutate a gene.

In some embodiments, the compositions provided herein may be used to introduce a genetic element of interest into a competent cell. In some embodiments, the compositions provided herein are used to remove a genomic target from a competent cell's genome. In some embodiments, the compositions provided herein are used to modify a genomic target within a competent cell's genome. In some embodiments, the compositions provided herein are used to replace a genomic target from a competent cell with a genetic element of interest. In some embodiments, the compositions described herein are used in the methods of the disclosure described in Sections III and IV of this disclosure.

In some embodiments, the compositions described herein comprise an extra-chromosomally replicating plasmid. An extra-chromosomally replicating plasmid can maintain replication of a plasmid independently of chromosomal replication. Plasmid replicators and transformation enhancers are DNA fragments optionally found on extra-chromosomally replicating plasmids which enable extrachromosomal maintenance of plasmids.

In some embodiments, the extra-chromosomally replicating plasmids described herein comprise one or more of a plasmid replicator, an autonomously replicating sequence (ARS), and a transformation enhancer.

In some embodiments, the extra-chromosomally replicating plasmid comprises a plasmid replicator. In some embodiments, the plasmid replicator is AMA1. AMA1 is described in detail in Aleksenko et al. Fungal Genetics and Biology 21, 373-387 (1997), which is incorporated by reference herein in its entirety. In some embodiments, the extra-chromosomally replicating plasmid comprises one of the two repeats of AMA1, as described by Fierro et al, and Sarkari et al., each of which is incorporated by reference herein in its entirety: Curr Genet. 1996 April; 29(5):482-9; Sarkari et al. Bioresour Technol. 2017 December; 245(Pt B):1327-1333.

In some embodiments, AMA1 has a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, AMA1 has a nucleic acid sequence comprising about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, AMA1 has a nucleic acid sequence comprising at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, AMA1 has about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 mutations, insertions, or deletions compared to the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, AMA1 has 1, up to 2, up to 3, up to 4, up to about 5, up to about 6, up to about 7, up to about 8, up to about 9, up to about 10, up to about 11, up to about 12, up to about 13, up to about 14, up to about 15, up to about 16, up to about 17, up to about 18, up to about 19, up to about 20, up to about 21, up to about 22, up to about 23, up to about 24, up to about 25, up to about 26, up to about 27, up to about 28, up to about 29, or up to about 30 mutations, insertions, or deletions compared to the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

In some embodiments, the extra-chromosomally replicating plasmid comprises a transformation enhancer. In some embodiments, the transformation enhancer is ANS1.

In some embodiments, the extra-chromosomally replicating plasmid comprises a selectable marker gene. In some embodiments, the selectable marker gene is selected from pvrG, hph, nat, amdS, nptII, niaD, and argB. In some embodiments, the selectable marker gene is an antibiotic resistance gene, for example, a chloramphenicol resistance gene, an ampicillin resistance gene, a tetracycline resistance gene, a Zeocin resistance gene, a spectinomycin resistance gene and a Km (Kanamycin resistance gene), tetA (tetracycline resistance gene), G418 (neomycin resistance gene), van (vancomycin resistance gene), tet (tetracycline resistance gene), ampicillin (ampicillin resistance gene), methicillin (methicillin resistance gene), penicillin (penicillin resistance gene), oxacillin (oxacillin resistance gene), erythromycin (erythromycin resistance gene), linezolid (linezolid resistance gene), puromycin (puromycin resistance gene) or a hygromycin (hygromycin resistance gene).

In some embodiments, the extra-chromosomally replicating plasmid comprises a suicide gene. Examples of suicide genes include, but are not limited to, herpes simplex virus thymidine kinase (HSV-TK), the cytoplasmic domain of Fas, a caspase such as caspase-8 or caspase-9, cytosine deaminase, E1A, FHIT, and other known suicide or apoptosis-inducing genes (Straathof et al., 2005, Blood 105:42474254; Cohen et al., 1999, Leuk. Lymphoma 34:473480; Thomis et al., 2001, Blood 97:1249-1257; Tey et al., 2007, Biol. Blood Marrow Transplant 13:913-924; and Di Stasi et al., 2011, N. Engl. J. Med. 365:1673-1683).

In some embodiments, the suicide gene is under control of an inducible promoter. In some embodiments, the inducible promoter is selected from an alcohol-regulated promoter, a tetracycline-regulated promoter, a steroid regulated promoter, a metal-regulated promoter, a pathogenesis regulated promoter, a carbon-regulated promoter, a heat shock promoter, a synthetic-transcription factor-dependent promoter, a xylose-regulated promoter, or a light-regulated promoter.

In some embodiments, the extra-chromosomally replicating plasmid comprises a carbon-regulated promoter. In some embodiments, the carbon-regulated promoter is controlled by xylose, glucose, sucrose, maltose, ethanol, glycerol, methanol, oleic acid, acetate, hexose, lactose, or galactose. Weinhandl et al. describes many carbon-regulated promoters and is incorporated by reference herein in its entirety: Weinhandl et al. Carbon source dependent promoters in yeasts.Microbial Cell Factories.2014. 13(5).

In some embodiments, the extra-chromosomally replicating plasmid comprises a barcode. A barcode is any sequence of nucleic acids. In some embodiments, a gene-editing complex or reagent herein recognizes the barcode on the extra-chromosomally replicating plasmid.

In some embodiments, the compositions and methods of the disclosure use competent cells. Competent cells are cells that take up nucleic acids like DNA. The competent cells utilized in the compositions and methods of the disclosure may be prokaryotic or eukaryotic cells. In some embodiments, the prokaryotic cells are bacteria, for example, species ofEscherichia, Klebsiella, Salmonella, Bacillus, Streptomyces, Streptococcus, Shigella, Staphylococcus, Corynebacterium, andPseudomonas. In some embodiments, the eukaryotic cells are animal cells, for example, human cells or insect cells. In some embodiments, the eukaryotic cells are fungi or yeast. In some embodiments, the eukaryotic cells are filamentous fungal cells. In some embodiments, the filamentous fungal cells are protoplasts.

Filamentous Fungal Cells

In some embodiments, mutants of the fungal species described herein are used in the compositions and methods of the disclosure. Examples of such mutants are strains that protoplast well: strains that produce primarily protoplasts with a single nucleus: strains that regenerate efficiently in microtiter plates, strains that regenerate faster and/or strains that take up polynucleotide (e.g., DNA) molecules efficiently, strains that have lost the ability to sporulate, slow-growing strains, and strains that produce cultures of low viscosity such as, for example, cells that produce hyphae in culture that are not so entangled as to prevent isolation of single clones and/or raise the viscosity of the culture, strains that have reduced random integration (e.g., disabled non-homologous end joining pathway) or combinations thereof.

In some embodiments, a mutant filamentous fungal strain lacks a selectable marker gene. In some embodiments, the mutant filamentous fungus strain is a uridine-requiring mutant strain. In some embodiments, the mutant strain is deficient in orotidine-5′-phosphate decarboxylase (OMPD), which is encoded by pyrG, or orotate p-ribosyl transferase (OPRT), which is encoded by pyrE. The following articles describe filamentous fungal strains and are incorporated by reference herein in their entirety: T. Goosen et al, Curr Genet. 1987, 11:499 503, J. Begueret et al., Gene. 1984 32:487 92.

In some embodiments, a mutant filamentous fungal strain possesses a compact cellular morphology characterized by shorter hyphae and a more yeast-like appearance. Examples of such mutants are filamentous fungal cells with altered gasl expression as described in U.S. Publication No. 2014/0220689, which is incorporated by reference herein in its entirety.

In some embodiments, a mutant filamentous fungal strain has an altered DNA repair system. In some embodiments, the altered DNA repair system is extremely efficient in homologous recombination and/or extremely inefficient in random integration. The efficiency of targeted integration of a genetic element of interest into the genome of the competent cell by homologous recombination, i.e. integration in a predetermined target locus, can be increased by augmented homologous recombination abilities and/or diminished non-homologous recombination abilities of the host cell. Augmentation of homologous recombination can be achieved by overexpressing one or more genes involved in homologous recombination (e.g., Rad51 and/or Rad52 protein). Removal, disruption or reduction in the activity of one or more non-homologous recombination pathways (e.g., the canonical non-homologous end joining (NHEJ) pathway, the Alternative NHEJ or microhomology-mediated end-joining (Ait-NHEJ/MMEJ) pathway and/or the polymerase theta mediated end-joining (TMEJ) pathway) in the competent cells of the present disclosure can be achieved by any method known in that art such as, for example, by use of an antibody, a chemical inhibitor, a protein inhibitor, a physical inhibitor, a peptide inhibitor, or an anti-sense or RNAi molecule directed against a component of a specific non-homologous recombination (NHR) pathway (e.g., the NHEJ pathway, the Alt-NHEJ/MMEJ pathway and/or the TMEJ pathway).

In some embodiments, the activity of a single non-homologous end joining pathway is inhibited or reduced. In some embodiments, the activity of a combination of non-homologous end-joining pathways is inhibited or reduced such that the activity of one of the non-homologous end-joining pathways remains intact. In some embodiments, the activity of every non-homologous end-joining pathway is reduced or inhibited.

Examples of components of the NHEJ pathway that can be targeted for inhibition or reduction of activity alone or in combination can include, but are not limited to yeast KU70 or yeast KU80 or homologues or orthologs thereof. Examples of components of the Alt-NHEJ/MMEJ pathway that can be targeted for inhibition or a reduction in activity alone or in combination can include, but are not limited to a Polq gene, a Mre11 gene, an XPF-ERCCI gene or homologues or orthologs thereof. An example of a component of the NHEJ/MMEJ pathway that can be targeted for inhibition or a reduction in activity can include, but is not limited to a Polq gene or a homologue or ortholog thereof. In some embodiments, the competent cell is deficient in one or more genes (e.g., yeast KU70, KU80 or homologues or orthologs thereof) of the NHEJ pathway. Examples of such mutants are cells with a deficient hdfA or hdfB gene as described in WO 05/95624, which is incorporated by reference herein in its entirety. In some embodiments, a host-cell for use in the methods provided herein can be deficient in one or more genes of the Alternative NHEJ or microhomology-mediated end-joining (Alt-NHEJ/MMEJ) pathway and/or TMEJ pathway. Examples of such mutants are cells that lack Polq gene or possess a mutant Polq gene as described in Wyatt et al. Essential roles for Polymerase θ mediated end-joining in repair of chromosome breaks Mol Cell. 2016 August 18; 63(4): 662-673.

In some embodiments, the methods and compositions described herein use fungal elements derived from filamentous fungi that may be readily separated from other such elements in a culture medium and are capable of reproducing. In some embodiments, the methods and compositions described herein use a fungal element selected from a spore, propagule, hyphal fragment, protoplast or micropellet.

Production of Protoplasts

In some embodiments, the filamentous fungi cell is a protoplast. A protoplast is a fungal cell without a cell wall. In some embodiments, protoplasts are generated from filamentous fungi cells using the methods described herein or any known method in the art. Suitable procedures for preparation of protoplasts are known in the art including, for example, those described in EP 238,023 and Yelton et al. (1984, Proc. Natl. Acad. Sci. USA 81:1470-1474), which are incorporated by reference herein in their entirety.

In some embodiments, protoplasts are generated by treating a culture of filamentous fungal cells with one or more lytic enzymes or a mixture thereof. The lytic enzymes can be a beta-glucanase and/or a polygalacturonase.

In some embodiments, after isolation of protoplasts, the protoplasts are cryopreserved. In some embodiments, the protoplasts are mixed with one or more cryoprotectants. The cryoprotectants can be glycols, dimethyl sulfoxide (DMSO), polyols, sugars, 2-Methyl-2,4-pentanediol (MPD), polyvinylpyrrolidone (PVP), methylcellulose, C-linked antifreeze glycoproteins (C-AFGP) or combinations thereof. Glycols for use as cryoprotectants in the methods and systems provided herein can be selected from ethylene glycol, propylene glycol, polypropylene glycol (PEG), glycerol, or combinations thereof. Polyols for use as cryoprotectants in the methods and systems provided herein can be selected from propane-1,2-diol, propane-1,3-diol, 1,1,1-tris-(hydroxymethyl)ethane (THME), and 2-ethyl-2-(hydroxymethyl)-propane-1,3-diol (EHMP), or combinations thereof. Sugars for use as cryoprotectants in the methods and systems provided herein can be selected from trehalose, sucrose, glucose, raffinose, dextrose or combinations thereof. In some embodiments, the protoplasts are mixed with DMSO. DMSO can be mixed with the protoplasts at a final concentration of at least, at most, less than, greater than, equal to, or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12.5%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% w/v or v/v. In some embodiments, the cryopreserved protoplasts are distributed to microtiter plates prior to storage. In some embodiments, the cryopreserved protoplasts are stored at a temperature from about −20° C. to about −80° C., for example about −20° C., about −22° C., about −24° C., about −26° C., about −28° C., about −30° C., about −32° C., about −34° C., about −36° C., about −38° C., about −40° C., about −42° C., about −44° C., about −46° C., about −48° C., about −50° C., about −52° C., about −54° C., about −56° C., about −58° C., about −60° C., about −62° C., about −64° C., about −66° C., about −68° C., about −70° C., about −72° C., about −74° C., about −76° C., about −78° C., or about −80° C.

C. Gene-Editing Complex that Recognizes a Genomic Target of a Competent Cell

In some embodiments, the compositions described herein comprise a gene-editing complex that recognizes a genomic target (used interchangeably herein with “genomic target site”) of a competent cell. As used herein, a “genomic target” refers to a nucleic acid within a competent cell.

In some embodiments, the gene-editing complex removes a genomic target from a competent cell.

In some embodiments, the gene-editing complex comprises a ribonucleoprotein (RNP). A RNP comprises a guide RNA (gRNA) and a nuclease. A gRNA is a nucleic acid that guides a nuclease to a target nucleic acid sequence (e.g. a location to be cleaved).

In some embodiments, the target nucleic acid sequence is a genomic target of a competent cell.

In some embodiments, the guide RNA is a single-molecule guide RNA (sgRNA). A sgRNA comprises a spacer sequence and a scaffold sequence. A spacer sequence is a short nucleic acid sequence used to target a nuclease (e.g., a Cas9 nuclease) to a specific nucleotide region of interest (e.g., a genomic DNA sequence to be cleaved). In some embodiments, the spacer may be about 17-24 base pairs in length, such as about 20 base pairs in length. In some embodiments, the spacer may be about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 base pairs in length. In some embodiments, the spacer may be at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 base pairs in length. In some embodiments, the spacer may be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs in length. In some embodiments, the spacer sequence has between about 40% to about 80% GC content.

In some embodiments, the spacer targets a site that immediately precedes a 5′ protospacer adjacent motif (PAM). The PAM sequence may be selected based on the desired nuclease. For example, the PAM sequence may be any one of the PAM sequences shown in Table 1 below, wherein N refers to any nucleic acid, R refers to A or G, Y refers to C or T. W refers to A or T, and V refers to A or C or G.

In some embodiments, a spacer may target a sequence of a mammalian gene, such as a human gene. In some embodiments, a spacer may target a sequence of a eukaryotic gene, such as a fungal gene. In some embodiments, the spacer may target a mutant gene. In some embodiments, the spacer may target a coding sequence. In some embodiments, the spacer may target an exonic sequence. In some embodiments, a spacer may target an intergenic or non-coding region.

The scaffold sequence is the sequence within the sgRNA that is responsible for nuclease (e.g., Cas9) binding. The scaffold sequence does not include the spacer/targeting sequence. In some embodiments, the scaffold may be about 1 to about 10, about 10 to about 20, about 20 to about 30, about 30 to about 40, about 40 to about 50, about 50 to about 60, about 60 to about 70, about 70 to about 80, about 80 to about 90, about 90 to about 100, about 100 to about 110, about 110 to about 120, or about 120 to about 130 nucleotides in length. In some embodiments, the scaffold may be about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 114, about 115, about 116, about 117, about 118, about 119, about 120, about 121, about 122, about 123, about 124, or about 125 nucleotides in length. In some embodiments, the scaffold may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 10, at least 110, at least 120, or at least 125 nucleotides in length.

In some embodiments, the gRNA is a dual-molecule guide RNA, e.g, crRNA and tracrRNA. In some embodiments, the gRNA is selected based on the source microorganism of a nuclease to be associated therewith. In some embodiments, the gRNA may further comprise a polyA tail.

In some embodiments, the gRNA is provided as a linear nucleic acid or as part of a plasmid.

In some embodiments, the nuclease is Cas9. The Cas9 protein may be an endonuclease derived fromStreptococcussp., for example,Streptococcus pyogenesorStaphylococcus aureus), but is not limited thereto. In some embodiments, the nuclease has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a Cas9 derived fromS. aureus(SaCas9) orS. pyogenes.

In some embodiments, the nuclease is Cpf1. In some embodiments, the nuclease has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a Cpf1. Examples of the Cpf1 protein include those derived from Parcubacteria bacterium, Lachnospiraceae bacterium,Butyrivibrio proleoclasticus, Peregrinibacteria bacterium,Acidaminococcussp.,Porphyromonas macacae, Lachnospiraceae bacterium.Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi, Smithellasp.,Leptospira inadai, Lachnospiraceae bacterium,Francisella novicida, CandidatusMethanoplasmatermitum, andEubacteriumeligens, but are not limited thereto.

In some embodiments, the nuclease is isolated from microorganisms. In some embodiments, the nuclease is produced through recombination or synthesis.

In some embodiments, the nuclease is a variant nuclease. A variant RNA-guided endonuclease (e.g, Cas9) has an amino acid sequence that differs by at least one amino acid (e.g, has a deletion, insertion, or substitution) when compared to the amino acid sequence of a wild type nuclease (e.g. Cas9). A variant nuclease may be truncated, fused to another protein (such as another nuclease), or catalytically inactivated. In some embodiments, the variant nuclease has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to a naturally occurring Cas9, Cas12a (Cpf1), Cas12b. Cas12c. Cas12d, Cas12e, Cas12h, Tnp-B like, Cas13a (C2c2), Cas13b, Cas13c, Cpf1, Cas14, or MAD7.

In some embodiments, the variant nuclease (e.g, Cas9) can cleave the complementary strand of a target nucleic acid but has reduced ability to cleave the non-complementary strand of a double stranded target nucleic acid.

In some embodiments, the variant nuclease (e.g, Cas9) has a mutation (amino acid substitution) that reduces the function of the RuvC domain of Cas9. As a non limiting example, in some embodiments, a variant Cas9 has a D10A mutation (e.g., aspartate to alanine at an amino acid position corresponding to position 10 of Cas9 encoded by the nucleic acid sequence of and can therefore cleave the complementary strand of a double stranded target nucleic acid but has reduced ability to cleave the non-complementary strand of a double stranded target nucleic acid (thus resulting in a single strand break (SSB) instead of a double strand break (DSB) when the variant Cas9 polypeptide cleaves a double stranded target nucleic acid) (see, for example, Jinek et ah, Science. 2012 Aug. 17; 337(6096):816-21).

In some embodiments, the variant nuclease (e.g., Cas9) can cleave the non-complementary strand of a double stranded target nucleic acid but has reduced ability to cleave the complementary strand of the target nucleic acid. For example, the variant nuclease (e.g., Cas9) can have a mutation (amino acid substitution) that reduces the function of the HNH domain of Cas9. As a non limiting example, in some embodiments, the variant Cas9 can have an H840A mutation (e.g., histidine to alanine at an amino acid position corresponding to position 840 ofStreptococcus pyogenesand can therefore cleave the non-complementary strand of the target nucleic acid but has reduced ability to cleave the complementary strand of the target nucleic acid (thus resulting in a single stranded break (SSB) instead of a double stranded break (DSB) when the variant Cas9 polypeptide cleaves a double stranded target nucleic acid).

In some embodiments, the compositions of the disclosure and methods disclosed herein can be used with a wild type nuclease (e.g., Cas9) having double-stranded nuclease activity, nuclease variants (e.g., Cas9 variants) that act as single-stranded nickases, or other mutants with modified nuclease activity. As such, a nuclease (e.g, Cas9) that is suitable for use in the subject invention can be an enzymatically active nuclease (e.g, Cas9 polypeptide), e.g, can make single- or double-stranded breaks in a target nucleic acid, or alternatively can have reduced enzymatic activity compared to a wild-type RNA-guided endonuclease polypeptide (e.g, Cas9 polypeptide).

The nuclease (e.g, Cas9) can be provided to, or in, a cell in a variety of suitable formats. In some embodiments, the nuclease is encoded by a plasmid. In some embodiments, the nuclease is provided as soluble protein. In some embodiments, the nuclease is provided using lentivirus or adeno-associated viruses.

In some embodiments, the nuclease comprises an element typically used for import into cell nuclei by nuclear transport in eukaryotes (e.g., a nuclear localization signal: NLS).

In some embodiments, the compositions and/or methods comprise a plasmid comprising a gRNA and a nuclease. In some cases, the plasmid or linear nucleic acid contains a sequence for negative selection (e.g. mazF, ccdB, gala-1, lacY, thyA, pheS, tetAR, rpsL, sacB, a temperature sensitive replication origin and the like) and/or flanking recombination sequences such as FLPs, loxP sequences, or the like, that can be activated at a later time for removal of the nuclease encoding sequence.

In some embodiments, the gene-editing complex comprises a transcription activator-like effector nuclease (TALEN) that cleaves a genomic target. A “TALEN” refers to a class of artificial restriction endonucleases that comprises a TAL effector DNA binding domain and a DNA cleavage domain. The target nucleic acid comprises a genomic target of the competent cell. A TALEN induces a site-specific double stranded DNA break in a genomic target.

In some embodiments, the TALEN is a monomeric TALEN that can cleave double stranded DNA without assistance from another TALEN. The term “TALEN” is also used to refer to one or both members of a pair of TALENs that are engineered to work together to cleave DNA at the same site. TALENs that work together can be referred to as a left-TALEN and a right-TALEN, which references the handedness of DNA.

In some embodiments, the DNA cleavage domain of the TALEN comprises any nuclease or fragment thereof described throughout this disclosure. In some embodiments, the DNA cleavage domain is derived from a class of non-specific DNA cleavage domains (e.g., the DNA cleavage domain of type II restriction enzymes). In certain embodiments, the DNA cleavage domain is derived from a type II restriction enzyme (FokI).

In some embodiments, the compositions of the disclosure encode an mRNA encoding for a TALEN. In some embodiments, the compositions comprise a plasmid encoding a TALEN. In some embodiments, the compositions comprise a soluble TALEN protein.

Zinc Finger Nuclease

In some embodiments, the gene-editing complex comprises a zinc-finger nuclease. As used herein, a “zinc-finger nuclease” or “ZFN” refers to a chimeric protein molecule comprising at least one zinc finger DNA binding domain linked to at least one nuclease capable of cleaving DNA. The zinc finger DNA binding domain recognizes a genomic target, and the nuclease cleaves the genomic target.

In some embodiments, the zinc finger DNA binding domain is at the N-terminus of the chimeric protein molecule and the DNA cleavage domain is located at the C-terminus of this molecule. In some embodiments, the zinc finger DNA binding domain is at the C-terminus of the chimeric protein molecule and the DNA cleavage domain is located at the N-terminus of this molecule.

In some embodiments, the DNA binding domain of the ZFN comprises at least one zinc finger DNA binding domain, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more zinc finger DNA binding domains. Each zinc finger DNA binding domain binds a genomic target. Two zinc finger DNA binding domains within a ZFN may recognize the same or different genomic targets. The region of DNA between the two zinc finger DNA binding domains is referred to as a “spacer.” In some embodiments, the spacer comprises between 1 and 300 base pairs of DNA.

The zinc finger domains of the present invention can be derived from any class or type of zinc finger. In certain embodiments, the zinc finger domain comprises a Cys2His2type zinc finger, typically represented by, for example, the zinc finger transcription factor TFIIIA or Sp1. DNA recognition specificity and/or binding specificity of ZFN may be varied to achieve the targeted genetic recombination at any genomic target. Such modifications could be accomplished using known molecular biological synthetic techniques and/or chemical synthesis techniques. ZFNs comprising zinc fingers with a wide variety of DNA recognition and/or binding specificities are within the scope of the present invention. In some embodiments, the zinc finger domain is a gag knuckle, a treble clef finger, a zinc ribbon, a Zn2/Cys6-like finger, a TAZ2-domain like, a short zinc-binding loop, or a metallothionein. Krishna et al. describes zinc finger domains in detail and is incorporated by reference herein in its entirety: Krishna et al. Nucleic Acids Res. 2003 Jan. 15; 31(2):532-50. Zinc finger binding domains can be “engineered” to bind to a predetermined nucleotide sequence. Non-limiting examples of methods for engineering zinc finger proteins are design and selection. A designed zinc finger protein is a protein not occurring in nature whose design/composition results principally from rational criteria. Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP designs and binding data.

ZFN DNA cleavage domains may comprise any nuclease or fragment thereof described throughout this disclosure. In some embodiments, the ZFN DNA cleavage domain is derived from a class of non-specific DNA cleavage domains (e.g., the DNA cleavage domain of type II restriction enzymes). In certain embodiments, the DNA cleavage domain is derived from a type II restriction enzyme (Fok1).

The linker between the cleavage domain of ZFN and the recognition domain, if present, contains a sequence of selected amino acid residues, so that the resulting linker is flexible. Alternatively, linkerless constructs are made for maximum target site specificity. A linker-free construct has strong preference for binding to recognition sites and then cleaving between recognition sites 6 bp apart. However, with a linker length between 0 and 18 amino acids long, ZFN-mediated cleavage is present between recognition sites 5-35 bp apart. For a given linker length, there are limitations on the distance between recognition sites that are consistent with both binding and dimerization. In some embodiments, there is no linker between the cleavage domain and the recognition domain, and the target position comprises two 9 nucleotide recognition sites separated by a 6 nucleotide spacer in an inverted orientation with respect to each other.

In some embodiments, the compositions of the disclosure encode an mRNA encoding for a ZFN. In some embodiments, the compositions comprise a plasmid encoding a ZFN. In some embodiments, the compositions comprise a soluble ZFN protein.

D. A Genetic Element of Interest

In some embodiments, the compositions of the disclosure comprise a genetic element of interest. As used herein, a “genetic element of interest” is a nucleic acid that is introduced at a genomic target site. In some embodiments, the genetic element of interest is a deoxyribonucleic acid (DNA). In some embodiments, the genetic element of interest is a ribonucleic acid (RNA). In some embodiments, the compositions do not comprise a genetic element of interest. In some embodiments, the compositions comprise between about 1 and about 100 genetic elements of interest. In some embodiments, the compositions comprise between about 1 and about 10 genetic elements of interest. For example, in some embodiments, the compositions comprise about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100 genetic elements of interest.

In some embodiments, the genetic element of interest comprises a region of homology to the genome of the competent cell (e.g., target genome). In some embodiments, a region of homology to the target genome of the genetic element of interest is found at the 5′ end of the genetic element of interest. In some embodiments, a region of homology to the target genome of the genetic element of interest is found at the 3′ end of the genetic element of interest. In some embodiments, a region of homology to the target genome of the genetic element of interest is found at the 5′ and 3′ end of the genetic element of interest.

In some embodiments, the genetic element of interest is introduced at a genomic target site. In some embodiments, the genetic element of interest replaces a genomic target site.

In some embodiments, the genomic target site is a promoter region. In some embodiments, the genomic target site is a terminator region. In some embodiments, the genomic target site is a coding region. In some embodiments, the genomic target site is a non-coding region.

In some embodiments, the genetic element of interest is selected from the group consisting of: a nucleic acid sequence, a gene of interest, a gene variant, a genetic edit, a single nucleotide polymorphism, a genetic regulatory sequence, a promoter, a non-coding nucleic acid sequence, a terminator, or any combination thereof. In some embodiments, the genetic element of interest is a biosynthetic gene cluster. A biosynthetic gene cluster is an organized group of genes responsible for the production of one or more compounds.

In some embodiments, the genetic element of interest is a nucleic acid sequence. Nucleic acids comprise nucleotides. In some embodiments, nucleotides contain ribose, deoxyribose, or analogs thereof, for example, 2-O-methyl, 2′-O-allyl, 2′-fluoro or 2′-Azidoribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogues such as methyl riboside. In some embodiments, one or more phosphodiester bonds of a nucleic acid may be replaced with alternative groups. Alternative groups include, but are not limited to P(O)S (“thioate”), P(S)S (“dithioate”), (O)NR2 (“amidate”), P(O)R, P(O)OR′, CO or CH2 (“formacetal”), in which each R or R′ is independently H or substituted or unsubstituted alkyl (1-20 C), optionally an ether-(—O—)-bond, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all bonds in a polynucleotide must be identical. The foregoing description is applicable to all of the nucleic acids referred to herein, including RNA and DNA.

In some embodiments, the nucleotide or nucleic acid is labeled. In some embodiments, a nucleotide is labeled according to methods known in the art. In some embodiments, the nucleotide is labeled with a dye and/or a detectable moiety such as a specific binding pair member (e.g. biotin-avidin). Labeled” dNTP or rNTP may also be indirect be marked by its attachment to, for example, a component to which a marker is/may be attached. A dNTP or rNTP may comprise a molecular moiety (for example, an amino group or hydrazide group) to which a label is attached. Non-limiting examples of labels include fluorescent dyes (e.g., fluorescein isothiocyanate, Texas Red, rhodamine, green fluorescent protein and the like), radioisotopes (e.g.3H,35S,32P,33P,125I or14C), enzymes (e.g. LacZ, horseradish peroxidase, alkaline phosphatase), digoxigenin, and colorimetric labels such as colloidal gold or colored glass or plastic beads (e.g., polystyrene, polypropylene, latex, etc.). Various anti-ligands and ligands may be used (as labels themselves or as a label attachment agent).

In some embodiments, the genetic element of interest is a promoter or a terminator sequence. The promoter sequence and/or terminator sequence can be endogenous or heterologous relative to the variant strain and/or the parental strain. Promoter sequences can be operably linked to the 5′ termini of the sequences to be expressed. A variety of known fungal promoters are likely to be functional in the disclosed host strains such as, for example, the promoter sequences of C1 endoglucanases, the 55 kDa cellobiohydrolase (CBHl), glyceraldehyde-3-phosphate dehydrogenase A.C. lucknowenseGARG 27K and the 30 kDa xylanase (XylF) promoters fromChrysosporium, as well as theAspergilluspromoters described in, e.g. U.S. Pat. Nos. 4,935,349; 5,198,345; 5,252,726; 5,705,358; and 5,965,384; and PCX application WO 93/07277. Terminator sequences can be operably linked to the 3′ termini of the sequences to be expressed. A variety of known fungal terminators are likely to be functional in the disclosed host strains. Examples are theA. nidulanstrpC terminator,A. nigeralpha-glucosidase terminator,A. nigerglucoamylase terminator,Mucor mieheicarboxyl protease terminator (see U.S. Pat. No. 5,578,463),Chrysosporiumterminator sequences, e.g. the EG6 terminator, and theTrichoderma reeseicellobiohydrolase terminator.

In some embodiments, the genetic element of interest is a gene edit. A gene edit may be an insertion of a genetic element of interest into the genome of a competent cell, substitution of a genomic target of a competent cell with a genetic element of interest, or generation of a single-nucleotide polymorphism within a competent cell.

In some embodiments, the genetic element of interest is a single nucleotide polymorphism.

In some embodiments, the genetic element of interest is a genetic regulatory sequence.

In some embodiments, the genetic element of interest is a non-coding nucleic acid sequence.

In some embodiments, the genetic element of interest is linear, single-stranded DNA. In some embodiments, the genetic element of interest is linear, double-stranded DNA. In some embodiments, the genetic element of interest comprises one or more sticky ends. As used herein, a “sticky end” is a region of unpaired nucleotides at the end of a DNA double helix. In some embodiments, the genetic element of interest is linear.

In some embodiments, the genetic element of interest is a vector. In some embodiments, a vector comprises a genetic element of interest. In some embodiments, the vector is an integrative vector. An integrative vector becomes integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. An integrative vector may integrate at random or at a predetermined genomic target site of a competent cell.

In some embodiments, the compositions of the disclosure comprise one or more additional reagents. In some embodiments, the additional reagent is utilized to remove an extra-chromosomally replicating plasmid from a competent cell. In some embodiments, the additional reagent maintains the pH of the composition. In some embodiments, the reagent is a recombinase, an integrase, an endonuclease, a RNP, a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease (ZFN), alcohol, a transcription factor, tetracycline, a steroid, a metal, heat, light, an antibiotic, a sugar, xylose, glucose, sucrose, maltose, ethanol, glycerol, methanol, oleic acid, acetate, hexose, lactose, galactose, a buffer, or a salt.

In some embodiments, the reagent recognizes a recombinatorial site, a restriction endonuclease site, a selectable marker gene, or another nucleotide sequence. In some embodiments, the reagent recognizes a recombinatorial site, restriction endonuclease site, a selectable marker gene, or another nucleotide sequence on an extra-chromosomally replicating plasmid.

In some embodiments, the compositions comprise a RNP that recognizes a site on the extra-chromosomally replicating plasmid. In some embodiments, the RNP recognizes the selectable marker gene. In some embodiments, the site is 500 base pairs or less from a terminus of the selectable marker gene on the extra-chromosomally replicating plasmid.

A RNP that recognizes a site on the extra-chromosomally replicating plasmid comprises a gRNA and a nuclease. Characteristics of an RNP that recognizes a site on the extra-chromosomally replicating plasmid are described in Section IIC of this disclosure. In some embodiments, the gRNA is an sgRNA. In some embodiments, the nuclease is Cas9.

In some embodiments, the compositions comprise a recombinase or an integrase. A recombinase is an enzyme which promotes genetic recombination. In some embodiments, integrases promote genetic recombination by integrating DNA into a cell. In some embodiments, genetic recombination is site-specific, e.g. the recombinase recognizes a specific sequence of DNA. In some embodiments, the recombinase recognizes a recombinatorial site.

Other Reagents

In some embodiments, the composition comprises an alcohol, a transcription factor, tetracycline, a steroid, a metal, heat, or light. In some embodiments, the compositions contain a reagent that regulates expression of a suicide gene. For example, the reagent may induce or enhance expression of a suicide gene. In some embodiments, the reagent is a carbon source. Non-limiting examples of carbon sources include xylose, glucose, sucrose, maltose, ethanol, glycerol, methanol, oleic acid, acetate, hexose, lactose, or galactose.

In some embodiments, the composition comprises agents that regulate pH (e.g. buffers).

In some embodiments, the composition comprises a salt. Non-limiting examples of salts include sodium chloride, potassium chloride, ammonium chloride, sodium acetate, sodium citrate, copper sulfate, sodium iodide, and sodium sulfate.

III. Methods for Recycling Extra-Chromosomally Replicating Plasmids

Extra-chromosomally replicating plasmids enable gene editing without integration of the selectable marker gene in the genome of the competent cell, so called ‘marker-free’ gene editing. However, the difficulties associated with removing extra-chromosomally replicating plasmids, limits their usefulness for making multiple genetic edits in a competent cell. Described herein are superior strategies for recycling extra-chromosomally replicating plasmids.

In some embodiments, the method for removing an extra-chromosomally replicating plasmid from a competent cell comprises administering a reagent to remove the extra-chromosomally replicating plasmid.

In some embodiments, the methods comprise removing an extra-chromosomally replicating plasmid from competent cells transformed with the compositions of the disclosure (e.g. those described in Section II of this disclosure). The method utilizes competent cells and extra-chromosomally replicating plasmids as described in Section II. For example, the competent cell may be a eukaryotic cell, a prokaryotic cell, a fungal cell, or a filamentous fungal cell (e.g., a protoplast), and the extra-chromosomally replicating plasmid may comprise any combination of a selectable marker gene, a recombinatorial site, an endonuclease site, and a suicide gene. In some embodiments, the extra-chromosomally replicating plasmid comprises a plasmid replicator. In some embodiments, the plasmid replicator is AMA1. In some embodiments, the competent cells are transformed according to the methods in Section IV of this disclosure. Transformation is evaluated by selecting for cells that comprise the extra-chromosomally replicating plasmid as described in Section IV of this disclosure.

In some embodiments, the method of recycling an extra-chromosomally replicating plasmid comprises administering a reagent to remove the extra-chromosomally replicating plasmid.

Recycling of Extra-Chromosomally Replicating Plasmid Through Introduction of an RNP

In some embodiments, the method of recycling an extra-chromosomally replicating plasmid comprises administering a RNP (FIG.1). In some embodiments, the RNP comprises a gRNA and a nuclease, wherein the gRNA recognizes a site on the extra-chromosomally replicating plasmid and directs cleavage of a target nucleic acid by a nuclease. In some embodiments, the target nucleic acid is a selectable marker gene on the extra-chromosomally replicating plasmid or fragment thereof. In some embodiments, the target nucleic acid is a nucleic acid within 500 base pairs of the selectable marker gene. In some embodiments, the target nucleic acid is about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, or about 100 nucleotides of a selectable marker gene. In some embodiments, addition of an RNP cleaves and destabilizes the extra-chromosomally replicating plasmid, facilitating its removal from a competent cell.

Non-limiting examples of nucleases include nucleases with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to a nuclease is selected from the group consisting of Cas9, Cas12a (Cpf1), Cas12b. Cas12c, Cas12d, Cas12e, Cas12h, Tnp-B like, Cas13a (C2c2), Cas13b, Cas13c, Cpf1, Cas14, and MAD7, or homologs, orthologs, or paralogs thereof.

Non-limiting examples of gRNAs include single-molecule guide RNA (sgRNA) or dual-molecule guide RNA, e.g, RNA which comprises a crRNA and tracrRNA.

Recycling of Extra-Chromosomally Replicating Plasmid Through Introduction of a Recombinase

In some embodiments, the method of recycling an extra-chromosomally replicating plasmid comprises administering a recombinase or an integrase (FIG.2). In some embodiments, a recombinase recognizes a recombinatorial site on the extra-chromosomally replicating plasmid. A recombinase catalyzes recombination of DNA between recombinatorial sites in a DNA molecule. In some embodiments, recombination destabilizes the extra-chromosomally replicating plasmid and facilitates its removal from a competent cell.

In some embodiments, the recombinatorial site is a loxP, a Frt, psi, dif, cer, attB, attP, attL, attR, att1, att2, or att site, or mutant, variant, or derivative thereof. In some embodiments, the recombinatorial site is recognized by a recombinase or integrase selected from the group of Cre recombinase, λ-integrase, XerC recombinase, XerD recombinase, flippase (Flp), Flp recombinase, Hin recombinase, Tre recombinase, RecA recombinase, Rad51 recombinase, gamma-delta resolvase, and Dmc1 recombinase. In some embodiments, the recombinatorial site is a loxP or a Frt site.

Recycling of Extra-Chromosomally Replicating Plasmid Through Introduction of an Endonuclease

In some embodiments, the method of recycling an extra-chromosomally replicating plasmid comprises administering an endonuclease (FIG.3). In some embodiments, an endonuclease recognizes a restriction site on the extra-chromosomally replicating plasmid. In some embodiments, the extra-chromosomally replicating plasmid comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more restriction sites. One or more endonucleases cut DNA (on the extra-chromosomally replicating plasmid) at one or more restriction sites, producing discrete DNA fragments or producing linear DNA from plasmid DNA consequently destabilizing the extra-chromosomally replicating plasmid and encouraging its loss from the cell or mycelium.

Recycling of Extra-Chromosomally Replicating Plasmid Through Induction of a Suicide Gene

In some embodiments, the method of recycling an extra-chromosomally replicating plasmid comprises removing the extra-chromosomally replicating plasmid via induction of a suicide gene which is under control of an inducible promoter. In some embodiments, the extra-chromosomally replicating plasmid comprises a suicide gene. Application of a reagent that induces expression of a suicide gene causes competent cells comprising the suicide gene to die.

In some embodiments, the suicide gene is under the control of an inducible promoter selected from the group consisting of an alcohol-regulated promoter, a tetracycline-regulated promoter, a steroid regulated promoter, a metal-regulated promoter, a pathogenesis regulated promoter, a heat shock promoter, a synthetic-transcription factor-dependent promoter, a carbon-regulated promoter, or a light-regulated promoter.

IV. Methods for Genome Editing

In some embodiments, the method comprises transforming a competent cell with a first composition comprising:

(a) an extra-chromosomally replicating plasmid, comprising a selectable marker gene; and

(b) a gene-editing complex that recognizes a genomic target of a competent cell. In some embodiments, the first composition comprises a genetic element of interest. In some embodiments, the first composition does not comprise a genetic element of interest. In some embodiments, the first composition comprises the extra-chromosomally replicating plasmid and a genetic element of interest.

In some embodiments, the extra-chromosomally replicating plasmid is the genetic element of interest. In some embodiments, the genetic element of interest is double stranded DNA. In some embodiments, the genetic element of interest is single stranded DNA. In some embodiments, the genetic element of interest is a plasmid. Various examples of genetic elements of interest are described in Section II of this disclosure. In some embodiments, the genetic element of interest is selected from the group consisting of: a nucleic acid sequence, a gene of interest, a gene variant, a genetic edit, a single nucleotide polymorphism, a genetic regulatory sequence, a promoter, a non-coding nucleic acid sequence, a terminator, or any combination thereof.

Upon transformation of the competent cell with the first composition, the RNP cleaves the genomic target from the competent cell via a CRISPR mechanism, and the genetic element of interest replaces the genomic target DNA. The following patent documents describe CRISPR and are incorporated by reference herein in their entirety: U.S. Pat. Nos. 8,697,359, 8,771,945, 8,795,965, 8,865,406, 8,871,445, 8,889,356, 8,895,308, 8,906,616, 8,932,814, 8,945,839, 8,993,233, 8,999,641, U.S. patent application Ser. No. 14/704,551, and U.S. patent application Ser. No. 13/842,859

Various methods for transformation are taught herein. In some embodiments, transformation of a competent cell involves heat-shock or electroporation. In some embodiments, transformation is automated. In some embodiments, competent cells are transformed using high-throughput electroporation systems, for example, the VWR®High-throughput Electroporation Systems, BTX™, Bio-Rad®, Gene Pulser MXcell™, or other multi-well electroporation systems. In some embodiments, transformation is mediated by polyethylene glycol (PEG).

In some embodiments, the method for gene editing comprises selecting for competent cells that comprise the extra-chromosomally replicating plasmid (e.g. transformed competent cells). In some embodiments, competent cells that comprise the extra-chromosomally replicating plasmid are selected by applying a selective agent to the competent cells.

Competent cells, extra-chromosomally replicating plasmids, genetic elements of interest, and gene-editing complexes that recognize a genomic target of a competent cell are described in Sections II and III of this disclosure. In some embodiments, the competent cell is a eukaryotic cell, a prokaryotic cell, a filamentous fungal cell, or a protoplast. In some embodiments, the extra-chromosomally replicating plasmid comprises a selectable marker gene, recombinatorial site, endonuclease site, suicide gene controlled by an inducible promoter, or combination thereof. In some embodiments, the gene-editing complex comprises a ribonucleoprotein (RNP), a TALEN, or a ZFN. In some embodiments, the first composition can be used to make between about 1 and about 100 genetic edits, for example about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, or about 100 genetic edits.

In some embodiments, the first composition comprises no genetic elements of interest.

In some embodiments, after RNP cleavage of the genomic target, genomic DNA is repaired by non-homologous end joining, homologous recombination, or micro-homology mediated repair.

In some embodiments, after selecting for cells that comprise a gene edit, an extra-chromosomally replicating plasmid is recycled according to the methods described in Section III of this disclosure.

In some embodiments, before each additional round, an extra-chromosomally replicating plasmid from a previous round is removed. Removal methods are described in Section III of this disclosure.

In some embodiments, each round of gene editing is numbered according to the order in which it is performed. For example, the first additional round is called the second round, and the second additional round is called the third round and so on.

In some embodiments, each additional round comprises transforming a competent cell with an additional composition. In some embodiments, each additional composition is numbered according to its round number. For example, the composition of the first additional round (e.g. the second round) is the second composition, and the composition of the second additional round (e.g. the third round) is the third composition and so on. Each additional composition comprises:

(a) a second extra-chromosomally replicating plasmid, wherein the extra-chromosomally replicating plasmid comprises a second selectable marker gene; and

(b) a gene-editing complex that recognizes a genomic target of a competent cell.

Characteristics of the second extra-chromosomally replicating plasmid and gene-editing complex are described in Section II of this disclosure.

In some embodiments, extra-chromosomally replicating plasmids of different rounds comprise the same selectable marker gene. In some embodiments, extra-chromosomally replicating plasmids of different rounds comprise different selectable marker genes.

In some embodiments, extra-chromosomally replicating plasmids comprise one or more of a recombinatorial site, a suicide gene, or a endonuclease site, as described in Sections II and III of this disclosure. In some embodiments, extra-chromosomally replicating plasmids from different rounds comprise different combinations of a recombinatorial site, a suicide gene, or an endonuclease site. In some embodiments, extra-chromosomally replicating plasmids from different rounds comprise the same combinations of a recombinatorial site, a suicide gene, or an endonuclease site.

EXAMPLES

Example 1. Use of RNPs to Recycle Extra-Chromosomally Replicating Plasmids for Gene-Editing

Fungal protoplasts are transformed with a composition comprising:

(a) an extra-chromosomally replicating plasmid, wherein the extra-chromosomally replicating plasmid comprises a selectable marker gene;

(b) At least one gene-editing complex that recognizes at least one genomic target of the competent cell; and

Protoplasts that comprise the composition are selected for by employing a selective agent. Protoplasts that grow in the presence of a selective agent have been transformed. The fungal protoplasts are subsequently transformed with a second composition. The second composition enables removal of the first extra-chromosomally replicating plasmid via a RNP. The second composition comprises:

(a) a second extra-chromosomally replicating plasmid, wherein the extra-chromosomally replicating plasmid comprises a selectable marker gene;

(b) At least one gene-editing complex that recognizes at least one genomic target of the competent cell, and

(d) an RNP that comprises Cas9 and a gRNA that recognizes the selectable marker gene of round 1 to remove the extra-chromosomally replicating plasmid utilized during round 1.

Protoplasts that grow in the presence of a selective agent have been transformed.

The fungal protoplasts are subsequently transformed with a third composition. The third composition enables removal of the second extra-chromosomally replicating plasmid via a RNP. The third composition comprises:

(a) a third extra-chromosomally replicating plasmid, wherein the extra-chromosomally replicating plasmid comprises a selectable marker gene;

(b) At least one gene-editing complex that recognizes at least one genomic target of the competent cell, and

(d) an RNP that comprises Cas9 and a gRNA that recognizes the selectable marker gene of round 2 to remove the extra-chromosomally replicating plasmid utilized during round 2 (FIG.1).

Example 2. Use of Recombinase to Recycle Extra-Chromosomally Replicating Plasmids for Gene-Editing

Fungal protoplasts are transformed with a composition comprising:

(a) An extra-chromosomally replicating plasmid, wherein the extra-chromosomally replicating plasmid comprises a selectable marker gene and at least two recombinatorial sites;

(b) At least one gene-editing complex that recognizes at least one genomic target of the competent cell; and

Protoplasts that comprise the composition are selected for by employing a selective agent. Protoplasts that grow in the presence of a selective agent have been transformed. The fungal protoplasts are transformed with a second composition. The second composition enables removal of the first extra-chromosomally replicating plasmid via a recombinase. The second composition comprises:

(a) a second extra-chromosomally replicating plasmid, wherein the extra-chromosomally replicating plasmid comprises a selectable marker gene;

(b) at least one gene-editing complex that recognizes at least one genomic target of the competent cell; and

(d) a recombinase that recognizes a recombinatorial site (e.g., Motif X) on the extra-chromosomally replicating plasmid.

Protoplasts that grow in the presence of a selective agent have been transformed. The fungal protoplasts are transformed with a third composition. The third composition enables removal of the second extra-chromosomally replicating plasmid via a recombinase. The third composition comprises:

(a) a third extra-chromosomally replicating plasmid, wherein the extra-chromosomally replicating plasmid comprises a selectable marker gene.

(b) at least one gene-editing complex that recognizes at least one genomic target of the competent cell; and

(d) a recombinase that recognizes a recombinatorial site (e.g., Motif Y) on the extra-chromosomally replicating plasmid of round 2. (FIG.2).

Example 3. Use of Endonuclease to Recycle Extra-Chromosomally Replicating Plasmids for Gene-Editing

Fungal protoplasts are transformed with a composition comprising:

(a) an extra-chromosomally replicating plasmid, wherein the extra-chromosomally replicating plasmid comprises a selectable marker gene;

(b) At least one gene-editing complex that recognizes at least one genomic target of the competent cell; and

Protoplasts that comprise the composition are selected for by employing a selective agent. Protoplasts that grow in the presence of a selective agent have been transformed. The fungal protoplasts are transformed with a second composition. The second composition enables removal of the first extra-chromosomally replicating plasmid via a restriction endonuclease. The second composition comprises:

(a) a second extra-chromosomally replicating plasmid, wherein the extra-chromosomally replicating plasmid comprises a selectable marker gene;

(b) At least one gene-editing complex that recognizes at least one genomic target of the competent cell; and

(c) optionally 1-100,000 genetic elements of interest; and

(d) a restriction endonuclease (e.g. a homing endonuclease) that recognizes the endonuclease site (e.g., Motif X) on the extra-chromosomally replicating plasmid of round 1.

(e) Protoplasts that grow in the presence of a selective agent have been transformed. The fungal protoplasts are transformed with a third composition. The third composition enables removal of the second extra-chromosomally replicating plasmid via a restriction endonuclease. The third composition comprises.

(f) a third extra-chromosomally replicating plasmid, wherein the extra-chromosomally replicating plasmid comprises a selectable marker gene;

(g) at least one gene-editing complex that recognizes at least one genomic target of the competent cell; and

(h) optionally 1-100,000 genetic elements of interest; and

(i) a restriction endonuclease (e.g. a homing endonuclease) that recognizes the endonuclease site (e.g., Motif Y) on the extra-chromosomally replicating plasmid of round 2 (FIG.3).

INCORPORATION BY REFERENCE

Additional Embodiments of the Disclosure

The following embodiments are also envisioned by the present disclosure:

1. A composition for gene editing, comprising:

(b) an extra-chromosomally replicating plasmid comprising a selectable marker gene; and

(c) a gene-editing complex that recognizes a genomic target of a competent cell.

2. The composition of embodiment 1, comprising a genetic element of interest.
3. The composition of embodiment 1, wherein the composition does not contain a genetic element of interest.
4. The composition of embodiment 2, wherein the genetic element of interest is selected from the group consisting of: a nucleic acid sequence, a gene of interest, a gene variant, a genetic edit, a single nucleotide polymorphism, a genetic regulatory sequence, a promoter, a non-coding nucleic acid sequence, a terminator, or any combination thereof.
5. The composition of embodiment 2, wherein the genetic element of interest is a promoter.
6. The composition of embodiment 2, wherein the genetic element of interest is a gene or fragment thereof.
7. The composition of any one of embodiments 1-6, wherein the gene-editing complex comprises a ribonucleoprotein (RNP).
8. The composition of embodiment 7, wherein the RNP comprises Cas9 and a guide RNA (gRNA) that recognizes the genomic target.
9. The composition of any one of embodiments 1-6, wherein the gene-editing complex comprises a transcription activator-like effector nuclease (TALEN).
10. The composition of any one of embodiments 1-6, wherein the gene-editing complex comprises a zinc-finger nuclease (ZFN).
11. The composition of any one of embodiments 1-10, wherein the competent cells are eukaryotic cells.
12. The composition of any one of embodiments 1-10, wherein the competent cells are prokaryotic cells.
13. The composition of any one of embodiments 1-10, wherein the competent cells are fungal cells.
14. The composition of any one of embodiments 1-10 or 13, wherein the competent cells are filamentous fungal cells.
15. The composition of any one of embodiments 1-10, 13, or 14, wherein the competent cells are protoplasts.
16. The composition of any one of embodiments 1-15, wherein the extra-chromosomally replicating plasmid comprises a plasmid replicator.
17. The composition of embodiment 16, wherein the plasmid replicator is AMA1.
18. The composition of any one of embodiments 1-17, wherein the selectable marker gene is selected from pyrG, hph, nat, amdS, nptII, niaD, and argB.
19. The composition of any one of embodiments 1-18, wherein the extra-chromosomally replicating plasmid comprises an endonuclease site.
20. The composition of any one of embodiments 1-19, wherein the extra-chromosomally replicating plasmid comprises a recombinatorial site.
21. The composition of embodiment 20, wherein the recombinatorial site is a loxP site or a Frt site.
22. The composition of any one of embodiments 1-21, comprising a RNP that recognizes the selectable marker gene.
23. The composition of embodiment 22, wherein the RNP comprises Cas9 and a gRNA.
24. The composition of any one of embodiments 1-23, comprising an endonuclease, which recognizes an endonuclease site.
25. The composition of any one of embodiments 1-14, comprising a recombinase, which recognizes a recombinatorial site.
26. The composition of any one of embodiments 1-25, wherein the extra-chromosomally replicating plasmid comprises a suicide gene, wherein the suicide gene is under control of an inducible promoter.
27. The composition of embodiment 26, wherein the inducible promoter is an alcohol-regulated promoter, a tetracycline-regulated promoter, a steroid regulated promoter, a metal-regulated promoter, a pathogenesis regulated promoter, a carbon-regulated promoter, a xylose-regulated promoter, a heat shock promoter, a synthetic-transcription factor-dependent promoter, or a light-regulated promoter.
28. The composition of embodiment 26, wherein expression of the suicide gene is induced by an alcohol, a transcription factor, tetracycline, a steroid, a metal, heat, light, an antibiotic, a sugar, xylose, glucose, sucrose, maltose, ethanol, glycerol, methanol, oleic acid, acetate, hexose, lactose, or galactose.
29. A method for gene editing, comprising: transforming a competent cell with a first composition comprising:

(a) an extra-chromosomally replicating plasmid comprising a selectable marker gene; and

(b) a gene-editing complex that recognizes a genomic target of a competent cell.

30. The method of embodiment 29, comprising a genetic element of interest.
31. The method of embodiment 29, wherein the composition does not contain a genetic element of interest.
32. The method of embodiment 30, wherein the genetic element of interest is selected from the group consisting of: a nucleic acid sequence, a gene of interest, a gene variant, a genetic edit, a single nucleotide polymorphism, a genetic regulatory sequence, a promoter, a non-coding nucleic acid sequence, a terminator, or any combination thereof.
33. The method of embodiment 30, wherein the genetic element of interest is a promoter.
34. The method of embodiment 30, wherein the genetic element of interest is a gene or fragment thereof.
35. The method of any one of embodiments 29-34, wherein the gene-editing complex comprises a ribonucleoprotein (RNP).
36. The method of embodiment 29, wherein the RNP comprises Cas9 and a guide RNA (gRNA) that recognizes the genomic target.
37. The method of any one of embodiments 29-36, wherein the gene-editing complex comprises a transcription activator-like effector nuclease (TALEN).
38. The method of any one of embodiments 29-37, wherein the gene-editing complex comprises a zinc-finger nuclease (ZFN).
39. The method of any one of embodiments 29-38, comprising selecting for competent cells that comprise the extra-chromosomally replicating plasmid.
40. The method of any one of embodiments 29-39, wherein the extra-chromosomally replicating plasmid comprises a plasmid replicator.
41. The method of embodiment 40, wherein the plasmid replicator is AMA1.
42. The method of any one of embodiments 29-41, wherein the selectable marker gene is selected from pyrG, hph, nat, amdS, nptII, niaD, and argB.
43. The method of any one of embodiments 29-42, wherein the extra-chromosomally replicating plasmid comprises a endonuclease site.
44. The method of any one of embodiments 29-43, wherein the extra-chromosomally replicating plasmid comprises a recombinatorial site.
45. The method of embodiment 44, wherein the recombinatorial site is a loxP site or a Fri site.
46. The method of any one of embodiments 29-45, comprising removing the extra-chromosomally replicating plasmid.
47. The method of any one of embodiments 29-46, comprising removing the extra-chromosomally replicating plasmid by applying a RNP to the competent cells comprising the extra-chromosomally replicating plasmid.
48. The method of embodiment 47, wherein the RNP comprises Cas9 and a gRNA that recognizes the selectable marker gene of the extra-chromosomally replicating plasmid.
49. The method of any one of embodiments 29-46, comprising removing the extra-chromosomally replicating plasmid by applying a recombinase to the competent cells comprising the extra-chromosomally replicating plasmid, wherein the recombinase recognizes a recombinatorial site on the extra-chromosomally replicating plasmid.
50. The method of any one of embodiments 29-42, comprising removing the extra-chromosomally replicating plasmid by applying an endonuclease to the competent cells comprising the extra-chromosomally replicating plasmid, wherein the endonuclease recognizes an endonuclease site on the extra-chromosomally replicating plasmid.
51. The method of any one of embodiments 29-50, wherein the genetic element of interest is introduced at the genomic target site.
52. The method of any one of embodiments 29-51, wherein the extra-chromosomally replicating plasmid comprises a suicide gene, wherein the suicide gene is under control of an inducible promoter.
53. The method of embodiment 52, wherein the inducible promoter is an alcohol-regulated promoter, a tetracycline-regulated promoter, a steroid regulated promoter, a metal-regulated promoter, a pathogenesis regulated promoter, a heat shock promoter, a carbon-regulated promoter, a xylose-regulated promoter, a synthetic-transcription factor-dependent promoter or a light-regulated promoter.
54. The method of any one of embodiments 29-53, comprising removing the extra-chromosomally replicating plasmid by inducing the promoter to express the suicide gene.
55. The method of embodiment 54, wherein inducing comprises introducing an alcohol, a transcription factor, tetracycline, a steroid, a metal, heat, light, an antibiotic, a sugar, xylose, glucose, sucrose, maltose, ethanol, glycerol, methanol, oleic acid, acetate, hexose, lactose, or galactose to the competent cells comprising the extra-chromosomally replicating plasmid.
56. The method of any one of embodiments 29-55, wherein the competent cell is a eukaryotic cell.
57. The method of any one of embodiments 29-56, wherein the competent cell is a prokaryotic cell.
58. The method of any one of embodiments 29-57, wherein the competent cell is a fungal cell.
59. The method of any one of embodiments 29-58, wherein the competent cell is a filamentous fungal cell.
60. The method of any one of embodiments 29-59, wherein the competent cell is a protoplast.
61. The method of any one of embodiments 29-60, comprising introducing a second composition, comprising:

(a) a second extra-chromosomally replicating plasmid, wherein the extra-chromosomally replicating plasmid comprises a second selectable marker gene; and

(b) a gene-editing complex that recognizes a genomic target of a competent cell.

62. The method of embodiment 61, wherein the second composition comprises a genetic element of interest.
63. The method of embodiment 61, wherein the second composition does not comprise a genetic element of interest.
64. The method of embodiment 62, wherein the genetic element of interest of the second composition is selected from the group consisting of: a nucleic acid sequence, a gene of interest, a gene variant, a genetic edit, a single nucleotide polymorphism, a genetic regulatory sequence, a promoter, a non-coding nucleic acid sequence, a terminator, or any combination thereof.
65. The method of embodiment 62, wherein the genetic element of interest of the second composition is a promoter.
66. The method of embodiment 62, wherein the genetic element of interest of the second composition is a gene or fragment thereof.
67. The method of any one of embodiments 61-66, wherein the gene-editing complex comprises a ribonucleoprotein (RNP).
68. The method of embodiment 67, wherein the RNP comprises Cas9 and a guide RNA (gRNA) that recognizes the genomic target.
69. The method of any one of embodiments 61-66, wherein the gene-editing complex comprises a transcription activator-like effector nuclease (TALEN).
70. The method of any one of embodiments 61-66, wherein the gene-editing complex comprises a zinc-finger nuclease (ZFN).
71. The method of any one of embodiments 61-70, comprising selecting for competent cells that comprise the second extra-chromosomally replicating plasmid.
72. The method of any one of embodiments 61-71, wherein the second extra-chromosomally replicating plasmid comprises a plasmid replicator.
73. The method of embodiment 72, wherein the plasmid replicator is AMA1.
74. The method of any one of embodiments 61-73, wherein the second selectable marker gene is selected from pyrG, hph, nat, amdS, nptII, niaD, and argB.
75. The method of any one of embodiments 61-74, wherein the second extra-chromosomally replicating plasmid comprises a endonuclease site.
76. The method of any one of embodiments 61-74, wherein the second extra-chromosomally replicating plasmid comprises a recombinatorial site.
77. The method of embodiment 76, wherein the recombinatorial site is a loxP site or a Frt site.
78. The method of any one of embodiments 61-77, comprising removing the second extra-chromosomally replicating plasmid.
79. The method of any one of embodiments 61-78, comprising removing the second extra-chromosomally replicating plasmid by applying a ribonucleoprotein (RNP) to the competent cells comprising the second extra-chromosomally replicating plasmid.
80. The method of embodiment 79, wherein the RNP comprises Cas9 and a gRNA that recognizes the selectable marker gene of the second extra-chromosomally replicating plasmid.
81. The method of any one of embodiments 61-78, comprising removing the second extra-chromosomally replicating plasmid by applying a recombinase to the competent cells comprising the second extra-chromosomally replicating plasmid, wherein the recombinase recognizes a recombinatorial site on the second extra-chromosomally replicating plasmid.
82. The method of any one of embodiments 61-78, comprising removing the extra-chromosomally replicating plasmid by applying an endonuclease to the competent cells comprising the extra-chromosomally replicating plasmid, wherein the endonuclease recognizes an endonuclease site on the extra-chromosomally replicating plasmid.
83. The method of any one of embodiments 61-82, wherein the genetic element of interest of the second composition is introduced at a genomic target site of the second composition.
84. The method of any one of embodiments 61-83, wherein the extra-chromosomally replicating plasmid comprises a suicide gene, wherein the suicide gene is under control of an inducible promoter.
85. The method of embodiment 84, wherein the inducible promoter is an alcohol-regulated promoter, a tetracycline-regulated promoter, a steroid regulated promoter, a metal-regulated promoter, a pathogenesis regulated promoter, a heat shock promoter, a carbon-regulated promoter, a xylose-regulated promoter, a synthetic-transcription factor-dependent promoter, or a light-regulated promoter.
86. The method of embodiment 84 or 85, comprising removing the extra-chromosomally replicating plasmid by inducing the promoter to express the suicide gene.
87. The method of embodiment 86, wherein inducing comprises introducing an alcohol, a transcription factor, tetracycline, a steroid, a metal, heat, light, an antibiotic, a sugar, xylose, glucose, sucrose, maltose, ethanol, glycerol, methanol, oleic acid, acetate, hexose, lactose, or galactose to the competent cells comprising the second extra-chromosomally replicating plasmid.
88. The method of any one of embodiments 61-87, wherein the competent cell is a eukaryotic cell.
89. The method of any one of embodiments 61-87, wherein the competent cell is a prokaryotic cell.
90. The method of any one of embodiments 61-87, wherein the competent cell is a fungal cell.
91. The method of any one of embodiments 61-87 or 90, wherein the competent cell is a filamentous fungal cell.
92. The method of any one of embodiments 61-87, 90, or 91, wherein the competent cell is a protoplast.
93. A method of removing an extra-chromosomally replicating plasmid comprising a selectable marker gene from a competent cell, comprising: administering a reagent to remove the extra-chromosomally replicating plasmid.
94. The method of embodiment 93, wherein the extra-chromosomally replicating plasmid comprises a plasmid replicator.
95. The method of embodiment 94, wherein the plasmid replicator is AMA1.
96. The method of any one of embodiments 93-95, wherein the selectable marker gene is selected from pyrG, hph, nat, amdS, nptII, niaD, and argB.
97. The method of any one of embodiments 93-96, wherein the competent cell is a eukaryotic cell.
98. The method of any one of embodiments 93-96, wherein the competent cell is a prokaryotic cell.
99. The method of any one of embodiments 93-96, wherein the competent cell is a fungal cell.
100. The method of any one of embodiments 93-96 or 99, wherein the competent cell is a filamentous fungal cell.
101. The method of any one of embodiments 93-96, 99, or 100, wherein the competent cell is a protoplast.
102. The method of any one of embodiments 93-101, wherein the reagent comprises a ribonucleoprotein (RNP), an endonuclease, or a recombinase.
103. The method of any one of embodiments 93-102, wherein the reagent comprises a ribonucleoprotein (RNP) that recognizes the selectable marker gene.
104. The method of embodiment 103, wherein the RNP comprises Cas9 and a gRNA.
105. The method of any one of embodiments 93-104, wherein the extra-chromosomally replicating plasmid comprises an endonuclease site.
106. The method of embodiment 105, wherein the reagent is an endonuclease that recognizes an endonuclease site.
107. The method of any one of embodiments 93-106, wherein the extra-chromosomally replicating plasmid comprises a recombinatorial site.
108. The method of embodiment 107, wherein the recombinatorial site is a loxP site or a Fri site.
109. The method of any one of embodiments 93-108, wherein the reagent is a recombinase that recognizes a recombinatorial site.
110. The method of any one of embodiments 93-109, wherein the extra-chromosomally replicating plasmid comprises a suicide gene, wherein the suicide gene is under control of an inducible promoter.
111. The method of embodiment 110, wherein the inducible promoter is an alcohol-regulated promoter, a tetracycline-regulated promoter, a steroid regulated promoter, a metal-regulated promoter, a pathogenesis regulated promoter, a heat shock promoter, a carbon-regulated promoter, a xylose-regulated promoter, a synthetic-transcription factor-dependent promoter, or a light-regulated promoter.
112. The method of embodiment 110 or 111, comprising introducing a reagent to induce expression of the suicide gene, wherein the reagent is selected from the group consisting of a metal, a transcription factor, heat, light, an antibiotic, a sugar, xylose, glucose, sucrose, maltose, ethanol, glycerol, methanol, oleic acid, acetate, hexose, lactose, and galactose.
113. A method for making markerless multiple genomic edits, comprising:

(a) transforming a competent cell with a first composition comprising:(i) an extra-chromosomally replicating plasmid comprising a selectable marker gene; and(ii) a gene-editing complex that recognizes a genomic target of a competent cell;

(b) selecting for competent cells that comprise the extra-chromosomally replicating plasmid of the first composition;

(c) removing the extra-chromosomally replicating plasmid of the first composition by administering a reagent;

(d) transforming a competent cell with a second composition comprising:(i) an extra-chromosomally replicating plasmid comprising a selectable marker gene; and(ii) a gene-editing complex that recognizes a genomic target of a competent cell;

(e) selecting for competent cells that comprise the extra-chromosomally replicating plasmid of the second composition; and

(f) removing the extra-chromosomally replicating plasmid of the second composition by administering a reagent.

114. The method of embodiment 113, wherein the first composition comprises a genetic element of interest.
115. The method of embodiment 113 or 114, wherein the second composition comprises a genetic element of interest.
116. The method of any one of embodiments 113-115, comprising:

(a) transforming the competent cell with a third composition comprising:(i) an extra-chromosomally replicating plasmid comprising a selectable marker gene; and(ii) a gene-editing complex that recognizes a genomic target of a competent cell.

(b) selecting for competent cells that comprise the extra-chromosomally replicating plasmid of the third composition; and

(c) removing the extra-chromosomally replicating plasmid of the third composition by administering a reagent.

117. The method of embodiment any one of embodiments 113-116, wherein the first extra-chromosomally replicating plasmid is removed by administering a recombinase that recognizes a recombinatorial site on the first extra-chromosomally replicating plasmid.
118. The method of any one of embodiments 113-117, wherein the second extra-chromosomally replicating plasmid is removed by administering a recombinase that recognizes a recombinatorial site on the second extra-chromosomally replicating plasmid.
119. The method of any one of embodiments 113-118, wherein the first extra-chromosomally replicating plasmid is removed by administering an endonuclease that recognizes an endonuclease site on the first extra-chromosomally replicating plasmid.
120. The method of any one of embodiments 113-119, wherein the second extra-chromosomally replicating plasmid is removed by administering an endonuclease that recognizes an endonuclease site on the second extra-chromosomally replicating plasmid.
121. The method of any one of embodiments 113-120, wherein the first extra-chromosomally replicating plasmid is removed by administering a RNP that recognizes a selectable marker gene on the first extra-chromosomally replicating plasmid.
122. The method of any one of embodiments 113-121, wherein the second extra-chromosomally replicating plasmid is removed by administering a RNP that recognizes a selectable marker gene on the second extra-chromosomally replicating plasmid.
123. The method of embodiment 121, wherein the RNP comprises a gRNA and Cas9.
124. The method of embodiment 122, wherein the RNP comprises a gRNA and Cas9.
125. The method of any one of embodiments 113-124, wherein the first extra-chromosomally replicating plasmid is removed by administering an inducer of a suicide gene on the first extra-chromosomally replicating plasmid.
126. The method of embodiments 113-125, wherein the second extra-chromosomally replicating plasmid is removed by administering an inducer of a suicide gene on the second extra-chromosomally replicating plasmid.