Method for selectively accumulating genetic modifications

Abstract

The invention provides a method for selectively accumulating genetic modifications in an offspring cell of a microbial species, wherein the microbial species has (i) a first mating type and (ii) a second mating type compatible with the first mating type; and wherein the method comprises a mating cycle comprising: a preparation stage comprising providing a first cell and a second cell of the microbial species, the first cell having the first mating type and the second cell having the second mating type, wherein the first cell and the second cell have a first locus, a second locus, a third locus, a fourth locus, and a fifth locus, wherein preferably at least the fourth locus and the fifth locus are chromosomal loci, and wherein: the first locus of the first cell encodes a first guide RNA, wherein the first guide RNA is configured to hybridize with a first target sequence, wherein the first locus of the second cell comprises the first target sequence; the second locus of the second cell encodes a second guide RNA, wherein the second guide RNA is configured to hybridize with a second target sequence, wherein the second locus of the first cell comprises the second target sequence; the third locus of the first cell and the third locus of the second cell encode an RNA-guided DNA endonuclease, wherein the RNA-guided DNA endonuclease is configured to functionally associate with the first guide RNA and with the second guide RNA; the fourth locus of the first cell comprises a first mating-type promoter configured to facilitate expression of a first selectable marker providing a first selectable trait in cells having the first mating type; the fourth locus of the second cell comprises a second mating-type promoter configured to facilitate expression of a second selectable marker providing a second selectable trait in cells having the second mating type; the fifth locus of the first cell comprises a third selectable marker providing a third selectable trait; the fifth locus of the second cell comprises a fourth selectable marker providing a fourth selectable trait; and preferably the first selectable trait, the second selectable trait, the third selectable trait and the fourth selectable trait differ from each other; a mating stage comprising bringing the first cell and the second cell in contact with each other to provide an offspring cell population; and a selection stage comprising selecting the offspring cell from the offspring cell population or from cells derived therefrom, wherein the offspring cell is selected based on at least two selectable traits, wherein preferably the at least two selectable traits comprise one or more of (a) at least the third selectable trait and the fourth selectable trait, (b) at least the first selectable trait and the fourth selectable trait, and (c) at least the second selectable trait and the third selectable trait.

Description

[0001] Method for selectively accumulating genetic modifications

[0002] FIELD OF THE INVENTION

[0003] The invention relates to a method for selectively accumulating genetic modifications in an offspring cell of a microbial species. The invention further relates to a recombinant cell.

[0004] BACKGROUND OF THE INVENTION

[0005] Methods for introducing genetic modifications into microbial species are known in the art. For instance, W02020014570 describes Multi-locus CRISPR-Cas gene drive systems, methods, and host cells involving integration of a CRISPR-Cas gene drive system into a diploid eukaryotic cell genome at multiple loci. The gene drive system comprises a plurality of distinct gene drive constructs, wherein at least one gene drive construct (and preferably only one gene drive construct within the system) encodes a functional CRISPR nuclease, along with two or more distinct gene drive constructs encoding respective sgRNA. Each of the gene drive constructs is configured for integration at separate, distinct loci in the genome that are non-adjacent to one another, and preferably at remote positions within the genome.

[0006] SUMMARY OF THE INVENTION

[0007] Gene drives are genetic elements whose proliferation through a population is enhanced via a specific molecular mechanism encoded by these elements, rather than being strictly dictated by Mendelian inheritance rules. Known examples in nature include B-chromosomes, meiotic drives, and transposable elements. Synthetic gene drives can be used to propagate a desired mutation within a population. For example, the prior art may describe approaches for using gene drives to (a) combat invasive or vector-carrying animals (e.g., mosquitoes, ticks) and (b) improve the resilience of agricultural crops against environmental challenges.

[0008] One class of synthetic gene drives is based on Homing Endonuclease Genes (HEGs), which encode proteins with homing activity. Inheritance of HEG-based gene drives is biased in a population because they induce homozygosity of the allele(s) they target. Specifically, the Homing Endonuclease, encoded by a HEG-containing allele, may induce a double-stranded break in a second, HEG-less allele. When the HEG-containing chromosome is used as a repair template, the HEG is copied into the other allele through homologous recombination. However, the flexibility and efficiency of HEG-based designs are relatively limited, e.g., due to their dependence on specific (hardcoded) target sequences (or “recognition sequences”). The prior art may further describe synthetic nuclease platforms based on the Transcription Activator-Like Effector Nuclease (TALEN) and Zinc Finger Nuclease (ZFN). However, the inherent repetitive nature of their coding sequences can result in genetic instability, potentially leading to a rapid decline in homing efficiency over generations.

[0009] The maturation of CRISPR-Cas technology may have revitalized interest in synthetic gene drives. In nature, CRISPR-Cas functions as an adaptive immune system, providing sequence-specific immunity against invading nucleic acids. In short, most DNA-targeting and DNA-cleaving CRISPR-Cas systems consist of CRISPR-associated genes (or “Cas genes”) and a CRISPR array. The CRISPR array is composed of repetitive sequences (repeats) separated by variable sequences called spacers. Expression and maturation of the CRISPR array result in the formation of mature CRISPR RNAs (crRNAs, or guide RNAs; gRNAs), which consist of a spacer sequence flanked by repeats. The gRNA is loaded into a single-protein or multi-subunit Cas protein complex, which guides the Cas protein(s) to bind and cleave DNA sequences (protospacers) that are complementary to the variable sequence of the gRNA. This system can be modified to target different genomic regions of interest by altering the variable sequence of the gRNA, making it suitable for HEG-based gene drive designs.

[0010] For instance, the prior art may describe the use of CRISPR-Cas nucleases in gene drive constructs to facilitate their propagation in sexually reproducing organisms, such as S. cerevisiae. The CRISPR-Cas ribonucleoprotein complex, comprising a nuclease and a functionally coupled guide RNA from an allele, may target an opposing gene drive-less allele, inducing a double-stranded break (DSB). The homologous, gene drive-containing chromosome may subsequently be used as a repair template, resulting in a cell that is homozygous for the gene drive construct. In S. cerevisiae, first-generation haploid offspring may have been generated using manual tetrad dissection to study the functionality of CRISPR-Cas9 based HEG gene drives. However, manual tetrad dissection can be quite laborious and effectively unsuitable for high-throughput experiments. Further, prior art methods may result in an effectively random distribution of genetic markers in the resulting haploids, which could complicate or even preclude further rounds of mating experiments.

[0011] The prior art may further describe an approach of providing multiple generations of S. cerevisiae, wherein markers are recycled using recombinase-based systems, such as the Cre-Lox system. However, the Cre-Lox system (and similar systems) can be complex to implement and may be unsuitable for use with gene drives, as their underlying working mechanisms may overlap.

[0012] The prior art may also describe crossing a haploid cell having a first gene drive with a library of haploid cells, each carrying a single secondary gene drive, to provide a library of diploid cells, each having the first gene drive and a secondary gene drive. This approach can be used to study the effects of double gene knockouts. However, these prior art approaches may also result in an effectively random distribution of genetic markers in further haploid offspring, complicating or preventing the accumulation of additional gene drives.

[0013] Further, prior art approaches may lack control over the mating type of offspring cells, complicating selective mating in later mating rounds.

[0014] Further, prior art approaches may require a separate marker gene for each CRISPR array, potentially limiting the number of gene drives that can be effectively combined and reducing flexibility in inter-crossing separately generated offspring cells.

[0015] Yet further, prior art methods may be relatively time-consuming.

[0016] Yet further, prior art approaches may also require a fully pre-defined mating scheme to produce a cell with a desired final genetic state, thereby lacking the flexibility to generate multiple branching mating lines from (intermediate) offspring cells.

[0017] Hence, it is an aspect of the invention to provide an alternative method for selectively accumulating genetic modifications in an offspring cell of a microbial species, which preferably further at least partly obviates one or more of above-described drawbacks. The present invention may have as object to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

[0018] Hence, the invention may provide a method for selectively accumulating genetic modifications in an offspring cell of a microbial species, especially in a microbial species having (i) a first mating type and (ii) a second mating type compatible with the first mating type. The microbial species may further be capable of homology directed repair. The method may comprise a mating cycle comprising one or more of a preparation stage, a mating stage and a selection stage. The preparation stage may comprise providing a first cell of the microbial species and a second cell of the microbial species. Especially, the first cell may have the first mating type and the second cell may have the second mating type. In embodiments, the first cell and the second cell may (both) have a first locus, a second locus, a third locus, a fourth locus, and a fifth locus, especially wherein at least the fourth locus and the fifth locus are chromosomal loci. The first locus of the first cell may encode a first guide RNA. The first guide RNA may be configured to hybridize with a first target sequence, especially wherein the first locus of the second cell comprises the first target sequence. Similarly, the second locus of the second cell may encode a second guide RNA. The second guide RNA may be configured to hybridize with a second target sequence, especially wherein the second locus of the first cell comprises the second target sequence. The third locus of the first cell and / or the third locus of the second cell may encode an RNA-guided DNA endonuclease. The RNA-guided DNA endonuclease may be configured to functionally associate with the first guide RNA and (separately) with the second guide RNA. The fourth locus of the first cell may comprise a first mating-type promoter configured to facilitate expression of a first selectable marker providing a first selectable trait in cells (of the microbial species) having the first mating type. Similarly, the fourth locus of the second cell may comprise a second mating-type promoter configured to facilitate expression of a second selectable marker providing a second selectable trait in cells (of the microbial species) having the second mating type. The fifth locus of the first cell may comprise a third selectable marker providing a third selectable trait, whereas the fifth locus of the second cell may comprise a fourth selectable marker providing a fourth selectable trait. In embodiments, the first selectable trait, the second selectable trait, the third selectable trait and the fourth selectable trait may all differ from each other. The mating stage may comprise bringing the first cell and the second cell in contact with each other to provide an offspring cell population. The selection stage may comprise selecting the offspring cell from the offspring cell population or from cells derived therefrom, wherein the offspring cell is selected based on (a combination of) at least two selectable traits, especially wherein the at least two selectable traits comprise one or more of (a) at least the third selectable trait and the fourth selectable trait, (b) at least the first selectable trait and the fourth selectable trait, and (c) at least the second selectable trait and the third selectable trait.

[0019] In specific embodiments, the invention may provide a method for selectively accumulating genetic modifications in an offspring cell of a microbial species, wherein the microbial species has (i) a first mating type and (ii) a second mating type compatible with the first mating type; and wherein the method comprises a mating cycle comprising: (I) a preparation stage comprising providing the first cell and the second cell of the microbial species, the first cell having the first mating type and the second cell having the second mating type, wherein the first cell and the second cell (each) have a first locus, a second locus, a third locus, a fourth locus, and a fifth locus, wherein at least the fourth locus and the fifth locus are chromosomal loci, and wherein: the first locus of the first cell encodes a first guide RNA, wherein the first guide RNA is configured to hybridize with a first target sequence, wherein the first locus of the second cell comprises the first target sequence; the second locus of the second cell encodes a second guide RNA, wherein the second guide RNA is configured to hybridize with a second target sequence, wherein the second locus of the first cell comprises the second target sequence; the third locus of the first cell and the third locus of the second cell encode an RNA-guided DNA endonuclease, wherein the RNA-guided DNA endonuclease is configured to functionally associate with the first guide RNA and with the second guide RNA; the fourth locus of the first cell comprises a first mating-type promoter configured to facilitate expression of a first selectable marker providing a first selectable trait in cells having the first mating type; the fourth locus of the second cell comprises a second mating-type promoter configured to facilitate expression of a second selectable marker providing a second selectable trait in cells having the second mating type; the fifth locus of the first cell comprises a third selectable marker providing a third selectable trait; the fifth locus of the second cell comprises a fourth selectable marker providing a fourth selectable trait; and the first selectable trait, the second selectable trait, the third selectable trait and the fourth selectable trait differ from each other; (II) a mating stage comprising bringing the first cell and the second cell in contact with each other to provide an offspring cell population; and (III) a selection stage comprising selecting the offspring cell from the offspring cell population or from cells derived therefrom, wherein the offspring cell is selected based on at least two selectable traits, wherein the at least two selectable traits comprise one or more of (a) at least the third selectable trait and the fourth selectable trait, (b) at least the first selectable trait and the fourth selectable trait, and (c) at least the second selectable trait and the third selectable trait.

[0020] The multiploid cell generated in the mating stage may initially be heterozygous for the first and second loci: for each it carries one allele with a guide RNA targeting the other corresponding allele. Due to the action of the guide RNA and the RNA-guided DNA endonuclease and subsequent homology directed repair, the multiploid cell will become homozygous for both the first and second loci, i.e., for each of these loci both alleles will encode the corresponding guide RNA. In embodiments, sporulation may be initiated in the multiploid cell to provide a haploid cell. As the multiploid cell was homozygous for the first and second locus, the haploid cell will also encode the first and second guide RNAs in the first and second locus, respectively. Further, as the multiploid cell was heterozygous for the genetic markers, the haploid cell will encode a combination of two of the four genetic markers, i.e., one genetic marker under control of a mating-type controller in the fourth locus and another genetic marker in the fifth locus. Thereby, the haploid offspring cell may be suitable for another mating cycle, which, again, may result in an offspring cell suitable for use in a further mating cycle. Thereby, the method of the invention facilitates selectively accumulating genetic modifications in an offspring cell.

[0021] Further, the method may facilitate easily generating offspring cells with different combinations of genetic modifications. For instance, a population of offspring cells carrying eight loci with respective homing guide RNAs may be generated, which offspring cell may subsequently be mated with a variety of other offspring cells carrying, for instance, two loci with respective homing guide RNAs, resulting in a library of offspring cells each having ten loci with respective homing guide RNAs. Hence, the method of the invention may provide a streamlined approach for strain engineering. The method may also be particularly suitable for gene drive research focused on monitoring gene drive systems over multiple generations.

[0022] Specifically, the double marker system provided by the fourth locus and the fifth locus can be used to cycle through the various stages of the sexual reproduction cycle of the microbial species, e.g., of S. cerevisiae, while maintaining control of the specific mating types of offspring cells. Effectively, the double marker system provides a recyclable system that can be re-used in consecutive mating cycles. The sexual life cycle selection system for parental haploids, diploids and haploid offspring was validated in S. cerevisiae (see experiments below). The ability to introduce and stack gene drive mutations via sexual life cycle iterations may have promising potential for a wide range of applications. First, it can provide information on how other factors, such as fitness or the presence of multiple gene drive constructs may affect proliferation over multiple generations, for instance in the context of the modification or eradication of detrimental species (e.g., malaria parasites) in a natural population. Second, starting from a single gene knockout (KO) library, the method may facilitate providing offspring carrying essentially any combination of KOs, as long as the combination of KOs results in a viable cell. Third, for industrial applications, the method can be used as a strain engineering approach to create a comprehensive permutation library that includes various mutation types beyond gene KOs, especially gain-of-function mutations or change-of-function mutations, such as modified gene expression levels, e.g., due to a different promoter, truncated genes, and replacement with a homologous gene, e.g., a replacement of a gene with a homologous gene from a (related) organism with a modified function, e.g., a coding for an enzyme with a higher turnover number or a higher heat tolerance.

[0023] In specific embodiments, the method of the invention may provide a multiploid offspring cell suitable for use as the first cell or as the second cell in a following mating cycle (see below). Such embodiments may be particularly fast compared to conventional engineering approaches as sporulation and selection of haploid for further iterations may be particularly time consuming.

[0024] As described above, the invention may provide a method for selectively accumulating genetic modifications in an offspring cell of a microbial species.

[0025] The term “genetic modification” may herein refer to essentially any genetic modification. For instance, a genetic modifications may be a so-called “knock-out”, i.e., the targeted removal or inactivation of a gene. Alternatively or additionally, a genetic modification may be a so-called “knock-in”, i.e., the insertion of a genetic sequence in a locus, especially an exogeneous genetic sequence, such as an exogeneous gene. A genetic modification may include a combination of a knock-out and a knock-in, i.e., the introduction of an exogeneous genetic sequence further causing the removal or disruption of a (natural) gene. The method of the invention may facilitate providing an offspring cell with a plurality of genetic modifications (relative to a wildtype cell), optionally including one or more knock-outs, and optionally including one or more knock-ins.

[0026] The term “offspring cell” may herein refer to a cell obtained using the method of the invention, especially obtained using the mating stage or the selection stage of the mating cycle. In embodiments, the offspring cell may be a haploid offspring cell, i.e., a cell having one copy of each chromosome. In further embodiments, the offspring cell may be a multiploid offspring cell, i.e., a cell having two or more copies of each chromosome, such as a diploid cell having two copies of each chromosome, or such as a polyploid cell having three or more copies of each chromosome.

[0027] The term “microbial species” may herein refer to any species of microorganisms. In embodiments, the microbial species may be a microbial eukaryotic species, especially a fungal species, such as a yeast species. Hence, in embodiments, the microbial species is a fungus, especially a yeast. In further embodiments, the microbial species may be selected from the group comprising Saccharomyces spp., Kluyveromyces spp., Schizosaccharomyces spp., Candida spp., Pichia spp., Yarrowia spp., Komagataella spp. and Aspergillus spp., especially from the group consisting of Saccharomyces spp., Kluyveromyces spp., Schizosaccharomyces spp., Candida spp., Pichia spp., Yarrowia spp., and Aspergillus spp..

[0028] In further embodiments, the microbial species may be a hybrid species, especially a hybrid strain, such as a hybrid species originating from protoplast fusion between Saccharomyces cerevisiae and Kluyveromyces lactis. In such embodiments, the hybrid species, such as the hybrid strain, may be derived from at least one microbial species selected from the group comprising Saccharomyces spp., Kluyveromyces spp., Schizosaccharomyces spp., Candida spp., Pichia spp., Yarrowia spp., Komagataella spp. and Aspergillus spp., especially from the group consisting of Saccharomyces spp., Kluyveromyces spp., Schizosaccharomyces spp., Candida spp., Pichia spp., Yarrowia spp., and Aspergillus spp.. In further embodiments, the hybrid species, such as the hybrid strain, may be derived from at least two microbial species selected from the group comprising Saccharomyces spp., Kluyveromyces spp., Schizosaccharomyces spp., Candida spp., Pichia spp., Yarrowia spp., Komagataella spp. and Aspergillus spp., especially from the group consisting of Saccharomyces spp., Kluyveromyces spp., Schizosaccharomyces spp., Candida spp., Pichia spp., Yarrowia spp., and Aspergillus spp..

[0029] Hence, in embodiments, the microbial species may belong to the division Ascomycota, especially to the class Saccharomycetes, such as to the order Saccharomycetales. In further embodiments, the microbial species may be a Saccharomyces spp., especially a species selected from the group comprising S. cerevisiae, S. pastorianus, S. chevalieri, S. paradoxus, S. uvaru, and S. bayanus, especially selected from the group comprising S. cerevisiae, S. pastorianus, S. paradoxus, S. uvaru, and S. bayanus.

[0030] The invention may herein for explanatory purposes primarily be discussed in the context of S. cerevisiae. It will be clear to the person skilled in the art, however, that the invention is not limited to such species.

[0031] For instance, in embodiments, the microbial species may be a Kluyveromyces spp., such as a species selected from the group comprising K. lactis^ K. marxianus, and K. thermotolerans. In further embodiments, the microbial species may be a Schizosaccharomyces spp., such as a species selected from the group comprising S. pombe and S. japonicus. In further embodiments, the microbial species may be a Candida spp., such as a species selected from the group comprising C. albicans, C. antarctica, C. rugosa, C. krusei, C. parapsilosis, C. tropicalis, C. dubliniensis and C. auris. In further embodiments, the microbial species may be a Pichia spp., such as a species selected from the group comprising P. pastoris (also known as Komagataella phaffi), P. stipites, P. terricola, P. kluyveri, P. membranifaciens. In further embodiments, the microbial species may be a Yarrowia spp., such as Y. lipolytica. In further embodiments, the microbial species may be a Komagataella spp., such as a species selected from the group comprising K. phaffi. In further embodiments, the microbial species may be an Aspergillus spp., such as a species selected from the group comprising A. niger, A nidulans, A.flavus, A. fumigatus, A. oryzae, A. sojae, and A. terreus. The microbial species may especially have (i) a first mating type and (ii) a second mating type compatible with the first mating type. In particular, the microbial species may be capable of sexual reproduction, wherein a cell of the first mating type mates with a cell of the second mating type. In particular, in embodiments, the first cell may have the first mating type and the second cell may have the second mating type.

[0032] For instance, in embodiments, the microbial species may be Saccharomyces cerevisiae, which species has the mating types MA Ta (or “a”) and MATa (or “a”). The mating type of an S. cerevisiae cell may be determined by the MAT locus; a cell that is homozygotic or hemizygotic for the MATa allele has the MATa mating type, whereas a cell that is homozygotic or hemizygotic for the MATa allele has the MATa mating type. A multiploid cell that is heterozygotic for MAT a and MA 'Ta. generally does not mate. Two cells both having mating type MA Ta cannot mate with one another, nor can two cells having mating type MA Ta. However, a cell having mating type MA Ta can mate with a cell having mating type MA Ta. For instance, a haploid cell having mating type MATa can mate with a haploid cell having mating type MA Ta to provide a diploid cell heterozygous at the MA T locus.

[0033] Hence, in embodiments wherein the microbial species comprises S. cerevisiae, the first mating type may comprise mating type MATa and the second mating type may comprise mating type MA Ta. Alternatively, in further embodiments, the first mating type may comprise mating type MATa and the second mating type may comprise mating type MATa.

[0034] It will be clear to the person skilled in the art that the generation of homozygous gene drive alleles (or “split drive alleles”) involves homology directed repair. Hence, the method of the invention may be applied to microbial species capable of homology directed repair, which species may include the vast majority of microbial species. Preferably, the microbial species may be efficient at homology directed repair, such as predominantly repair DNA cleavage with homology directed repair over non-homologous end joining (NHEJ). Hence, in embodiments, the microbial species, especially the first cell and the second cell of the microbial species, may be capable of homology directed repair.

[0035] The method of the invention may especially comprise one or more mating cycles, wherein each mating cycle comprises a preparation stage, a mating stage, and a selection stage. In short, the preparation stage may comprise providing suitable parent cells, the mating stage may comprise mating between the parent cells to provide an offspring cell population, and the selection stage may comprise selecting a suitable offspring cell from the offspring cell population based on genetic markers, especially wherein the offspring cell is suitable for use as a parent cell in the preparation stage of a later mating cycle. In embodiments, the preparation stage may comprise providing a first cell (or “first parent cell”) and a second cell (or “second parent cell”) of the microbial species. The first cell and the second cell may thus both belong to the same microbial species. The first cell and the second cell may both be recombinant cells, i.e., both may comprise one or more genetic modifications relative to a wildtype cell of the microbial species. In particular, both the first cell and the second cell may comprise a plurality of genetic modifications relative to a wildtype cell of the microbial species (also see below). The terms “first cell” and “second cell” may herein also refer to a plurality of first cells and a plurality of second cells, respectively.

[0036] The first cell and the second cell may both have a first locus, a second locus, a third locus, a fourth locus, and a fifth locus.

[0037] The phrase “the first cell and the second cell may both have a first locus” and similar phrases used herein indicate that both the first cell and the second cell have a same specific (physical) location in their genetic code, such as a specific region of a chromosome, e.g., a specific gene-coding region. Optionally, the first cell and the second cell have the same allele at the first locus. Alternatively, the first cell and the second cell may have different alleles at the first locus.

[0038] The term “locus” may herein refer to a location in a chromosome, especially a natural chromosome, or especially in a synthetic chromosome, or in a plasmid. The term “chromosomal locus” may herein specifically refer to a location in a chromosome, especially in a natural chromosome, or especially in a synthetic chromosome. The term “natural chromosome” may herein refer to a chromosome present in wildtype cells of the microbial species. The term “synthetic chromosome” may herein refer to a chromosome that does not occur naturally and was made by humans, e.g., a chromosome built from synthetic building blocks, e.g., from DNA fragments isolated from purified DNA from other organisms and replicated by PCR. The term “plasmidic locus” may herein specifically refer to a location in a plasmid.

[0039] The term “allele” may herein refer to one of two or more versions of a DNA sequence (a single base or a segment of bases) at a given locus, i.e., at a given location in the genetic code of an organism.

[0040] In embodiments, at least the fourth locus and the fifth locus may be chromosomal loci. The fourth and fifth loci being chromosomal loci may facilitate providing haploid offspring cells carrying only a single allele for each locus, i.e., only a single genetic marker for these loci. Thereby, the two-marker system may be maintained, thereby facilitating multiple iterations of the mating cycle. The first locus and the second locus may especially correspond to targets locations for a knock-out or a knock-in. Hence, the first locus and the second locus may be selected in view of a (strain) engineering goal. Typically, the target loci may be chromosomal loci. Hence, in embodiments, the chromosomal loci may (further) include the first locus and / or the second locus, especially the first locus and the second locus. However, the target loci may also include plasmidic loci. Hence, in further embodiments, the first locus and / or the second locus may be plasmidic loci. Especially, (both) the first locus and the second locus may be plasmidic loci.

[0041] The third locus may encode an RNA-guided DNA endonuclease (also see below). In view of biosafety (regulations), it may be preferred for the RNA-guided DNA endonuclease not to spread through a wildtype population as a gene drive would, also considering the guide RNAs would replicate themselves as long as a corresponding nuclease is available, but rather based on Mendelian genetics, which may mitigate potential risks associated to accidental release. In particular, autonomous gene drive elements may require that all necessary elements (Cas nuclease gene, CRISPR array(s) and homologous regions) are subjected to homing activity. However, the homing activity itself may only require the presence of a CRISPR-Cas ribonucleoprotein complex, i.e., the guide RNA and the nuclease may be separately provided, such as in separate loci. Splitting the sequence encoding the Cas nuclease and the genomic region subjected to homing activity provides a so-called “split gene drive system” (systems where the genomic sequence subjected to homing activity lacks one or more required components), which may be preferred for limiting, especially preventing, propagation of the gene drive in natural populations upon an accidental release.

[0042] In embodiments, the third locus may (also) comprise a chromosomal locus, which may provide a more stable performance and may avoid or reduce plasmidic burden in the offspring cell. A chromosomal third locus may be particularly suitable for embodiments of the method involving iterative mating cycles.

[0043] In further embodiments, the third locus may comprise a plasmidic locus, which may be beneficial in terms of biosafety (regulations) as micro-organisms may relatively quickly shed plasmids providing no competitive advantage, especially plasmids providing a competitive disadvantage, in the absence of a selective pressure.

[0044] Hence, in embodiments, the first locus, the second locus, the fourth locus, and the fifth locus may be chromosomal loci, especially wherein the third locus is a plasmidic locus. In further embodiments, the first locus, the second locus, the third locus, the fourth locus, and the fifth locus may be chromosomal loci. The first locus of the first cell may encode a first guide RNA. The first guide RNA may be configured to hybridize with a first target (DNA) sequence, especially wherein the first locus of the second cell comprises the first target sequence. Similarly, the second locus of the second cell may encodes a second guide RNA. The second guide RNA may be configured to hybridize with a second target sequence, especially wherein the second locus of the first cell comprises the second target sequence.

[0045] The phrase “a locus may encode X” and similar phrases used herein may refer to the locus containing a DNA sequence encoding for X, wherein X is obtainable via transcription of the DNA sequence , optionally transcription followed by translation or pre crRNA processing (see below). For instance, if transcription of the DNA sequence results in a (functional) RNA molecule, such as in a guide RNA, or such as in an mRNA molecule, the locus encodes the RNA molecule. Further, if transcription of the DNA sequence results in an mRNA molecule and subsequent translation of the mRNA molecule results in a protein, the locus (also) encodes the protein. Similarly, if transcription of the DNA sequence results in a pre-crRNA and subsequent processing of the pre-crRNA results in a guide RNA, the locus encodes the guide RNA.

[0046] The term “guide RNA” may herein refer to an RNA molecule configured (a) to functionally associate with an RNA-guided DNA endonuclease, and (b) to hybridize with a target DNA sequence. In particular, the guide RNA, especially a spacer region of the guide RNA (see below), may be configured to hybridize with the target DNA sequence along a complementary region. In further embodiments, especially wherein the RNA-guided DNA endonuclease comprises a Cas9 endonuclease, the guide RNA may comprise a single guide RNA (sgRNA).

[0047] The third locus of the first cell and / or the third locus of the second cell may encode an RNA-guided DNA endonuclease, especially an RNA-guided dsDNA endonuclease. In embodiments, at least the third locus of the first cell may encode the RNA-guided DNA endonuclease. In further embodiments, at least the third locus of the second cell may encode the RNA-guided DNA endonuclease. Especially, the third locus of (both of) the first cell and the third locus of the second cell may encode the RNA-guided DNA endonuclease. The term “RNA-guided DNA endonuclease” may herein refer to an endonuclease that may functionally associate with a guide RNA, especially a crRNA, which may guide the endonuclease to a target DNA sequence, wherein the endonuclease is configured to subject the target DNA sequence to endonuclease activity, i.e., wherein the endonuclease is configured to cleave the target DNA sequence. The term “RNA-guided dsDNA endonuclease” may herein specifically refer to an endonuclease configured to cleave double stranded DNA. The RNA-guided DNA endonuclease may especially be configured to functionally associate with the first guide RNA and (separately) with the second guide RNA.

[0048] In particular, the first guide RNA and the RNA-guided DNA endonuclease (see below) may be configured to functionally associate to provide a first ribonucleoprotein. The first ribonucleoprotein may be configured to (locate and) cleave a DNA strand comprising the first target sequence. Hence, when the first ribonucleoprotein and the first locus of the second cell come in contact - after mating between the first cell and the second cell - the first ribonucleoprotein may cleave the first locus that originated from the second cell.

[0049] Similarly, the second guide RNA and the RNA-guided DNA endonuclease may be configured to functionally associate to provide a second ribonucleoprotein. The second ribonucleoprotein may be configured to (locate and) cleave a DNA strand comprising the second target sequence.

[0050] In principle, in embodiments wherein the third locus of both the first cell and the second cell encode an RNA-guided DNA endonuclease, two different RNA-guided DNA endonuclease may be encoded, i.e., the third locus of the first cell encodes a first RNA-guided DNA endonuclease and the third locus of the second cell encodes a second RNA-guided endonuclease. However, generally, the same endonuclease may be used as this may facilitate providing consistent results across parallelly and successively executed mating cycles. Hence, in embodiments, the third locus of the first cell and the third locus of the second cell encode (essentially) the same RNA-guided DNA endonuclease.

[0051] In further embodiments, the RNA-guided DNA endonuclease may be selected from the group comprising a Cas9 endonuclease and a Casl2a endonuclease, especially a Cas9 endonuclease, or especially a Casl2a endonuclease. The Cas9 endonuclease may be a naturally occurring Cas9 protein or an engineered version. Similarly, the Casl2a endonuclease may be a naturally occurring Cast 2a protein or an engineered version.

[0052] In embodiments, the RNA-guided DNA endonuclease may comprise a Cas9 endonuclease. In further embodiments, a Cas9 amino acid sequence of the Cas9 endonuclease may have at least 5% sequence identity to SEQ ID NO: 1 with respect to a (Cas9) sequence alignment between the Cas9 amino acid sequence and SEQ ID NO: 1, such as 10% or more, 20% or more, 30% or more, 40% or more, or at least 50% sequence identity, especially wherein the sequence alignment has a length of at least 80% of a sequence length of SEQ ID NO: 1. In further embodiments, the Cas9 amino acid sequence may have at least 60% sequence identity with the Cas9 amino acid sequence of SEQ ID NO: 1, such as at least 70% sequence identity, especially > 80%, such as > 85%, especially > 90%, such as > 93%, especially > 95%, such as > 97%, including 100% sequence identity, with respect to a sequence alignment between the Cas9 amino acid sequence and SEQ ID NO: 1. The sequence alignment may especially have a length > 50% of the sequence length of SEQ ID NO: 1, such as > 60%, especially > 70%, such as > 80%, especially > 90%, such as > 99%, including 100%. In particular, the Cas9 endonuclease may be an RNA-guided DNA endonuclease, especially with properties similar to SEQ ID NO: 1.

[0053] In embodiments, the RNA-guided DNA endonuclease may comprise a Cast 2a endonuclease. In further embodiments, a Cast 2a amino acid sequence of the Cast 2a endonuclease may have at least 5% sequence identity to SEQ ID NO:2 with respect to a (Casl2a) sequence alignment between the Casl2a amino acid sequence and SEQ ID NO:2, such as 10% or more, 20% or more, 30% or more, or at least 40% sequence identity, especially wherein the sequence alignment has a length of at least 80% of a sequence length of SEQ ID NO:2, such as at least 90%. In further embodiments, the Casl2a amino acid sequence may have at least 50% sequence identity with SEQ ID NO:2, especially at least 60% sequence identity, such as at least 70% sequence identity, especially > 80%, such as > 85%, especially > 90%, such as > 93%, especially > 95%, such as > 97%, including 100% sequence identity, with respect to a sequence alignment between the Casl2a amino acid sequence and SEQ ID NO:2. The sequence alignment may especially have a length > 40% of the sequence length of SEQ ID NO:2, especially > 50%, such as > 60%, especially > 70%, such as > 80%, especially > 90%, such as > 99%, including 100%. In particular, the Casl2a endonuclease may be an RNA-guided DNA endonuclease, especially with properties similar to SEQ ID NO:2. In further embodiments, the Cast 2a amino acid sequence may have at least 40% sequence identity with the Casl2a reference amino acid sequence of SEQ ID NO:2 with respect to a sequence alignment between the Casl2a amino acid sequence and SEQ ID NO:2, wherein the sequence alignment has a length > 80% of the sequence length of SEQ ID NO:2, such as > 90%.

[0054] Amino acid sequence alignments may especially be obtained using BLASTp at the website of the National Center for Biotechnology Information (NCBI). Two sequences may be aligned via BLASTp, especially using default algorithm parameters, such as using a BLOSUM62 matrix with a gap cost of 11 : 1 (existence:extension).

[0055] In further embodiments, a sequence alignment obtained using BLASTp with default algorithm parameters between the Cas9 amino acid sequence of the Cas9 endonuclease and SEQ ID NO: 1 may have an E-value < 10'6especially < 10'5, such as < 10'4. In further embodiments, a sequence alignment obtained using BLASTp with default algorithm parameters between the Casl2a amino acid sequence of the Casl2a endonuclease and SEQ ID NO:2 may have an E-value < 10'6especially < 10'5, such as < 10'4.

[0056] The Casl2a endonuclease may be particularly suitable in the context of the method of the invention. In particular, the Casl2a protein may be configured to catalyze the maturation of pre-crRNA into crRNAs (see below), which may simplify practical applications as no separate protein may be required for pre-crRNA processing, and which may facilitate multiplexing the guide RNAs in CRISPR arrays. The multiplexing may include targeting a same allele with multiple guide RNAs, which may reduce the odds of ‘escapes’ due to the occurrence of non-homologous end joining (NHEJ) instead of homology directed repair. The multiplexing may further facilitate targeting multiple loci from a single locus with guide RNAs. For instance, the first locus may encode, besides the first guide RNA, an off-locus guide RNA, wherein the off-locus guide RNA is configured to hybridize with a target DNA sequence of a different locus, such as a mating-type locus (see below).

[0057] In particular, in embodiments, the first locus may comprise a first CRISPR array, wherein the first CRISPR array encodes at least the first guide RNA. Especially, the first CRISPR array may encode a plurality of (different) first guide RNAs, wherein each first guide RNA is configured to hybridize with the first target sequence or a variation thereof, e.g., accounting for degeneracy of the first target sequence at the first locus and / or accounting for NHEJ-events resulting in slight modifications at the first locus. Similarly, the second locus may encode a second CRISPR array, wherein the second CRISPR array encodes at least the second guide RNA. Especially, the second CRISPR array may encode a plurality of (different) second guide RNAs, wherein each second guide RNA may be configured to hybridize with the second target sequence or a variation thereof.

[0058] The term “CRISPR array” may herein refer to a sequence comprising identical sequences (“repeats”) that are separated by variable sequences (“spacers”). The CRISPR array may be transcribed to a pre-crRNA (“pre-CRISPR RNA”), which may be processed, especially by a Casl2a endonuclease, into a plurality of guide RNAs. In particular, each guide RNA from a pre-crRNA may comprise a repeat region corresponding to (at least part of) a repeat of the CRISPR array and a spacer region corresponding to a spacer of the CRISPR array, wherein the spacer region is configured for hybridizing with a (corresponding) target DNA sequence. Hence, the target DNA sequence(s) may be defined (at least partially) through the spacers included in the CRISPR array. Specifically, the spacer may be complementary to a so-called ‘protospacer’ and the RNA-guided DNA endonuclease will cleave a DNA sequence comprising the protospacer and an adjacent ‘protospacer adjacent motif (or “PAM”). Hence, the target DNA sequence(s) may be defined based on the spacer (i.e., complementary to the spacer) and the presence of a PAM-sequence. The PAM-sequence may vary for different RNA-guided DNA endonucleases. With respect to guide RNAs provided from a CRISPR array, the term “spacer region” may refer to a part of the guide RNA originating from a spacer in the CRISPR array.

[0059] As described above, the first cell and the second cell may further comprise a fourth locus. The fourth locus of the first cell may comprise a first mating-type promoter, especially controlling the expression of a first selectable marker providing a first selectable trait. The first mating-type promoter may be configured to facilitate expression of the first selectable marker in cells (of the microbial species) having the first mating type. Similarly, the fourth locus of the second cell may comprise a second mating-type promoter, especially controlling the expression of a second selectable marker providing a second selectable trait. The second mating-type promoter may be configured to facilitate expression of the second selectable marker in cells (of the microbial species) having the second mating type.

[0060] The term “promoter” may herein refer to a sequence of DNA to which proteins may bind to initiate transcription of an RNA transcript downstream of the promoter. The term “mating-type promoter” may herein specifically refer to a promoter configured to control the transcription downstream of the promoter in dependence of a mating type of the encompassing cell. For instance, a first mating-type promoter controlling expression of a first marker may be configured to (essentially) only express the first marker in cells of the first mating type, whereas a second mating-type promoter controlling expression of a second marker may be configured to (essentially) only express the second marker in cells of the second mating type.

[0061] For instance, for S. cerevisiae, the first mating type promoter may be selected from the group comprising pMFAl, pMFA2 and pSTE2. Further, in S. cerevisiae, the second mating type promoter may be selected from the group comprising pMFal, pMFa2, and pSTE3. As will be known to the person skilled in the art, the leading ‘p’ in, for example, pMFA2 and pSTE2 refers to the promoter of the mentioned genes, i.e., ‘pMFA2 ’ may refer to the promoter of Axe,MFA2 gene.

[0062] In further embodiments, the fifth locus of the first cell may comprise a third selectable marker providing a third selectable trait. Similarly, the fifth locus of the second cell may comprise a fourth selectable marker providing a fourth selectable trait.

[0063] The term “selectable marker” may herein refer to a gene that, when expressed, provides a protein conferring a trait suitable for (artificial) selection. Specifically, cells comprising the selectable marker may be distinguished from cells not comprising the selectable marker by exposing the cells to an (artificial) selective pressure. For instance, a selectable marker encoding a protein providing resistance to an antibiotic, thereby providing an antibiotic resistance trait, may facilitate selection of cells comprising that marker by exposing the cells to the antibiotic. Similarly, a selectable marker encoding an enzyme complementing an auxotrophy, thereby providing a prototrophic trait, may facilitate selection of cells comprising that marker by cultivating the cells in a growth medium lacking a nutrient corresponding to the auxotrophy.

[0064] In principle, multiple distinct selectable markers could provide the same selectable trait. For instance, the resistance to an antibiotic may have evolved separately multiple times and, similarly, multiple distinct enzymes may complement an auxutrophy, optionally even by catalyzing different reactions. However, for distinguishing between the parental cells, multiploid offspring cells, and haploid offspring cells of the method of the invention, and for facilitating multiple successive iterations of the mating cycle, four distinct selectable traits may be required.

[0065] Hence, in embodiments, the first selectable trait, the second selectable trait, the third selectable trait and the fourth selectable trait may differ from each other, i.e., each selectable trait may be distinct from each other selectable trait.

[0066] Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequential orders than explicitly described or illustrated herein. Specifically, in the context of successive mating cycles (see below), it will be clear to the person skilled in the art that the first cell and the second cell of each mating cycle each comprise two distinct selectable markers in the fourth locus and the fifth locus such that the cells together comprise four different selectable markers with four different selectable traits in the fourth locus and the fifth locus. It will be clear to the person skilled in the art that the selectable markers used in successive mating cycles are not necessarily the same selectable markers, i.e., the third selectable marker of an earlier mating cycle may differ from the third selectable marker used in a later mating cycle.

[0067] Hence, in embodiments, in each mating cycle, each of (a) the fourth locus of the first cell, (b) the fourth locus of the second cell, (c) the fifth locus of the first cell, and (d) the fifth locus of the second cell, may comprise a selectable marker different from the rest of (a) the fourth locus of the first cell, (b) the fourth locus of the second cell, (c) the fifth locus of the first cell, and (d) the fifth locus of the second cell. Specifically, each selectable marker of (a) the fourth locus of the first cell, (b) the fourth locus of the second cell, (c) the fifth locus of the first cell, and (d) the fifth locus of the second cell may provide a selectable trait different from the other selectable markers.

[0068] In further embodiments, especially in each mating cycle, the selectable traits of the first selectable marker, the second selectable marker, the third selectable marker and the fourth selectable marker (or “the first selectable trait, the second selectable trait, the third selectable trait and the fourth selectable trait) are (each) selected from the group comprising antibiotic resistance traits, morphological traits, optically observable traits, apoptosis induction traits, growth inhibition traits, and prototrophic traits, such as from the group consisting of antibiotic resistance traits, morphological traits, optically observable traits, apoptosis induction traits, growth inhibition traits, and prototrophic traits, especially from the group consisting of antibiotic resistance traits and prototrophic traits. Antibiotic resistance traits and prototrophic traits may be particularly convenient as they provide a competitive advantage under a selective pressure, which may facilitate selecting suitable cells.

[0069] For instance, in specific embodiments, wherein the microbial species comprises S. cerevisiae, the selectable traits of the first selectable marker, the second selectable marker, the third selectable marker and the fourth selectable marker are selected from the group comprising uracil auxotrophy, uracil prototrophy, lysine prototrophy, leucine prototrophy, tryptophan prototrophy, methionine prototrophy, histidine prototrophy, hygromycin resistance, nourseothricin resistance, zeocin resistance, kanamycin and G418 resistance, especially from the group comprising uracil prototrophy, lysine prototrophy, leucine prototrophy, tryptophan prototrophy, methionine prototrophy, histidine prototrophy, hygromycin resistance, nourseothricin resistance, zeocin resistance, kanamycin and G418 resistance.

[0070] As describe above, the mating cycle may further comprise a mating stage. The mating stage may comprise bringing the first cell and the second cell in contact (with each other) to provide an offspring cell population. For instance, the mating stage may comprise providing the first cell and the second cell, especially a plurality of first cells and a plurality of second cells, to the same location on an agar plate, e.g., on a plate comprising yeast extract peptone dextrose medium (also “YEPD medium” or “YPD medium”. Alternatively, the mating stage may comprise providing the first cell and the second cell, especially a plurality of first cells and a plurality of second cells, into a (suitable) liquid growth medium. It will be clear to the person skilled in the art that the conditions for mating and the time needed for mating, and optionally for growth of thereby generated multiploid cells, may vary for different microbial species. For instance, for S. cerevisiae, mating of cells of compatible mating types may be essentially spontaneous and may occur in (nutrient)-poor or (nutrient-)rich growth media, although the mating may be more efficient in rich growth media. In contrast, Schizosaccharomyces pombe may require starvation conditions for sexual differentiation and mating.

[0071] In embodiments, the mating stage may further comprise providing suitable growth conditions for (multiploid cells of) the microbial species. Further, the mating stage may comprise allowing an offspring cell population to grow, such as into a (visible) colony on an agar plate, or such as into a suitable cell count in a liquid culture, e.g., determined based on optical density.

[0072] The offspring cell population, as provided via the mating cycle, may comprise a multiploid cell population. In particular, a multiploid cells in this multiploid cell population may result from mating between a first cell and a second cell. The multiploid cell may thus comprise the chromosomes and, if applicable, plasmids of both the first cell and the second cell. The multiploid cell may, initially, be heterozygous for (at least) the first locus, the second locus, the fourth locus and the fifth locus. However, as the allele of the first locus originating from the first cell contains a guide RNA targeting the allele of the first locus originating from the second cell, and as the third locus encodes an RNA-guided DNA endonuclease, the multiploid cell may become homozygous for the first locus following cleavage of the allele originating from the second cell and subsequent homology directed repair with the allele originating from the first cell as repair template. Similarly, the multiploid cell may become homozygous for the second locus following cleavage and subsequent homology directed repair at the allele originating from the first cell. Following the homing activity from the first and second loci, the multiploid cell may thus be (a) homozygous for at least the first locus and the second locus and (b) heterozygous for at least the fourth locus and for the fifth locus.

[0073] The selection stage (of the mating cycle) may comprise selecting the offspring cell from the offspring cell population or from cells derived therefrom. The offspring cell may be a multiploid offspring cell, which may generally be directly selected from the multiploid cell population, or may be a haploid offspring cell selected following sporulation of a multiploid offspring cell from the offspring cell population.

[0074] In particular, a multiploid cell originating from a successful mating event may be selected based on the third selectable trait and the fourth selectable trait. As the third selectable trait originates from the third selectable marker of the first cell and the fourth selectable trait originated from the fourth selectable marker of the second cell, a combined selective pressure based on the third and fourth selectable traits specifically selects multiploid cells resulting from a successful mating event. Of note, as a multiploid generally (see exception below) does not having a mating type, the multiploid offspring cell will not have the first selectable trait or the second selectable trait due to the corresponding mating type promoters selectively controlling expression in dependence of the mating type of the cell.

[0075] Further, an offspring cell from a successful mating event may be selected based on (a) the first selectable trait and the fourth selectable trait or (b) the second selectable trait and the third selectable trait. As the first and third selectable traits originate from the first cell and as the second and fourth selectable traits originate from the second cell, the combination of first and fourth selectable trait and the combination of second and third selectable trait specifically selecting for offspring cells resulting from successful mating, wherein the offspring cells have the first mating type or the second mating type, respectively.

[0076] In particular, in embodiments, the selecting stage may comprise initiating sporulation in a multiploid offspring cell, optionally selected based on the third selectable trait and the fourth selectable trait, to provide a haploid offspring cell population. A haploid offspring cell originating from a multiploid cell resulting from a successful mating event may be selected from the haploid cell population based on (a) the first selectable trait and the fourth selectable trait or (b) the second selectable trait and the third selectable trait. In particular, by selecting the haploid offspring cell based on the first selectable trait a haploid offspring cell having the first mating type is selected. Similarly, by selecting the haploid offspring cell based on the second selectable trait, a haploid offspring cell having the second mating type is selected.

[0077] The term “cells derived therefrom” in the phrase “the offspring cell population or cells derived therefrom” may especially refer to cells from the haploid offspring cell population. Additionally, the term “cells derived therefrom” may refer to cells cultivated from cells isolated from the offspring cell population.

[0078] Hence, in embodiments, the selection stage may comprise selecting the offspring cell based on (a combination of) at least two selectable traits, wherein the at least two selectable traits comprise one or more of (a) at least the third selectable trait and the fourth selectable trait, (b) at least the first selectable trait and the fourth selectable trait, and (c) at least the second selectable trait and the third selectable trait.

[0079] Generally, in embodiments wherein the offspring cell comprises a haploid offspring cell, it may be preferred to first select the multiploid offspring cell based on the third selectable trait and the fourth selectable trait, to initiate sporulation in the multiploid offspring cell, and to subsequently select the haploid offspring cell based on (a) the first selectable trait and optionally the fourth selectable trait, and (b) the second selectable trait and optionally the third selectable trait. The first selection may facilitate an initial enrichment of multiploid offspring cells, which may subsequently grow in the absence of competition from other cells, e.g., non-mated first cells and second cells, and the second selection may facilitate selecting specific haploid offspring cells for a later mating cycle. Such a double selection in the selection stage may reduce the likelihood of the (unlikely) selection of a cell not resulting from a successful mating between the first cell and the second cell. Hence, in embodiments, the selection stage may comprise (a) selecting a multiploid offspring cell based on the third selectable trait and the fourth selectable trait, (b) initiating sporulation in the multiploid offspring cell to provide a haploid offspring cell population, and (c) selecting a haploid offspring cell from the haploid offspring cell population based on (cl) the first selectable trait, especially based on the first selectable trait and the fourth selectable trait, or (c2) the second selectable trait, especially based on the second selectable trait and the third selectable trait.

[0080] As described above, the fourth locus of the first cell and of the second cell may comprise a mating-type promoter controlling the expression of the corresponding selectable marker. It will be clear to the person skilled in the art that the other loci may also contain promoters upstream of their coding sequences to facilitate the transcription of the coding sequence. Especially the other loci may contain non-mating type promoters. Further, the loci may comprise terminators arranged downstream of the coding sequences configured to stop transcription. In particular, the loci may contain so-called ‘expression cassettes’ containing a promoter, a coding sequence (or “open reading frame”), and a terminator, wherein the coding sequence encodes the corresponding gRNA and / or protein.

[0081] The terms “upstream” and “downstream” may relate to an arrangement of genetic features, especially relative to the reading direction of a coding sequence, or especially to the thereby encoded amino acid sequence. Hence, an element arranged at the 5’ end relative to another genetic feature (especially with respect to the reading direction of a gene) may be referred to as “upstream”, such as a promotor being upstream of the respective gene, whereas an element arranged at the 3’ end relative to another genetic feature, such as a terminator being downstream of the respective gene, may be referred to as “downstream”.

[0082] In embodiments, the first locus of the first cell may contain a first expression cassette comprising a first coding sequence. The first coding sequence may encode the first gRNA, especially the first CRISPR array. The first expression cassette may further contain a first promoter arranged upstream of the first coding sequence and / or a first terminator arranged downstream from the first coding sequence, especially both the first promoter and the first terminator. In further embodiments, the first promoter may be a constitutive promoter. In further embodiments, the first promoter may be an inducible promoter.

[0083] The term “constitutive promoter” may herein refer to a promoter configured to facilitate continual transcription of a coding sequence downstream of the promoter, i.e., the promoter may essentially be unregulated. The person skilled in the art will be capable of selecting suitable constitutive promoters for the microbial species.

[0084] The term “inducible promoter” may herein refer to a promoter that (only) facilitates transcription of a coding sequence downstream of the promoter in dependence of an external stimulus, i.e., the promoter may be regulated in dependence of an external stimulus. The external stimulus may, for instance, be the presence of a specific compound, such a specific nutrient, in the extracellular environment, such as the presence of Isopropyl P-D-l -thiogalactopyranoside (IPTG) to stimulate the inducible lac promoter. The stimulus may further be an environmental temperature or light, especially light of a specific wavelength. The person skilled in the art will be capable of selecting suitable inducible promoters for the microbial species.

[0085] In further embodiments, the second locus of the second cell may contain a second expression cassette comprising a second coding sequence. The second coding sequence may encode the second gRNA, especially the second CRISPR array. The second expression cassette may further contain a second promoter arranged upstream of the second coding sequence and / or a second terminator arranged downstream from the second coding sequence, especially both the second promoter and the second terminator. In further embodiments, the second promoter may be a constitutive promoter. In further embodiments, the second promoter may be an inducible promoter.

[0086] In further embodiments, the third locus of the first cell may contain a third expression cassette comprising a third coding sequence. The third coding sequence may encode the RNA-guided DNA endonuclease. The third expression cassette may further contain a third promoter arranged upstream of the third coding sequence and / or a third terminator arranged downstream from the third coding sequence, especially both the third promoter and the third terminator. In further embodiments, the third promoter may be a constitutive promoter. In further embodiments, the third promoter may be an inducible promoter.

[0087] In further embodiments, the third locus of the second cell may contain a secondary third expression cassette comprising a secondary third coding sequence. The secondary third coding sequence may encode the RNA-guided DNA endonuclease. The secondary third expression cassette may further contain a secondary third promoter arranged upstream of the secondary third coding sequence and / or a secondary third terminator arranged downstream from the secondary third coding sequence, especially both the secondary third promoter and the secondary third terminator. In further embodiments, the secondary third promoter may be a constitutive promoter. In further embodiments, the secondary third promoter may be an inducible promoter.

[0088] In further embodiments, the fourth locus of the first cell may contain a fourth expression cassette comprising the first mating-type promoter and a fourth coding sequence downstream from the first mating-type promoter. The fourth coding sequence may comprise the first selectable marker. The fourth expression cassette may further contain a fourth terminator arranged downstream from the fourth coding sequence.

[0089] In further embodiments, the fourth locus of the second cell may contain a secondary fourth expression cassette comprising the second mating-type promoter and a secondary fourth coding sequence downstream from the second mating-type promoter. The secondary fourth coding sequence may comprise the second selectable marker. The fourth expression cassette may further contain a secondary fourth terminator arranged downstream from the secondary fourth coding sequence.

[0090] In further embodiments, the fifth locus of the first cell may contain a fifth expression cassette comprising a fifth coding sequence. The fifth coding sequence may comprise the third selectable marker. The fifth expression cassette may further contain a fifth promoter arranged upstream of the fifth coding sequence and / or a fifth terminator arranged downstream from the fifth coding sequence, especially both the fifth promoter and the fifth terminator. In further embodiments, the fifth promoter may be a constitutive promoter. In further embodiments, the fifth promoter may be an inducible promoter.

[0091] In further embodiments, the fifth locus of the second cell may contain a secondary fifth expression cassette comprising a secondary fifth coding sequence. The secondary fifth coding sequence may comprise the third selectable marker. The secondary fifth expression cassette may further contain a secondary fifth promoter arranged upstream of the secondary fifth coding sequence and / or a secondary fifth terminator arranged downstream from the secondary fifth coding sequence, especially both the secondary fifth promoter and the secondary fifth terminator. In further embodiments, the secondary fifth promoter may be a constitutive promoter. In further embodiments, the secondary fifth promoter may be an inducible promoter. As described above, the offspring cell selected during the selection stage may especially be suitable for use in another mating cycle, thereby facilitating accumulating desired genetic modifications in a (further) offspring cell.

[0092] Hence, in embodiments, the method may comprise a plurality of mating cycles. Especially, the offspring cell of an earlier mating cycle is used as the first cell or as the second cell in a later mating cycle. In specific embodiments, the earlier mating cycle may directly precede the later mating cycle. Especially, in embodiments, the offspring cell of the mating cycle as defined above may be used as the first cell or as the second cell in a second mating cycle.

[0093] For instance, in embodiments, the preparation stage of the later mating cycle may comprise providing the offspring cell of the earlier mating cycle and a third cell of the microbial species. The third cell may (also) have the first locus, the second locus, the third locus, the fourth locus, and the fifth locus. Further, the offspring cell of the earlier mating cycle and the third cell may (both) have a sixth locus. Hence, the offspring cell of the earlier mating cycle and the third cell may have the first locus, the second locus, the third locus, the fourth locus, the fifth locus, and the sixth locus, especially wherein at least the fourth locus and the fifth locus are chromosomal loci. Further, the offspring cell of the earlier mating cycle and the third cell may differ in mating type. In embodiments, the first locus of the third cell may comprise the first target sequence. Similarly, in embodiments, the second locus of the third cell may comprise the second target sequence. In further embodiments, the sixth locus of the third cell may encode a third guide RNA, wherein the third guide RNA is configured to hybridize with a third target sequence. Especially, the sixth locus of the offspring cell of the earlier mating cycle may comprise the third target sequence. The mating stage of the later mating cycle may comprise bringing the offspring cell of the earlier mating cycle in contact with the third cell to provide an offspring cell population of the later mating cycle.

[0094] Hence, in specific embodiments, in the later mating cycle: the preparation stage may comprise providing the offspring cell of the earlier mating cycle and a third cell of the microbial species, wherein the third cell has the first locus, the second locus, the third locus, the fourth locus, and the fifth locus, and wherein the offspring cell of the earlier mating cycle and the third cell have a sixth locus, wherein the offspring cell and the third cell differ in mating type, wherein: the first locus of the third cell comprises the first target sequence; the second locus of the third cell comprises the second target sequence; and the sixth locus of the third cell encodes a third guide RNA, wherein the third guide RNA is configured to hybridize with a third target sequence, wherein the sixth locus of the offspring cell of the earlier mating cycle comprises the third target sequence; and the mating stage comprising bringing the offspring cell of the earlier mating cycle in contact with the third cell to provide an offspring cell population of the later mating cycle.

[0095] In further embodiments, the sixth locus of the third cell may contain a sixth expression cassette comprising a sixth coding sequence. The sixth coding sequence may encode the sixth gRNA, especially the sixth CRISPR array. The sixth expression cassette may further contain a sixth promoter arranged upstream of the sixth coding sequence and / or a sixth terminator arranged downstream from the sixth coding sequence, especially both the sixth promoter and the sixth terminator. In further embodiments, the sixth promoter may be a constitutive promoter. In further embodiments, the sixth promoter may be an inducible promoter.

[0096] In particular, in the later mating cycle, the offspring cell of the earlier mating cycle and the third cell may be used as first cell and as second cell, respectively, or vice versa. In particular, the offspring cell of the earlier mating cycle may have a locus encoding a guide RNA suitable to target the corresponding locus in the third cell and the third cell may have a locus encoding a guide RNA suitable to target the corresponding locus in the offspring cell of the earlier mating cycle.

[0097] Further, as also described above, the offspring cell of the earlier mating cycle and the third cell may each encode two distinct genetic markers in the fourth locus and the fifth locus. In particular, the fourth locus of the offspring cell of the earlier mating cycle, the fourth locus of the third cell, the fifth locus of the offspring cell of the earlier mating cycle, and the fifth locus of the third cell differ from each other in selectable markers, wherein the encoded selectable markers differ from each other in selectable traits.

[0098] In embodiments, the selection stage of the earlier mating cycle may comprise selecting a haploid offspring cell. Especially, the selection stage of the earlier mating cycle may comprise initiating sporulation in a multiploid offspring cell from the offspring cell population, especially in a plurality of offspring cells from the offspring cell population, to provide a haploid offspring cell population.

[0099] In further embodiments, the selection stage may comprise selecting the offspring cell from the haploid offspring cell population based on the first selectable trait and the fourth selectable trait. Selection based on the first selectable trait, which selectable marker is under control of a first mating type promoter, may provide a haploid offspring cell having the first mating type. Hence, in such embodiments, the preparation stage of the later mating cycle may comprise providing the haploid offspring cell of the earlier mating cycle as the first cell. In further embodiments, the selection stage may comprise selecting the offspring cell from the haploid offspring cell population based on the second selectable trait and the third selectable trait. Selection based on the second selectable trait, which selectable marker is under control of a second mating type promoter, may provide a haploid offspring cell having the second mating type. Hence, in such embodiments, the preparation stage of the later mating cycle may comprise providing the haploid offspring cell of the earlier mating cycle as the second cell.

[0100] Further, in such embodiments, the first cell and the second cell (in the earlier mating cycle) may especially be haploid. Especially, the first cell and the second cell of the later mating cycle, such as the (haploid) offspring cell of the earlier mating cycle and the third cell, may (also) be haploid. Hence, in such embodiments, the method may comprise providing haploid (parent) cells, mating to a diploid cell to accumulate the genetic modifications, and sporulating to provide haploid offspring cells, which may be suitable for use as parent cells in a future mating cycle.

[0101] In embodiments wherein the offspring cell population is a polyploid cell population (also see below), sporulation may result in multiploid offspring cells rather than in haploid offspring cells. For instance, sporulation of a polyploid S. cerevisiae cell having a ploidy of 4 (4n polyploidy), sporulation may typically result in diploid cells (2n). A second iteration of sporulation of these diploid cells may result in haploid cells. Hence, in embodiments, the selection stage may comprise executing one or more rounds of sporulation starting from one or more multiploid offspring cells of the offspring cell population to provide haploid offspring cells.

[0102] In further embodiments, the selection stage (of the earlier mating cycle) may comprise (a) selecting a multiploid offspring cell from the offspring cell population based on the third selectable trait and the fourth selectable trait; and (b) initiating sporulation in the multiploid offspring cell to provide a haploid offspring cell population; and (c) selecting the offspring cell from the haploid offspring cell population based on the first selectable trait or the second selectable trait. In such embodiments, successful mating between the first cell and the second cell (in the earlier mating cycle) may be confirmed through selection of a multiploid cell having the first selectable trait and the third selectable trait and, following sporulation, a haploid offspring cell of a desired mating type is selected, e.g., for use in a later mating cycle.

[0103] Sporulation in the selection stage may facilitate providing a haploid offspring cell for a subsequent mating cycle, which may facilitate maintaining a consistent performance across mating cycles as each mating cycle is started with two haploid cells. However, sporulation may be a time-intensive step and / or may typically require manual procedures, such as to prevent (direct) mating between compatible offspring cells. .For instance, tetrad dissection may be used to select haploid offspring cells, but may be a time-intensive and manual process, which may complicate or even prevent automation of successive mating cycles. It may thus be beneficial to circumvent sporulation by using a multiploid offspring cell for further mating cycles. Generally, however, multiploid cells may not have a mating type and may be unsuitable for mating. For instance, a diploid S. cerevisiae cell obtained from mating a haploid MA T cell and a haploid MA Ta cell will be heterozygous for the MAT gene and will not mate.

[0104] In embodiments, the method of the invention may provide multiploid offspring cells suitable for mating. Specifically, one of the gRNA-encoding loci may encode an additional mating-type gRNA configured to cleave a mating type gene. Following homology directed repair, the multiploid offspring cell may then be heterozygous for the mating type gene and may be suitable for mating. For instance, in the context of S. cerevisiae, a gRNA may be configured to (selectively) hybridize with the MA T locus (and not with the MA 'Ta locus) such that a diploid cell heterozygous for the MA T gene becomes homozygous for MA 'Ta following cleavage and homology directed repair using the MA 'Ta locus as a template.

[0105] Sporulation and subsequent selection may, however, take multiple days depending on the microbial species. For instance, for S. cerevisiae strain CEN.PK, sporulation may take about 3 days and may typically be the longest step in strain engineering. In further embodiments of the method of the invention, a mating-type locus is thus targeted to cause mating-type switching, thereby allowing the multiploid yeast cell to mate and circumventing the need for sporulation.

[0106] Hence, in embodiments, the first cell and the second cell may (further) have a mating-type locus, especially a chromosomal mating-type locus. Especially, the first locus of the first cell may (further) encode a first mating-type guide RNA, wherein the first mating-type guide RNA is configured to hybridize with a second mating-type target sequence. In such embodiments, the chromosomal mating-type locus of the second cell may comprise the second mating-type target sequence. Thereby, multiploid offspring cells having the first mating type may be provided. Alternatively, the second locus of the second cell may (further) encode a second mating-type guide RNA, wherein the second mating-type guide RNA is configured to hybridize with a first mating-type target sequence. In such embodiments, the chromosomal mating-type locus of the first cell may comprise the first mating-type target sequence. Thereby, multiploid offspring cells having the second mating type may be provided. For instance, in S. cerevisiae, the mating-type locus may be the MA T locus. The MAT locus may correspond to the locus with ID SGD:S000029699 in the Saccharomyces Genome Database (SGD).

[0107] It may, especially in the above context, be relatively challenging to include a coding sequence for the mating-type guide RNA directly in the mating-type locus (of the first cell or of the second cell), also given that the intention may be to preserve the function of a mating-type gene, e.g., of MA T or of MATa. Hence, in further embodiments, the first locus and the second locus may be different from the mating-type locus. In further embodiments, the first locus may be the mating-type locus. Alternatively, in further embodiments, the second locus may be the mating-type locus.

[0108] In specific embodiments, the selection stage of the earlier mating cycle may comprise selecting the offspring cell from the offspring cell population based on the third selectable trait and the fourth selectable trait, wherein the offspring cell comprises a multiploid offspring cell, and wherein: (I) the first locus of the first cell further encodes a first mating-type guide RNA, wherein the first mating-type guide RNA is configured to hybridize with a second mating-type target sequence, wherein a chromosomal mating-type locus of the second cell comprises the second mating-type target sequence, wherein the offspring cell of the earlier mating cycle has the first mating type, wherein the offspring cell of the earlier mating cycle is used as the first cell in the later mating cycle; or (II) the second locus of the second cell further encodes a second mating-type guide RNA, wherein the second mating-type guide RNA is configured to hybridize with a first mating-type target sequence, wherein a chromosomal mating-type locus of the first cell comprises the first mating-type target sequence, wherein the offspring cell of the earlier mating cycle has the second mating type, wherein the offspring cell of the earlier mating cycle is used as the second cell in the later mating cycle.

[0109] In specific embodiments, the selection stage of the earlier mating cycle may comprise selecting the offspring cell from the offspring cell population based on the third selectable trait and the fourth selectable trait, wherein the offspring cell comprises a multiploid offspring cell, and wherein: (I) the first locus of the first cell further encodes a first mating-type guide RNA, wherein the first mating-type guide RNA is configured to hybridize with a second mating-type target sequence, wherein a chromosomal mating-type locus of the second cell comprises the second mating-type target sequence, wherein the offspring cell of the earlier mating cycle has the first mating type, wherein the offspring cell of the earlier mating cycle is used as the first cell in the later mating cycle; or (II) the second locus of the second cell further encodes a second mating-type guide RNA, wherein the second mating-type guide RNA is configured to hybridize with a first mating-type target sequence, wherein a chromosomal mating-type locus of the first cell comprises the first mating-type target sequence, wherein the offspring cell of the earlier mating cycle has the second mating type, wherein the offspring cell of the earlier mating cycle is used as the second cell in the later mating cycle.

[0110] In such embodiments, the first locus of the first cell or the second locus of the second cell may encode a marker guide RNA targeting the fourth marker gene or the third marker gene, respectively, such that the resulting multiploid offspring cell will (also) be homozygous for the fifth locus, which may facilitate selection in further mating cycles.

[0111] The generation of the first (or second) mating-type guide RNA may be controlled by a constitutive or by an inducible promoter.

[0112] The use of a constitutive promoter for controlling the expression of the mating-type guide RNA may be simpler in use, but may result in multiple generations of mating within a single mating stage. For instance, when a population of haploid cells having mating type A are brought in contact with a population of haploid cells having mating type B, initially multiploid cells heterozygous at the mating locus, i.e., AB cells, may be provided. If these cells subsequently become homozygous at the mating locus (see above), e.g., if they become AA cells, these cells mag again mate with haploid B cells, initially resulting in AAB cells. This process may repeat until no B cells remain. Hence, in a single mating stage starting from haploid cells, multiploid cells having a ploidy of 3 or more (3+n) may be generated. Such polyploid cells may provide interesting insights in the effects of additional copy numbers of (exogeneous) genes.

[0113] The use of an inducible promoter for controlling the expression of the mating-type guide RNA may provide control over the timing of the formation of the guide RNA. Hence, in such embodiments, the guide RNA may be generated after the mating stage has ended, optionally after selection of successfully mated cells based on the third selectable trait and the fourth selectable trait, to control the chromosome copy number.

[0114] Similarly, the generation of the marker guide RNA may be controlled by a constitutive or by an inducible promoter. The constitutive promoter may be easier in use, but may complicate selection of multiploid cells resulting from successful mating if the cleavage of the targeted allele of the fifth locus occurs prior to the selection stage. Hence, in embodiments, the generation of the marker guide RNA may be controlled by an inducible promoter.

[0115] In particular, in embodiments, the preparation stage of the (earlier) mating cycle may comprise providing the first cell and the second cell such that: the first locus of the first cell further encodes a first mating-type guide RNA, wherein the first mating-type guide RNA is configured to hybridize with a second mating-type target sequence, wherein a chromosomal mating-type locus of the second cell comprises the second mating-type target sequence. In such embodiments, the selection stage of the (earlier) mating cycle may comprise selecting the offspring cell from the offspring cell population based on the third selectable trait and the fourth selectable trait, wherein the offspring cell comprises a multiploid offspring cell, and wherein the offspring cell of the earlier mating cycle has the first mating type. The multiploid offspring cell may especially be homozygous for a first mating type gene. Especially, the offspring cell of the earlier mating cycle may be used as the first cell in the later mating cycle.

[0116] In further embodiments, the first locus may comprise a first CRISPR array. The first CRISPR array may encode one or more of the first gRNA, the first mating-type guide RNA, and a first marker guide RNA, especially the first gRNA and the first mating-type guide RNA, or especially the first gRNA and the first marker guide RNA, or especially the first gRNA, the first mating-type guide RNA, and the first marker guide RNA. In further embodiments, the first locus may comprise a first promoter upstream of the CRISPR array, especially wherein the first promoter is an inducible promoter, or especially wherein the first promoter is a constitutive promoter.

[0117] In further embodiments, the first marker guide RNA may be configured to hybridize with a first marker target sequence, wherein the fifth locus of the second cell comprises the first marker target sequence.

[0118] Alternatively, in further embodiments, the preparation stage of the (earlier) mating cycle may comprise providing the second cell and the first cell such that: the second locus of the second cell further encodes a second mating-type guide RNA, wherein the second mating-type guide RNA is configured to hybridize with a first mating-type target sequence, wherein a chromosomal mating-type locus of the first cell comprises the first mating-type target sequence. In such embodiments, the selection stage of the (earlier) mating cycle may comprise selecting the offspring cell from the offspring cell population based on the third selectable trait and the fourth selectable trait, wherein the offspring cell comprises a multiploid offspring cell, and wherein the offspring cell of the earlier mating cycle has the second mating type. The multiploid offspring cell may especially be homozygous for a second mating type gene. Especially, the offspring cell of the earlier mating cycle may be used as the second cell in the later mating cycle.

[0119] In further embodiments, the second locus may comprise a second CRISPR array. The second CRISPR array may encode one or more of the second gRNA, the second mating-type guide RNA, and a second marker guide RNA, especially the second gRNA and the second mating-type guide RNA, or especially the second gRNA and the second marker guide RNA, or especially the second gRNA, the second mating-type guide RNA, and the second marker guide RNA. In further embodiments, the second locus may comprise a second promoter upstream of the CRISPR array, especially wherein the second promoter is an inducible promoter, or especially wherein the second promoter is a constitutive promoter.

[0120] In further embodiments, the second marker guide RNA may be configured to hybridize with a second marker target sequence, wherein the fifth locus of the first cell comprises the second marker target sequence.

[0121] The invention may thus facilitate providing a multiploid offspring cell as first cell or as second cell in the preparation stage of a mating cycle. In such embodiments, the multiploid offspring cell may especially be homozygous for at least the first locus and for the second locus. Preferably, the multiploid offspring cell may be homozygous for the first locus, the second locus, the fourth locus and the fifth locus.

[0122] Hence, in embodiments, the first cell may be homozygous or hemizygous for at least the first locus, the second locus, the fourth locus and the fifth locus. In further embodiments, the second cell may be homozygous or hemizygous for at least the first locus, the second locus, the fourth locus and the fifth locus.

[0123] As described above, a guide RNA may be configured to hybridize with a respective target sequence. In particular, the guide RNA, especially a spacer region of the guide RNA, may be configured to hybridize with the respective target sequence along a first complementary region. In embodiments, the first complementary region may have a length of at least k nucleotides, wherein k is at least 15, especially at least 16, such as at least 18, especially at least 19, such as at least 20. It will be clear to the person skilled in the art that the hybridization, and following cleavage, may not require the guide RNA and the respective target sequence to be fully complementary. In particular, in embodiments, the first guide RNA and the first target sequence may have at least k-2 complementary nucleotides in the first complementary region, especially at least k-1 complementary nucleotides, such as k complementary nucleotides.

[0124] In further embodiments, the first guide RNA may be configured to hybridize with the first target sequence along a first complementary region, wherein the first complementary region has a length of at least m nucleotides, wherein m is at least 18, such as at least 19, especially at least 20. In further embodiments, the first guide RNA and the first target sequence have at least m-2 complementary nucleotides in the first complementary region, such as at least m-1 complementary nucleotides, especially m complementary nucleotides. In embodiments wherein a first CRISPR array encodes a plurality of first guide RNAs, each first guide RNA may have at least m-3 complementary nucleotides in the first complementary region with the first target sequence, especially at least m-2 complementary nucleotides, such as at least m-1 complementary nucleotides.

[0125] In further embodiments, the second guide RNA may be configured to hybridize with the second target sequence along a second complementary region, wherein the second complementary region has a length of at least m nucleotides, wherein n is at least 18, such as at least 19, especially at least 20. In further embodiments, the second guide RNA and the second target sequence have at least n-2 complementary nucleotides in the second complementary region, such as at least n-1 complementary nucleotides, especially n complementary nucleotides. In embodiments wherein a second CRISPR array encodes a plurality of second guide RNAs, each second guide RNA may have at least n-3 complementary nucleotides in the second complementary region with the second target sequence, especially at least n-2 complementary nucleotides, such as at least n-1 complementary nucleotides.

[0126] In a further aspect, the invention may provide a method of mating using the double marker system as such. Specifically, the invention may provide a method for providing an offspring cell of a microbial species, wherein the microbial species has (i) a first mating type and (ii) a second mating type compatible with the first mating type; and wherein the method comprises a mating cycle comprising: (I) a preparation stage comprising providing the first cell and the second cell of the microbial species, the first cell having the first mating type and the second cell having the second mating type, wherein the first cell and the second cell (each) have a first marker locus (or “fourth locus”), and a second marker locus (or “fifth locus”), wherein the first marker locus and the second marker locus are chromosomal loci, and wherein: the first marker locus of the first cell comprises a first mating-type promoter configured to facilitate expression of a first selectable marker providing a first selectable trait in cells having the first mating type; the first marker locus of the second cell comprises a second mating-type promoter configured to facilitate expression of a second selectable marker providing a second selectable trait in cells having the second mating type; the second marker locus of the first cell comprises a third selectable marker providing a third selectable trait; the second marker locus of the second cell comprises a fourth selectable marker providing a fourth selectable trait; and the first selectable trait, the second selectable trait, the third selectable trait and the fourth selectable trait differ from each other; (II) a mating stage comprising bringing the first cell and the second cell in contact with each other to provide an offspring cell population; and (III) a selection stage comprising selecting the offspring cell from the offspring cell population or from cells derived therefrom, wherein the offspring cell is selected based on at least two selectable traits, wherein the at least two selectable traits comprise one or more of (a) at least the third selectable trait and the fourth selectable trait, (b) at least the first selectable trait and the fourth selectable trait, and (c) at least the second selectable trait and the third selectable trait.

[0127] In a further aspect, the invention may provide a recombinant cell of a microbial species. The recombinant cell may be a cell suitable for use in the method of the invention, e.g., a first cell or a second cell as defined above. The recombinant cell may further comprise an offspring cell obtainable with the method of the invention, e.g., a haploid offspring cell or a multiploid offspring cell as defined above. In embodiments, the microbial species may be a fungus, especially a yeast, or especially a species selected from the group consisting of Saccharomyces spp. , Kluyveromyces spp. , Schizosaccharomyces spp. , Candida spp. , Pichia spp., Yarrow ia spp., and Aspergillus spp., more especially Saccharomyces cerevisiae. The microbial species may have a first mating type and a second mating type compatible with the first mating type. Further, the microbial species may be capable of homology directed repair. The microbial species, especially the recombinant cell, may have a first locus, a third locus, a fourth locus, and a fifth locus, especially wherein at least the fourth locus and the fifth locus are chromosomal loci. In embodiments, the first locus of the recombinant cell may encode a first guide RNA, wherein the first guide RNA is configured to hybridize with a first target sequence. Especially, the first target sequence may comprise (or be comprised by) a first wildtype sequence of the first locus. In further embodiments, the third locus of the recombinant cell may encode an RNA-guided DNA endonuclease, especially wherein the RNA-guided DNA endonuclease is configured to functionally associate with the first guide RNA, thereby especially forming a first ribonucleoprotein. In further embodiments, the fourth locus of the recombinant cell comprises a mating-type promoter configured to facilitate expression of a first selectable marker providing a first selectable trait (a) in cells (of the microbial species) having the first mating type or (b) in cells (of the microbial species) having the second mating type. In further embodiments, the fifth locus of the recombinant cell comprises a third selectable marker providing a third selectable trait. Especially, the first selectable trait and the third selectable trait differ from each other.

[0128] Hence, in specific embodiments, the invention may provide a recombinant cell of a microbial species, especially a recombinant cell obtainable with the method of the invention, wherein the microbial species has a first mating type and a second mating type compatible with the first mating type, and wherein the microbial species has a first locus, a third locus, a fourth locus, and a fifth locus, wherein at least the fourth locus and the fifth locus are chromosomal loci; and wherein, wherein: the first locus of the recombinant cell encodes a first guide RNA, wherein the first guide RNA is configured to hybridize with a first target sequence, wherein the first target sequence comprises a first wildtype sequence of the first locus; the third locus of the recombinant cell encodes an RNA-guided DNA endonuclease, wherein the RNA-guided DNA endonuclease is configured to functionally associate with the first guide RNA; the fourth locus of the recombinant cell comprises a mating-type promoter configured to facilitate expression of a first selectable marker providing a first selectable trait (a) in cells having the first mating type or (b) in cells having the second mating type; the fifth locus of the recombinant cell comprises a third selectable marker providing a third selectable trait; and the first selectable trait and the third selectable trait differ.

[0129] A benefit of the recombinant cell may be its suitability for the method of the invention. Hence, the recombinant cell may be used to mate with other (recombinant) cells in order to selectively accumulate genetic modifications in (further) offspring cells.

[0130] Further, in embodiments wherein the recombinant cell was obtained using the method of the invention, the recombinant cell may comprise a plurality of genetic modifications. For instance, the recombinant cell may comprise at least two loci encoding a guide RNA targeting a wildtype sequence of the corresponding locus, such as at least 4 of such loci, especially at least 8, such as at least 16.

[0131] The term “recombinant cell” may herein refer to a cell that has been genetically modified or has been derived from a genetically modified cell. In particular, the term “recombinant cell” may refer to cells containing one or more genetic modifications relative to wildtype cells of the corresponding microbial species.

[0132] As described above, the microbial species may be a fungus, especially a yeast. Especially, the microbial species may be selected from the group comprising Saccharomyces spp., Kluyveromyces spp., Schizosaccharomyces spp., Candida spp., Pichia spp., Yarrow ia spp., Komagataella spp. and Aspergillus spp. In further embodiments, the microbial species may belong to the division Ascomycota, especially to the class Saccharomycetes, such as to the order Saccharomycetales. In further embodiments, the microbial species may be a Saccharomyces spp., especially a species selected from the group comprising S. cerevisiae, S. pastorianus, S. chevalieri, S. paradoxus, S. uvaru, and S. bayanus. The first locus of the recombinant cell may encode a first guide RNA, wherein the first guide RNA is configured to hybridize with a first target sequence. Especially, the first target sequence may comprise a first wildtype sequence of the first locus.

[0133] The phrase “a first wildtype sequence of the first locus” and similar phrases used herein may refer to a sequence that is (naturally) present in the first locus in a wildtype cell of the microbial species. Hence, if such a recombinant cell, further having the third locus as defined above, would mate with a wildtype cell (of the microbial species, the resulting multiploid cell would become homozygous for the first locus.

[0134] In further embodiments, the microbial species, especially the recombinant cell, further has a second locus. The second locus of the recombinant cell may encode a second guide RNA, wherein the second guide RNA is configured to hybridize with a second target sequence. Especially, the second target sequence may comprise a second wildtype sequence of the second locus. Such a recombinant cell may be obtainable with (a single mating cycle of) the method of the invention. In further embodiments, the recombinant cell may especially be haploid

[0135] In further embodiments, the recombinant cell may be multiploid, especially wherein the recombinant cell is homozygous for at least the first locus and the second locus and heterozygous for at least the fourth locus and the fifth locus. In further embodiments, the second locus of the recombinant cell may encode a second guide RNA, wherein the second guide RNA is configured to hybridize with a second target sequence, wherein the second target sequence comprises a second wildtype sequence of the second locus. In further embodiments, a fourth locus first allele of the recombinant cell may comprise a first mating-type promoter configured to facilitate expression of the first selectable marker providing the first selectable trait in cells having the first mating type; a fourth locus second allele of the recombinant cell comprises a second mating-type promoter configured to facilitate expression of a second selectable marker providing a second selectable trait in cells having the second mating type; a fifth locus first allele of the recombinant cell comprises the third selectable marker providing the third selectable trait; and a fifth locus second allele of the recombinant cell comprises a fourth selectable marker providing a fourth selectable trait. Especially, the first selectable trait, the second selectable trait, the third selectable trait, and the fourth selectable trait differ from each other.

[0136] The terms “a fourth locus first allele” and “a fourth locus second allele” may herein especially refer to two different alleles of the fourth locus. Similarly, the terms “a fifth locus first allele” and “a fifth locus second allele” may herein especially refer to two different alleles of the fifth locus.

[0137] As described above, the first locus may comprise a first CRISPR array. The first CRISPR array may encode one or more of the first gRNA, the first mating-type guide RNA, and a first marker guide RNA. Hence, in further embodiments, the recombinant cell may be multiploid, wherein the recombinant cell is homozygous for at least the first locus and the second locus and for one or more of the fourth locus and the fifth locus, especially homozygous for the first locus, the second locus, the fourth locus, and the fifth locus. In further embodiments, the second locus of the recombinant cell may encode a second guide RNA, wherein the second guide RNA is configured to hybridize with a second target sequence, wherein the second target sequence comprises a second wildtype sequence of the second locus.

[0138] BRIEF DESCRIPTION OF THE DRAWINGS

[0139] Embodiments of the invention will now be described, by way of example only, with reference to the accompanying schematic drawings in which corresponding reference symbols indicate corresponding parts, and in which: Fig.lA-C schematically depict embodiments of the method and of the recombinant cell. Fig. 2A-B schematically depict a later mating cycle of embodiments of the method. Fig. 3 schematically depicts experimental results obtained using the method of the invention. The schematic drawings are not necessarily on scale.

[0140] DETAILED DESCRIPTION OF THE EMBODIMENTS

[0141] Fig. 1 A schematically depicts an embodiment of the method of the invention. In the depicted embodiment, a first cell 110 having a first mating type 101 and a second cell 120 having a second mating type are depicted. In particular, the first cell 110 and the second cell 120 may be S. cerevisiae cells, the first mating type 101 is the MATa mating type (or “a”) and the second mating type 102 is the MATa mating type (or “a”), which are compatible mating types. Specifically, Fig. 1A schematically depicts a single mating cycle, wherein the top part on the first page corresponds to the (parental) first cell 110 and the second cell 120, the bottom part on the first page corresponds to a multiploid offspring cell 152, and the part continued on the second page corresponds to two haploid offspring cells 151. To the right of each cell, one or more rows of genetic elements are indicated. Specifically, for each haploid cell a single row of genetic elements is indicated and for the multiploid offspring cell 152, specifically a diploid offspring cell, two rows of genetic elements are indicated as each locus may be represented twice in the diploid offspring cell.

[0142] In the depicted embodiment, the preparation stage comprises providing the first cell 110 and the second cell 120 of the microbial species, wherein the first cell 110 and the second cell 120 (each) have a first locus 10, a second locus 20, a third locus 30, a fourth locus 40, and a fifth locus 50. In embodiments, at least the fourth locus 40 and the fifth locus 50 may be chromosomal loci. In the depicted embodiments, each of the first locus 10, the second locus 20, the third locus 30, the fourth locus 40, and the fifth locus 50 may be chromosomal loci. In the depicted embodiment, the first cell 110 and the second cell 120 are both haploid. Hence, in the depicted embodiment, the first cell 110 is hemizygous for the first locus 10, the second locus 20, the third locus 30 the fourth locus 40 and the fifth locus 50, and, similarly, the second cell 120 may is for the first locus 10, the second locus 20, the third locus 30, the fourth locus 40 and the fifth locus 50.

[0143] The first locus 10 of the first cell 110 encodes a first guide RNA 15, wherein the first guide RNA 15 is configured to hybridize with a first target sequence 16, wherein the first locus 10 of the second cell 120 comprises the first target sequence 16. The first guide RNA 15 may especially be configured to hybridize with the first target sequence 16 along a first complementary region 14. Similarly, the second locus 20 of the second cell 120 encodes a second guide RNA 25, wherein the second guide RNA 25 is configured to hybridize with a second target sequence 27, wherein the second locus 20 of the first cell 110 comprises the second target sequence 27. The second guide RNA 25 may especially be configured to hybridize with the second target sequence 27 along a second complementary region 24. The single arrow between a first coding sequence 215 of the first locus 10 and the first guide RNA 15represents the guide RNA being a result of transcription, and optionally pre-crRNA processing (see above), and no translation. Further, in the depicted embodiment, the third locus 30 of the first cell 110 and the third locus 30 of the second cell 120 encode an RNA-guided DNA endonuclease 35. The RNA-guided DNA endonuclease 35 may especially be configured to functionally associate with the first guide RNA 15 and (separately) with the second guide RNA 25. The double arrows between a third coding sequence 235 of the third locus 30 and the RNA-guided DNA endonuclease 35 represents the RNA-guided DNA endonuclease 35 being a result of transcription and subsequent translation. The fourth locus 40 of the first cell 110 comprises a first mating-type promoter 41 configured to facilitate expression of a first selectable marker 201 providing a first selectable trait in cells having the first mating type 101. Similarly, the fourth locus 40 of the second cell 120 comprises a second mating-type promoter 42 configured to facilitate expression of a second selectable marker 202 providing a second selectable trait in cells having the second mating type 102. The fifth locus 50 of the first cell 110 comprises a third selectable marker 203 providing a third selectable trait and the fifth locus 50 of the second cell 120 comprises a fourth selectable marker 204 providing a fourth selectable trait. As described above, the first selectable trait, the second selectable trait, the third selectable trait and the fourth selectable trait may all differ from each other.

[0144] Fig. 1A further schematically depicts the first locus 10 of the first cell 110 to comprise a first promoter 11 arranged upstream of the first coding sequence 215, wherein the first coding sequence 215 encodes the first guide RNA 15. Similarly, in the depicted embodiments, the second locus 20 of the second cell 120 comprises a second promoter 12 arranged upstream of the second coding sequence 225, wherein the second coding sequence encodes the second guide RNA 25. In the depicted embodiment, the third locus 30 of the first cell 110 and of the second cell 120 comprise a third promoter 31 or a secondary third promoter 32 arranged upstream of a third coding sequence 235, respectively, wherein the third coding sequences encodes the RNA-guided DNA endonuclease 35. Further, Fig. 1A schematically depicts: the fourth locus 40 of the first cell 110 to comprise the first mating-type promoter 41 controlling the expression of the first selectable marker 201, expression of which results in a corresponding first selectable protein 1; the fourth locus 40 of the second cell 120 to comprise the second mating-type promoter 42 controlling the expression of the second selectable marker 202, expression of which results in a corresponding second selectable protein 2; the fifth locus 50 of the first cell 110 to comprise a fifth promoter 51 controlling the expression of the third selectable marker 203, expression of which results in a corresponding third selectable protein 3; and the fifth locus 50 of the second cell 120 to comprise a secondary fifth promoter 51 controlling the expression of the fourth selectable marker 204, expression of which results in a corresponding fourth selectable protein 4.

[0145] As schematically depicted in Fig. 1 A, the first cell 110 and the second cell 120 may be mated to provide an offspring cell 150, especially a multiploid offspring cell 152. As indicated within the depicted cell, the multiploid offspring cell 152 comprises both the MA T A and the MATa alleles (a / a) and is thus not capable of mating. As the multiploid offspring cell 152 has neither the first mating type 101 nor the second mating type 102, the first-mating type promoter 41 and the second mating type-promoter 42 do not facilitate, especially prevent, expression of the first selectable marker 201 and the second selectable marker 202, respectively, as indicated with the cross through the schematically depicted promoters and the absence of the first and second selectable proteins 1,2. Given that the third selectable marker 203 and the fourth selectable marker 204 are active, the multiploid offspring cell 152 may be selected based on the third selectable trait and the fourth selectable trait. Due to the expression of the first coding sequence 215, the second coding sequence 225 and the third coding sequence 235 as described above, the multiploid offspring cell 152 has become homozygous for the first locus 10 and for the second locus 20 (also see Fig. 1C).

[0146] In the depicted embodiment, the selection stage comprises sporulation of the multiploid offspring cell 152 to provide haploid offspring cells 151 (depicted on the second page of Fig. 1A). The depicted haploid offspring cells 151 each have a first locus 10 corresponding to the first (parent) cell 101 and a second locus 20 corresponding to the second (parent) cell 120. Further, as these haploid offspring cells 151 both have a mating type matching their respective mating-type promoters, the mating-type promoters are depicted as active. Specifically, the haploid offspring cell 151 having the first mating type 101 may be selected based on selectable traits 1 and 4, whereas the haploid offspring cell 151 having the second mating type 102 may be selected based on selectable traits 2 and 3. As these haploid offspring cells 151 again have two different selectable markers in their fourth locus 40 and fifth locus 50, these haploid offspring cells 151 may be suitable for use as a first cell 110 or as a second cell 120 in a later mating cycle (also see Fig. 2).

[0147] Hence, the selectable traits facilitate specifically selecting each of the first cell 110, the second 120, the multiploid offspring cell 152, the haploid offspring cell 151 of the first mating type 101, and the haploid offspring cell 151 of the second mating type 102 from the rest based on two selectable markers.

[0148] Fig. 1A further schematically depicts an embodiment of the recombinant cell 170 of the invention. Specifically, Fig. 1A schematically depicts five different embodiments of the recombinant cell 170, wherein the depicted first cell 110, second cell 120, and the haploid offspring cells 151 are (directly) suitable for use as first cell 110 or as second cell 120 in a mating cycle of the method of the invention. Further, the depicted multiploid offspring cell 152 and the depicted haploid offspring cells 151 may be recombinant cells 170 obtainable using the method of the invention.

[0149] Fig. IB schematically depicts an embodiment wherein the preparation stage comprises providing a plurality of first cells 110 having a first mating type 101, especially the MATa mating type and a plurality of second cells 120 having a second mating type 102, especially the MATa mating type. In the mating stage, the first cells 110 and the second cells 120 are brought in contact with each other to provide an offspring cell population 160, especially a multiploid offspring cell population 162. The hyphened arrows in Fig. IB schematically depict an optional step in the generation of haploid offspring cells 151. Specifically, the hyphened arrows depict the selection of an offspring cell 150, especially a multiploid offspring cell 152, from the offspring cell population 160 based on the third selectable trait and the fourth selectable trait, i.e., based on the presence of the third selectable marker 203 and the fourth selectable marker 204 in the multiploid offspring cell 152.

[0150] Fig. IB further schematically depicts sporulation of the multiploid cell population 162 and sporulation of the selected multiploid offspring cell 152. Either path may be followed to provide a haploid offspring cell population 161. Fig. IB further schematically depict haploid offspring cells 151 and, schematically depicted below the cells, the corresponding selectable markers. Hence, from left to right, the haploid offspring cells 151 may be selected based on (1) the first and third selectable traits, (2) the first and fourth selectable traits, (3) the second and third selectable traits, and (4) the second and fourth selectable traits. Specifically, the selection based on the fist and third selectable traits and the selection based on the second and fourth selectable traits may (essentially) guarantee the selection of a haploid offspring cell 151 derived from a successful mating event between the first cell 110 and the second cell 120.

[0151] Fig. 1C schematically depicts the homing activity of the second locus 20 of the second cell 120. Specifically, Fig. 1C schematically depicts the mating between a first cell 110 and a second cell 120, thereby (initially) providing a multiploid offspring cell 152 heterozygous for the second locus. As schematically depicted, the second guide RNA 25 and the RNA-guided DNA endonuclease 35 form a second ribonucleoprotein 37 configured to (locate and) cleave the second target sequence 27, as comprised by the allele of the second locus 20 originating from the first cell 110. Following cleavage of the second target sequence 27, the multiploid offspring cell 152 may repair the resulting cleaved DNA fragments based on homology direct repair using the allele of the second locus 20 originating from the second cell 120 as a repair template. As a result, the multiploid offspring cell 152 becomes homozygous for the second locus 20.

[0152] In the depicted embodiment, the second guide RNA 25 may be configured to hybridize with the second target sequence 27 along a second complementary region 24. The second complementary region 24 may have a length of at least n nucleotides, especially wherein n is at least 18, such as at least 19, especially at least 20. In further embodiments, the second guide RNA 25 and the second target sequence 27 may have at least n-2 complementary nucleotides in the second complementary region 24, such as at least n-1 complementary nucleotides, especially n complementary nucleotides.

[0153] Specifically, in the depicted embodiment, the RNA-guided DNA endonuclease 35 may comprises a Casl2a endonuclease. Further, in the depicted embodiment, the second guide RNA 25 may be configured to functionally associate with the RNA-guided DNA endonuclease 35 to form a second ribonucleoprotein 37, wherein the second ribonucleoprotein 37 is configured to cleave the second locus 20 at the second target sequence 27.

[0154] Fig. 2 schematically depicts a later mating cycle following the earlier mating cycle schematically depicted in Fig. 1A. In the depicted embodiment, an offspring cell 150 of the mating cycle schematically depicted in Fig. 1A, specifically the haploid offspring cell 151 having the first mating type 101, is used as the first cell 110 in the later mating cycle schematically depicted in Fig. 2. Alternatively, the haploid offspring cell 151 having the second mating type 102 could be used as the second cell 120 in the later mating cycle (with a different first cell 110).

[0155] In the depicted embodiment, the preparation stage of the second mating cycle comprises providing the offspring cell 150 of the earlier mating cycle and a third cell 130 of the microbial species, specifically as the first cell 110 and as the second cell 120 of the later mating cycle, respectively. Hence, the offspring cell 150 of the earlier mating cycle has the first mating type 101 and the third cell 130 has the second mating type 102. In the depicted embodiment, the third cell (also) has the first locus 10, the second locus 20, the third locus 30, the fourth locus 40, and the fifth locus 50. Further, offspring cell 150 of the earlier mating cycle and the third cell 130 (both) have a sixth locus 60. In the depicted embodiment: the first locus 10 of the third cell 130 comprises the first target sequence 16; the second locus 20 of the third cell 130 comprises the second target sequence 27; and the sixth locus 60 of the third cell 130 encodes a third guide RNA 65, wherein the third guide RNA 65 is configured to hybridize with a third target sequence 68, wherein the sixth locus 60 of the offspring cell 150 of the earlier mating cycle comprises the third target sequence 68. In embodiments, the mating stage may comprise bringing the offspring cell 150 of the earlier mating cycle in contact with the third cell 130 to provide an offspring cell population 160 of the later mating cycle. As the offspring cell 150 comprises the first selectable marker 201 and the fourth selectable marker 204 in the fourth and fifth locus respectively, the third cell 130 may comprise the second selectable marker 202 and the third selectable marker 203 in the fourth and fifth locus, respectively. As noted above, in the context of successive mating cycles, it will be clear to the person skilled in the art that the first cell 110 and the second cell 120 of each mating cycle each comprise two distinct selectable markers in the fourth locus 40 and the fifth locus 50 such that the cells together comprise four different selectable markers with four different selectable traits in the fourth locus and the fifth locus. It will be clear to the person skilled in the art that the selectable markers used in successive mating cycles are not necessarily the same selectable markers, i.e., the third selectable marker 203 of an earlier mating cycle may differ from the third selectable marker 203 used in a later mating cycle.

[0156] Hence, in specific embodiments, the fifth locus 50 of the first cell 110 of the earlier mating cycle may comprise a third selectable marker 203, whereas the fifth locus 50 of the first cell 110 of the later mating cycle may comprise a fourth selectable marker 204. In such embodiments, the fifth locus 50 of the second cell 120 of the earlier mating cycle may comprise a fourth selectable marker 204, whereas the fifth locus 50 of the second cell 120 of the later mating cycle may comprise a third selectable marker 203 or a further selectable marker, e.g., a fifth selectable marker.

[0157] Fig. 1A and Fig. 2 may together schematically depict an embodiment wherein the earlier mating cycle comprises initiating sporulation in a multiploid offspring cell 152 from the offspring cell population 160 to provide a haploid offspring cell population 161, and subsequently selecting a haploid offspring cell 161 from the haploid offspring cell population 161 for use in the later mating cycle. Specifically, Fig. 1 A and 2 may schematically depict an embodiment wherein the selection stage of the earlier mating cycle comprises initiating sporulation in a multiploid offspring cell 152 from the offspring cell population 160 to provide a haploid offspring cell population 161, and wherein: in the earlier mating cycle, the selection stage comprises selecting the offspring cell 150 from the haploid offspring cell population 161 based on the first selectable trait and the fourth selectable trait, and in the later mating cycle the preparation stage comprises providing the haploid offspring cell 150 of the earlier mating cycle as the first cell 110. In further embodiments, the selection stage of the earlier mating cycle may comprise selecting the offspring cell 150 from the haploid offspring cell population 161 based on the second selectable trait and the third selectable trait, and in the later mating cycle the preparation stage may comprise providing the haploid offspring cell 150 of the earlier mating cycle as the second cell 120.

[0158] Fig. 2B schematically depicts an embodiment wherein the selection stage of the earlier mating cycle comprises selecting the offspring cell 150 from the offspring cell population 160 based on the third selectable trait and the fourth selectable trait, wherein the offspring cell 150 comprises a multiploid offspring cell 152. Specifically, in the depicted embodiment, the first cell 110 and the second cell 120 further have a (chromosomal) mating-type locus 90. Cells of the microbial species homozygous or hemizygous at the mating-type locus 90 may have a mating type. As the depicted first cell 110 and second cell 120 are haploid cells and are thus hemizygous for the mating-type locus 90, these cells correspondingly have the first mating type 101 and the second mating type 102, respectively. Further, in the depicted embodiment, the first locus 10 of the first cell 110 comprises a first coding sequence 215, wherein the first coding sequence 215 comprises a first CRISPR array. Specifically, in the depicted embodiment, the first CRISPR array encodes for a first mating-type guide RNA 115 and for the first guide RNA 15. The first mating-type guide RNA may be configured to hybridize with a second mating-type target sequence 92. In the depicted embodiment, the chromosomal mating-type locus 90 of the second cell 120 comprises the second mating-type target sequence 92. Following mating between the first cell 110 and the second 120, a multiploid offspring cell 152 is provided that, initially, is heterozygous at the first locus 10 and at the mating-type locus 90. As schematically depicted, the first guide RNA 15 and the RNA-guided endonuclease 35 form a first ribonucleoprotein 36. The first ribonucleoprotein 36 is configured to cleave the allele of the first locus 10 originating from the second cell 120 at the first target sequence 16. Similarly, the first mating-type guide RNA 15 and the RNA-guided endonuclease 35 form a first mating-type ribonucleoprotein 39. The first mating-type ribonucleoprotein 39 is configured to cleave the allele of the mating-type locus 90 originating from the second cell 120 at the second mating-type target sequence 92. Following the cleavage of the alleles of the first locus 10 and of the mating-type locus 90 originating from the second cell 120 and the subsequent homology directed repair based on the alleles of the first locus 10 and of the mating-type locus 90 originating from the first cell 110, a multiploid offspring cell 150,152 homozygous for the first locus 10 and the mating-type locus 90 is provided. As the multiploid offspring cell 150,152 is homozygous for the allele of the mating-type locus 90 originating from the first cell 110, the multiploid offspring cell 150,152 has the first mating type 101.

[0159] Fig. 2B further schematically depicts using the multiploid offspring cell 150,152 having the first mating type 101 as a first cell 110 in a later mating cycle. As the depicted multiploid offspring cell 150,152 is diploid and as the second cell 120 in the later mating cycle is haploid, the resulting multiploid offspring cell 150,152 is tripl oid. As schematically depicted, the first locus 10 and the mating-type locus 90 of the multiploid offspring cell 150,152 of the later mating cycle will again be cleaved and repaired using homology directed repair. Afterwards, the multiploid offspring cell 150,152 of the later mating cycle will be homozygous for the first locus 10 and for the mating-type locus 90 and will have the first mating type 101, which may allow its use as a first cell 110 in a yet later mating cycle.

[0160] In further embodiments, the first coding sequence 215 may further encode a first marker guide RNA targeting a marker gene in the fifth locus 50 of the second cell 120, e.g., the fourth marker gene 204. Thereby, the resulting multiploid offspring cell 150, 152 will (also) be homozygous for the fifth locus, which may facilitate selection in further mating cycles. In such embodiments, the first promoter 11 may especially be an inducible promoter.

[0161] In alternative embodiments, the second locus 20 of the second cell 120, especially a second coding sequence 225 may further encode a second mating-type guide RNA 125. The second mating-type guide RNA 125 may be configured to hybridize with a first mating-type target sequence 91, wherein a chromosomal mating-type locus 90 of the first cell 110 comprises the first mating-type target sequence 91. In such embodiments, the offspring cell 150, especially the multiploid offspring cell 152, of the (earlier) mating cycle may have the second mating type and may be used as the second cell 120 in a later mating cycle.

[0162] In further embodiments, the second coding sequence 225 may further encode a first marker guide RNA targeting a marker gene in the fifth locus 50 of the first cell 110, e.g., the third marker gene 203. Thereby, the resulting multiploid offspring cell 150, 152 will (also) be homozygous for the fifth locus, which may facilitate selection in further mating cycles. In such embodiments, the second promoter 12 may especially be an inducible promoter.

[0163] Experiments

[0164] Unless specified otherwise, the experiments described herein were performed using the following materials and methods.

[0165] Media composition - S. cerevisiae strains were cultivated using YPD (10 g-L'1yeast extract, 20 g-L'1peptone, 20 g-L'1glucose) supplemented with Hygromycin B 300 mg-L'1, nourseothricin 100 mg-L'1and / or G418 200 mg-L'1where appropriate. In case selection included an auxotrophic marker, Yeast Nitrogen Base (YNB) supplemented with yeast synthetic drop-out medium (Sigma) and urea (2.3 g-L'1) was used. For solid media 20 g-L'1agar was added. To facilitate phenotypic screening of red colonies (due to the absence of a functional ADE2 gene), YNB plates containing 10 mg-L'1of Adenine instead of 18 mg-L'1were used.

[0166] Mating and Sporulation - Haploid strains of opposing mating types were allowed to mate in liquid YPD. Equivalent number of cells (calculated by OD600 measurements) were added to fresh media and left overnight shaking at 30 °C. This culture was inoculated in liquid YPD supplemented with hygromycin and nourseothricin to select for diploids. Diploid cultures were subsequently sporulated in minimal media containing 10 g-L'1potassium acetate, 1 g-L'1yeast extract, 0.5 g-L'1maltose and appropriate amino acids at 30 °C for a minimum of three days until sufficient tetrads were observed (above about 5%). Sporulation media was treated with Y-PER and resuspended in selective media lacking either leucine or tryptophan and supplemented with either nourseothricin or hygromycin.

[0167] Growth experiments - Overnight cultures were diluted and resuspended in fresh YNB media. OD measurements were performed in 96-well format plates using a Biotek Synergy MX microplate reader. Overnight cultures were used to inoculate fresh YNB media at an initial OD600 of 0.1. Cultures were incubated at 30 °C with double orbital shaking and growth was monitored through measurement of OD600 every 10 minutes for 30 hours.

[0168] Plasmids - The following plasmids were used in the experiments described below:

[0169] Primers - The following primers were used in the experiments describes below:

[0170]

[0171] Molecular biology techniques - For cloning and sequencing PCR products were generated using Q5 polymerase (NEB). For screening purposes, Phire polymerase (Fisher) was used.

[0172] Platform strains - Markers were introduced by transforming linear fragments with homology regions via the lithium acetate protocol as described in GIETZ et al., High-efficiency yeast transformation using the LiAc / SS carrier DNA / PEG method, Nature Protocols, 2007, Vol. 2, pages 31-34, which is hereby herein incorporated by reference. The transformation was followed by selection of the integrated marker. The strain CEN.PK113-7D (GenBank ID JRIV00000000) was used for amplification of promoters and markers (see below). Strains CEN.PK113-6B and CEN.PK113-7B, which are auxotrophic for leucine, uracil and tryptophane, were used for the experiments described below. Strains CEN.PK113-6B and CEN.PK113-7B have the MATa and MATa. mating types. A strain collection comprising CEN.PK113-6B and CEN.PK113-7B is described in ENTIAN and KOTTER, 25 Yeast Genetic Strain and Plasmid Collections, Methods In Microbiology, 2007, vol. 36.

[0173] Linear fragments were PCR amplified from genomic DNA or plasmids using primers with homology overhangs and were assembled in vivo. First, the constitutively expressed hygromycin and nourseothricin resistance markers were PCR amplified from plasmids pMEL12 with primers BG19878 and BG19879 and plasmid pYTK078(47) with primers BG20474 and BG21763, respectively. Two 500 bp fragments up- and downstream of the HO locus were PCR amplified to create linear fragments with homology arms. Primers BG16835 and BG21782 or BG21764 were used to create the upstream fragment for respectively the HygR or NatR marker. Primers BG21765 or BG21729 combined with primer BG16838 were used for the downstream fragment. A FnCasl2a nuclease was amplified from pUDE731 using primers BG21301 and BG16331 and was integrated in the trpl-289 locus using klURA3 as a selection marker. Specifically, the coding sequence of FnCasl2a encoded the Casl2a endonuclease with three nuclear localization signals (NLS tags), together correspond to SEQ ID NO:50. klURA3 was amplified from pUDE731 with primers BG22785 and BG22786. Two linear fragments serving as homology arms up- and downstream of the trp 1-289 locus were PCR amplified from genomic DNA using primers BG21934, BG22109, BG21304 and BG21305. Mating type specific selection markers were integrated in the non-functional Leu2-3,112 locus. Linear fragments serving as homology arms were amplified from the genome using primers BG21934, BG22109, BG22117 and BG22118. Mating type specific promoters of genes STE2 (primers BG22110 and BG22111), STE3 (primers BG22112 and BG22113) and marker TRP1 (BG22115 and BG22116) were amplified from genomic DNA, whereas plasmid pHLUM was used to amplify LEU2 with primers BG16730 and BG16731. Appropriate homology arms were added by using primers with homology overhangs. Plasmids were cured by repeated re-inoculation in liquid media and testing resulting individual colonies for plasmid presence.

[0174] Split drive and knockout mutants - Regular knockout, knock-in and split drive mutants were created by co-transforming plasmids carrying a crRNA with linear fragments serving as repair templates. All repair templates provided at least 50 bp overhang, i.e., the homology arms for homology directed repair were at least 50 bp long. For ADE2 mutants, PL-crRNA-ade2, a plasmid carrying a crRNA targeting the ADE2 region was used and co-transformed to either create a clean knockout or a non-functional ADE2 due to insertion of the crRNA. For the clean knockout, repair templates were PCR amplified from genomic DNA using primers BG23784, BG23785, BG23786 and BG23787. To integrate the split drive mutant, repair templates were PCR amplified from PL-crRNA-ade2 using primers BG23789 and BG23792 and from genomic DNA using primers BG23784, BG23788, BG23793 and BG23787. For the superfolder green fluorescent protein (sfGFP) knock-in mutants, repair templates were created using plasmid pCfB2791 (primers BG25380 and BG17378) and PL-crRNA-intl (primers BG25378 and BG25379) and co-transformed with plasmid PL-crRNA-intl, a plasmid carrying a crRNA targeting the / N77 locus.

[0175] Experiment 1

[0176] Two haploid S. cerevisiae strains were generated, each carrying a constitutive dominant marker for downstream diploid selection (HygR or NatR) in combination with a marker to enable mating-type-regulated auxothropy (or “MRA”) based on LEU2 or TRP1. This double marker system was integrated in the genome of S. cerevisiae haploid strains with the CEN.PK background, specifically in CEN.PK113-6B and CEN.PK113-7B (see above), to enable selection for the different stages of the sexual reproduction life cycle of S. cerevisiae. Constitutively expressed dominant markers in haploids of opposing mating types (initially a hygromycin (HygR) marker for MAT and a nourseothricin (NatR) marker for MA Ta) were introduced in chromosome IV, at the locus harboring a non-functional Homothallic switching endonuclease (HO locus) and, as mentioned, served as diploid selection markers., i.e., as the third selectable marker 203 and as the fourth selectable marker 204. Mating type specific auxotrophic markers were integrated on the same chromosome that carries the mating-type locus 90 in S. cerevisiae (chromosome III). The selectable marker LEU2 was used for mating type MA Tu, and the selectable marker TRP1 was used for mating type MA Ta, i.e., LEU2 and TRP1 were used as first selectable marker 201 and as second selectable marker 202. These markers were placed under the mating type specific promoters of the genes coding for pheromone receptors: STE2 (MATa.) and STE3 (MATa).

[0177] Specifically, the HygR and NatR selectable markers provide resistance to the antibiotics hygromycin and nourseothricin, respectively. The Leu2 and TRP1 selectable markers complement leucine and tryptophane auxotrophy, respectively. Hence, HygR and NatR are antibiotic resistance markers providing antibiotic resistance traits and Leu2 and TRP1 are prototrophic markers providing prototrophic traits. Hence, in embodiments, the selectable traits of the first selectable marker 201, the second selectable marker 202, the third selectable marker 203 and the fourth selectable marker 204 are selected from the group consisting of antibiotic resistance traits and prototrophic traits.

[0178] This setup allowed for the selection of three different stages of the sexual reproduction life cycle (i.e., parental haploids, diploid and haploid offspring), as schematically depicted in Fig. 3. Specifically, five strains carrying different marker combinations were grown in selective media: four haploid strains carrying the four possible combinations of a mating type specific marker (LEU2 or TRP 1) and a diploid marker (HygR or NatR), and a diploid strain carrying all four markers.

[0179] Specifically, Fig. 3 schematically depicts the growth of the first (parent) cells 110 having selectable markers 201 and 203, second (parent) cells 120 having selectable markers 202 and 204, multiploid offspring cells 150,152, and haploid offspring cells 150,151 having the first mating type 101 and haploid offspring cells 150,151 having the second mating type 102 in six different growth media. Specifically, each growth curve indicates the optical density OD (in a.u.) against time T (in hours). Each growth curve represents the average and standard deviation (see shading) of three replicates in a growth medium suitable for selecting for the selectable markers indicated above the corresponding growth curve. The lines indicate the following: line Luo indicates the growth of the first cells 110; line Lno indicates the growth of the second parent cells 120; line L152 indicates the growth of the multiploid offspring cells 150,152; line Lisia indicates the growth of the haploid offspring cells 150,151 having the first mating type 101; and line Lisia indicates the growth of the haploid offspring cells 150,151 having the second mating type 102. Hence, for example, in the selectable medium for selecting for the second selectable marker 202 and for the fourth selectable marker 204 (bottom left panel), effectively only the second cells 120 successfully grow, with the onset of growth of the multiploid offspring cells 150,152 being observed after about 24 hours. All cells were capable of growing in the growth medium without selective pressures (bottom middle panel).

[0180] Without being bound by theory, the observed growth of the multiploid offspring cell 150,152 is speculated to result from the presence of a small fraction of diploids with a MAT^IA, MA TfjJa, MATaJ- or MATal- genotype due to loss of heterozygosity at chromosome III. If needed for haploid selection, a post-sporulation enrichment step may be used during which vegetative diploid cells are selectively killed.

[0181] Hence, as may be observed in the growth curves of Fig. 3, the double marker system of the method of the invention may facilitate selecting for specific parent cells and offspring cells 150.

[0182] To test the suitability of the method of the invention for gene drive research, several ADE2 knock-out strains were created. ADE2 KO mutants are auxotrophic for adenine and may have a distinct red color due to accumulation of a purine precursor. In diploids, only homozygous mutants may turn red, whereas heterozygous mutants may be cream colored, facilitating an easy phenotypical evaluation of the efficiency of the gene drive system. The previously created split drive platform strains were used to create a strain carrying a clean ADE2 knockout (KO) and a strain where the ADE2 gene was replaced by a CRISPR array expressing a single crRNA ADE2 -targeting spacer (representing the split drive, SD). When the SD strain mates with a yeast cell carrying the wild type ADE2 locus (WT), this locus will be targeted by ribonucleoprotein, here especially by the Cast 2a nuclease-guide complex. Repair of the double-stranded DNA break via homology directed repair using the SD ADE2 locus as a repair template results in a diploid homozygous for the SD ADE2 mutation. Strains carrying either ADE2 (WT), Aade2 (KO) or Aade2::crRNA (SD) were mated with a WT strain to evaluate homing efficiencies. After a diploid selection step, cultures were allowed to sporulate for 72 hours, followed by an asci-enrichment step and resuspension in selective media for haploid offspring cells 150,151. Presence of the double marker system facilitates iterative rounds through the sexual life cycle. Therefore, haploid offspring cells 150, 151 were mated again with a WT strain, followed by a diploid selection step to generate a second generation of diploid offspring cells 150, 152. Cultures containing first-generation diploids, haploid offspring and second-generation diploids were genetically screened via PCR, using primers that bind outside of the ADE2 locus resulting in amplicons of different sizes for the three alleles. The results of the PCR experiment are summarized in the following table:

[0183] As expected, diploids resulting from the WT x KO cross, resulted in two bands, representing the WT and KO locus. On the other hand, the WT x SD cross only shows a single amplicon with a size corresponding to that of the SD, indicating that homing activity had occurred with high efficiency. Similar results were obtained for second generation diploids, indicating maintained homing activity after crossing WT x SD offspring with WT. Phenotypical screening was also performed by spotting cultures on selective agar plates. Only crosses involving SD displayed a uniform red color, indicating the absence of a functional ADE2 gene. Offspring of the WT x KO cross showed a mixed pattern of red and cream-colored colonies, whereas diploids remained cream colored, indicating that at least one of the two ADE2 alleles was still functional. The phenotype assessment was therefore in perfect agreement with the genetic screening.

[0184] To test whether an integrated gene encoding a Cas nuclease could drive multiple crRNA-encoding alleles, and whether other types of mutations could be driven as well, another split drive mutant strain was created. To create a gain-of-function split drive mutant (GoF SD), a sequence containing either a heterologous sfGFP gene or a heterologous sfGFP gene with a crRNA-encoding allele was integrated in a non-coding region on chromosome XIV (INTI locus). The gain-of-function SD mutant was mated with the Aade2::crRNA strain described above. PCR screening of the resulting diploid culture on both the ADE2 region on chromosome XV and the INTI locus (or “INTI region”) on chromosome XIV showed a single amplicon corresponding to the SD drives. These results demonstrate that the method of the invention can also be used to drive gain-of-function mutations and that homing activity is maintained with no apparent loss of efficiency when multiple split drives are combined. The term “plurality” refers to two or more. Furthermore, the terms “a plurality of’ and “a number of’ may be used interchangeably.

[0185] The terms “substantially” or “essentially” herein, and similar terms, will be understood by the person skilled in the art. The terms “substantially” or “essentially” may also include embodiments with “entirely”, “completely”, “all”, etc. Hence, in embodiments the adjective substantially or essentially may also be removed. Where applicable, the term “substantially” or the term “essentially” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. Moreover, the terms ’’about” and “approximately” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. For numerical values it is to be understood that the terms “substantially”, “essentially”, “about”, and “approximately” may also relate to the range of 90% - 110%, such as 95%-105%, especially 99%-101% of the values(s) it refers to.

[0186] The term “comprise” also includes embodiments wherein the term “comprises” means “consists of’.

[0187] The term “and / or” especially relates to one or more of the items mentioned before and after “and / or”. For instance, a phrase “item 1 and / or item 2” and similar phrases may relate to one or more of item 1 and item 2. The term "comprising" may in an embodiment refer to "consisting of' but may in another embodiment also refer to "containing at least the defined species and optionally one or more other species".

[0188] The term “further embodiment” and similar terms may refer to an embodiment comprising the features of the previously discussed embodiment, but may also refer to an alternative embodiment.

[0189] It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the appended claims.

[0190] In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim.

[0191] Use of the verb "to comprise" and its conjugations does not exclude the presence of elements or steps other than those stated in a claim. Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, “include”, “including”, “contain”, “containing” and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”. The article "a" or "an" preceding an element does not exclude the presence of a plurality of such elements.

[0192] The invention further applies to a device, apparatus, or system comprising one or more of the characterizing features described in the description and / or shown in the attached drawings. The invention further pertains to a method or process comprising one or more of the characterizing features described in the description and / or shown in the attached drawings. Moreover, if a method or an embodiment of the method is described being executed in a device, apparatus, or system, it will be understood that the device, apparatus, or system is suitable for or configured for (executing) the method or the embodiment of the method, respectively.

[0193] The invention may be implemented by means of hardware comprising several distinct elements, and by means of a suitably programmed computer. In a device claim, or an apparatus claim, or a system claim, enumerating several means, several of these means may be embodied by one and the same item of hardware. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

[0194] The invention also provides a control system that may control the device, apparatus, or system, or that may execute the herein described method or process. Yet further, the invention also provides a computer program product, when running on a computer which is functionally coupled to or comprised by the device, apparatus, or system, controls one or more controllable elements of such device, apparatus, or system.

[0195] The devices, apparatus, or systems may herein amongst others be described during operation. As will be clear to the person skilled in the art, the invention is not limited to methods of operation, or devices, apparatus, or systems in operation.

[0196] The various aspects discussed in this patent can be combined in order to provide additional advantages. Further, the person skilled in the art will understand that embodiments can be combined, and that also more than two embodiments can be combined. Furthermore, some of the features can form the basis for one or more divisional applications.

Claims

CLAIMS:

1. A method for selectively accumulating genetic modifications in an offspring cell(150) of a microbial species, wherein the microbial species has (i) a first mating type (101) and (ii) a second mating type (102) compatible with the first mating type (101); and wherein the method comprises a mating cycle comprising: a preparation stage comprising providing a first cell (110) and a second cell (120) of the microbial species, the first cell (110) having the first mating type (101) and the second cell (120) having the second mating type (102), wherein the first cell (110) and the second cell (120) have a first locus (10), a second locus (20), a third locus (30), a fourth locus (40), and a fifth locus (50), wherein preferably at least the fourth locus (40) and the fifth locus (50) are chromosomal loci, and wherein: the first locus (10) of the first cell (110) encodes a first guide RNA (15), wherein the first guide RNA (15) is configured to hybridize with a first target sequence (16), wherein the first locus (10) of the second cell (120) comprises the first target sequence (16); the second locus (20) of the second cell (120) encodes a second guide RNA (25), wherein the second guide RNA (25) is configured to hybridize with a second target sequence (27), wherein the second locus (20) of the first cell (110) comprises the second target sequence (27); the third locus (30) of the first cell (110) and the third locus (30) of the second cell (120) encode an RNA-guided DNA endonuclease (35), wherein the RNA-guided DNA endonuclease (35) is configured to functionally associate with the first guide RNA (15) and with the second guide RNA (25); the fourth locus (40) of the first cell (110) comprises a first mating-type promoter (41) configured to facilitate expression of a first selectable marker (201) providing a first selectable trait in cells having the first mating type (101); the fourth locus (40) of the second cell (120) comprises a second mating-type promoter (42) configured to facilitate expression of a second selectable marker (202) providing a second selectable trait in cells having the second mating type (102); the fifth locus (50) of the first cell (110) comprises a third selectable marker (203) providing a third selectable trait;the fifth locus (50) of the second cell (120) comprises a fourth selectable marker (204) providing a fourth selectable trait; and preferably the first selectable trait, the second selectable trait, the third selectable trait and the fourth selectable trait differ from each other; a mating stage comprising bringing the first cell (110) and the second cell (120) in contact with each other to provide an offspring cell population (160); and a selection stage comprising selecting the offspring cell (150) from the offspring cell population (160) or from cells derived therefrom, wherein the offspring cell (150) is selected based on at least two selectable traits, wherein preferably the at least two selectable traits comprise one or more of (a) at least the third selectable trait and the fourth selectable trait, (b) at least the first selectable trait and the fourth selectable trait, and (c) at least the second selectable trait and the third selectable trait.

2. The method according to claim 1, comprising a plurality of mating cycles as defined in claim 1, wherein the offspring cell (150) of an earlier mating cycle is used as the first cell (110) or as the second cell (120) in the later mating cycle.

3. The method according to claim 2, wherein in the later mating cycle: the preparation stage comprises providing the offspring cell (150) of the earlier mating cycle and a third cell (130) of the microbial species, wherein the third cell has the first locus (10), the second locus (20), the third locus (30), the fourth locus (40), and the fifth locus (50), and wherein the offspring cell (150) of the earlier mating cycle and the third cell have a sixth locus (60), wherein the offspring cell (150) and the third cell (130) differ in mating type, wherein: the first locus (10) of the third cell (130) comprises the first target sequence (16); the second locus (20) of the third cell (130) comprises the second target sequence (27); and the sixth locus (60) of the third cell (130) encodes a third guide RNA (65), wherein the third guide RNA (65) is configured to hybridize with a third target sequence (68), wherein the sixth locus (60) of the offspring cell (150) of the earlier mating cycle comprises the third target sequence (68); the mating stage comprising bringing the offspring cell (150) of the earlier mating cycle in contact with the third cell (130) to provide an offspring cell population (160) of the later mating cycle.

4. The method according to claim 2 or 3, wherein the selection stage of the earlier mating cycle comprises initiating sporulation in a multiploid offspring cell (152) from the offspring cell population (160) to provide a haploid offspring cell population (161), and wherein: in the earlier mating cycle, the selection stage comprises selecting the offspring cell (150) from the haploid offspring cell population (161) based on the first selectable trait and the fourth selectable trait, and in the later mating cycle the preparation stage comprises providing the haploid offspring cell (150) of the earlier mating cycle as the first cell (110); or in the earlier mating cycle, the selection stage comprises selecting the offspring cell (150) from the haploid offspring cell population (161) based on the second selectable trait and the third selectable trait, and in the later mating cycle the preparation stage comprises providing the haploid offspring cell (150) of the earlier mating cycle as the second cell (120).

5. The method according to claim 2 or 3, wherein the selection stage of the earlier mating cycle comprises selecting the offspring cell (150) from the offspring cell population (160) based on the third selectable trait and the fourth selectable trait, wherein the offspring cell (150) comprises a multiploid offspring cell (152), and wherein: the first locus (10) of the first cell (110) further encodes a first mating-type guide RNA (115), wherein the first mating-type guide RNA (115) is configured to hybridize with a second mating-type target sequence (92), wherein a chromosomal mating-type locus (90) of the second cell (120) comprises the second mating-type target sequence (92), wherein the offspring cell (150) of the earlier mating cycle has the first mating type, wherein the offspring cell (150) of the earlier mating cycle is used as the first cell (110) in the later mating cycle; or the second locus (20) of the second cell (120) further encodes a second mating-type guide RNA, wherein the second mating-type guide RNA is configured to hybridize with a first mating-type target sequence (91), wherein a chromosomal mating-type locus (90) of the first cell (110) comprises the first mating-type target sequence (91), wherein the offspring cell (150) of the earlier mating cycle has the second mating type, wherein the offspring cell (150) of the earlier mating cycle is used as the second cell (120) in the later mating cycle.

6. The method according to any one of the preceding claims, wherein the chromosomal loci further include the first locus and the second locus, wherein the first cell (110) is homozygous or hemizygous for at least the first locus (10), the second locus (20), thefourth locus (40) and the fifth locus (50), and / or wherein the second cell (120) is homozygous or hemizygous for at least the first locus (10), the second locus (20), the fourth locus (40) and the fifth locus (50).

7. The method according to any one of the preceding claims, wherein the first cell (110) and the second cell (120) are haploid.

8. The method according to any one of the preceding claims, wherein the microbial species is a fungus, preferably a yeast, more preferably selected from the group consisting of Saccharomyces spp., Kluyveromyces spp., Schizosaccharomyces spp., Candida spp., Pichia spp., Yarrow ia spp., and Aspergillus spp.

9. The method according to any claim 8, wherein the microbial species is Saccharomyces cerevisiae, and wherein: the first mating type comprises mating type MATa and the second mating type comprises mating type MATa:, or the first mating type comprises mating type MATa and the second mating type comprises mating type MATa.

10. The method according to any one of the preceding claims, wherein the selectable traits of the first selectable marker (201), the second selectable marker (202), the third selectable marker (203) and the fourth selectable marker (204) are selected from the group consisting of antibiotic resistance traits and prototrophic traits.

11. The method according to any one of the preceding claims, wherein: the first guide RNA (15) is configured to hybridize with the first target sequence (16) along a first complementary region (14), wherein the first complementary region (14) has a length of at least m nucleotides, wherein m is at least 18, and wherein the first guide RNA (15) and the first target sequence (16) have at least m-2 complementary nucleotides in the first complementary region (14); and the second guide RNA (25) is configured to hybridize with the second target sequence (27) along a second complementary region (24), wherein the second complementary region (24) has a length of at least n nucleotides, wherein n is at least 18, and wherein thesecond guide RNA (25) and the second target sequence (27) have at least n-2 complementary nucleotides in the second complementary region (24).

12. The method according to any one of the preceding claims, wherein the RNA-guided DNA endonuclease (35) comprises a Casl2a endonuclease, wherein: the first guide RNA (15) is configured to functionally associate with the RNA-guided DNA endonuclease (35) to form a first ribonucleoprotein (36), wherein the first ribonucleoprotein (36) is configured to cleave the first locus (10) at the first target sequence (16); and the second guide RNA (25) is configured to functionally associate with the RNA-guided DNA endonuclease (35) to form a second ribonucleoprotein (37), wherein the second ribonucleoprotein (37) is configured to cleave the second locus (20) at the second target sequence (27).

13. A recombinant cell (170) of a microbial species, wherein the microbial species has a first mating type and a second mating type compatible with the first mating type, wherein the microbial species has a first locus (10), a third locus (30), a fourth locus (40), and a fifth locus (50), wherein preferably at least the fourth locus (40) and the fifth locus (50) are chromosomal loci; and wherein: the first locus (10) of the recombinant cell (170) encodes a first guide RNA (15), wherein the first guide RNA (15) is configured to hybridize with a first target sequence (16), wherein the first target sequence (16) comprises a first wildtype sequence of the first locus (10); the third locus (30) of the recombinant cell (170) encodes an RNA-guided DNA endonuclease (35), wherein the RNA-guided DNA endonuclease (35) is configured to functionally associate with the first guide RNA (15); the fourth locus (40) of the recombinant cell (170) comprises a mating-type promoter (41,42) configured to facilitate expression of a first selectable marker (201) providing a first selectable trait (a) in cells having the first mating type (101) or (b) in cells having the second mating type (102); the fifth locus (50) of the recombinant cell (170) comprises a third selectable marker (203) providing a third selectable trait; and preferably the first selectable trait and the third selectable trait differ.

14. The recombinant cell (170) of claim 13, obtainable using the method according to any one of the preceding claims 1-12.

15. The recombinant cell (170) of claim 13 or 14, wherein the microbial species is as defined in claim 8 or 9.

16. The recombinant cell (170) of any one of claims 13-15, wherein the microbial species further has a second locus (20), wherein the recombinant cell (170) is haploid, and wherein: the second locus (20) of the recombinant cell (170) encodes a second guide RNA (25), wherein the second guide RNA (25) is configured to hybridize with a second target sequence (27), wherein the second target sequence (27) comprises a second wildtype sequence of the second locus (20).

17. The recombinant cell (170) according to any one of claims 13-16 being multiploid, wherein the recombinant cell (170) is homozygous for at least the first locus (10) and the second locus (20) and heterozygous for at least the fourth locus (40) and the fifth locus (50), wherein: the second locus (20) of the recombinant cell (170) encodes a second guide RNA (25), wherein the second guide RNA (25) is configured to hybridize with a second target sequence (27), wherein the second target sequence (27) comprises a second wildtype sequence of the second locus (20); a fourth locus first allele (46) of the recombinant cell (170) comprises a first mating-type promoter (41) configured to facilitate expression of the first selectable marker (201) providing the first selectable trait in cells having the first mating type (101); a fourth locus second allele (47) of the recombinant cell (170) comprises a second mating-type promoter (42) configured to facilitate expression of a second selectable marker (202) providing a second selectable trait in cells having the second mating type (102); a fifth locus first allele (56) of the recombinant cell (170) comprising the third selectable marker (203) providing the third selectable trait; a fifth locus second allele (57) of the recombinant cell (170) comprising a fourth selectable marker (204) providing a fourth selectable trait; and preferably the first selectable trait, the second selectable trait, the third selectable trait, and the fourth selectable trait differ from each other.

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