WO2018053457A1

WO2018053457A1 - Methods of genetically altering yeast to produce yeast variants

Info

Publication number: WO2018053457A1
Application number: PCT/US2017/052128
Authority: WO
Inventors: Christian Schroeder KAAS; Alejandro Chavez; Xiaoge GUO
Original assignee: Harvard University
Current assignee: Harvard University
Priority date: 2016-09-19
Filing date: 2017-09-19
Publication date: 2018-03-22
Anticipated expiration: 2019-03-19
Also published as: WO2018053457A9

Abstract

A method of genetically altering a cell is provided including combining the cell including a target genomic sequence with a vector including an oligonucleotide sequence including a unique gene drive and nucleic acid sequences that target the target genomic sequence, wherein the unique gene drive is inserted into the target genomic sequence to produce a genetically altered cell.

Description

METHODS OF GENETICALLY ALTERING YEAST TO PRODUCE YEAST

VARIANTS

RELATED APPLICATION DATA

This application claims priority to U.S. Provisional Application No. 62/396,395 filed on September 19, 2016 which is hereby incorporated herein by reference in its entirety for all purposes.

STATEMENT OF GOVERNMENT INTERESTS

This invention was made with government support under HG008525 and 5T32CA009216-34 awarded by National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

Gene drives are generally known as genetic elements that skew the natural odds in their favor of being inherited and passed on by progeny. Examples include homing endonuclease genes that copy themselves into chromosomes lacking them, segregation distorters that destroy competing chromosomes during meiosis, transposons that insert copies of themselves elsewhere in the genome, Medea elements that eliminate competing siblings who do not inherit them, and maternally heritable microorganisms such as Wolbachia that induce cytoplasmic incompatibility to favor the spread of infected individuals. Because they circumvent the normal rules of natural selection, all of these elements have been considered as potential "gene drive" systems capable of spreading engineered modifications through insect vector populations to block the spread of disease. Homing endonuclease based gene drives have been proposed as a means of genetically controlling malaria mosquito populations. See Windbichler et al., Nature, doi:10.1038/nature09937 (2011). Site-specific selfish genes have been proposed as tools for the control and genetic engineering of natural populations. See Burt, Proc. R. Soc. Lond. B (2003) 270, 921-928 (2003). However, such proposed gene drives are limited in their ability to rapidly and efficiently introduce genetically altered variants in a given population. A need therefore exists to develop gene drives which can target any desired gene and allow for rapid and efficient generation of genetically altered variants in a given population.

SUMMARY

The present disclosure provides for methods of making a plurality of genetically altered proliferating cells based on gene drive, which has been shown to violate Mendelian inheritance allowing for dominant inheritance of drives upon mating of the cells.

Aspects of the present disclosure are directed to engineered foreign nucleic acid sequences containing RNA guided gene drives. These foreign nucleic acid sequences are synthesized as substrate bound oligonucleotide sequences which are stably introduced into the genomes of proliferating cells, such as yeast cells. Through rounds of mating and sporulation, a plurality of genetically altered yeast cell variants are generated. As a result of the foreign nucleic acid sequence being stably entered into the genome of the cell progeny, the cell progeny may have one or more desired traits resulting from expression of the foreign nucleic acid.

According to certain aspects, the foreign nucleic acid sequence encodes at least an RNA guided DNA binding protein, such as one or more of an RNA guided DNA binding protein nuclease, an RNA guided DNA binding protein nickase or a nuclease null RNA guided DNA binding protein fused to a cleavage domain such as a nuclease or nickase domain, and one or more or a plurality of guide RNAs (ribonucleic acids). A guide RNA is complementary to DNA (deoxyribonucleic acid), such as a target DNA in the genome of a proliferating cell. The foreign nucleic acid sequence also encodes at least one or more promoters such that the proliferating cell may express the RNA guided DNA binding protein and the guide RNAs or any other nucleic acid sequence or gene which may be in the foreign nucleic acid sequence. One of skill will readily be able to identify suitable promoters based on the present disclosure and the particular cell. The foreign nucleic acid sequence may also include any other nucleic acid sequence or sequences known to those of skill in the art to be required for expression of the foreign nucleic acid sequence by a proliferating cell. The foreign nucleic acid sequence may also include any other gene sequence or gene sequences desired to be expressed by the cell. Such a gene sequence or such gene sequences may be referred to as "cargo sequence" or "cargo DNA." It is to be understood that one of skill will readily be able to identify one or more gene sequences depending upon the desired trait one of skill wishes to be exhibited by the cell or the organism developed from the cell when the cell expresses the foreign nucleic acid sequence. The foreign nucleic acid sequence also encodes at least two flanking sequences which flank at least the RNA guided DNA binding protein nuclease and the one or more guide RNAs. As known to those of skill in the art, flanking sequences are placed at opposite ends of a particular nucleic acid sequence such that the particular nucleic acid sequence is between the flanking sequences. According to one aspect, the flanking sequences include at least a sequence which is identical to a corresponding sequence on a selected chromosome. According to one aspect, such flanking sequences allow a cell to insert the foreign nucleic acid sequence into its genomic DNA at a cut site using well-understood mechanisms such as homologous recombination or nonhomologous end joining.

According to certain aspects, when the foreign nucleic acid sequence is expressed by the cell, one or more of an RNA guided DNA binding protein and one or more or a plurality of guide RNAs are produced. The RNA guided DNA binding protein and a guide RNA produces a complex of the RNA guided DNA binding protein, the guide RNA and a double stranded DNA target sequence. In this aspect, the RNA is said to guide the DNA binding protein to the double stranded DNA target sequence for binding thereto. This aspect of the present disclosure may be referred to as co-localization of the RNA and DNA binding protein to or with the double stranded DNA.

DNA binding proteins within the scope of the present disclosure may include those which create a double stranded break (which may be referred to as a DNA binding protein nuclease), those which create a single stranded break (referred to as a DNA binding protein nickase) or those which have no nuclease activity (referred to as a nuclease null DNA binding protein) but otherwise bind to target DNA. According to certain aspects, the foreign nucleic acid sequence may encode one or more of a DNA binding protein nuclease, a DNA binding protein nickase or a nuclease null DNA binding protein fused to a cleavage domain such as a nuclease or a nickase domain. The foreign nucleic acid sequence may also encode one or more transcriptional regulator proteins or domains or one or more donor nucleic acid sequences that are intended to be inserted into the genomic DNA. According to one aspect, the foreign nucleic acid sequence encoding an RNA guided nuclease-null DNA binding protein which is fused to a cleavage domain may further encode the transcriptional regulator protein or domain fused to the RNA guided nuclease-null DNA binding protein. According to one aspect, the foreign nucleic acid sequence encoding one or more RNAs further encodes a target of an RNA-binding domain and the foreign nucleic acid encoding the transcriptional regulator protein or domain further encodes an RNA-binding domain fused to the transcriptional regulator protein or domain.

Accordingly, expression of a foreign nucleic acid sequence by a cell may result in a double stranded break, a single stranded break and/or transcriptional activation or repression of the genomic DNA. Donor DNA may be inserted at the break site by cell mechanisms such as homologous recombination or nonhomologous end joining. It is to be understood that expression of a foreign nucleic acid sequence as described herein may result in a plurality of double stranded breaks or single stranded breaks at various locations along target genomic DNA, including one or more or a plurality of gene sequences, as desired.

Aspects of the present disclosure are directed to using the foreign nucleic acid sequence as a gene drive. The concept of a gene drive is known to those of skill in the art and refers to a foreign nucleic acid sequence which when expressed is capable of inserting itself into the genome of the cell into which it has been introduced. The concept of a gene drive is provided in Windbichler et al., Nature, doi:10.1038/nature09937 (2011) and Burt, Proc. R. Soc. Lond. B (2003) 270, 921-928 (2003) each of which is hereby incorporated by reference in their entireties.

According to one aspect of the present disclosure, the foreign nucleic acid sequences described herein act as gene drives when introduced into a cell. In one aspect, the foreign nucleic acid sequence is expressed by the cell to produce an RNA guided DNA binding protein and a guide RNA. The guide RNA is complementary to a target DNA sequence on a chromosome. The RNA guided DNA binding protein and the guide RNA co-localize to the target DNA, and the target DNA is cleaved in a site specific manner. The target DNA may be a target DNA site on one or both chromosomes of a chromosome pair. The foreign nucleic acid sequence is then inserted into the genomic DNA at the target DNA cut site, for example, by homologous recombination. The foreign nucleic acid sequence may be inserted into the genomic DNA at one or both chromosomes of a chromosome pair if each chromosome has been cleaved in a site specific manner by the RNA guided DNA binding protein. If inserted into both chromosomes of a chromosome pair, then the cell is homozygous for the foreign nucleic acid sequence. In an alternate embodiment, the foreign nucleic acid sequence is inserted into a first chromosome of a chromosome pair. The inserted foreign nucleic acid sequence is then expressed by the cell and the RNA guided DNA binding protein and the guide

RNA co-localize at or to a second chromosome of a chromosome pair which is then cleaved in a site specific manner, just as was the first chromosome. The cleaved target DNA in the second chromosome is then repaired, for example by homologous recombination, using the first chromosome as a template. In this manner, the second chromosome is repaired to include the foreign nucleic acid sequence resulting in a cell that is homozygous for the foreign nucleic sequence, i.e., the foreign nucleic acid sequence is present in both the first and second chromosome of the chromosome pair. The mechanisms by which cells repair damaged, cleaved or cut genomic DNA are well known. Aspects of the present disclosure take advantage of these cell mechanisms in combination with DNA binding protein nucleases or nickases to create a gene drive with desired foreign genetic material that inserts into the genomic DNA of cells wherein the cell becomes homozygous for the foreign genetic material. A population of transgenic organisms having one or more desired traits can be generated when the foreign genetic material is introduced into a germline cell of the organism. In the case of yeast, through rounds of mating and sporulation, a plurality of genetically altered yeast cell variants are generated.

According to the present disclosure, a method of making a plurality of substrate bound oligonucleotide sequences for insertion into corresponding vectors is provided. The method includes synthesizing the plurality of oligonucleotide sequences with each oligonucleotide sequence including at least one unique guide RNA spacer sequence and a plurality of endonuclease restriction sites, wherein each oligonucleotide sequence includes first flanking outer endonuclease restriction sites, second flanking inner endonuclease restriction sites, flanking random primer binding sites and a unique gene drive component flanked by the flanking random primer binding sites, wherein the unique gene drive component includes in series, an upstream target nucleic acid flanking sequence, a third endonuclease restriction site, the at least one unique guide RNA spacer sequence having a sequence complementary to a target nucleic acid sequence, a fourth endonuclease restriction site and a downstream target nucleic acid flanking sequence, and wherein the first flanking outer endonuclease restriction sites, the second flanking inner endonuclease restriction sites, the third endonuclease restriction site, and the fourth endonuclease restriction site are different.

According to the present disclosure, a method of making a plurality of vectors with each vector of the plurality including a unique gene drive component is provided. The method includes removing a plurality of bound oligonucleotide sequences from a substrate using a first endonuclease, wherein each substrate bound oligonucleotide sequence includes at least one unique guide RNA spacer sequence and a plurality of endonuclease restriction sites, wherein each substrate bound oligonucleotide sequence includes first flanking outer endonuclease restriction sites, second flanking inner endonuclease restriction sites, flanking random primer binding sites and the unique gene drive component flanked by the flanking random primer binding sites, wherein the unique gene drive component includes in series, an upstream target nucleic acid flanking sequence, a third endonuclease restriction site, the at least one unique guide RNA spacer sequence having a sequence complementary to a target nucleic acid sequence, a fourth endonuclease restriction site and a downstream target nucleic acid flanking sequence, wherein the first flanking outer endonuclease restriction sites, the second flanking inner endonuclease restriction sites, the third endonuclease restriction site, and the fourth endonuclease restriction site are different, wherein the first endonuclease corresponds to the first flanking outer endonuclease restriction sites and cuts the bound oligonucleotide sequence at the first flanking outer endonuclease restriction sites to produce a plurality of unbound oligonucleotide sequences, and inserting each unbound oligonucleotide sequence into a corresponding vector to produce the plurality of vectors each with a unique gene drive component.

According to the present disclosure, a method of making a plurality of vectors with each vector of the plurality including a unique gene drive is provided. The method includes providing a plurality of vectors with each vector of the plurality including an oligonucleotide sequence including first flanking endonuclease restriction sites, flanking random primer binding sites and a unique gene drive component flanked by the flanking random primer binding sites, wherein the unique gene drive component includes in series, an upstream target nucleic acid flanking sequence, a second endonuclease restriction site, at least one unique guide RNA spacer sequence having a sequence complementary to a target nucleic acid sequence, a third endonuclease restriction site and a downstream target nucleic acid flanking sequence, wherein the first flanking endonuclease restriction sites, the second endonuclease restriction site, and the third endonuclease restriction site are different, and creating the plurality of vectors with each vector of the plurality including a unique gene drive by (1) cutting each oligonucleotide sequence within its corresponding vector at the second endonuclease restriction site using a corresponding second endonuclease and inserting a guide RNA promotor and, optionally, a selection marker therein, and (2) cutting each oligonucleotide sequence within its corresponding vector at the third endonuclease restriction site using a corresponding third endonuclease and inserting a spacer tail nucleic acid sequence encoding a tracr mate sequence and a tracr sequence therein to create at least one guide RNA and optionally inserting a cargo sequence therein.

According to the present disclosure, a method of making a plurality of genetically altered proliferating cells is provided. The method includes combining a plurality of proliferating cells including a target gene sequence with a plurality of vectors with each vector of the plurality including an oligonucleotide sequence including first flanking endonuclease restriction sites, flanking random primer binding sites and a unique gene drive flanked by the flanking random primer binding sites, wherein the unique gene drive includes in series, an upstream target genomic nucleic acid flanking sequence, a guide RNA promoter, at least one unique guide RNA sequence having a spacer sequence complementary to the target genomic nucleic acid sequence, an optional cargo sequence, and a downstream target genomic nucleic acid flanking sequence, wherein corresponding cells of the plurality of proliferating cells each receive a single vector, wherein the corresponding cells each include an RNA guided DNA binding protein, and wherein the guide RNA is produced and a colocalization complex of the guide RNA and the RNA guided DNA binding protein forms at the target genomic nucleic acid sequence and the target genomic nucleic acid sequence is cut and the unique gene drive is inserted therein to produce the plurality of genetically altered proliferating cells.

The disclosure provides a substrate having a plurality of substrate bound oligonucleotide sequences for insertion into corresponding vectors, with each oligonucleotide sequence including at least one unique guide RNA spacer sequence and a plurality of endonuclease restriction sites, wherein each oligonucleotide sequence includes first flanking outer endonuclease restriction sites, second flanking inner endonuclease restriction sites, flanking random primer binding sites and a unique gene drive component flanked by the flanking random primer binding sites, wherein the unique gene drive component includes in series, an upstream target nucleic acid flanking sequence, a third endonuclease restriction site, the at least one unique guide RNA spacer sequence having a sequence complementary to a target nucleic acid sequence, a fourth endonuclease restriction site and a downstream target nucleic acid flanking sequence, and wherein the first flanking outer endonuclease restriction sites, the second flanking inner endonuclease restriction sites, the third endonuclease restriction site, and the fourth endonuclease restriction site are different.

The disclosure further provides a modified donor method for improved chromosomal integration efficiency of gene drives into cells such as yeast cells. In an exemplary embodiment,

Two linear fragments are prepared from a donor plasmid vector including a gene drive. The two linear fragments are transformed into the proliferating cells. The two linear fragments undergo homologous recombination to generate a stably inherited circular plasmid after being transformed into the cells. The modified donor method results in improved transformation efficiency of about 10, 100, to 1000 fold. The modified donor method results in 100% integration efficiency of the gene drive at the desired locus.

In one embodiment, a method of making a genetically altered proliferating cell is provided. The method comprises providing to the proliferating cell including a target gene sequence with a vector including an oligonucleotide sequence including first flanking endonuclease restriction sites, flanking random primer binding sites and a unique gene drive flanked by the flanking random primer binding sites, wherein the unique gene drive includes in series, an upstream target genomic nucleic acid flanking sequence, a guide RNA promoter, at least one unique guide RNA sequence having a spacer sequence complementary to the target genomic nucleic acid sequence, an optional cargo sequence, and a downstream target genomic nucleic acid flanking sequence, wherein the proliferating cell includes an RNA guided DNA binding protein, and wherein the guide RNA is produced and a colocalization complex of the guide RNA and the RNA guided DNA binding protein forms at the target genomic nucleic acid sequence and the target genomic nucleic acid sequence is cut and the unique gene drive is inserted therein to produce the genetically altered proliferating cell. In one embodiment, the vector is provided to the proliferating cell in the form of two linear fragments. In another embodiment, the two linear fragments undergo homologous recombination to generate a stably inherited circular plasmid after being transformed into the cell.

The disclosure provides a plurality of vectors with each vector of the plurality includes an oligonucleotide sequence according to the present disclosure. The disclosure further provides a cell including a vector including an oligonucleotide sequence according to the present disclosure. Further features and advantages of certain embodiments of the present invention will become more fully apparent in the following description of embodiments and drawings thereof, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. The foregoing and other features and advantages of the present embodiments will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings in which:

FIGS. 1A-1B show the difference of Mendelian and biased inheritance in Yeast (Saccharomyces cerevisiae). Gene drives are able to selfishly drive themselves through a population of cells thus when two haploid cells each with drives mate to produce a diploid cell and the diploid cell subsequently sporulates and produces haploid cells, all of their progeny will inherit both drives, in contrast to standard Mendelian inheritance in which only 25% of the progeny receive both genes (FIG. 1A). This feature is what enables gene drives to rapidly propagate through a population of sexually reproducing cells at high efficiency (FIG. IB).

FIG. 2 is a graph depicting variant generation built up through mating of yeast cells each containing different gene drives. If a population of yeast cells contains 20 different gene drives, each containing one unique gene drive per yeast cell, then following mating of the yeast cell, each yeast cell will contain two gene drives (20x19 = 380) variants. If these yeast cells are mated once again each cell will contain up to four gene drives (20x19x18x17 = 116,280) and so forth. An enormous diversity of variant cells can thus be generated targeting specific pathways for optimization of a given trait such as growth, production/degradation of a metabolite, etc.).

FIGS. 3A-3G show a schematic of a general strategy for construction of gene drive from OLS array. FIG. 3A. An ssDNA oligo is synthesized containing the variable sequence constituting a gene drive including e.g., upstream flanking sequence, targeting sequence and downstream flanking sequence. FIG. 3B. This oligo is amplified into a dsDNA PCR product. FIG. 3C. The dsDNA PCR product is then digested with a restriction enzyme insertion of the oligo into a plasmid backbone. FIGS. 3D-3E. The plasmid is then linearized by another restriction enzyme to insert cargo sequence and promoter sequence for expression of the guide RNA and then linearized again by a 3^rd enzyme to insert the guide RNA scaffold and cargo sequence. FIGS. 3F-3G. The final gene drive construct is then amplified using standard primers flanking the entire gene drive and subsequent enzymatic digestion remove the general sequence flanking the gene drive exposing the flanking sequence.

FIG. 4 depicts general building blocks for insertion into oligo based gene drives. Promoter (left) and guide RNA scaffold + terminator (right) sequences are shown with different cargo examples. The sequences are flanked by type 2 restriction sites for standardized insertion into plasmids.

FIGS. 5A-5C show integration of a gene drive for promoter swapping in yeast cells. FIG. 5A shows a schematic of a gene drive designed for targeting upstream of the initiation codon of the ADE2 gene in a yeast cell expressing Cas9. Following the introduction of a double stranded break the gene drive is wedged in between the native promoter and the ADE2 coding region. FIG. 5B shows that if the gene drive does not contain a selection marker inside the gene drive element, a low insertion efficiency is observed as compared to inserting a selection marker inside the gene drive (red colonies represent successful integration). FIG. 5C show yeast cells containing no gene drives display white phenotype on both rich media (YPD) and minimal media with galactose (MM+Galactose). In yeast cells containing a gene drive upstream of ADE2 without a new promoter being added the phenotype is red (indicating the cells do not express ADE2) on both types of media. In yeast cells containing a gene drive where the gene drive inserts a GAL7 promoter upstream of the ADE2 gene the phenotype is red on YPD and white on minimal media with galactose (indicating the cells do no express ADE2 on YPD media but will produce ADE2 on galactose media).

FIGS. 6A-6C show enriching for haploid and diploid cells using counter selectable markers. FIG. 6A is a graph that shows that in order to cycle gene drives into yeast cells, the haploid yeast cells are mated to yield diploid yeast cells, which will subsequently sporulate and produce haploid yeast cells. FIG. 6B shows that enriching is done by using haploid specific promoters which select for haploid yeast cells in media without Tryptophan (due to inactive transcription of TRPl) and uracil (due to inactive transcription of URA3); whereas diploid yeast cells can be selected for in media with 5-FOA (toxic to cells expressing URA3) and 5- FAA (toxic to cells expressing TRPl). FIG. 6C shows that the selection is shown to be most efficient when both 5-FAA and 5-FOA are added in the same mixture.

FIGS. 7A-7B show a comparison of transformation efficiency between yeast transformed with circular plasmid (Fig. 7A) and yeast transformed with linearized fragments (Fig. 7B).

DETAILED DESCRIPTION

The present disclosure provides methods of making a plurality of genetically altered proliferating cells based on the technology of gene drive. Gene drives have been shown to violate Mendelian inheritance allowing for biased inheritance of drives upon mating. Yeast cell , e.g., Saccharomyces cerevisiae, is able to grow as haploid with either mating type a or a. Yeast cells of opposite mating types can be mated giving rise to diploid yeast cells that can later be sporulated to produce haploid yeast cells again. In the case where a diverse population of yeast cells each containing one gene drive are allowed one cycle of mating and sporulation each member of the population would then contain two gene drives. After one additional mating and sporulation cycle, there will be four gene drives in each member of the population. The number of gene drives in each member of the subsequent cycle of mating and sporulation will grow exponentially, enabling users to rapidly generate high levels of programmable genetic diversity with minimal intervention required.

According to the present disclosure, gene drives can be engineered to alter and edit target genes and/or sequences of a cell or an organism that mainly reproduce sexually, such as yeast, fungi, insects, animals and plants. The disclosure provides that a particular gene drive component can be synthesized in an oligonucleotide sequence. In addition to the gene drive component, the oligonucleotide sequence includes additional sequences such as endonuclease restriction sites and primer binding sites for cloning and amplification. Therefore, in one embodiment, the disclosure provides a method of making a plurality of substrate bound oligonucleotide sequences for insertion into corresponding vectors including synthesizing the plurality of oligonucleotide sequences with each oligonucleotide sequence including at least one unique guide RNA spacer sequence and a plurality of endonuclease restriction sites, wherein each oligonucleotide sequence includes first flanking outer endonuclease restriction sites, second flanking inner endonuclease restriction sites, flanking random primer binding sites and a unique gene drive component flanked by the flanking random primer binding sites, wherein the unique gene drive component includes in series, an upstream target nucleic acid flanking sequence, a third endonuclease restriction site, the at least one unique guide RNA spacer sequence having a sequence complementary to a target nucleic acid sequence, a fourth endonuclease restriction site and a downstream target nucleic acid flanking sequence, and wherein the first flanking outer endonuclease restriction sites, the second flanking inner endonuclease restriction sites, the third endonuclease restriction site, and the fourth endonuclease restriction site are different.

According to certain embodiments, the plurality of oligonucleotide sequences is made by array-based oligonucleotide synthesis including but are not limited to semiconductor-based electrochemical-synthesis process, photolithographic techniques, inkjet printing, and successively reacting nucleotide monomers. According to one embodiment, the plurality of oligonucleotide sequences is made using monomer by monomer oligonucleotide synthesis.

The disclosure provides that sequences amenable for cloning of the gene drive component through vectors can be included in the oligonucleotide sequences containing the gene drive component. These additional sequences can include but are not limited to endonuclease restriction sites. According the some embodiments, each endonuclease restriction site of the plurality of endonuclease restriction sites is a member selected from the group consisting of type II restriction endonucleases such as Acul, Alwl, Bael, Bbsl, Bbvl, Bed, BceAI, Bcgl, BciVI, BcoDI, BfuAI, Bmrl, Bpml, BpuEI, Bsal, BsaXI, BseRI, Bsgl, BsmAI, BsmBI, BsmFI, Bsml, BspCNI, BspMI, BspQI, BsrDI, Bsrl, BtgZI, BtsCI, Btsl, BtsIMutI, CspCI, Earl, Ecil, Faul, Fokl, Hgal, Hphl, HpyAV, MboII, Mlyl, Mmel, Mnll, NmeAIII, Plel, Sapl, and SfaNI.

The present disclosure contemplates inserting the oligonucleotide sequence containing the gene drive into the target genome. According to one embodiment, the insertion is via homologous recombination. According to one embodiment, the upstream target nucleic acid flanking sequence and the downstream target nucleic acid flanking sequence are complementary to corresponding sequences between an endogenous promoter and a corresponding endogenous gene within a target cell. According to another embodiment, the upstream target nucleic acid flanking sequence and the downstream target nucleic acid flanking sequence are complementary to corresponding sequences between gene coding regions of an endogenous gene within a target cell. The disclosure provides that the number of oligonucleotide sequences depends on the particular gene drives and the size of the target genome and target genes. In certain embodiments, the plurality of oligonucleotide sequences includes between 2 and 250,000, between 10 and 100,000, between 20 and 6,000, between 50 and 1,000, and between 100 and 500 oligonucleotide sequences.

The present disclosure provides that the oligonucleotides containing the gene drive component will be cloned into vectors for additional engineering and amplification of the gene drive. According to one embodiment, a method of making a plurality of vectors with each vector of the plurality including a unique gene drive component is provided including removing a plurality of bound oligonucleotide sequences from a substrate using a first endonuclease, wherein each substrate bound oligonucleotide sequence includes at least one unique guide RNA spacer sequence and a plurality of endonuclease restriction sites, wherein each substrate bound oligonucleotide sequence includes first flanking outer endonuclease restriction sites, second flanking inner endonuclease restriction sites, flanking random primer binding sites and the unique gene drive component flanked by the flanking random primer binding sites, wherein the unique gene drive component includes in series, an upstream target nucleic acid flanking sequence, a third endonuclease restriction site, the at least one unique guide RNA spacer sequence having a sequence complementary to a target nucleic acid sequence, a fourth endonuclease restriction site and a downstream target nucleic acid flanking sequence, wherein the first flanking outer endonuclease restriction sites, the second flanking inner endonuclease restriction sites, the third endonuclease restriction site, and the fourth endonuclease restriction site are different, wherein the first endonuclease corresponds to the first flanking outer endonuclease restriction sites and cuts the bound oligonucleotide sequence at the first flanking outer endonuclease restriction sites to produce a plurality of unbound oligonucleotide sequences, and inserting each unbound oligonucleotide sequence into a corresponding vector to produce the plurality of vectors each with a unique gene drive component. The disclosure provides that the vector is a plasmid or any other genetic element that can be propagated in a bacterial host.

The present disclosure provides a method of making a plurality of vectors with each vector of the plurality including a unique gene drive including providing a plurality of vectors with each vector of the plurality including an oligonucleotide sequence including first flanking endonuclease restriction sites, flanking random primer binding sites and a unique gene drive component flanked by the flanking random primer binding sites, wherein the unique gene drive component includes in series, an upstream target nucleic acid flanking sequence, a second endonuclease restriction site, at least one unique guide RNA spacer sequence having a sequence complementary to a target nucleic acid sequence, a third endonuclease restriction site and a downstream target nucleic acid flanking sequence, wherein the first flanking endonuclease restriction sites, the second endonuclease restriction site, and the third endonuclease restriction site are different, and creating the plurality of vectors with each vector of the plurality including a unique gene drive by (1) cutting each oligonucleotide sequence within its corresponding vector at the second endonuclease restriction site using a corresponding second endonuclease and inserting a guide RNA promotor and, optionally, a selection marker therein, and (2) cutting each oligonucleotide sequence within its corresponding vector at the third endonuclease restriction site using a corresponding third endonuclease and inserting a spacer tail nucleic acid sequence encoding a tracr mate sequence and a tracr sequence therein to create at least one guide RNA and optionally inserting a cargo sequence therein. According to one embodiment, the spacer tail nucleic acid sequence further encodes a transcriptional modulator to create a guide RNA with the transcriptional modulator bound thereto.

The disclosure provides that the engineered gene drive will be delivered into proliferating cells to alter the cell's genome and to produce a plurality of variants. According to the present disclosure, a method of making a plurality of genetically altered proliferating cells is provided including combining a plurality of proliferating cells including a target gene sequence with a plurality of vectors with each vector of the plurality including an oligonucleotide sequence including first flanking endonuclease restriction sites, flanking random primer binding sites and a unique gene drive flanked by the flanking random primer binding sites, wherein the unique gene drive includes in series, an upstream target genomic nucleic acid flanking sequence, a guide RNA promoter, at least one unique guide RNA sequence having a spacer sequence complementary to the target genomic nucleic acid sequence, an optional cargo sequence, and a downstream target genomic nucleic acid flanking sequence, wherein corresponding cells of the plurality of proliferating cells each receive a single vector, wherein the corresponding cells each include an RNA guided DNA binding protein, and wherein the guide RNA is produced and a colocalization complex of the guide RNA and the RNA guided DNA binding protein forms at the target genomic nucleic acid sequence and the target genomic nucleic acid sequence is cut and the unique gene drive is inserted therein to produce the plurality of genetically altered proliferating cells. In one embodiment, the method further includes a first round step of mating the plurality of genetically altered proliferating cells among themselves to produce a plurality of first round variant cells with each variant having two unique gene drives. In another embodiment, the method further includes a second round step of mating the plurality of first round variant cells among themselves to produce a plurality of second round variant cells with each variant having four unique gene drives. In yet another embodiment, the method further includes a third round step of mating the plurality of second round variant cells among themselves to produce a plurality of third round variant cells with each variant having eight unique gene drives. In still another embodiment, the method further includes a fourth round step of mating the plurality of third round variant cells among themselves to produce a plurality of fourth round variant cells with each variant having sixteen unique gene drives. In yet another embodiment, the method further includes subsequent rounds of mating the plurality of previous round variant cells among themselves to produce a plurality of variant cells with each variant having amplified gene drives before equilibrium is attained.

In certain embodiments, the proliferating cell type is a member of the group consisting of genus Saccharomyces, genus Schizosaccharomyces), genus Kluveromyces, genus Candida and Pichia pastoris. In other embodiments, the proliferating cell type is a member of the group consisting of Aspergillus nidulans, A. oryza, A. niger, and A. sojae.

According to the present disclosure, a substrate having a plurality of substrate bound oligonucleotide sequences containing the gene drive are provided according to the disclosure.

The disclosure provides a plurality of vectors with each vector of the plurality including an oligonucleotide sequence containing the gene drive is provided according to the disclosure. In some embodiments, the vector is a plasmid or any other genetic element that can be propagated in a bacterial host. In other embodiments, each vector of the plurality including an oligonucleotide sequence including first flanking endonuclease restriction sites, flanking random primer binding sites and a unique gene drive flanked by the flanking random primer binding sites, wherein the unique gene drive includes in series, an upstream target genomic nucleic acid flanking sequence, a guide RNA promoter, at least one unique guide RNA sequence having a spacer sequence complementary to the target genomic nucleic acid sequence, an optional cargo sequence, and a downstream target genomic nucleic acid flanking sequence.

The disclosure provides a cell including a vector including an oligonucleotide sequence containing the gene drive according to the disclosure.

EXEMPLARY PROLIFERATING CELL TYPES

Proliferating cell types or organisms useful in the methods described herein are those which proliferate at a rate sufficient to carry out experiments in a desirable period of time. According to one aspect, exemplary proliferating cell types are capable of switching between a haploid and a diploid state.

Exemplary eukaryotic cells include yeast strains or fungus strains.

Exemplary yeast strains include Saccharomyces cerevisia (and subtypes such as S288C, CEN.PK etc), genus Saccharomyces (e.g., S. cerevisiae, S. bayanus, S. boulardii, S. pastorianus, S. rouxii and S. uvarum), Schizosaccharomyces (e.g., S. pombe), Kluveromyces (e.g., K. lactis and K. fragilis), genus Candida (C. albicans, C. krusei and C. tropicalis) and Pichia pastoris and the like.

Exemplary fungus strains include Aspergillus nidulans, A. oryza, A. niger, A. sojae and the like.

Exemplary eukaryotic organisms potentially include all sexually mating eukaryotic organisms which include but are not limited to Drosophila melanogaster, Caenorhabditis elegans, Mus musculus, Rattus norvegicus and the like.

EXEMPLARY CARGO SEQUENCES Exemplary cargo sequences can be used to insert, delete, and/or modulate the target gene or sequences in the cell or for screening and/or selection of the cells containing the gene drive. In one embodiment, the cargo sequence is a target gene promoter. In another embodiment, the cargo sequence is a target gene. In one embodiment, the cargo sequence is a nucleic acid sequence encoding an RNA guided DNA binding protein. In another embodiment, the cargo sequence is a nucleic acid sequence encoding a fluorescent protein allowing for screening of organism carrying the gene drive. In one embodiment, the cargo sequence is a nucleic acid sequence encoding a fluorescent protein fused to a target protein at the C-terminal or N-terminal region. In another embodiment, the cargo sequence is a nucleic acid sequence encoding a scaffold domain fused to a target protein at the C-terminal or N-terminal region, wherein the scaffold domain confers binding property to the target protein for phenotype analysis. In one embodiment, the cargo sequence is a nucleic acid sequence encoding a regulatory subunit fused to a target protein at the C-terminal or N-terminal region, wherein the regulatory subunit creates novel regulatory phenotype of the target gene expression. In another embodiment, the cargo sequence is a nucleic acid sequence containing restriction sites allowing removal of the cargo sequence at a later stage. In one embodiment, the cargo sequence is a nucleic acid sequence encoding an altered endogenous untranslated region of a target gene changing the transcription and/or translation efficiency of the target gene. In one embodiment, the cargo sequence is a nucleic acid sequence encoding a Cas9 protein. In another embodiment, the cargo sequence is a nucleic acid sequence encoding a Cas9 enzyme, a Cas9 nickase, a nuclease null Cas9 fused to a cleavage domain, a nuclease null Cas9 or a nuclease null Cas9 with a transcriptional modulator attached thereto.

EXEMPLARY METHODS OF GENETICALLY MODIFYING A CELL WITH A GENE DRIVE The disclosure provides that the engineered gene drive oligonucleotide sequences will be delivered into a sexually proliferating cell type where the RNA guided DNA binding protein such as endonuclease Cas9 will cut the chromosomes at a specific site. The cell will repair the damage by copying the drive sequence onto the damaged chromosome. This is derived from genome editing techniques and similarly relies on the fact that double strand breaks are most frequently repaired by homologous recombination if a template is present, and less often by non-homologous end joining. The cell then has two copies of the drive sequence. By targeting the gene drive to a gene coding sequence, this gene will be inactivated; additional sequences can be introduced in the gene drive to encode new functions. As an example, yeast cells may be genetically modified using methods known to those of skill in the art including by LiAc, Electroporation, Biolistic transformation as described in Kawai S, Hashimoto W, Murata K. Transformation of Saccharomyces cerevisiae and other fungi: Methods and possible underlying mechanism. Bioengineered Bugs. 2010;l(6):395-403 hereby incorporated by reference in its entirety.

According to certain aspects, the RNA guided DNA binding protein can be provided to a cell by genetically modifying the cell to include a nucleic acid encoding the RNA guided DNA binding protein or otherwise providing a vector or plasmid encoding the RNA guided DNA binding protein wherein the nucleic acid is expressed to produce the RNA guided DNA binding protein. The RNA guided DNA binding protein may also be provided to the cell as a native protein, i.e. not as a product of expression of a nucleic acid sequence. Methods of providing an RNA guided DNA binding protein to a cell are known in the art.

AMPLIFICATION METHODS

Nucleic acids within cells of a pool of proliferating cells, such as yeast or fungi, may be amplified using methods known to those of skill in the art. Exemplary amplification methods include contacting a nucleic acid with one or more primers that specifically hybridize to the nucleic acid under conditions that facilitate hybridization and chain extension. Exemplary methods for amplifying nucleic acids include the polymerase chain reaction (PCR) (see, e.g., Mullis et al. (1986) Cold Spring Harb. Symp. Quant. Biol. 51 Pt 1 :263 and Cleary et al. (2004) Nature Methods 1 :241; and U.S. Patent Nos. 4,683,195 and 4,683,202), anchor PCR, RACE PCR, ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241: 1077-1080; and Nakazawa et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91 :360-364), self sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87: 1874), transcriptional amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86: 1173), Q-Beta Replicase (Lizardi et al. (1988) BioTechnology 6: 1197), recursive PCR (Jaffe et al. (2000) /. Biol. Chem. 275:2619; and Williams et al. (2002) /. Biol. Chem. 277:7790), the amplification methods described in U.S. Patent Nos. 6,391,544, 6,365,375, 6,294,323, 6,261,797, 6,124,090 and 5,612,199, isothermal amplification (e.g., rolling circle amplification (RCA), hyperbranched rolling circle amplification (HRCA), strand displacement amplification (SDA), helicase-dependent amplification (HDA), PWGA) or any other nucleic acid amplification method using techniques well known to those of skill in the art.

SEQUENCING METHODS

Nucleic acids within cells of a pool of proliferating cells, such as yeast or fungi, may be sequenced using methods known to those of skill in the art such as high throughput disclosed in Mitra (1999) Nucleic Acids Res. 27(24):e34; pp.1-6. Sequencing methods useful in the present disclosure include Shendure et al., Accurate multiplex polony sequencing of an evolved bacterial genome, Science, vol. 309, p. 1728-32. 2005; Drmanac et al., Human genome sequencing using unchained base reads on self-assembling DNA nanoarrays, Science, vol. 327, p. 78-81. 2009; McKernan et al., Sequence and structural variation in a human genome uncovered by short-read, massively parallel ligation sequencing using two-base encoding, Genome Res. , vol. 19, p. 1527-41. 2009; Rodrigue et al., Unlocking short read sequencing for metagenomics, PLoS One, vol. 28, el l840. 2010; Rothberg et al., An integrated semiconductor device enabling non-optical genome sequencing, Nature, vol. 475, p. 348-352. 2011; Margulies et al., Genome sequencing in microfabricated high-density picolitre reactors, Nature, vol. 437, p. 376-380. 2005; Rasko et al. Origins of the E. coli strain causing an outbreak of hemolytic- uremic syndrome in Germany, N. Engl. J. Med., Epub. 2011; Hutter et al., Labeled nucleoside triphosphates with reversibly terminating aminoalkoxyl groups, Nucleos. Nucleot. Nucl , vol. 92, p. 879-895. 2010; Seo et al., Four-color DNA sequencing by synthesis on a chip using photocleavable fluorescent nucleotides, Proc. Natl. Acad. Sci. USA. , Vol. 102, P. 5926-5931 (2005); Olejnik et al.; Photocleavable biotin derivatives: a versatile approach for the isolation of biomolecules, Proc. Natl. Acad. Sci. U.S.A. , vol. 92, p. 7590-7594. 1995; US 2009/0062129 and US 2009/0191553.

Exemplary next generating sequencing methods known to those of skill in the art include Massively parallel signature sequencing (MPSS), Polony sequencing, pyrosequencing (454), Illumina (Solexa) sequencing by synthesis, SOLiD sequencing by ligation, Ion semiconductor sequencing (Ion Torrent sequencing), DNA nanoball sequencing, chain termination sequencing (Sanger sequencing), Heliscope single molecule sequencing, Single molecule real time (SMRT) sequencing (Pacific Biosciences) and nanopore sequencing such as is described at world wide website nanoporetech.com.

EXEMPLARY RNA-GUIDED DNA BINDING PROTEINS The RNA-guided DNA binding protein includes an RNA-guided DNA binding protein nuclease, a thermophilic RNA-guided DNA binding protein nuclease, an RNA-guided DNA binding protein nickase, or a nuclease null RNA-guided DNA binding protein fused to a cleavage domain such as a nuclease or a nickase domain. According to one aspect, the RNA- guided DNA binding protein includes a Cas nuclease, a Cas nickase or a nuclease null Cas protein fused to a cleavage domain such as a nuclease or a nickase domain. A Cas nickase or a nuclease-null Cas protein is provided where one or more amino acids in Cas, such as Cas9, are altered or otherwise removed to provide a Cas nickase or a nuclease null Cas protein fused to a cleavage domain such as a nuclease or a nickase domain. According to one aspect, the amino acids include D10 and H840 of Cas9. See Jinek et al., Science 337, 816-821 (2012). RNA guided DNA binding proteins are readily known to those of skill in the art to bind to DNA for various purposes. Such DNA binding proteins may be naturally occurring. DNA binding proteins having nuclease activity are known to those of skill in the art, and include naturally occurring DNA binding proteins having nuclease activity, such as Cas9 proteins present, for example, in Type II CRISPR systems. Such Cas9 proteins and Type II CRISPR systems are well documented in the art. See Makarova et al., Nature Reviews, Microbiology, Vol. 9, June 2011, pp. 467-477 including all supplementary information hereby incorporated by reference in its entirety.

A Cas as described herein may be any Cas known to those of skill in the art that may be directed to a target nucleic acid using an RNA as known to those of skill in the art. The Cas may be wild type or a homolog or ortholog thereof, such as Cpfl (See, Zetsche, Bernd et al., Cpf 1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System, Cell, Volume 163, Issue 3, pgs 759 - 771, hereby incorporated by reference in its entirety). The Cas may be nonnaturally occurring, such as an engineered Cas as disclosed in Slaymaker, I.M., Gao, L., Zetsche, B., Scott, D.A., Yan, W.X. and Zhang, F., 2016. Rationally engineered Cas9 nucleases with improved specificity. Science, 351 (6268), pp.84-88 hereby incorporated by reference in its entirety. The Cas may have one or more nucleolytic domains altered to prevent nucleolytic activity, such as with a Cas nickase or nuclease null or "dead" Cas. Aspects of the present disclosure utilize nicking to effect cutting of one strand of the target nucleic acid. A nuclease null or "dead" Cas may have a nuclease attached thereto to effect cutting, cleaving or nicking of the target nucleic acid. Such nucleases are known to those of skill in the art.

According to one aspect, the RNA-guided DNA binding protein includes a Cas9 nuclease, a Cas9 nickase or a nuclease null Cas9 protein. According to one aspect, the RNA- guided DNA binding protein includes a spCas9 nuclease, a spCas9 nickase or a nuclease null spCas9 protein. According to one aspect, the RNA-guided DNA binding proteins includes S. pyogenes Cas9, S. thermophilis Cas9, N. meningitides Cas9, T. denticola Cas9, or S. aureus Cas9. According to one aspect, the RNA-guided DNA binding protein includes a Cpfl nuclease, a Cpfl nickase or a nuclease null Cpfl protein.

According to one aspect, the Cas9 protein, Cas9 protein nickase or nuclease null Cas9 includes homologs and orthologs thereof which retain the ability of the protein to bind to the DNA and be guided by the RNA. According to one aspect, the Cas9 protein includes the sequence as set forth for naturally occurring Cas9 from S. thermophiles or S. pyogenes or N. meningitides or T. denticola or S. aureus or Cpfl or NgAgo or C2C2 or protein sequences having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% homology thereto and being a DNA binding protein, such as an RNA guided DNA binding protein.

An exemplary CRISPR system includes the S. thermophiles Cas9 nuclease (ST1 Cas9) (see Esvelt KM, et al., Orthogonal Cas9 proteins for RNA-guided gene regulation and editing, Nature Methods., (2013) hereby incorporated by reference in its entirety).An exemplary CRISPR system includes the S. pyogenes Cas9 nuclease (Sp. Cas9), an extremely high-affinity (see Steinberg, S.H., Redding, S., Jinek, M., Greene, E.C. & Doudna, J.A. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507, 62-67 (2014) hereby incorporated by reference in its entirety), programmable DNA-binding protein isolated from a type II CRISPR-associated system (see Garneau, J.E. et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468, 67-71 (2010) and Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816-821 (2012) each of which are hereby incorporated by reference in its entirety). According to certain aspects, a nuclease null or nuclease deficient Cas 9 can be used in the methods described herein. Such nuclease null or nuclease deficient Cas9 proteins are described in Gilbert, L.A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442-451 (2013); Mali, P. et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature biotechnology 31, 833-838 (2013); Maeder, M.L. et al. CRISPR RNA- guided activation of endogenous human genes. Nature methods 10, 977-979 (2013); and Perez- Pinera, P. et al. RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nature methods 10, 973-976 (2013) each of which are hereby incorporated by reference in its entirety. The DNA locus targeted by Cas9 (and by its nuclease-deficient mutant, "dCas9" precedes a three nucleotide (nt) 5'-NGG-3 ' "PAM" sequence, and matches a 15-22-nt guide or spacer sequence within a Cas9-bound RNA cofactor, referred to herein and in the art as a guide RNA. Altering this guide RNA is sufficient to target Cas9 or a nuclease deficient Cas9 to a target nucleic acid. In a multitude of CRISPR-based biotechnology applications (see Mali, P., Esvelt, K.M. & Church, G.M. Cas9 as a versatile tool for engineering biology. Nature methods 10, 957-963 (2013); Hsu, P.D., Lander, E.S. & Zhang, F. Development and Applications of CRISPR-Cas9 for Genome Engineering. Cell 157, 1262-1278 (2014); Chen, B. et al. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155, 1479-1491 (2013); Shalem, O. et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343, 84-87 (2014); Wang, T., Wei, J.J., Sabatini, D.M. & Lander, E.S. Genetic screens in human cells using the CRISPR-Cas9 system. Science 343, 80- 84 (2014); Nissim, L., Perli, S.D., Fridkin, A., Perez-Pinera, P. & Lu, T.K. Multiplexed and Programmable Regulation of Gene Networks with an Integrated RNA and CRISPR/Cas Toolkit in Human Cells. Molecular cell 54, 698-710 (2014); Ryan, O.W. et al. Selection of chromosomal DNA libraries using a multiplex CRISPR system. eLife 3 (2014); Gilbert, L.A. et al. Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Cell (2014); and Citorik, R.J., Mimee, M. & Lu, T.K. Sequence- specific antimicrobials using efficiently delivered RNA-guided nucleases. Nature biotechnology (2014) each of which are hereby incorporated by reference in its entirety), the guide is often presented in a so-called sgRNA (single guide RNA), wherein the two natural Cas9 RNA cofactors (gRNA and tracrRNA) are fused via an engineered loop or linker.

Cas9 proteins are known to exist in many Type II CRISPR systems including the following as identified in the supplementary information to Makarova et al., Nature Reviews, Microbiology, Vol. 9, June 2011, pp. 467-477: Methanococcus maripaludis C7; Corynebacterium diphtheriae; Corynebacterium efficiens YS-314; Corynebacterium glutamicum ATCC 13032 Kitasato; Corynebacterium glutamicum ATCC 13032 Bielefeld; Corynebacterium glutamicum R; Corynebacterium kroppenstedtii DSM 44385; Mycobacterium abscessus ATCC 19977; Nocardia farcinica IFM10152; Rhodococcus erythropolis PR4; Rhodococcus jostii RHA1; Rhodococcus opacus B4 uid36573; Acidothermus cellulolyticus 11B; Arthrobacter chlorophenolicus A6; Kribbella flavida DSM 17836 uid43465; Thermomonospora curvata DSM 43183; Bifidobacterium dentium Bdl; Bifidobacterium longum DJO10A; Slackia heliotrinireducens DSM 20476; Persephonella marina EX HI ; Bacteroides fragilis NCTC 9434; Capnocytophaga ochracea DSM 7271 ; Flavobacterium psychrophilum JIP02 86; Akkermansia muciniphila ATCC BAA 835; Roseiflexus castenholzii DSM 13941; Roseiflexus RSI; Synechocystis PCC6803; Elusimicrobium minutum Peil91; uncultured Termite group 1 bacterium phylotype Rs D17; Fibrobacter succinogenes S85; Bacillus cereus ATCC 10987; Listeria innocua;Lactobacillus casei; Lactobacillus rhamnosus GG; Lactobacillus salivarius UCC118; Streptococcus agalactiae A909; Streptococcus agalactiae NEM316; Streptococcus agalactiae 2603; Streptococcus dysgalactiae equisimilis GGS 124; Streptococcus equi zooepidemicus MGCS10565; Streptococcus gallolyticus UCN34 uid46061; Streptococcus gordonii Challis subst CHI; Streptococcus mutans NN2025 uid46353; Streptococcus mutans; Streptococcus pyogenes Ml GAS; Streptococcus pyogenes MGAS5005; Streptococcus pyogenes MGAS2096; Streptococcus pyogenes MGAS9429; Streptococcus pyogenes MGAS 10270; Streptococcus pyogenes MGAS6180; Streptococcus pyogenes MGAS315; Streptococcus pyogenes SSI-1; Streptococcus pyogenes MGAS 10750; Streptococcus pyogenes NZ131; Streptococcus thermophiles CNRZ1066; Streptococcus thermophiles LMD-9; Streptococcus thermophiles LMG 18311; Clostridium botulinum A3 Loch Maree; Clostridium botulinum B Eklund 17B; Clostridium botulinum Ba4 657; Clostridium botulinum F Langeland; Clostridium cellulolyticum H10; Finegoldia magna ATCC 29328; Eubacterium rectale ATCC 33656; Mycoplasma gallisepticum; Mycoplasma mobile 163K; Mycoplasma penetrans; Mycoplasma synoviae 53; Streptobacillus moniliformis DSM 12112; Bradyrhizobium BTAil; Nitrobacter hamburgensis X14; Rhodopseudomonas palustris BisB18; Rhodopseudomonas palustris BisB5; Parvibaculum lavamentivorans DS-1; Dinoroseobacter shibae DFL 12; Gluconacetobacter diazotrophicus Pal 5 FAPERJ; Gluconacetobacter diazotrophicus Pal 5 JGI; Azospirillum B510 uid46085; Rhodospirillum rubrum ATCC 11170; Diaphorobacter TPSY uid29975; Verminephrobacter eiseniae EFOl-2; Neisseria meningitides 053442; Neisseria meningitides alphal4; Neisseria meningitides Z2491; Desulfovibrio salexigens DSM 2638; Campylobacter jejuni doylei 269 97; Campylobacter jejuni 81116; Campylobacter jejuni; Campylobacter lari RM2100; Helicobacter hepaticus; Wolinella succinogenes; Tolumonas auensis DSM 9187; Pseudoalteromonas atlantica T6c; Shewanella pealeana ATCC 700345; Legionella pneumophila Paris; Actinobacillus succinogenes 130Z; Pasteurella multocida; Francisella tularensis novicida U112; Francisella tularensis holarctica; Francisella tularensis FSC 198; Francisella tularensis tularensis; Francisella tularensis WY96-3418; and Treponema denticola ATCC 35405. The Cas9 protein may be referred by one of skill in the art in the literature as Csnl. An exemplary S. pyogenes Cas9 protein sequence is provided in Deltcheva et al., Nature All, 602-607 (2011) hereby incorporated by reference in its entirety.

Modification to the Cas protein is a representative embodiment of the present disclosure. CRISPR systems useful in the present disclosure are described in R. Barrangou, P. Horvath, CRISPR: new horizons in phage resistance and strain identification. Annual review of food science and technology 3, 143 (2012) and B. Wiedenheft, S. H. Sternberg, J. A. Doudna, RNA-guided genetic silencing systems in bacteria and archaea. Nature 482, 331 (Feb 16, 2012) each of which are hereby incorporated by reference in their entireties.

According to one aspect, the RNA-guided DNA binding protein includes an effector moiety or group attached thereto to affect or alter or modulate a target nucleic acid. The RNA- guided DNA binding protein may be a nuclease null RNA-guided DNA binding protein including an effector moiety or group attached thereto. An effector moiety or group includes a modulator moiety or group. Modulating may refer to the function of the effector group or moiety attached to the RNA-guided DNA binding protein or guide RNA. A target nucleic acid may be modulated by being cut or nicked by the RNA-guided DNA binding protein. A target nucleic acid may be modulated by being bound by the RNA-guided DNA binding protein. A target nucleic acid may be modulated by the function of the effector group or moiety attached to the RNA-guided DNA binding protein or the guide RNA. A target nucleic acid may be modulated by being bound by the RNA-guided DNA binding protein and the function of the effector group or moiety attached to the RNA-guided DNA binding protein or the guide RNA.

Additional exemplary RNA-guided DNA binding proteins includes Cas9 proteins include Cas9 proteins attached to, bound to or fused or connected or tethered with a functional protein or effector group or modulator such as transcriptional regulators, such as transcriptional activators or repressors, a Fok-domain, such as Fok 1, an aptamer, a binding protein, PP7, MS2 and the like. The nuclease null Cas9 protein and the guide RNA colocalize to the target nucleic acid or the nucleic acid encoding the guide RNA resulting in binding but not cleaving of the target nucleic acid as encoded by cargo sequences can be included. The activity or transcription of the target nucleic acid is regulated by such binding. The Cas9 protein can further comprise a transcriptional regulator or DNA modifying protein attached thereto. According to one aspect, the transcriptional regulator protein or domain is a transcriptional activator. According to one aspect, the transcriptional regulator protein or domain upregulates expression of the target nucleic acid. According to one aspect, the transcriptional regulator protein or domain is a transcriptional repressor. According to one aspect, the transcriptional regulator protein or domain downregulates expression of the target nucleic acid. Transcriptional activators and transcriptional repressors can be readily identified by one of skill in the art based on the present disclosure. Exemplary transcriptional regulators are known to a skilled in the art and include VPR, VP16, VP64, P65 and RTA. See Zhang et al., Nature Biotechnology 29, 149-153 (2011) hereby incorporated by reference in its entirety. The transcriptional regulatory domains correspond to targeted loci. Accordingly, aspects of the present disclosure include methods and materials for localizing transcriptional regulatory domains to targeted loci of target nucleic acids by fusing, connecting or joining such domains to an RNA-guided DNA binding protein such as Cas or a guide RNA. Exemplary effector groups or moieties include a detectable moiety, a transcriptional regulator, a protein domain, a nuclease, a phosphatase, deaminase, kinase, polynucleotide kinase, Uracil-DNA glycosylase, nuclease, endonuclease, exonuclease, site-specific nuclease, ligase, polymerase, recombinase, methyl-transferase, fluorescent protein, beta-galactosidase, antibody, scFv single-chain variable fragment of an antibody, nanobody, transcriptional activator, transcriptional repressor, biotin, streptavidin, aptamer, nanoparticle, gold nanoparticle, quantum dot, magnetic bead, paramagnetic particle, or oligonucleotide. Exemplary DNA-modifying enzymes are known to a skilled in the art and include Cytidine deaminases, APOBECs, Fokl, endonuc leases and DNases.

EXEMPLARY GUIDE RNA

Embodiments of the present disclosure are directed to the use of a RNA-guided DNA binding protein/guide RNA system, such as a CRISPR/Cas system and, in particular, a guide RNA which may include one or more of a spacer sequence, a tracr mate sequence and a tracr sequence. The term spacer sequence is understood by those of skill in the art and may include any polynucleotide having sufficient complementarity with a target nucleic acid sequence to hybridize with the target nucleic acid sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. According to certain aspects, an exemplary spacer sequence is between 10 and 30 nucleotides in length. According to certain aspects, an exemplary spacer sequence is between 15 and 25 nucleotides in length. An exemplary spacer sequence is between 18 and 22 nucleotides in length. An exemplary spacer sequence is 20 nucleotides in length.

The guide RNA may be formed from a spacer sequence covalently connected to a tracr mate sequence (which may be referred to as a crRNA) and a separate tracr sequence, wherein the tracr mate sequence is hybridized to a portion of the tracr sequence. According to certain aspects, the tracr mate sequence and the tracr sequence are connected or linked such as by covalent bonds by a linker sequence, which construct may be referred to as a fusion of the tracr mate sequence and the tracr sequence. The linker sequence referred to herein is a sequence of nucleotides, referred to herein as a nucleic acid sequence, which connect the tracr mate sequence and the tracr sequence. Accordingly, a guide RNA may be a two component species (i.e., separate crRNA and tracr RNA which hybridize together) or a unimolecular species (i.e., a crRNA-tracr RNA fusion, often termed an sgRNA).

Tracr mate sequences and tracr sequences are known to those of skill in the art, such as those described in US 2014/0356958 and as shown in Fig. 2. An exemplary tracr mate sequence and tracr sequence is N20 to N8- gttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttttt with N20-8 being the number of nucleotides complementary to a target locus of interest. According to certain aspects, the tracr mate sequence is between about 17 and about 27 nucleotides in length. According to certain aspects, the tracr sequence is between about 65 and about 75 nucleotides in length. According to certain aspects, the linker nucleic acid sequence is between about 4 and about 6. According to certain methods, two or more or a plurality of guide RNAs may be used in the practice of certain embodiments. According to certain aspects, the guide RNA is between about 10 to about 500 nucleotides. According to one aspect, the guide RNA is between about 20 to about 100 nucleotides. According to certain aspects, the spacer sequence is between about 10 and about 500 nucleotides in length and particularly between about 14 and about 22 nucleotides in length. According to certain aspects, the tracr mate sequence is between about 10 and about 500 nucleotides in length. According to certain aspects, the tracr sequence is between about 10 and about 100 nucleotides in length. According to certain aspects, the linker nucleic acid sequence is between about 4 and about 100 nucleotides in length, and particularly between about 4 and about 6 nucleotides in length. According to one aspect, the guide RNA includes an effector moiety or group attached thereto. An effector moiety or group includes a modulator moiety or group. Exemplary effector groups or moieties include a detectable moiety, a transcriptional regulator, a protein domain, a nuclease, a phosphatase, deaminase, kinase, polynucleotide kinase, Uracil-DNA glycosylase, nuclease, endonuclease, exonuclease, site-specific nuclease, ligase, polymerase, recombinase, methyl-transferase, fluorescent protein, beta-galactosidase, antibody, scFv single-chain variable fragment of an antibody, nanobody, transcriptional activator, transcriptional repressor, biotin, streptavidin, aptamer, nanoparticle, gold nanoparticle, quantum dot, magnetic bead, paramagnetic particle, or oligonucleotide.

EXEMPLARY TARGET NUCLEIC ACID

Target nucleic acids as described herein include any nucleic acid sequence to which a co-localization complex as described herein can be useful to either cut, nick or regulate or modulate. Target nucleic acids include nucleic acid sequences, such as genomic nucleic acids, such as genes, capable of being expressed into proteins. For purposes of the present disclosure, a co-localization complex can bind to or otherwise co-localize with the target nucleic acid at or adjacent or near the target nucleic acid and in a manner in which the co-localization complex may have a desired effect on the target nucleic acid. One of skill based on the present disclosure will readily be able to identify or design guide RNAs and Cas9 proteins which co- localize to a target nucleic acid. One of skill will further be able to identify effector groups or modulators or transcriptional regulator proteins or domains which likewise co-localize to a target nucleic acid.

EXAMPLES

The present disclosure provides a gene drive based method of making a plurality of genetically altered proliferating cells. A "gene drive" is an inheritance-biasing element that skews Mendelian inheritance in order to favor its passage to subsequent generations. Gene drives based on RNA guided endonucleases such as Cas9 can copy themselves at high efficiency making it virtually impossible for a diploid cell to be heterozygote for the particular gene drive. As shown in FIGS. 1A-1C, gene drives thus display a pattern of inheritance different from Mendelian inheritance. It has been envisioned that gene drives can act as agents allowing one or several specific traits to pass through an entire population and reach equilibrium when all organisms in a given population contain the particular gene drive(s). The disclosure provides methods of using gene drives to induce variations in a given population by releasing a multitude of gene drives at the same time point and using the variations that will occur within the first couple of generations before the population reach equilibrium for all gene drives and thus are genetically identical again. For example, if 20 gene drives are released in a yeast population, all the yeast cells would contain two gene drives after the first round of mating and sporulation, giving rise to -400 different yeast genotypes, which would then reach >100,000 unique genotypes after the second round of mating and sporulation when each yeast cell would contain at most 4 gene drives (FIG. 2). Thus, utilizing gene drives, which do not need a selection marker to be maintained in the genome, one can quickly generate an enormous amount of variants in a short period of time due to the near exponential growth of gene drives per cell per round of mating.

In an exemplary designed system according to the present disclosure, a gene drive consists of at least five fragments: 1) A sequence homologous to the DNA upstream of the insertion site of the drive (upstream flanking sequence), 2) a promoter sequence ensuring the transcription of the targeting and guide sequences (guide RNA promoter), 3) a targeting sequence (for guide RNA sequence) specific for the insertion site in the host genome 4) a spacer tail sequence and transcriptional terminator allowing the transcribed RNA to interact with Cas9 or other programmable nuclease, and 5) a sequence homologous to the DNA downstream of the insertion site (downstream flanking sequence). A limiting parameter for utilization of this concept would be the cost of synthesis of the gene drives which are -100 USD per drive for a 500bp gene drive cassette. However, of the five fragments mentioned above, only 1 , 3 and 5 are specific for a given drive whereas 2 and 4 are constant for each and every drive (although they constitute 80% of the sequence to be synthesized). According to certain aspect as disclosed herein, the disclosure provides an outline of a scheme for the synthesis of gene drives from oligonucleotide pools allowing for a drive to be made for a low cost of ~5 cents and subsequently inserting the constant sequence to create standardized low cost gene drives. In certain embodiments, the gene drives can be made to target a region just upstream of a translational initiation ATG site of any given gene and thus creating the possibility of making promoter swaps or performing epitope tagging of genes. Under the same strategy, the gene drives according to the present disclosure also allow one skilled in the art to perform simultaneous genetic manipulations including but not limited to knock out, repression, activation, induction, or protein fusion etc., in the same system in a high throughput manner at a low cost. In summary, the present disclosure provides the use of gene drives for rapid and efficient generation of combinatorial genetic diversity and methods for cost efficient generation of gene drives at library scales.

Example I

Constructing a gene drive in a standardized method using short oligo DNA sequences

The present disclosure provides a method for constructing a gene drive using short oligo DNA sequences in a standardized method. A schematic of a general strategy for constructing gene drives from oligos according to an exemplary embodiment is shown in FIGS. 3A-3G. The oligo (or library of oligos) is PCR amplified based on the primer sequences flanking the pre-drive sequence (FIGS. 3A-3B). The pre-drive oligo is inserted into a minimal vector from which guide RNA promoter sequence can be inserted into on one side of the targeting sequence. The plasmid vector is then reopened on the other side of the targeting sequence to insert the guide RNA and terminator sequences (FIGS. 3C-D). The final gene drive is PCR amplified and subject to enzymatic digestion to remove the PCR primer flanking sequences (FIGS. 3F-3G).

According to some embodiments, the present disclosure provides that as the system is standardized, it is possible to insert additional cargo sequences flanking the necessary target sequence promoter and guide RNA sequence (FIG. 4). For example, it is possible to add a selection marker on one side and an inducible promoter on the other side, although any other desired genetic manipulations can be added to either side depending on the needs of a skilled in the art. The general building blocks as shown in FIG. 4 can be synthesized and inserted into standardized plasmids allowing quick PCR amplification of a particular gene drive that is needed for a given experiment.

Example II

A gene drive can be efficiently used to switch the promoter of an endogenous gene

The present disclosure provides as an exemplary embodiment a method for switching the promoter of an endogenous gene using a gene drive. Gene drives can target an insertion at any position in the genome flanked by the Cas9 NGG motif. According to certain

embodiments, the gene drive method allows for knocking out an endogenous gene by removing the gene and replacing it with the gene drive. According to alternative

embodiments, the gene drive can be wedged in between the native promoter and coding region of a target gene (FIG. 5A). This strategy would allow the decoupling of the native promoter to the coding region of a target gene and swap the native promoter with any desired promotor. In the promoter swapping experiment shown in FIGS. 5A-5C, three gene drives were constructed. 1) A gene drive was constructed without a selection marker; 2) A gene drive was constructed with a HIS 3 marker on one side and only the guide RNA terminator on the other side and 3) A gene drive with a HIS3 marker on one side and guide RNA terminator plus an inducible GAL7 promoter on the other side. It was evident that the presence of a selection marker within the drive leads to -100% insertion frequency in yeast cells, whereas co-transfection with a marker outside of the drive leads to much lower insertion frequency (FIG. 5B). In the case where the inducible GAL7 promoter was not supplied as cargo in the gene drive, the target gene was effectively silenced and showed the phenotype of a "knock out" (FIG. 5C). On the contrary, in the case where the inducible GAL7 promoter was supplied as cargo, the yeast cell displayed the "knock out" phenotype on glucose containing media (YPD) but showed the wild type phenotype when grown on galactose inducible media (FIG. 5C).

Example III

Yeast expressing haploid specific ura3 and trpl can be used for liquid cyclic mating

The present disclosure provides a method for enriching for haploid or diploid yeast cells using selectable markers. In order to create yeast pools that contain multiple gene drives, it is desirable to have an efficient system of liquid mating where the majority of cells are mated and sporulated (FIG. 6A). In an exemplary embodiment, this was achieved through expressing the TRP1 gene under the control of the haploid- specific HO promoter and the URA3 gene under the control of the haploid-specific STE5 promoter. Both of these promoters are only strong and active in haploid cells but are repressed in diploid cells (FIG. 6B). By growing the haploid and diploid cells in media lacking tryptophan and uracil, the diploid cells showed a severe growth defect (due to neither the TRP1 or URA3 genes being expressed), while the haploid cells grew well. In contrast, when haploid cells are grown in media containing 5-Fluoroorotic acid (5-FOA) and Fluoroanthranilic acid (5-FAA), which is toxic to cells that express TRPl and URA3, respectively, they showed poor growth, while the diploid cells that express neither gene grew well (FIG. 6C). Therefore, using the disclosed selection method, a skilled in the art is readily able to select between haploid and diploid states of yeast cells to facilitate sequential rounds of mating and haploid isolation.

EXAMPLE IV

A modified donor method for improved chromosomal integration efficiency of gene drives into yeast

Integration of gene drives into yeast chromosomes is a crucial first step for downstream propagation of the drives. Current standard method of yeast transformation is inefficient in that transformation efficiency occurs at about 1 in 10⁶ cells and integration may happen at undesirable off-target sites (Baudin, A. et al. A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. Nucleic Acids Res 21(14). 3329-3330 (1993)). The present disclosure provides a modified donor DNA method in which a plasmid donor containing a desired gene drive is provided to the cell in the form of two linear fragments. Following transformation of the yeast cells with the two linear fragments, the two linear fragments have to undergo homologous recombination within the cell in order to generate a stably inherited circular plasmid. Using this modified donor DNA method, increased transformation efficiency of about 10² fold was observed (FIGS. 7A-7B). Further, integration of the gene drive at the desired locus occurred at a frequency of 100% (Table 1). Using this new method, yeast strains were constructed containing gene drives targeting autonomous replicating sequence (ARS) elements within the yeast genome to investigate the number of drives that can be present in a cell and function at high efficiency.

Cloning of gene drive fragments into backbone vector According to one embodiment of the present disclosure, the gene drive-containing fragments were synthesized as gBlocks® Gene Fragments by IDT (Coralville, Iowa) and cloned into a pADE2 -backbone vector via Gibson Assembly (Gibson et al, Enzymatic assembly of DNA molecules up to several hundred kilobases, Nature Methods 2009, 6:343- 345). Briefly, the backbone vector was digested by Sacl and Kpnl followed by gel purification of a 6.4 kb fragment. The gBlock was then fused into the SacI-KpnI-linearized backbone in a standard overnight Gibson reaction. Subsequently, 1 μΐ of product was transformed into chemically competent DH5alpha E.coli cells.

Preparation of modified donor for yeast transformation

To achieve high gene drive integration efficiency, two linear DNA fragments were derived from the pADE2-gene drive plasmid generated above. The first fragment was obtained via digestion of the pADE2-gene drive plasmid with Aflll and Aatll followed by gel purification of a 6.8kb fragment to create a gap at the ADE2 sequence in the plasmid. The second fragment was a -730 bp PCR fragment amplified off the pADE2-gene drive plasmid using primers TGAGAAGTGACGCAAGCATC and ATGACCACGTTAATGGCTCC. Upon transformation into yeast, the homologous sequences on both ends of the plasmid

(TGAGAAGTGACGCAAGCATCAATGGTATAATGTCCAGAGTTGTGAGGCCTTGGG

GCAATTTCGTTAATAAGCAATTCCCCTGTTTCTAAATAGAACATTTCCACACCAA

ATATACCACAACCGGGAAAAGATTTGATTGCATTTTCTGCCAACAACTTCGC and

TCACTGGCTTGTTCCACAGGAACACTTTGGGTAACTGCTATACCATTTTTGATTAA

ATGCTCTTTTTGAATATATTTGTCTTGTATCAATCTGATTGTTTCTGGAGAAGGGT

AAATTTTTAATTTGGGATGTTTTACTTGAAGATTCTTTAGTGTAGGAACATCAACA

TGCTCAATCTCAATCGTTAGCACATCACATTTTTCAGCTAGTTTTTCGATATCAAG

AGGATTGGAAAAGGAGCCATTAACGTGGTCAT) were used to recombine into an intact circular plasmid for stable inheritance and propagation into the progeny yeast cells. Yeast transformation

400 ng of each DNA fragment were simultaneously delivered into yeast using standard LiAc method as previously described (Gietz RD and Schiest RH, High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method, Nat Protoc. 2007, 2(l):31-4) and plated onto SC media lacking adenine. All strains were grown at at 30°C.

Sequence of the pADE2-backbone vector gacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttaatatgatccaatatcaaaggaaatgatag cattgaaggatgagactaatccaattgaggagtggcagcatatagaacagctaaagggtagtgctgaaggaagcatacgataccccg catggaatgggataatatcacaggaggtactagactacctttcatcctacataaatagacgcatataagtacgcatttaagcataaacac gcactatgccgttcttctcatgtatatatatatacaggcaacacgcagatataggtgcgacgtgaacagtgagctgtatgtgcgcagctc gcgttgcattttcggaagcgctcgttttcggaaacgctttgaagttcctattccgaagttcctattctctagaaagtataggaacttcagagc gcttttgaaaaccaaaagcgctctgaagacgcactttcaaaaaaccaaaaacgcaccggactgtaacgagctactaaaatattgcgaat accgcttccacaaacattgctcaaaagtatctctttgctatatatctctgtgctatatccctatataacctacccatccacctttcgctccttga acttgcatctaaactcgacctctacattttttatgtttatctctagtattactctttagacaaaaaaattgtagtaagaactattcatagagtgaat cgaaaacaatacgaaaatgtaaacatttcctatacgtagtatatagagacaaaatagaagaaaccgttcataattttctgaccaatgaaga atcatcaacgctatcactttctgttcacaaagtatgcgcaatccacatcggtatagaatataatcggggatgcctttatcttgaaaaaatgc acccgcagcttcgctagtaatcagtaaacgcgggaagtggagtcaggctttttttatggaagagaaaatagacaccaaagtagccttctt ctaaccttaacggacctacagtgcaaaaagttatcaagagactgcattatagagcgcacaaaggagaaaaaaagtaatctaagatgctt tgttagaaaaatagcgctctcgggatgcatttttgtagaacaaaaaagaagtatagattctttgttggtaaaatagcgctctcgcgttgcatt tctgttctgtaaaaatgcagctcagattctttgtttgaaaaattagcgctctcgcgttgcatttttgttttacaaaaatgaagcacagattcttcg ttggtaaaatagcgctttcgcgttgcatttctgttctgtaaaaatgcagctcagattctttgtttgaaaaattagcgctctcgcgttgcatttttg ttctacaaaatgaagcacagatgcttcgttcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattc aaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcg cccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgc acgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcactttt aaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttg gttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataa cactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgcc ttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgc gcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttc tgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactgggg ccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgaga taggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaagga tctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatc aaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggat caagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggc caccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgt cttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagctt ggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcgga caggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctg tcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggc ctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgag tgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaa accgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgc aattaatgtgagttacctcactcattaggcaccccaggctttacactttatgcttccggctcctatgttgtgtggaattgtgagcggataaca atttcacacaggaaacagctatgaccatgattacgccaagcgcgcaattaaccctcactaaagggaacaaaagctggagctcTAA

TAATAATACTATTGATCGTAGTGAAAGCAAAACACGACTGTACTTGAGTTATAAA

TTTTGTTGCTGTAGAAATTTCAGTGATCATTCATTAGCAACTTATTAAGATGACGA

TTATATCATATGTTAATTAAGGCGCGCCtctttgaaaagataatgtatgattatgctttcactcatatttatacaga aacttgatgttttctttcgagtatatacaaggtgattacatgtacgtttgaagtacaactctagattttgtagtgccctcttgggctagcggtaa aggtgcgcattttttcacaccctacaatgttctgttcaaaagattttggtcaaacgctgtagaagtgaaagttggtgcgcatgtttcggcgtt cgaaacttctccgcagtgaaagataaatgatcATTTTAATTAAGATACAATTGTTTTAGAGCTAGAA

ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAG

TCGGTGGTGCTTTTTTTGTTTTTTATGTCTGGTTCACCCGAACGTTATCATATATAC

GCTCATATTATTCCAAGAAAGACACATTTGTCTAGATAGGTTTCGGTAAAGGCGT

CCGTAGTTAGAGAGTAGATGGCACTAACTTGAAAGCAGggtacccaattcgccctatagtgagtc gtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatcc ccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgcg acgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgccc gctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgattta gtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgcccttt gacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataaGG

TATATCATTTTATAATTATTTGCTGTACAAGTATATCAATAAACTTATATATTACT

TGTTTTCTAGATAAGCTTCGTAACCGACAGTTTCTAACTTTTGTGCTTTGACAAGA

ACTTCTTCTTCTTGCTTTAATAAAAACTGTTCCATTTTCGTTGTATAACTTGAATC

ATAAGCGCCAAGCAGTCTGACAGCCAACAGCGCAGCGTTCGTACTATTATTAAT

AGCGACGGTAGCTACTGGAACACCTCTAGGCATTTGCACAATTGAATGTAAAGA

ATCTACTCCATCTAGACAAGAACCTTTTACGGGCACACCGATGACAGGAAGTGG

TGTCATTGCAGCCACCATACCTGGCAAGTGAGCAGCCCCACCAGCTCCAGCGAT

AATTGTTTTAATTCCACGCTTGCTTGCGGAAATAGCATATGCTGACATCCTATGTG

GAGTTCTATGAGCAGAGACTATTGTCACTTCAAATGGAACGCCAAAATCTTTTAA

AACCGCACATGCGGCAGACATTACCGGCAAGTCAGAGTCTGATCCCATGATGAT

TCCAACCAATGGTTTGACCATTGCTTCCAAGTCCAACTTTTGAGCGACAGAGATT

TTGATTGGAATATCAGTTCTACCTGTAATGTAGTTCAGCCTTTGTTCACATTCCGC CATACTGGAGGCAATAATATTTATGTGACCTACTTTTCTGTTAGGTCTAGACTCTT

TTCCATATAAGTACACTGAGGAACCTGGAGTCGCCAATGCTCTTTCGCAAGTTTC

TAGCTCTTTATCTTTTGTATGTTTGTCTCCAAGAACATTTAGCATAATGGCGTTCG

TTGTAATGGTGGAGAAAGATGTGAAATTCTTTGGCATTGGCAAATCCAATATTGA

TCTCAAATGAGCTTCAAATTGAGAAGTGACGCAAGCATCAATGGTATAATGTCC

AGAGTTGTGAGGCCTTGGGGCAATTTCGTTAATAAGCAATTCCCCTGTTTCTAAA

TAGAACATTTCCACACCAAATATACCACAACCGGGAAAAGATTTGATTGCATTTT

CTGCCAACAACTTCGCCTTAAGTTGAACGGAGTCCGGAACTCTAGCAGGCGCAT

AACATAAGTCACAAATATTGTCCTTGTGGATAGTCTCTACAATTGGGTAAGAAAA

CACTAAACCGTTAACAGATCTCACAATCATGACTGCTAATTCTTTAGTAAATGGT

GCCCATTTTTCGGCGTACAAAGGACGATCCTTCAGTACTTCCAAAGCTTCCGGAA

TCATTTCCTTATTCTTTACAACGAAGTTACCTCTTCCATCGTATGCCAAAGTCCTC

GACTTCAAGACGAATGGAAAACCCAAATCTCTTCCAACATTCAATAGGGACGTC

TCACTGGCTTGTTCCACAGGAACACTTTGGGTAACTGCTATACCATTTTTGATTAA

ATGCTCTTTTTGAATATATTTGTCTTGTATCAATCTGATTGTTTCTGGAGAAGGGT

AAATTTTTAATTTGGGATGTTTTACTTGAAGATTCTTTAGTGTAGGAACATCAACA

TGCTCAATCTCAATCGTTAGCACATCACATTTTTCAGCTAGTTTTTCGATATCAAG

AGGATTGGAAAAGGAGCCATTAACGTGGTCATTGGAGTTGCTTATTTGTTTGGCA

GGAGAATTTTCAGCATCTAGTATTACCGTCTTAATGTTGAGCCTGTTTGCTGCCTC

AACAATCATACGTCCCAATTGTCCCCCTCCTAATATACCAACTGTTCTAGAATCC

ATACTTGATTGTTTTGTCCGATTTTCTTGTTTTTCTTGATTGTTATAGTAGGATGTA

CTTAGAAGAGAGATCCAACGATTTTACGCACCAATTTATACATGAAATGCTCCAT

AATATTGTCCATTTAGTTCTTAATAAAAGGTCAGCAAGAGcacgtgctcaatagtcaccaatgc cctccctcttggccctctccttttcttttttcgaccgaattaattcttaatcggcaaaaaaagaaaagctccggatcaagattgtacgtaaggt gacaagctatttttcaataaagaatatcttccactactgccatctggcgtcataactgcaaagtacacatatattacgatgctgtctattaaat gcttcctatattatatatatagtaatgtcgtttatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacaccc gccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcat gtgtcagaggttttcaccgtcatcaccgaaacgcgcga

Table 1. Integration efficiency of gene drives using the modified donor method.

EXAMPLE V

Materials and Methods

The present disclosure utilizes the following material and methods in one or more of the Examples described herein.

Design of pre-drives from small oligos and amplification

The sequence upstream of the translational initiation ATG for each gene in the yeast genome was mined for the presence of the Cas9 NGG motif using a custom batch script. The distance from the ATG to the double- stranded break was minimized but all spacer sequences not unique in the genome for the 13 bp upstream of the NGG was discarded. The 34bp downstream flanking sequence of the double stranded break was denoted as DOWN for the drive and the 34bp upstream flanking sequence of the ATG was denoted as UP for the drive. Using a custom script the oligo was designed as: Primer upstream sequence (GC AGTCCGTCTTGCCATC) , UP sequence

(AATCAAGAAAAACAAGAAAATCGGACAAAACAATCAA), Bsal-cassette

(TCTTAGAGACCGGTCTCCGATC), spacer sequence (TCGGACAAAACAATCAAGTA), Sapl cassette (GTT AG A AG AGCTCTTCT) , DOWN sequence

(ATGGATTCTAGAACAGTTGGTATATTAGGAGGGGGACA) and primer downstream sequence (GTCTACGCGATTATCTGC). The pre-drive was amplified using the primers CATGCGTCTCTCCTAGTGCAGTCCGTCTTGCCATC and

GCATCGTCTCTCTCAGTGCAGATAATCGCGTAGAC at 72°C elongation using Phusion polymerase. The oligo was either ordered as ssDNA oligo (IDT) or as part of an oligo pool (Custom array).

Preparing a background vector

The background vector was made by amplifying a 2.7kb backbone fragment from pAG60 vector (Addgene #35128) using the primers

CATGCGTCTCTTGAGCAGCTCACTCAAAGGCG and

GCATCGTCTCTTAGGTTGCTCAAAAGTATCTCTTTGCTAT. The template DNA was digested using 10U of Dpnl in the PCR buffer for 10 minutes at 37°C. The PCR product was ligated using T4 ligase in the PCR buffer for lh and Ιμΐ was used to transform chemically competent DH5alpha chemically competent E. coli.

The minimal backbone vector was digested using 1U of Ahdl and Rsal and the 2.3kb fragment was purified from agarose gel. A gBlock was designed containing mutations of Bsal, BsrDI and Btsl sites in the ampicillin coding region creating silent mutations. The gBlock was fused to the backbone by adding 40ng of gBlock, 40ng of backbone in 6μ1 total volume and 6μ1 of 2x Gibson Assembly Master Mix (New England Biolabs). The mixture was left to react for 2h at 50°C and Ιμΐ was transformed into DH5alpha chemically competent E. coli.

Vector construction using Golden gate cloning

400 pmoles of pre-drive PCR amplified oligo and 40 pmol of background vector were mixed in a 20μ1 reaction containing 2μ1 of buffer 3.1 (NEB), Ιμΐ BsmBI enzyme (NEB), 2μ1 ATP, Ιμΐ T4 DNA ligase (NEB) and were placed in a thermocyclers for 12 hours cycling between 37°C for 2 minutes and 16°C for 2 minutes. Following heat inactivation at 65 °C for 20 minutes, the mixture were given an additional Ιμΐ of BsmBI enzyme was then added to each reaction and allowed to digest at 55°C for an additional hour. Golden gate reactions were transformed into DH5 alpha chemically competent E. coli and plated on LB plates containing ampicillin selection. The colonies were harvested after 18h from the plate and used as input for a standard miniprep plasmid extraction (Qiagen #27106).

An oligo containing the His3 selection marker flanked by the guide-RNA promoter was synthesized and an oligo only containing the guide-RNA promoter were amplified using primers GCCTTTTTACGGTTCCTGGC and GTGACCTGTTCGTTGCAACA. 400 pmoles of insertion oligo and 40 pmol of background miniprep DNA were mixed in a 20μ1 reaction containing 2μ1 of Cutsmart buffer (NEB), Ιμΐ Bsal-HF enzyme (NEB), 2μ1 ATP, Ιμΐ T4 DNA ligase (NEB) and were placed in a thermocyclers for 12 hours cycling between 37°C for 2 minutes and 16°C for 2 minutes. Following heat inactivation at 65 °C for 20 minutes, the mixture were given an additional Ιμΐ of Bsal-HF enzyme to each reaction and allowed to digest at 37°C for an additional hour. Golden gate reactions were transformed into DH5alpha chemically competent E. coli and plated on LB plates containing ampicillin selection. The colonies were harvested after 18h from the plate and used as input for a standard miniprep plasmid extraction (Qiagen #27106). An oligo containing the Guide sequence flanked by Sapl restriction sites and an oligo containing guide sequence and the Gal7 promoter sequence flanked by Sapl restriction sites were synthesized and amplified using primers GCCTTTTTACGGTTCCTGGC and

GTGACCTGTTCGTTGCAACA. 400 pmoles of insertion oligo and 40 pmol of background miniprep DNA were mixed in a 20μ1 reaction containing 2μ1 of Cutsmart buffer (NEB), Ιμΐ Sapl enzyme (NEB), 2μ1 ATP, Ιμΐ T4 DNA ligase (NEB) and were placed in a

thermocyclers for 12 hours cycling between 37°C for 2 minutes and 16°C for 2 minutes. Following heat inactivation at 65 °C for 20 minutes, the mixture were given an additional Ιμΐ of Sapl enzyme to each reaction and allowed to digest at 37°C for an additional hour. Golden gate reactions were transformed into DH5alpha chemically competent E. coli and plated on LB plates containing ampicillin selection. The colonies were harvested after 18h from the plate and used as input for a standard miniprep plasmid extraction (Qiagen #27106).

Creating yeast background strains

A cas9 containing yeast background strain was created by transforming BY4741/4742 strain background with the two linear fragments 1) pNEB 193-HO-TRPl- STE5p-CaURA3- nopl-cas9-natMX-HO plasmid linearized using Pmel (NEB) and 2) knocking out TRP1 using LEU2. The two strains were mated for 24h in YPD media and switched to minimal media with Histidine+Uracil+Trypthophan to select for diploid cells. These cells were used for all experiments and are from here on referred to as MATa cycle-competent (based on BY4741), MATalpha cycle-competent (based on BY4742) and diploid cycle-competent (mate of MATa and MATalpha cycle-competent).

Transforming drives into yeast

The plasmids containing drive with and without the Gal7 promoter and His3 marker sequence were amplified using the general primers GCGAAAGGTGGATGGGTAG CCCTGATTCTGTGGATAACCG for 30 cycles using Phusion polymerase (NEB) and subsequently adding 1U of Bsgl (NEB) into the PCR buffer. The reaction was allowed to react at 37C for lh before enzymatic purification using DNA Clean & Concentrator- 5 (Zymo research).

Yeast cells were transformed using the Li- Ac method as previously described (Gietz RD and Schiest RH, High-efficiency yeast transformation using the LiAc/SS carrier

DNA/PEG method, Nat Protoc, 2007, 2(l):31-4) and plated on SC media lacking histidine. The yeast transformed with the drive not containing the His3 marker was co-transformed with a plasmid containing the His3 marker. All strains were grown at 30°C in YDP or minimal media with appropriate auxotrophic supplementation.

Transformants were screened on YPD, SC-His+ galactose and SC-His+glucose containing media.

Growth phenotype of diploid and haploid yeast

25 μΐ of MATa and MATalpha cycle-competent cells from saturated cultures and 50μ1 of diploid cycle-competent cells were used to inoculate 5ml of SC media, SC media containing 600ng/ml of 5-FOA (30μ1 of 100X DMSO dilution), SC media containing 800ng/ml of 5 -FA A (40μ1 of 100X DMSO dilution), SC media containing 600ng/ml of 5- FOA and 5-FAA and SC-Trp-Ura. The cultures were set up in biological triplicates and grown at 30C on shake for 22h. The OD was measured in a 96well OD reader (Biotek synergy HT) at Oh and 22h.

Media used for selection system

YPD: 24 g Bacto agar, 20 g Bacto peptone, 10 g Yeast extract, lOg glucose, 950 mL H2O. Autoclave before use. SC: 3.4g base w/o amino acids, lOg glucose,0.36g DO supplement -URA (Clontech, cat: 630416), 5ml 1% Uracil, Fill to 500ml of H₂0. Autoclave before use.

SC+FOA+FAA: SC media made above autoclaved. Create stock 5-FOA: dissolve O.lg of 5-FOA in 1ml of DMSO. Store at -20°C. Create stock 5 -FAA: dissolve O.lg of 5- FAA in 1ml of DMSO. Make fresh. Add 30μ1 of each mix to a 5ml growth culture of SC and vortex to dissolve.

SC-Trp-Ura: 3.4g base w/o amino acids, lOg glucose,0.36g DO supplement -TRP/- URA (Clontech, cat: 630427), Fill to 500ml of H₂0. Autoclave before use.

PreSpo (Pre-sporulation media, make fresh for each use or store max 2 weeks): 5 g yeast extract (1%), lOg Peptone (2%), 5g potassium acetate (1%), Fill to 500ml of ¾0. Autoclave before use.

Spo (Sporulation media make fresh for each use or store max 2 weeks): 5g potassium acetate (1%), 0.04g 0.36g DO supplement -URA (Clontech, cat: 630416) (10% of normal concentration), 500μ1 1% Uracil (10% of normal concentration), Fill to 500ml of ¾0. Autoclave before use.

Protocol for liquid mating of yeast with selection of haploid and diploid yeast

Passage 1 : MATa and MATalpha cycle competent yeast were mated in YPD media overnight. Typically add 50μ1 of each saturated culture.

Passage 2: Cells were washed twice in water to remove the additives found in the YPD media. ΙΟΟμΙ of saturated cells were used to inoculate a 5ml SC media culture to allow the cells to adjust to minimal media for a minimum of 4 hours. Passage 3: Cells were spun down and resuspended in SC + FAA + FOA. Run in two to three different concentrations to get enough cells within the given time.

Passage 4: Cells were passage to SC+FAA+FOA to continue the selection for diploid cells.

Passage 5: Cells were inoculate in 8ml of pre-sporulation media with i.e. 100/500μ1 of cells. OD was measured after 4-6h culture in exponential grown phase with an OD of 0.7-1.0 was used.

Passage 6: Cells were spin down and washed twice in water. The cells were resuspended in sporulation media and allowed to incubate for 48-96h at 30C. Sporulation were verified using a microscope.

Isolation of haploid yeast: Sporulated cells were centrifuged and resuspended in 50 μΐ_^ of a stock solution of zymolyase (50 μg/mL in 1M sorbitol) and incubated at 30C for 10 minutes. Trie cells were added to 5 ml of 1.5% Nonidet P-40 (Sigma- Aldrich) and left on ice for 15 minutes. Sonicate the tube for 30 sec at 50% to 75% full power, then set on ice 2 min. Repeat twice. Centrifuge spores 10 min at 1200 x g. Aspirate or pour off supernatant and resuspend in 10ml of PBS. Vortex vigorously. Repeat twice resuspending in 5ml of SC- URA-TRP media in the end.

Cells were allowed to grow in SC-URA-TRP culture for 24h or until OD = 1.0. The protocol can be repeated for subsequent rounds of mating or cells can be plated on Ura/Trp- plates and pick colonies. Identify yeast mating type for colony using PCR using primers FW_MATalpha: GCACGGAATATGGGACTACTT / FW_MATa:

ACTCCACTTCAAGTAAGAGTTTGG / RV_general:

AGTCACATCAAGATCGTTTATGG. Add all three primers for colony PCR at 58°C. Yield 544 bp bands for MATa 404 bp band for MATalpha and both for diploid. Identify drive for a given colony. PCR amplify the N20 + flanking sequencing using fw:

TGGGCTAGCGGTAAAGGTG and rv: ACGGACTAGCCTTATTTTAACTTGCT at 60°C for a 202bp band.

Supplemental Sequences:

ADE2 pre-drive oligo:

GCAGAAAGCCTGTCTGGTAATCAAGAAAAACAAGAAAATCGGACAAAACAATC AATCTTAGAGACCGGTCTCCGATCTCGGACAAAACAATCAAGTAGTTAGAAGAG CTCTTCTATGGATTCTAGAACAGTTGGTATATTAGGAGGGGGACAACCGGCACTA CATTCTGC

General oligo for His3 selection marker and SNR52promoter for guide expression flanked by Bsal

GCCTTTTTACGGTTCCTGGCGGTCTCATCTTACCACGGCATTAGTCAGGGAAGTC

ATAACACAGTCCTTTCCCGCAATTTTCTTTTTCTATTACTCTTGGCCTCCTCTAGTA

CACTCTATATTTTTTTATGCCTCGGTAATGATTTTCATTTTTTTTTTTCCACCTAGC

GGATGACTCTTTTTTTTTCTTAGCGATTGGCATTATCACATAATGAATTATACATT

ATATAAAGTAATGTGATTTCTTCGAAGAATATACTAAAAAATGAGCAGGCAAGA

TAAACGAAGGCAAAGATGACAGAGCAGAAAGCCCTAGTAAAGCGTATTACAAA

TGAAACCAAGATTCAGATTGCGATCTCTTTAAAGGGTGGTCCCCTAGCGATAGAG

CACTCGATCTTCCCAGAAAAAGAGGCAGAAGCAGTAGCAGAACAGGCCACACA

ATCGCAAGTGATTAACGTCCACACAGGTATAGGGTTTCTGGACCATATGATACAT

GCTCTGGCCAAGCATTCCGGCTGGTCGCTAATCGTTGAGTGCATTGGTGACTTAC

ACATAGACGACCATCACACCACTGAAGACTGCGGGATTGCTCTCGGTCAAGCTTT TAAAGAGGCCCTACTGGCGCGTGGAGTAAAAAGGTTTGGATCAGGATTTGCGCC

TTTGGATGAGGCACTTTCCAGAGCGGTGGTAGATCTTTCGAACAGGCCGTACGCA

GTTGTCGAACTTGGTTTGCAAAGGGAGAAAGTAGGAGATCTCTCTTGCGAGATG

ATCCCGCATTTTCTTGAAAGCTTTGCAGAGGCTAGCAGAATTACCCTCCACGTTG

ATTGTCTGCGAGGCAAGAATGATCATCACCGTAGTGAGAGTGCGTTCAAGGCTCT

TGCGGTTGCCATAAGAGAAGCCACCTCGCCCAATGGTACCAACGATGTTCCCTCC

ACCAAAGGTGTTCTTATGTAGTGACACCGATTATTTAAAGCTGCAGCATACGATA

TATATACATGTGTATATATGTATACCTATGAATGTCAGTAAGTATGTATACGAAC

AGTATGATACTGAAGATGACAAGGTAATGCATCATTCTATACGTGTCATTCTGAA

CGAGGCGCGCTTTCCTTTTTTCTTTTTGCTTTTTCTTTTTTTTTCTCTTGAACTCGAC

GGATCTATGCGGCGATTCTTTGAAAAGATAATGTATGATTATGCTTTCACTCATA

TTTATACAGAAACTTGATGTTTTCTTTCGAGTATATACAAGGTGATTACATGTACG

TTTGAAGTACAACTCTAGATTTTGTAGTGCCCTCTTGGGCTAGCGGTAAAGGTGC

GCATTTTTTCACACCCTACAATGTTCTGTTCAAAAGATTTTGGTCAAACGCTGTAG

AAGTGAAAGTTGGTGCGCATGTTTCGGCGTTCGAAACTTCTCCGCAGTGAAAGAT

AAATGATCGATCAGAGACCTGTTGCAACGAACAGGTCAC

General oligo for guide sequence and the Gal7 promoter flanked by Sapl sites

GCCTTTTTACGGTTCCTGGCGCTCTTCAGTTTTAGAGCTAGAAATAGCAAGTTAA

AATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGGTGCTTT

TTTTGTTTTTTATGTCTATTTGCCAGCTTACTATCCTTCTTGAAAATATGCACTCTA

TATCTTTTAGTTCTTAATTGCAACACATAGATTTGCTGTATAACGAATTTTATGCT

ATTTTTTAAATTTGGAGTTCAGTGATAAAAGTGTCACAGCGAATTTCCTCACATG

TAGGGACCGAATTGTTTACAAGTTCTCTGTACCACCATGGAGACATCAAAAATTG

AAAATCTATGGAAAGATATGGACGGTAGCAACAAGAATATAGCACGAGCCGCG

GAGTTCATTTCGTTACTTTTGATATCACTCACAACTATTGCGAAGCGCTTCAGTGA AAAAATCATAAGGAAAAGTTGTAAATATTATTGGTAGTATTCGTTTGGTAAAGTA

GAGGGGGTAATTTTTCCCCTTTATTTTGTTCATACATTCTTAAATTGCTTTGCCTCT

CCTTTTGGAAAGCTATACTTCGGAGCACTGTTGAGCGAAGGCTCATTAGATATAT

TTTCTGTCATTTTCCTTAACCCAAAAATAAGGGAAAGGGTCCAAAAAGCGCTCGG

ACAACTGTTGACCGTGATCCGAAGGACTGGCTATACAGTGTTCACAAAATAGCC

AAGCTGAAAATAATGTGTAGCTATGTTCAGTTAGTTTGGCTAGCAAAGATATAAA

AGCAGGTCGGAAATATTTATGGGCATTATTATGCAGAGCATCAACATGATAAAA

AAAAACAGTTGAATATTCCCTCAAAAATGTGAAGAGCTGTTGCAACGAACAGGT

CACTGTTGCAACGAACAGGTCAC

General oligo for guide sequence flanked by Sapl sites

GCCTTTTTACGGTTCCTGGCGCTCTTCAGTTTTAGAGCTAGAAATAGCAAGTTAA AATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGGTGCTTT TTTTGTTTTTTATGTCTATGTGAAGAGC TGTTGCAACGAACAGGTCAC

ADE2 pre-drive oligo with His3 marker and SNR52promoter for guide expression

AATCAAGAAAAACAAGAAAATCGGACAAAACAATCAATCTTACCACGGCATTAG

TCAGGGAAGTCATAACACAGTCCTTTCCCGCAATTTTCTTTTTCTATTACTCTTGG

CCTCCTCTAGTACACTCTATATTTTTTTATGCCTCGGTAATGATTTTCATTTTTTTT

TTTCCACCTAGCGGATGACTCTTTTTTTTTCTTAGCGATTGGCATTATCACATAAT

GAATTATACATTATATAAAGTAATGTGATTTCTTCGAAGAATATACTAAAAAATG

AGCAGGCAAGATAAACGAAGGCAAAGATGACAGAGCAGAAAGCCCTAGTAAAG

CGTATTACAAATGAAACCAAGATTCAGATTGCGATCTCTTTAAAGGGTGGTCCCC

TAGCGATAGAGCACTCGATCTTCCCAGAAAAAGAGGCAGAAGCAGTAGCAGAAC

AGGCCACACAATCGCAAGTGATTAACGTCCACACAGGTATAGGGTTTCTGGACC

ATATGATACATGCTCTGGCCAAGCATTCCGGCTGGTCGCTAATCGTTGAGTGCAT TGGTGACTTACACATAGACGACCATCACACCACTGAAGACTGCGGGATTGCTCTC

GGTCAAGCTTTTAAAGAGGCCCTACTGGCGCGTGGAGTAAAAAGGTTTGGATCA

GGATTTGCGCCTTTGGATGAGGCACTTTCCAGAGCGGTGGTAGATCTTTCGAACA

GGCCGTACGCAGTTGTCGAACTTGGTTTGCAAAGGGAGAAAGTAGGAGATCTCT

CTTGCGAGATGATCCCGCATTTTCTTGAAAGCTTTGCAGAGGCTAGCAGAATTAC

CCTCCACGTTGATTGTCTGCGAGGCAAGAATGATCATCACCGTAGTGAGAGTGCG

TTCAAGGCTCTTGCGGTTGCCATAAGAGAAGCCACCTCGCCCAATGGTACCAACG

ATGTTCCCTCCACCAAAGGTGTTCTTATGTAGTGACACCGATTATTTAAAGCTGC

AGCATACGATATATATACATGTGTATATATGTATACCTATGAATGTCAGTAAGTA

TGTATACGAACAGTATGATACTGAAGATGACAAGGTAATGCATCATTCTATACGT

GTCATTCTGAACGAGGCGCGCTTTCCTTTTTTCTTTTTGCTTTTTCTTTTTTTTTCTC

TTGAACTCGACGGATCTATGCGGCGATTCTTTGAAAAGATAATGTATGATTATGC

TTTCACTCATATTTATACAGAAACTTGATGTTTTCTTTCGAGTATATACAAGGTGA

TTACATGTACGTTTGAAGTACAACTCTAGATTTTGTAGTGCCCTCTTGGGCTAGCG

GTAAAGGTGCGCATTTTTTCACACCCTACAATGTTCTGTTCAAAAGATTTTGGTCA

AACGCTGTAGAAGTGAAAGTTGGTGCGCATGTTTCGGCGTTCGAAACTTCTCCGC

AGTGAAAGATAAATGATCGATCTCGGACAAAACAATCAAGTAGTTAGAAGAGCT

CTTCTATGGATTCTAGAACAGTTGGTATATTAGGAGGGGGACA

ADE2 drive containing His3 selection marker, SNR52promoter, spacer sequence, guide sequence and a Gal7 promoter

AATCAAGAAAAACAAGAAAATCGGACAAAACAATCAATCTTACCACGGCATTAG

TCAGGGAAGTCATAACACAGTCCTTTCCCGCAATTTTCTTTTTCTATTACTCTTGG

CCTCCTCTAGTACACTCTATATTTTTTTATGCCTCGGTAATGATTTTCATTTTTTTT

TTTCCACCTAGCGGATGACTCTTTTTTTTTCTTAGCGATTGGCATTATCACATAAT

GAATTATACATTATATAAAGTAATGTGATTTCTTCGAAGAATATACTAAAAAATG AGCAGGCAAGATAAACGAAGGCAAAGATGACAGAGCAGAAAGCCCTAGTAAAG

CGTATTACAAATGAAACCAAGATTCAGATTGCGATCTCTTTAAAGGGTGGTCCCC

TAGCGATAGAGCACTCGATCTTCCCAGAAAAAGAGGCAGAAGCAGTAGCAGAAC

AGGCCACACAATCGCAAGTGATTAACGTCCACACAGGTATAGGGTTTCTGGACC

ATATGATACATGCTCTGGCCAAGCATTCCGGCTGGTCGCTAATCGTTGAGTGCAT

TGGTGACTTACACATAGACGACCATCACACCACTGAAGACTGCGGGATTGCTCTC

GGTCAAGCTTTTAAAGAGGCCCTACTGGCGCGTGGAGTAAAAAGGTTTGGATCA

GGATTTGCGCCTTTGGATGAGGCACTTTCCAGAGCGGTGGTAGATCTTTCGAACA

GGCCGTACGCAGTTGTCGAACTTGGTTTGCAAAGGGAGAAAGTAGGAGATCTCT

CTTGCGAGATGATCCCGCATTTTCTTGAAAGCTTTGCAGAGGCTAGCAGAATTAC

CCTCCACGTTGATTGTCTGCGAGGCAAGAATGATCATCACCGTAGTGAGAGTGCG

TTCAAGGCTCTTGCGGTTGCCATAAGAGAAGCCACCTCGCCCAATGGTACCAACG

ATGTTCCCTCCACCAAAGGTGTTCTTATGTAGTGACACCGATTATTTAAAGCTGC

AGCATACGATATATATACATGTGTATATATGTATACCTATGAATGTCAGTAAGTA

TGTATACGAACAGTATGATACTGAAGATGACAAGGTAATGCATCATTCTATACGT

GTCATTCTGAACGAGGCGCGCTTTCCTTTTTTCTTTTTGCTTTTTCTTTTTTTTTCTC

TTGAACTCGACGGATCTATGCGGCGATTCTTTGAAAAGATAATGTATGATTATGC

TTTCACTCATATTTATACAGAAACTTGATGTTTTCTTTCGAGTATATACAAGGTGA

TTACATGTACGTTTGAAGTACAACTCTAGATTTTGTAGTGCCCTCTTGGGCTAGCG

GTAAAGGTGCGCATTTTTTCACACCCTACAATGTTCTGTTCAAAAGATTTTGGTCA

AACGCTGTAGAAGTGAAAGTTGGTGCGCATGTTTCGGCGTTCGAAACTTCTCCGC

AGTGAAAGATAAATGATCGATCTCGGACAAAACAATCAAGTAGTTTTAGAGCTA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACC

GAGTCGGTGGTGCTTTTTTTGTTTTTTATGTCTATTTGCCAGCTTACTATCCTTCTT

GAAAATATGCACTCTATATCTTTTAGTTCTTAATTGCAACACATAGATTTGCTGTA TAACGAATTTTATGCTATTTTTTAAATTTGGAGTTCAGTGATAAAAGTGTCACAG

CGAATTTCCTCACATGTAGGGACCGAATTGTTTACAAGTTCTCTGTACCACCATG

GAGACATCAAAAATTGAAAATCTATGGAAAGATATGGACGGTAGCAACAAGAAT

ATAGCACGAGCCGCGGAGTTCATTTCGTTACTTTTGATATCACTCACAACTATTG

CGAAGCGCTTCAGTGAAAAAATCATAAGGAAAAGTTGTAAATATTATTGGTAGT

ATTCGTTTGGTAAAGTAGAGGGGGTAATTTTTCCCCTTTATTTTGTTCATACATTC

TTAAATTGCTTTGCCTCTCCTTTTGGAAAGCTATACTTCGGAGCACTGTTGAGCGA

AGGCTCATTAGATATATTTTCTGTCATTTTCCTTAACCCAAAAATAAGGGAAAGG

GTCCAAAAAGCGCTCGGACAACTGTTGACCGTGATCCGAAGGACTGGCTATACA

GTGTTCACAAAATAGCCAAGCTGAAAATAATGTGTAGCTATGTTCAGTTAGTTTG

GCTAGCAAAGATATAAAAGCAGGTCGGAAATATTTATGGGCATTATTATGCAGA

GCATCAACATGATAAAAAAAAACAGTTGAATATTCCCTCAAAAATGGATTCTAG

AACAGTTGGTATATTAGGAGGGGGACA

ADE2 drive containing His3 selection marker, SNR52promoter, spacer sequence, guide sequence

AATCAAGAAAAACAAGAAAATCGGACAAAACAATCAATCTTACCACGGCATTAG

TCAGGGAAGTCATAACACAGTCCTTTCCCGCAATTTTCTTTTTCTATTACTCTTGG

CCTCCTCTAGTACACTCTATATTTTTTTATGCCTCGGTAATGATTTTCATTTTTTTT

TTTCCACCTAGCGGATGACTCTTTTTTTTTCTTAGCGATTGGCATTATCACATAAT

GAATTATACATTATATAAAGTAATGTGATTTCTTCGAAGAATATACTAAAAAATG

AGCAGGCAAGATAAACGAAGGCAAAGATGACAGAGCAGAAAGCCCTAGTAAAG

CGTATTACAAATGAAACCAAGATTCAGATTGCGATCTCTTTAAAGGGTGGTCCCC

TAGCGATAGAGCACTCGATCTTCCCAGAAAAAGAGGCAGAAGCAGTAGCAGAAC

AGGCCACACAATCGCAAGTGATTAACGTCCACACAGGTATAGGGTTTCTGGACC

GGTCAAGCTTTTAAAGAGGCCCTACTGGCGCGTGGAGTAAAAAGGTTTGGATCA

GGATTTGCGCCTTTGGATGAGGCACTTTCCAGAGCGGTGGTAGATCTTTCGAACA

GGCCGTACGCAGTTGTCGAACTTGGTTTGCAAAGGGAGAAAGTAGGAGATCTCT

CTTGCGAGATGATCCCGCATTTTCTTGAAAGCTTTGCAGAGGCTAGCAGAATTAC

CCTCCACGTTGATTGTCTGCGAGGCAAGAATGATCATCACCGTAGTGAGAGTGCG

TTCAAGGCTCTTGCGGTTGCCATAAGAGAAGCCACCTCGCCCAATGGTACCAACG

ATGTTCCCTCCACCAAAGGTGTTCTTATGTAGTGACACCGATTATTTAAAGCTGC

AGCATACGATATATATACATGTGTATATATGTATACCTATGAATGTCAGTAAGTA

TGTATACGAACAGTATGATACTGAAGATGACAAGGTAATGCATCATTCTATACGT

GTCATTCTGAACGAGGCGCGCTTTCCTTTTTTCTTTTTGCTTTTTCTTTTTTTTTCTC

TTGAACTCGACGGATCTATGCGGCGATTCTTTGAAAAGATAATGTATGATTATGC

TTTCACTCATATTTATACAGAAACTTGATGTTTTCTTTCGAGTATATACAAGGTGA

TTACATGTACGTTTGAAGTACAACTCTAGATTTTGTAGTGCCCTCTTGGGCTAGCG

GTAAAGGTGCGCATTTTTTCACACCCTACAATGTTCTGTTCAAAAGATTTTGGTCA

AACGCTGTAGAAGTGAAAGTTGGTGCGCATGTTTCGGCGTTCGAAACTTCTCCGC

AGTGAAAGATAAATGATCGATCTCGGACAAAACAATCAAGTAGTTAGAAGAGCT

CTTCTATGGATTCTAGAACAGTTGGTATATTAGGAGGGGGACA

ADE2 pre-drive oligo inside minimal vector

TAGAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCT

GCATTTTTACAGAACAGAAATGCAACGCGAAAGCGCTATTTTACCAACGAAGAA

TCTGTGCTTCATTTTTGTAAAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAA

ACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAGAGCGCTA

TTTTACCAACAAAGAATCTATACTTCTTTTTTGTTCTACAAAAATGCATCCCGAGA

GCGCTATTTTTCTAACAAAGCATCTTAGATTACTTTTTTTCTCCTTTGTGCGCTCTA TAATGCAGTCTCTTGATAACTTTTTGCACTGTAGGTCCGTTAAGGTTAGAAGAAG

GCTACTTTGGTGTCTATTTTCTCTTCCATAAAAAAAGCCTGACTCCACTTCCCGCG

TTTACTGATTACTAGCGAAGCTGCGGGTGCATTTTTTCAAGATAAAGGCATCCCC

GATTATATTCTATACCGATGTGGATTGCGCATACTTTGTGAACAGAAAGTGATAG

CGTTGATGATTCTTCATTGGTCAGAAAATTATGAACGGTTTCTTCTATTTTGTCTC

TATATACTACGTATAGGAAATGTTTACATTTTCGTATTGTTTTCGATTCACTCTAT

GAATAGTTCTTACTACAATTTTTTTGTCTAAAGAGTAATACTAGAGATAAACATA

AAAAATGTAGAGGTCGAGTTTAGATGCAAGTTCAAGGAGCGAAAGGTGGATGGG

TAGGTTATATAGGGATATAGCACAGAGATATATAGCAAAGAGATACTTTTGAGC

AACCTAGCAGAAAGCCTGTCTGGTAATCAAGAAAAACAAGAAAATCGGACAAA

ACAATCAATCTTAGAGACCGGTCTCCGATCTCGGACAAAACAATCAAGTAGTTA

GAAGAGCTCTTCTATGGATTCTAGAACAGTTGGTATATTAGGAGGGGGACAACC

GGCACTACATTCTGCACTGAGCAGCTCACTCAAAGGCGGTAATACGGTTATCCAC

AGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGG

CCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCC

TGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGG

ACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTT

CCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGG

CGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCC

AAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCG

GTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGC

AGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTT

CTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGC

GCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCA

AACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCG CAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCT

CAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGG

ATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTA

TATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTAT

CTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGA

TAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGC

GAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAA

GGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAA

TTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTT

GTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATT

CAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAA

AAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAG

TGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCC

GTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGT

GTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCC

ACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAA

ACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCA

CCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAA

CAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGA

ATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCT

CATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCC

GCGCACATTTCCCCGAAAAGTGCCACCTGAACGAAGCATCTGTGCTTCATTTTG gBlock containing sequence to remove common restriction sites in the amp coding region AGAATTATGCAGAGCTGCCATAACCATGAGTGATAACACAGCGGCCAACTTACT

TCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGG

GGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACC

AAACGACGAGCGTGACACCACGATGCCTGTAGCTATGGCAACAACGTTGCGCAA

ACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGG

ATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCT

GGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCACGCGGTATCATAGC

AGCA pNEB193-HO-TRPl- STE5p-CaURA3- nopl-cas9-natMX-HO

GAGCTCTCTCAAATAGCTCAATTGGTTTCTAGATAGCTAAATAGAATATAATCTT

ACTGTCCTCCGTTCTGTAAAATTCACGCTCTTAGTCCCTTTTCATAATTCCTTAAC

TTTTTGCGTACAAAATGATATGTTTATTATATTTTTCTTTTTTTTTTTTTAAATTTTT

TCTTTTTCTTGAAAAATTTTTCAAATTGGAAAGCTCATCTCTCTTGAATGTATAAT

ACTTTCTTCCTCTAACTTTCAAAAAGTTTTACATAGCCAAGAAGTTTTCCTTACAT

CGGTATACTACTGTTATATAAGTTATTCTTCGAGAAACAATTAGATATCATTCATC

GGATAAATCTAAGTTGCCCATTGCTTTCAATAACTCCGATCAAATTAACTCAAAT

CAACTAAAACAGTAACTAGTGGATCCCCCATCACAAGTTTGTACAAAAAAGCAG

GCTACAAAATGGACAAGAAGTACTCCATTGGGCTCGATATCGGCACAAACAGCG

TCGGCTGGGCCGTCATTACGGACGAGTACAAGGTGCCGAGCAAAAAATTCAAAG

TTCTGGGCAATACCGATCGCCACAGCATAAAGAAGAACCTCATTGGCGCCCTCCT

GTTCGACTCCGGGGAGACGGCCGAAGCCACGCGGCTCAAAAGAACAGCACGGC

GCAGATATACCCGCAGAAAGAATCGGATCTGCTACCTGCAGGAGATCTTTAGTA

ATGAGATGGCTAAGGTGGATGACTCTTTCTTCCATAGGCTGGAGGAGTCCTTTTT

GGTGGAGGAGGATAAAAAGCACGAGCGCCACCCAATCTTTGGCAATATCGTGGA

CGAGGTGGCGTACCATGAAAAGTACCCAACCATATATCATCTGAGGAAGAAGCT TGTAGACAGTACTGATAAGGCTGACTTGCGGTTGATCTATCTCGCGCTGGCGCAT

ATGATCAAATTTCGGGGACACTTCCTCATCGAGGGGGACCTGAACCCAGACAAC

AGCGATGTCGACAAACTCTTTATCCAACTGGTTCAGACTTACAATCAGCTTTTCG

AAGAGAACCCGATCAACGCATCCGGAGTTGACGCCAAAGCAATCCTGAGCGCTA

GGCTGTCCAAATCCCGGCGGCTCGAAAACCTCATCGCACAGCTCCCTGGGGAGA

AGAAGAACGGCCTGTTTGGTAATCTTATCGCCCTGTCACTCGGGCTGACCCCCAA

CTTTAAATCTAACTTCGACCTGGCCGAAGATGCCAAGCTTCAACTGAGCAAAGAC

ACCTACGATGATGATCTCGACAATCTGCTGGCCCAGATCGGCGACCAGTACGCA

GACCTTTTTTTGGCGGCAAAGAACCTGTCAGACGCCATTCTGCTGAGTGATATTC

TGCGAGTGAACACGGAGATCACCAAAGCTCCGCTGAGCGCTAGTATGATCAAGC

GCTATGATGAGCACCACCAAGACTTGACTTTGCTGAAGGCCCTTGTCAGACAGCA

ACTGCCTGAGAAGTACAAGGAAATTTTCTTCGATCAGTCTAAAAATGGCTACGCC

GGATACATTGACGGCGGAGCAAGCCAGGAGGAATTTTACAAATTTATTAAGCCC

ATCTTGGAAAAAATGGACGGCACCGAGGAGCTGCTGGTAAAGCTTAACAGAGAA

GATCTGTTGCGCAAACAGCGCACTTTCGACAATGGAAGCATCCCCCACCAGATTC

ACCTGGGCGAACTGCACGCTATCCTCAGGCGGCAAGAGGATTTCTACCCCTTTTT

GAAAGATAACAGGGAAAAGATTGAGAAAATCCTCACATTTCGGATACCCTACTA

TGTAGGCCCCCTCGCCCGGGGAAATTCCAGATTCGCGTGGATGACTCGCAAATCA

GAAGAGACCATCACTCCCTGGAACTTCGAGGAAGTCGTGGATAAGGGGGCCTCT

GCCCAGTCCTTCATCGAAAGGATGACTAACTTTGATAAAAATCTGCCTAACGAAA

AGGTGCTTCCTAAACACTCTCTGCTGTACGAGTACTTCACAGTTTATAACGAGCT

CACCAAGGTCAAATACGTCACAGAAGGGATGAGAAAGCCAGCATTCCTGTCTGG

AGAGCAGAAGAAAGCTATCGTGGACCTCCTCTTCAAGACGAACCGGAAAGTTAC

CGTGAAACAGCTCAAAGAAGACTATTTCAAAAAGATTGAATGTTTCGACTCTGTT

GAAATCAGCGGAGTGGAGGATCGCTTCAACGCATCCCTGGGAACGTATCACGAT CTCCTGAAAATCATTAAAGACAAGGACTTCCTGGACAATGAGGAGAACGAGGAC

ATTCTTGAGGACATTGTCCTCACCCTTACGTTGTTTGAAGATAGGGAGATGATTG

AAGAACGCTTGAAAACTTACGCTCATCTCTTCGACGACAAAGTCATGAAACAGC

TCAAGAGGCGCCGATATACAGGATGGGGGCGGCTGTCAAGAAAACTGATCAATG

GGATCCGAGACAAGCAGAGTGGAAAGACAATCCTGGATTTTCTTAAGTCCGATG

GATTTGCCAACCGGAACTTCATGCAGTTGATCCATGATGACTCTCTCACCTTTAA

GGAGGACATCCAGAAAGCACAAGTTTCTGGCCAGGGGGACAGTCTTCACGAGCA

CATCGCTAATCTTGCAGGTAGCCCAGCTATCAAAAAGGGAATACTGCAGACCGT

TAAGGTCGTGGATGAACTCGTCAAAGTAATGGGAAGGCATAAGCCCGAGAATAT

CGTTATCGAGATGGCCCGAGAGAACCAAACTACCCAGAAGGGACAGAAGAACA

GTAGGGAAAGGATGAAGAGGATTGAAGAGGGTATAAAAGAACTGGGGTCCCAA

ATCCTTAAGGAACACCCAGTTGAAAACACCCAGCTTCAGAATGAGAAGCTCTAC

CTGTACTACCTGCAGAACGGCAGGGACATGTACGTGGATCAGGAACTGGACATC

AATCGGCTCTCCGACTACGACGTGGATCATATCGTGCCCCAGTCTTTTCTCAAAG

ATGATTCTATTGATAATAAAGTGTTGACAAGATCCGATAAAAATAGAGGGAAGA

GTGATAACGTCCCCTCAGAAGAAGTTGTCAAGAAAATGAAAAATTATTGGCGGC

AGCTGCTGAACGCCAAACTGATCACACAACGGAAGTTCGATAATCTGACTAAGG

CTGAACGAGGTGGCCTGTCTGAGTTGGATAAAGCCGGCTTCATCAAAAGGCAGC

TTGTTGAGACACGCCAGATCACCAAGCACGTGGCCCAAATTCTCGATTCACGCAT

GAACACCAAGTACGATGAAAATGACAAACTGATTCGAGAGGTGAAAGTTATTAC

TCTGAAGTCTAAGCTGGTCTCAGATTTCAGAAAGGACTTTCAGTTTTATAAGGTG

AGAGAGATCAACAATTACCACCATGCGCATGATGCCTACCTGAATGCAGTGGTA

GGCACTGCACTTATCAAAAAATATCCCAAGCTTGAATCTGAATTTGTTTACGGAG

ACTATAAAGTGTACGATGTTAGGAAAATGATCGCAAAGTCTGAGCAGGAAATAG

GCAAGGCCACCGCTAAGTACTTCTTTTACAGCAATATTATGAATTTTTTCAAGAC CGAGATTACACTGGCCAATGGAGAGATTCGGAAGCGACCACTTATCGAAACAAA

CGGAGAAACAGGAGAAATCGTGTGGGACAAGGGTAGGGATTTCGCGACAGTCC

GGAAGGTCCTGTCCATGCCGCAGGTGAACATCGTTAAAAAGACCGAAGTACAGA

CCGGAGGCTTCTCCAAGGAAAGTATCCTCCCGAAAAGGAACAGCGACAAGCTGA

TCGCACGCAAAAAAGATTGGGACCCCAAGAAATACGGCGGATTCGATTCTCCTA

CAGTCGCTTACAGTGTACTGGTTGTGGCCAAAGTGGAGAAAGGGAAGTCTAAAA

AACTCAAAAGCGTCAAGGAACTGCTGGGCATCACAATCATGGAGCGATCAAGCT

TCGAAAAAAACCCCATCGACTTTCTCGAGGCGAAAGGATATAAAGAGGTCAAAA

AAGACCTCATCATTAAGCTTCCCAAGTACTCTCTCTTTGAGCTTGAAAACGGCCG

GAAACGAATGCTCGCTAGTGCGGGCGAGCTGCAGAAAGGTAACGAGCTGGCACT

GCCCTCTAAATACGTTAATTTCTTGTATCTGGCCAGCCACTATGAAAAGCTCAAA

GGGTCTCCCGAAGATAATGAGCAGAAGCAGCTGTTCGTGGAACAACACAAACAC

TACCTTGATGAGATCATCGAGCAAATAAGCGAATTCTCCAAAAGAGTGATCCTC

GCCGACGCTAACCTCGATAAGGTGCTTTCTGCTTACAATAAGCACAGGGATAAG

CCCATCAGGGAGCAGGCAGAAAACATTATCCACTTGTTTACTCTGACCAACTTGG

GCGCGCCTGCAGCCTTCAAGTACTTCGACACCACCATAGACAGAAAGCGGTACA

CCTCTACAAAGGAGGTCCTGGACGCCACACTGATTCATCAGTCAATTACGGGGCT

CTATGAAACAAGAATCGACCTCTCTCAGCTCGGTGGAGACAGCAGGGCTGACCC

CAAGAAGAAGAGGAAGGTGTGAAACCCAGCTTTCTTGTACAAAGTGGTGATGGG

CTGCAGGAATTCGATATCAAGCTTATCGATACCGTCGACCTCGAGTCATGTAATT

AGTTATGTCACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAG

GAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTT

AGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCG

TGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTC

GAAGGCTTTAATTTGCGGCCGGTACCCAATTCGCCCTATAGTGAGTCGTATTACA TATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCG

CCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCT

TCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGG

GTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTC

GAGCTCGGTACCCGGGGGCGCGCCGGATCCTTAATTAAGGCCGCCAGCTGAAGC

TTCGTACGCTGCAGGTCGACGGATCCCCGGGTTAATTAAGGCGCGCCAGATCTGT

TTAGCTTGCCTTGTCCCCGCCGGGTCACCCGGCCAGCGACATGGAGGCCCAGAAT

ACCCTCCTTGACAGTCTTGACGTGCGCAGCTCAGGGGCATGATGTGACTGTCGCC

CGTACATTTAGCCCATACATCCCCATGTATAATCATTTGCATCCATACATTTTGAT

GGCCGCACGGCGCGAAGCAAAAATTACGGCTCCTCGCTGCAGACCTGCGAGCAG

GGAAACGCTCCCCTCACAGACGCGTTGAATTGTCCCCACGCCGCGCCCCTGTAGA

GAAATATAAAAGGTTAGGATTTGCCACTGAGGTTCTTCTTTCATATACTTCCTTTT

AAAATCTTGCTAGGATACAGTTCTCACATCACATCCGAACATAAACAACCATGG

GTACCACTCTTGACGACACGGCTTACCGGTACCGCACCAGTGTCCCGGGGGACG

CCGAGGCCATCGAGGCACTGGATGGGTCCTTCACCACCGACACCGTCTTCCGCGT

CACCGCCACCGGGGACGGCTTCACCCTGCGGGAGGTGCCGGTGGACCCGCCCCT

GACCAAGGTGTTCCCCGACGACGAATCGGACGACGAATCGGACGACGGGGAGG

ACGGCGACCCGGACTCCCGGACGTTCGTCGCGTACGGGGACGACGGCGACCTGG

CGGGCTTCGTGGTCGTCTCGTACTCCGGCTGGAACCGCCGGCTGACCGTCGAGGA

CATCGAGGTCGCCCCGGAGCACCGGGGGCACGGGGTCGGGCGCGCGTTGATGGG

GCTCGCGACGGAGTTCGCCCGCGAGCGGGGCGCCGGGCACCTCTGGCTGGAGGT

CACCAACGTCAACGCACCGGCGATCCACGCGTACCGGCGGATGGGGTTCACCCT

CTGCGGCCTGGACACCGCCCTGTACGACGGCACCGCCTCGGACGGCGAGCAGGC

GCTCTACATGAGCATGCCCTGCCCCTAATCAGTACTGACAATAAAAAGATTCTTG

TTTTCAAGAACTTGTCATTTGTATAGTTTTTTTATATTGTAGTTGTTCTATTTTAAT CAAATGTTAGCGTGATTTATATTTTTTTTCGCCTCGACATCATCTGCCCAGATGCG

AAGTTAAGTGCGCAGAAAGTAATATCATGCGTCAATCGTATGTGAATGCTGGTCG

CTATACTGCTGTCGATTCGATACTAACGCCGCCATCCAGTGTCGAAAACGAGCTC

GAATTCATCGATGATATCAGATCCACTAGTGGCCTATGCGGCAATGTGTATATTA

GTTTAAAAAGTTGTATGTAATAAAAGTAAAATTTAATATTTTGGATGAAAAAAAC

CATTTTTAGACTTTTTCTTAACTAGAATGCTGGAGTAGAAATACGCCATCTCAAG

ATACAAAAAGCGTTACCGGCACTGATTTGTTTCAACCAGTATATAGATTATTATT

GGGTCTTGATCAACTTTCCTCAGACATATCAGTAACAGTTATCAAGCTAAATATT

TACGCGAAAGAAAAACAAATATTTTAATTGTGATACTTGTGAATTTTATTTTATT

AAGGATACAAAGTTAAGAGAAAACAAAATTTATATACAATATAAGTAATATTCA

TATATATGTGATGAATGCAGTCTTAACGAGAAGACATGGCCTTGGTGACAACTCT

CTTCAAACCAACTTCAGCCTTTCTCAATTCATCAGCAGATGGGTCTTCGATTTGCA

AAGCAGCCAAAGCATCGGACAAAGCAGCTTCAATCTTGGACTTGGAAGTTTAAA

CATTGCTATTGAGTAAGTTCGATCCGTTTGGCGTCTTTTGGGGTGTAACGCCAAA

CTTATTACTTTTCCTATTTGAGGTTGGTATTGATTGTTGTCAAAGAATGAAAATAT

ACACAAACGCCACAATATACGTACCAGGTTCACGAAAACTGATCGTATGGTTCA

TACCCTGACTTGGCAAACCTAATGTGACCGTCGCTGATTAGCGGATCACGAAAA

GTGATCTCGATACAATTAGAGGATCCACGAAAATGATGTGAATGAATACATGAA

AGATTCATGAGATCTGACAACATGGTAGACGTGTGTGTCTCATGGAAATTGATGC

AGTTGAAGACATGTGCGTCACGAAAAAAGAAATCAATCCTACACAGGGCTTAAG

GGCAAATGTATTCATGTGTGTCACGAAAAGTGATGTAACTAAATACACGATTACC

ATGGAAATTAACGTACCTTTTTTGTGCGTGTATTGAAATATTATGACATATTACA

GAAAGGGTTCGCAAGTCCTGTTTCTATGCCTTTCTCTTAGTAATTCACGAAATAA

ACCTATGGTTTACGAAATGATCCACGAAAATCATGTTATTATTTACATCAACATA

TCGCGAAAATTCATGTCATGTCCACATTAACATCATTGCAGAGCAACAATTCATT TTCATAGAGAAATTTGCTACTATCACCCACTAGTACTACCATTGGTACCTACTAC

TTTGAATTGTACTACCGCTGGGCGTTATTAGGTGTGAAACCACGAAAAGTTCACC

ATAACTTCGAATAAAGTCGCGGAAAAAAGTAAACAGCTATTGCTACTCAAATGA

GGTTTGCAGAAGCTTGTTGAAGCATGATGAAGCGTTCTAAACGCACTATTCATCA

TTAAATATTTAAAGCTCATAAAATTGTATTCAATTCCTATTCTAAATGGCTTTTAT

TTCTATTACAACTATTAGCTCTAAATCCATATCCTCATAAGCAGCAATCAATTCTA

TCTATACTTTAAAATGTCTGTTATTAATTTCACAGGTAGTTCTGGTCCATTGGTGA

AAGTTTGCGGCTTGCAGAGCACAGAGGCCGCAGAATGTGCTCTAGATTCCGATG

CTGACTTGCTGGGTATTATATGTGTGCCCAATAGAAAGAGAACAATTGACCCGGT

TATTGCAAGGAAAATTTCAAGTCTTGTAAAAGCATATAAAAATAGTTCAGGCACT

CCGAAATACTTGGTTGGCGTGTTTCGTAATCAACCTAAGGAGGATGTTTTGGCTC

TGGTCAATGATTACGGCATTGATATCGTCCAACTGCATGGAGATGAGTCGTGGCA

AGAATACCAAGAGTTCCTCGGTTTGCCAGTTATTAAAAGACTCGTATTTCCAAAA

GACTGCAACATACTACTCAGTGCAGCTTCACAGAAACCTCATTCGTTTATTCCCT

TGTTTGATTCAGAAGCAGGTGGGACAGGTGAACTTTTGGATTGGAACTCGATTTC

TGACTGGGTTGGAAGGCAAGAGAGCCCCGAAAGCTTACATTTTATGTTAGCTGGT

GGACTGACGCCAGAAAATGTTGGTGATGCGCTTAGATTAAATGGCGTTATTGGTG

TTGATGTAAGCGGAGGTGTGGAGACAAATGGTGTAAAAGACTCTAACAAAATAG

CAAATTTCGTCAAAAATGCTAAGAAATAGGTTATTACTGAGTAGTATTTATTTAA

GTATTGTTTGTGCACTTGCCGATCTATGCGGTGTGAAATACCGCACAGATGCTTC

CTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCT

CACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAG

AACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTT

GCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGC

TCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCC CCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCT

GTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGT

ATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCC

CGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCG

GTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGA

GCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGC

TACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCG

GAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTG

GTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAG

ATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTA

AGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAAT

TAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACA

GTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCA

TCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTAC

CATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAG

ATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTG

CAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAG

TAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTG

GTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAA

GGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCC

TCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCA

GCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGG

TGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCT

TGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTG

CTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGT TGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTT

TACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAA

AAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAA

TATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAAT

GTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGC

CACCTGACGTCTTATAATTGGCCAGTCTTTTTCAAATAAGCATTCCAACCAGCAT

CTCTATACCTTTTACCTTCAATATCTGGATCTCTTCCTTTACCAAACAATCCTCTA

CCAACAATGATAATATCAGTTCCAGTGCTAACAACTTCATCAACAGTTCTATATT

GTTGTCCTAATCCATCACCTTTATCATCTAATCCAACTCCAGGTGTCATAATAAGC

CAATCAAATCCTTCTTCTTGTCCACCCATATCACGTTGGGCAATAAATCCAATAA

CAAATTCCTTATCGGATTTAGCAATTTCAACAGTTTTTTGAGAATATTCTCCATAT

GCTAATGATCCCACTGATGATAATTCAGCTAACATCAATAACCCTCTTGGCTCTT

GGTTGGTGGTGGTTTCTTTAGCTCCCTGTTTTAATCCTTCAACTACTCCATTCCCA

GTGACACCATGAGCATTAGTAATATCTGCCCAACTACTAATTTTATAAACTCCAC

CAATATATTGTTTCTTCACGGTATTACCAATATCAGCAAATTTTCTATCTTCAAAA

ATCATAAATTGATGTTTACGTGAAAGTTCTAATAATGGTTCAATAGTGGATTCAT

AGGAAAAATCATTGATTATATCAATATGAGTCTTGATTAAGCATACATAAGGACC

CAATTTATCAATTAATTCAAGGAATTCCTTAGTGGTATCAACATCAATTGATGCA

CATAAATTGGTTTTCTTCAGTTCCATTAATCGAAATAATCGTTGTGCTACTGGTGA

GGCATGAGTTTCTGCTCTCTCACTATAGGTCTTAGTGTTGACTGTCATTTAAAAGT

TGTTTCCGCTGTATCCTGTATCTTCCTTTTTTAGCGCGGATGCTAGATGGGTTAAA

ACAGTTTTAAAATATTTTCAAATTACTTTTTTATGCCTTTCTTTTGTCGTCAACTTT

TCGAGGAAATTCTCGGTATTTTCTTTAGCTACTATCGCCTTTCATATTTTGATGCA

GAAAAGTACATGATGTGTAAATTAACAAGCTACATATCGCCGCAGACGTCAATT

AACCCTCACTAAAGGGAACAAAAGCTG Fragment for knocking out TRP1 gene replacing it with LEU2

ACGGAAGAGGAGTAGGGAATATTACTGGCTGAAAATAAGTCTTGAATGAACGTA

TACGCGTATATTTCTACCAATCTCTCAACACTGAGTAATGGTAGTTATAAGAAAG

AGACCGAGTTAGGGACAGTTAGAGGCGGTGGAGATATTCCTTATGGCATGTCTG

GCGATGATAAAACTTTTCAAACGGCAGCCCCGATCTAAAAGAGCTGACAGGGAA

ATGGTCAGAAAAAGAAACGTGCACCCGCCCGTCTGGACGCGCCGCTCACCCGCA

CGGCAGAGACCAATCAGTAAAAATCAACGGTTAACGACATTACTATATATATAA

TATAGGAAGCATTTAATAGAACAGCATCGTAATATATGTGTACTTTGCAGTTATG

ACGCCAGATGGCAGTAGTGGAAGATATTCTTTATTGAAAAATAGCTTGTCACCTT

ACGTACAATCTTGATCCGGAGCTTTTCTTTTTTTGCCGATTAAGAATTCGGTCGAA

AAAAGAAAAGGAGAGGGCCAAGAGGGAGGGCATTGGTGACTATCCACGACTCA

TCTCCATGCAGTTGGACGATCGATGATAAGCTGTCAAACATGAGAATTAATTCTA

CCCTATGAACATATTCCATTTTGTAATTTCGTGTCGTTTCTATTATGAATTTCATTT

ATAAAGTTTATGTACAAATATCATAAAAAAAGAGAATCTTTTTAAGCAAGGATTT

TCTTAACTTCTTCGGCGACAGCATCACCGACTTCGGTGGTACTGTTGGAACCACC

TAAATCACCAGTTCTGATACCTGCATCCAAAACCTTTTTAACTGCATCTTCAATG

GCCTTACCTTCTTCAGGCAAGTTCAATGACAATTTCAACATCATTGCAGCAGACA

AGATAGTGGCGATAGGGTCAACCTTATTCTTTGGCAAATCTGGAGCAGAACCGT

GGCATGGTTCGTACAAACCAAATGCGGTGTTCTTGTCTGGCAAAGAGGCCAAGG

ACGCAGATGGCAACAAACCCAAGGAACCTGGGATAACGGAGGCTTCATCGGAG

ATGATATCACCAAACATGTTGCTGGTGATTATAATACCATTTAGGTGGGTTGGGT

TCTTAACTAGGATCATGGCGGCAGAATCAATCAATTGATGTTGAACCTTCAATGT

AGGAAATTCGTTCTTGATGGTTTCCTCCACAGTTTTTCTCCATAATCTTGAAGAGG

CCAAAACATTAGCTTTATCCAAGGACCAAATAGGCAATGGTGGCTCATGTTGTAG

GGCCATGAAAGCGGCCATTCTTGTGATTCTTTGCACTTCTGGAACGGTGTATTGTT CACTATCCCAAGCGACACCATCACCATCGTCTTCCTTTCTCTTACCAAAGTAAAT

ACCTCCCACTAATTCTCTGACAACAACGAAGTCAGTACCTTTAGCAAATTGTGGC

TTGATTGGAGATAAGTCTAAAAGAGAGTCGGATGCAAAGTTACATGGTCTTAAG

TTGGCGTACAATTGAAGTTCTTTACGGATTTTTAGTAAACCTTGTTCAGGTCTAAC

ACTACCTGTACCCCATTTAGGACCACCCACAGCACCTAACAAAACGGCATCAAC

CTTCTTGGAGGCTTCCAGCGCCTCATCTGGAAGTGGGACACCTGTAGCATCGATA

GCAGCACCACCAATTAAATGATTTTCGAAATCGAACTTGACATTGGAACGAACA

TCAGAAATAGCTTTAAGAACCTTAATGGCTTCGGCTGTGATTTCTTGACCAACGT

GGTCACCTGGCAAAACGACGATCTTCTTAGGGGCAGACATAGGGGCAGACATTA

GAATGGTATATCCTTGAAATATATATATATATTGCTGAAATGTAAAAGGTAAGAA

AAGTTAGAAAGTAAGACGATTGCTAACCACCTATTGGAAAAAACAATAGGTCCT

TAAATAATATTGTCAACTTCAAGTATTGTGATGCAAGCATTTAGTCATGAACGCT

TCTCTATTCTATATGAAAAGCCGGTTCCGGCCTCTCACCTTTCCTTTTTCTCCCAA

TTTTTCAGTTGAAAAAGGTATATGCGTCAGGCGACCTCTGAAATTAACAAAAAAT

TTCCAGTCATCGAATTTGATTCTGTGCGATAGCGCCCCTGTGTGTTCTCGTTATGT

TGAGGAAAAAAATAATGGTTGCTAAGAGATTCGAACTCTTGCATCTTACGATACC

TGAGTATTCCCACAGTTAATTCTTGAAGACGAAAGGGCCTCGTGACTTTTACCAT

TTCACCGCAATGGAATCAAACTTGTTGAAGAGAATGTTCACAGGCGCATACGCT

ACAATGACCCGATTCTTGCTAGCCTTTTCTCGGTCTTGCAAACAACCGCCGGCAG

CTTAGTATATAAATACACATGTACATACCTCTCTCCGTATCCTCGTAATCATTTTC

TTGTATTTATCGTCTTTTCGCTGTAAAAACTTTATCACACTTATCTCAAATACACT

TATTAACCGCTTTTACTATTATCTTCTACGCTGACAGTAATATCAAACAGTGACA

CATATTAAACACAGTGGTTTCTTTGCATAAACACCATCAGCCTCAAGTCGTCAAG

TAAAGATTTCGTGTTCATGCAGATAGATAACAATCTATATGTTGATAATTAGCGT

TGCCTCATCAATGCGAGATCCGTTTAACCGGACCCTAGTGCACTTACCCCACGTT CGGTCCACTGTGTGCCGAACATGCTCCTTCACTATTTTAACATGTGGAATTCTTGA AAGAATGAAATCGCCATGCCAAGCCATCACACGGTCTTTTATGCAATTGATTGAC CGCCTGCAACACATAGGCAGTAAAATTTTTACTGAAACGTATATAATCATCATAA GCGACAAGTGAGGCAACACCTTTGTTACCACATTGACAACCCCAGGTATTCATAC TTCCTATTAGCGGAATCAGGAGTGCA

Claims

1. A method of making a plurality of substrate bound oligonucleotide sequences for insertion into corresponding vectors comprising synthesizing the plurality of oligonucleotide sequences with each oligonucleotide sequence including at least one unique guide RNA spacer sequence and a plurality of endonuclease restriction sites, wherein each oligonucleotide sequence includes first flanking outer endonuclease restriction sites, second flanking inner endonuclease restriction sites, flanking random primer binding sites and a unique gene drive component flanked by the flanking random primer binding sites, wherein the unique gene drive component includes in series, an upstream target nucleic acid flanking sequence, a third endonuclease restriction site, the at least one unique guide RNA spacer sequence having a sequence complementary to a target nucleic acid sequence, a fourth endonuclease restriction site and a downstream target nucleic acid flanking sequence, and wherein the first flanking outer endonuclease restriction sites, the second flanking inner endonuclease restriction sites, the third endonuclease restriction site, and the fourth endonuclease restriction site are different.

2. The method of claim 1 wherein the plurality of oligonucleotide sequences is made by array-based oligonucleotide synthesis including semiconductor-based electrochemical-synthesis process, photolithographic techniques, inkjet printing, and successively reacting nucleotide monomers.

3. The method of claim 1 wherein the plurality of oligonucleotide sequences is made using monomer by monomer oligonucleotide synthesis.

4. The method of claim 1 wherein each endonuclease restriction site of the plurality of endonuclease restriction sites is a member selected from the group consisting of type II restriction endonucleases such as Acul, Alwl, Bael, Bbsl, Bbvl, Bed, BceAI, Bcgl, BciVI, BcoDI, BfuAI, Bmrl, Bpml, BpuEI, Bsal, BsaXI, BseRI, Bsgl, BsmAI, BsmBI, BsmFI, Bsml, BspCNI, BspMI, BspQI, BsrDI, Bsrl, BtgZI, BtsCI, Btsl, BtsIMutI, CspCI, Earl, Ecil, Faul, Fokl, Hgal, Hphl, HpyAV, MboII, Mlyl, Mmel, Mnll, NmeAIII, Plel, Sapl, and SfaNI.

5. The method of claim 1 wherein the upstream target nucleic acid flanking sequence and the downstream target nucleic acid flanking sequence are complementary to corresponding sequences between an endogenous promoter and a corresponding endogenous gene within a target cell.

6. The method of claim 1 wherein the upstream target nucleic acid flanking sequence and the downstream target nucleic acid flanking sequence are complementary to corresponding sequences between gene coding regions of an endogenous gene within a target cell.

7. The method of claim 1 wherein the plurality of oligonucleotide sequences includes between 2 and 250,000 oligonucleotide sequences.

8. The method of claim 1 wherein the plurality of oligonucleotide sequences includes between 10 and 100,000 oligonucleotide sequences.

9. The method of claim 1 wherein the plurality of oligonucleotide sequences includes between 20 and 6,000 oligonucleotide sequences.

10. The method of claim 1 wherein the plurality of oligonucleotide sequences includes between 50 and 1,000 oligonucleotide sequences.

11. The method of claim 1 wherein the plurality of oligonucleotide sequences includes between 100 and 500 oligonucleotide sequences.

12. A method of making a plurality of vectors with each vector of the plurality including a unique gene drive component comprising removing a plurality of bound oligonucleotide sequences from a substrate using a first endonuclease, wherein each substrate bound oligonucleotide sequence includes at least one unique guide RNA spacer sequence and a plurality of endonuclease restriction sites, wherein each substrate bound oligonucleotide sequence includes first flanking outer endonuclease restriction sites, second flanking inner endonuclease restriction sites, flanking random primer binding sites and the unique gene drive component flanked by the flanking random primer binding sites, wherein the unique gene drive component includes in series, an upstream target nucleic acid flanking sequence, a third endonuclease restriction site, the at least one unique guide RNA spacer sequence having a sequence complementary to a target nucleic acid sequence, a fourth endonuclease restriction site and a downstream target nucleic acid flanking sequence, wherein the first flanking outer endonuclease restriction sites, the second flanking inner endonuclease restriction sites, the third endonuclease restriction site, and the fourth endonuclease restriction site are different, wherein the first endonuclease corresponds to the first flanking outer endonuclease restriction sites and cuts the bound oligonucleotide sequence at the first flanking outer endonuclease restriction sites to produce a plurality of unbound oligonucleotide sequences, and inserting each unbound oligonucleotide sequence into a corresponding vector to produce the plurality of vectors each with a unique gene drive component.

13. The method of claim 12 wherein each endonuclease restriction site of the plurality of endonuclease restriction sites is a member selected from the group consisting of type II restriction endonucleases such as Acul, Alwl, Bael, Bbsl, Bbvl, Bed, BceAI, Bcgl, BciVI, BcoDI, BfuAI, Bmrl, Bpml, BpuEI, Bsal, BsaXI, BseRI, Bsgl, BsmAI, BsmBI, BsmFI, Bsml, BspCNI, BspMI, BspQI, BsrDI, Bsrl, BtgZI, BtsCI, Btsl, BtsIMutI, CspCI, Earl, Ecil, Faul, Fokl, Hgal, Hphl, HpyAV, MboII, Mlyl, Mmel, Mnll, NmeAIII, Plel, Sapl, and SfaNI.

14. The method of claim 12 wherein the upstream target nucleic acid flanking sequence and the downstream target nucleic acid flanking sequence are complementary to corresponding sequences between an endogenous promoter and a corresponding endogenous gene within a target cell.

15. The method of claim 12 wherein the upstream target nucleic acid flanking sequence and the downstream target nucleic acid flanking sequence are complementary to corresponding sequences between gene coding regions of an endogenous gene within a target cell.

16. The method of claim 12 wherein the plurality of oligonucleotide sequences includes between 2 and 250,000 oligonucleotide sequences.

17. The method of claim 12 wherein the plurality of oligonucleotide sequences includes between 10 and 100,000 oligonucleotide sequences.

18. The method of claim 12 wherein the plurality of oligonucleotide sequences includes between 20 and 6,000 oligonucleotide sequences.

19. The method of claim 12 wherein the plurality of oligonucleotide sequences includes between 50 and 1,000 oligonucleotide sequences.

20. The method of claim 12 wherein the plurality of oligonucleotide sequences includes between 100 and 500 oligonucleotide sequences.

21. The method of claim 1 wherein the vector is a plasmid or any other genetic element that can be propagated in a bacterial host.

22. A method of making a plurality of vectors with each vector of the plurality including a unique gene drive comprising providing a plurality of vectors with each vector of the plurality including an oligonucleotide sequence including first flanking endonuclease restriction sites, flanking random primer binding sites and a unique gene drive component flanked by the flanking random primer binding sites, wherein the unique gene drive component includes in series, an upstream target nucleic acid flanking sequence, a second endonuclease restriction site, at least one unique guide RNA spacer sequence having a sequence complementary to a target nucleic acid sequence, a third endonuclease restriction site and a downstream target nucleic acid flanking sequence, wherein the first flanking endonuclease restriction sites, the second endonuclease restriction site, and the third endonuclease restriction site are different, and creating the plurality of vectors with each vector of the plurality including a unique gene drive by (1) cutting each oligonucleotide sequence within its corresponding vector at the second endonuclease restriction site using a corresponding second endonuclease and inserting a guide RNA promotor and, optionally, a selection marker therein, and (2) cutting each oligonucleotide sequence within its corresponding vector at the third endonuclease restriction site using a corresponding third endonuclease and inserting a spacer tail nucleic acid sequence encoding a tracr mate sequence and a tracr sequence therein to create at least one guide RNA and optionally inserting a cargo sequence therein.

23. The method of claim 22 wherein each endonuclease restriction site of the plurality of endonuclease restriction sites is a member selected from the group consisting of type II restriction endonucleases such as Acul, Alwl, Bael, Bbsl, Bbvl, Bed, BceAI, Bcgl, BciVI, BcoDI, BfuAI, Bmrl, Bpml, BpuEI, Bsal, BsaXI, BseRI, Bsgl, BsmAI, BsmBI, BsmFI, Bsml, BspCNI, BspMI, BspQI, BsrDI, Bsrl, BtgZI, BtsCI, Btsl, BtsIMutI, CspCI, Earl, Ecil, Faul, Fokl, Hgal, Hphl, HpyAV, MboII, Mlyl, Mmel, Mnll, NmeAIII, Plel, Sapl, and SfaNI.

24. The method of claim 22 wherein the upstream target nucleic acid flanking sequence and the downstream target nucleic acid flanking sequence are complementary to corresponding sequences between an endogenous promoter and a corresponding endogenous gene within a target cell.

25. The method of claim 22 wherein the upstream target nucleic acid flanking sequence and the downstream target nucleic acid flanking sequence are complementary to corresponding sequences between gene coding regions of an endogenous gene within a target cell.

26. The method of claim 22 wherein the plurality of oligonucleotide sequences includes between 2 and 250,000 oligonucleotide sequences.

27. The method of claim 22 wherein the plurality of oligonucleotide sequences includes between 10 and 100,000 oligonucleotide sequences.

28. The method of claim 22 wherein the plurality of oligonucleotide sequences includes between 20 and 6,000 oligonucleotide sequences.

29. The method of claim 22 wherein the plurality of oligonucleotide sequences includes between 50 and 1,000 oligonucleotide sequences.

30. The method of claim 22 wherein the plurality of oligonucleotide sequences includes between 100 and 500 oligonucleotide sequences.

31. The method of claim 22 wherein the vector is a plasmid or any other genetic element that can be propagated in a bacterial host.

34. The method of claim 22 wherein the cargo sequence is a target gene promoter.

35. The method of claim 22 wherein the cargo sequence is a target gene.

36. The method of claim 22 wherein the cargo sequence is a nucleic acid sequence encoding an RNA guided DNA binding protein.

37. The method of claim 22 wherein the cargo sequence is a nucleic acid sequence encoding a fluorescent protein allowing for screening of organism carrying the gene drive.

38. The method of claim 22 wherein the cargo sequence is a nucleic acid sequence encoding a fluorescent protein fused to a target protein at the C-terminal or N-terminal region.

39. The method of claim 22 wherein the cargo sequence is a nucleic acid sequence encoding a scaffold domain fused to a target protein at the C-terminal or N-terminal region, wherein the scaffold domain confers binding property to the target protein for phenotype analysis.

40. The method of claim 22 wherein the cargo sequence is a nucleic acid sequence encoding a regulatory subunit fused to a target protein at the C-terminal or N-terminal region, wherein the regulatory subunit creates novel regulatory phenotype of the target gene expression.

41. The method of claim 22 wherein the cargo sequence is a nucleic acid sequence containing restriction sites allowing removal of the cargo sequence at a later stage.

42. The method of claim 22 wherein the cargo sequence is a nucleic acid sequence encoding an altered endogenous untranslated region of a target gene changing the transcription and/or translation efficiency of the target gene.

43. The method of claim 22 wherein the cargo sequence is a nucleic acid sequence encoding a Cas9 protein.

44. The method of claim 22 wherein the cargo sequence is a nucleic acid sequence encoding a Cas9 enzyme, a Cas9 nickase, a nuclease null Cas9 fused to a nuclease or nickase domain, or a nuclease null Cas9 fused to a nuclease or nickase domain with a transcriptional modulator attached thereto.

45. The method of claim 22 wherein the spacer tail nucleic acid sequence further encodes a transcriptional modulator to create a guide RNA with the transcriptional modulator bound thereto.

46. A method of making a plurality of genetically altered proliferating cells comprising combining a plurality of proliferating cells including a target gene sequence with a plurality of vectors with each vector of the plurality including an oligonucleotide sequence including first flanking endonuclease restriction sites, flanking random primer binding sites and a unique gene drive flanked by the flanking random primer binding sites, wherein the unique gene drive includes in series, an upstream target genomic nucleic acid flanking sequence, a guide RNA promoter, at least one unique guide RNA sequence having a spacer sequence complementary to the target genomic nucleic acid sequence, an optional cargo sequence, and a downstream target genomic nucleic acid flanking sequence, wherein corresponding cells of the plurality of proliferating cells each receive a single vector, wherein the corresponding cells each include an RNA guided DNA binding protein, and wherein the guide RNA is produced and a colocalization complex of the guide RNA and the RNA guided DNA binding protein forms at the target genomic nucleic acid sequence and the target genomic nucleic acid sequence is cut and the unique gene drive is inserted therein to produce the plurality of genetically altered proliferating cells.

47. The method of claim 46 further including a first round step of mating the plurality of genetically altered proliferating cells among themselves to produce a plurality of first round variant cells with each variant having two unique gene drives.

48. The method of claim 47 further including a second round step of mating the plurality of first round variant cells among themselves to produce a plurality of second round variant cells with each variant having four unique gene drives.

49. The method of claim 48 further including a third round step of mating the plurality of second round variant cells among themselves to produce a plurality of third round variant cells with each variant having eight unique gene drives.

50. The method of claim 49 further including a fourth round step of mating the plurality of third round variant cells among themselves to produce a plurality of fourth round variant cells with each variant having sixteen unique gene drives.

51. The method of claim 50 further including subsequent rounds of mating the plurality of previous round variant cells among themselves to produce a plurality of variant cells with each variant having amplified gene drives before equilibrium is attained.

52. The method of claim 46 wherein the proliferating cell type is a member of the group consisting of genus Saccharomyces, genus Schizosaccharomyce ), genus Kluveromyces, genus Candida and Pichia pastoris.

53. The method of claim 46 wherein the proliferating cell type is a member of the group consisting of Aspergillus nidulans, A. oryza, A. niger, and A. sojae.

54. A substrate having a plurality of substrate bound oligonucleotide sequences according to claim 1.

55. A plurality of vectors with each vector of the plurality including an oligonucleotide sequence according to claim 22.

56. The vector of claim 55 wherein the vector is a plasmid or any other genetic element that can be propagated in a bacterial host.

57. The plurality of vectors of claim 53 wherein each vector of the plurality including an oligonucleotide sequence including first flanking endonuclease restriction sites, flanking random primer binding sites and a unique gene drive flanked by the flanking random primer binding sites, wherein the unique gene drive includes in series, an upstream target genomic nucleic acid flanking sequence, a guide RNA promoter, at least one unique guide RNA sequence having a spacer sequence complementary to the target genomic nucleic acid sequence, an optional cargo sequence, and a downstream target genomic nucleic acid flanking sequence.

58. A cell including a vector of claim 22.

59. A method of making a genetically altered proliferating cell comprising providing to the proliferating cell including a target gene sequence with a vector including an oligonucleotide sequence including first flanking endonuclease restriction sites, flanking random primer binding sites and a unique gene drive flanked by the flanking random primer binding sites, wherein the unique gene drive includes in series, an upstream target genomic nucleic acid flanking sequence, a guide RNA promoter, at least one unique guide RNA sequence having a spacer sequence complementary to the target genomic nucleic acid sequence, an optional cargo sequence, and a downstream target genomic nucleic acid flanking sequence, wherein the proliferating cell includes an RNA guided DNA binding protein, and wherein the guide RNA is produced and a colocalization complex of the guide RNA and the RNA guided DNA binding protein forms at the target genomic nucleic acid sequence and the target genomic nucleic acid sequence is cut and the unique gene drive is inserted therein to produce the genetically altered proliferating cell.

60. The method of claim 59 wherein the vector is provided to the proliferating cell in the form of two linear fragments.

61. The method of claim 60 wherein the two linear fragments undergo homologous recombination to generate a stably inherited circular plasmid after being transformed into the cell.