Multiplex RNA-guided genome editing

Abstract

The steps of DNA modification are repeated or iterated on the cells to generate cells with multiple DNA modifications within the cells. [Solution] A method for multiple genome modification in cells using Cas9 is provided, which includes a cycle of introducing into a cell a first foreign nucleic acid encoding one or more types of RNA that are complementary to the target DNA and guide an enzyme to the target DNA, wherein the one or more types of RNA and the enzyme are components of a co-localized complex to the target DNA, and introducing into the cell a second foreign nucleic acid sequence encoding one or more types of donor nucleic acid sequences, and this cycle is repeated a desired number of times to perform multiple DNA modifications in the cell.

Description

[Technical field]

[0001] Related Application Data This application claims priority to U.S. Provisional Patent Application No. 61 / 844,168, filed July 9, 2013, which is incorporated by reference in its entirety for all purposes.

[0002] Government Benefits Explained This invention was made with Federal support under Department of Energy Grant No. DE-FG02-02ER63445, National Science Foundation Grant No. NSF-SynBERC, and National Science Foundation Grant No. SA5283-11210. The United States Government has certain rights in this invention. [Background technology]

[0003] Bacterial and archaeal CRISPR-Cas systems rely on short guide RNAs complexed with Cas proteins to degrade complementary sequences present in invading foreign nucleic acids. Deltcheva, E. et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471, 602-607 (2011); Gasiunas, G., Barrangou, R., Horvath, P. & Siksnys, V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proceedings of the National Academy of Sciences of the United States of America 109, E2579-2586 (2012); Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816-821 (2012); Sapranauskas, R. et al. The Streptococcus thermophilus CRISPR / Cas system provides immunity in Escherichia coli. Nucleic acids research 39, 9275-9282 (2011); and Bhaya, D., Davison, M. & Barrangou, R. CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. Annual review of genetics 45, 273-297 (2011).Recently, in vitro reconstitution of the type II CRISPR system in S. pyogenes demonstrated that a crRNA ("CRISPR RNA") fused to a tracrRNA ("trans-activating CRISPR RNA"), normally encoded in trans, is sufficient to direct the Cas9 protein to sequence-specifically cleave a target DNA sequence that matches the crRNA. Expression of a gRNA homologous to the target site recruits Cas9 and degrades the target DNA. See H. Deveau et al, Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. Journal of Bacteriology 190, 1390 (Feb, 2008). Summary of the Invention [Problem to be solved by the invention]

[0004] Aspects of the present disclosure relate to multiple modification of DNA in a cell using one or more guide RNAs (ribonucleic acids) to guide an enzyme with nuclease activity expressed by the cell, such as a DNA binding protein with nuclease activity, to a target location on the DNA, where the enzyme cleaves the DNA and an exogenous donor nucleic acid is inserted into the DNA, such as by homologous recombination. Aspects of the present disclosure include repeating or iterating the steps of DNA modification on a cell to create a cell with multiple DNA modifications in the cell. The modification may include the insertion of an exogenous donor nucleic acid. [Means for solving the problem]

[0005] Multiple insertions of exogenous nucleic acids can be achieved by a single step of introducing multiple RNA-encoding nucleic acids and multiple exogenous donor nucleic acids into a cell expressing an enzyme, such as by co-transformation. In this step, the RNAs are expressed, each of the multiple RNAs guides an enzyme to a specific site in the DNA, the enzyme cleaves the DNA, and one of the multiple exogenous nucleic acids is inserted into the cleaved site of the DNA. According to this embodiment, multiple changes or modifications are made to the DNA in the cell in a single cycle.

[0006] Multiple insertions of exogenous nucleic acids can be achieved in a cell by repeated steps or cycles of introducing one or more nucleic acids encoding one or more RNAs and one or more exogenous nucleic acids into a cell expressing an enzyme, in which the RNA is expressed, the enzyme is guided to a specific site in the DNA, the enzyme cleaves the DNA, and the exogenous nucleic acid is inserted into the cleaved site of the DNA, resulting in a cell having multiple changes or insertions of exogenous DNA in the DNA of the cell. According to certain embodiments, the cell expressing the enzyme may be a cell that naturally expresses the enzyme, or may be a cell that has been genetically altered to express the enzyme, such as by introducing into the cell a nucleic acid that encodes the enzyme and can be expressed by the cell. Thus, embodiments of the present disclosure include repeated steps of introducing RNA into a cell expressing the enzyme, introducing an exogenous donor nucleic acid into the cell, expressing the RNA, forming a co-localized complex of the RNA, enzyme, and DNA, enzymatically cleaving the DNA with the enzyme, and inserting the donor nucleic acid into the DNA. Repetition or repetition of the above steps results in multiple genetic modifications of cells at multiple loci, i.e., cells carrying multiple genetic alterations.

[0007] According to one aspect, a method is provided for increasing the homologous recombination rate by the above-mentioned repeating method. According to one embodiment, Cas9-dependent genomic DNA cleavage dramatically increases the homologous recombination rate to stimulate exogenous DNA. According to another aspect, the exogenous donor nucleic acid includes a homologous sequence or arm adjacent to the cleavage site. According to another aspect, the exogenous donor nucleic acid includes a sequence for removing the cleavage sequence. According to another aspect, the exogenous donor nucleic acid includes a homologous sequence or arm adjacent to the cleavage site and a sequence for removing the cleavage site. In this way, Cas9 can be used as a negative selection for cells that do not incorporate exogenous donor DNA. Thus, a negative selection method is provided for identifying cells with high recombination frequency.

[0008] According to an embodiment, the DNA binding protein or enzyme within the scope of the present disclosure includes a protein that forms a complex with a guide RNA, and the guide RNA guides the complex to a double-stranded DNA sequence, where the complex binds to the DNA sequence. According to an embodiment, the enzyme may be an RNA guided DNA binding protein, such as the RNA guided DNA binding protein of the type II CRISPR system that binds to DNA and is guided by RNA. According to an embodiment, the RNA guided DNA binding protein is a Cas9 protein.

[0009] This aspect of the disclosure can be referred to as co-localization of RNA and DNA binding proteins to double-stranded DNA, or co-localization of RNA and DNA binding proteins with double-stranded DNA. In this way, the DNA binding protein-guide RNA complex can be used to cleave multiple sites in double-stranded DNA to create cells with multiple genetic modifications, such as multiple insertions of exogenous donor DNA.

[0010] According to one embodiment, a method is provided for producing multiple alterations in a target DNA in a cell expressing an enzyme that forms a co-localized complex with a complementary RNA and site-specifically cleaves the target DNA, the method comprising: (a) introducing into the cell a first exogenous nucleic acid encoding one or more RNAs that are complementary to the target DNA and guide the enzyme to the target DNA, wherein the one or more RNAs and the enzyme are components of a co-localized complex to the target DNA, introducing into the cell a second exogenous nucleic acid encoding one or more donor nucleic acid sequences, wherein the one or more RNAs and the one or more donor nucleic acid sequences are expressed, the one or more RNAs and the enzyme co-localize to the target DNA, the enzyme cleaves the target DNA, and the donor nucleic acid is inserted into the target DNA to produce altered DNA in the cell, and repeating step (a) multiple times to produce multiple alterations in DNA in the cell.

[0011] In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a yeast cell, a plant cell, or an animal cell. In some embodiments, the cell is a mammalian cell.

[0012] In one embodiment, the RNA is about 10 to about 500 nucleotides In one embodiment, the RNA is about 20 to about 100 nucleotides.

[0013] In one embodiment, the one or more RNAs are guide RNAs. In one embodiment, the one or more RNAs are tracrRNA-crRNA fusions.

[0014] According to certain embodiments, the DNA is genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA.

[0015] Other features and advantages of particular embodiments of the present invention will become more fully apparent in the following description of the embodiments and the drawings thereof, and from the claims.

[0016] The above 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. [Brief description of the drawings]

[0017] [Figure 1] Schematic diagram of Cas9-mediated RNA-guided genome cleavage. [Diagram 2] FIG. 1 is a schematic diagram showing multiple genome engineering in yeast using Cas9. [Diagram 3] FIG. 1 is a schematic showing allelic replacement using oligonucleotides targeting four loci important for thermotolerance in yeast. [Figure 4] Graph showing the number of modifications per cell after 1 cycle and after 2 cycles. [Figure 5A] 1 is a table of strains with mutations. [Figure 5B] FIG. 1 shows thermotolerance to heat shock for various strains. [Figure 6A] FIG. 1 shows graphical data on transformation frequencies. [Figure 6B] Graphical data for individual recombination frequencies are shown. [Figure 6C] FIG. 1 shows graphical data on co-recombination frequencies at the can1 and KanMX loci. [Figure 7] FIG. 1 shows graphical data for the incorporation of multiple linear cassettes at two loci. [Figure 8A] FIG. 1 shows graphical data for fold change in doubling time at 30° C. [Figure 8B] FIG. 1 shows graphical data for fold change in doubling time at 37° C. [Figure 8C]FIG. 13 shows graphical data for fold change in doubling time at 42° C., where cells were plated from late stationary phase cultures. [Figure 8D] FIG. 13 shows graphical data for fold change in doubling time at 42° C., where cells were plated from a late log phase culture. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The embodiment of the present disclosure is based on the repeated use of foreign DNA, nuclease enzymes such as DNA binding proteins, and guide RNAs to co-localize with DNA and digest or cleave the DNA to insert foreign DNA by homologous recombination or the like. Those skilled in the art will easily understand such DNA binding proteins that bind to DNA for various purposes. Such DNA binding proteins may be natural. DNA binding proteins within the scope of the present disclosure include proteins that can be guided by RNA, referred to herein as guide RNA. According to this aspect, guide RNA and RNA-guided DNA binding proteins form a co-localized complex in DNA. Such DNA binding proteins with nuclease activity are known to those skilled in the art, and include natural DNA binding proteins with nuclease activity, such as Cas9 protein, present in type II CRISPR system. Such Cas9 protein and type II CRISPR system are well documented in the art. See Makarova et al., Nature Reviews, Microbiology, Vol. 9, June 2011, pp. 467-477, including all additional information, which is incorporated by reference in its entirety.

[0019] Exemplary DNA binding proteins with nuclease activity function to form nicks in double-stranded DNA or to cut double-stranded DNA. Such nuclease activity can be derived from DNA binding proteins with one or more polypeptide sequences that exhibit nuclease activity. Such exemplary DNA binding proteins may have two separate nuclease domains, each of which is involved in cutting or forming nicks in a specific strand of double-stranded DNA. Exemplary polypeptide sequences with nuclease activity known to those skilled in the art include McrA-HNH nuclease-associated domain and RuvC-like nuclease domain. Thus, exemplary DNA binding proteins are natural proteins that include one or more of McrA-HNH nuclease-associated domain and RuvC-like nuclease domain.

[0020] An exemplary DNA binding protein is the RNA-guided DNA binding protein of type II CRISPR system.An exemplary DNA binding protein is Cas9 protein.

[0021] In S. pyogenes, Cas9 creates a blunt-ended double-stranded break 3 bp upstream of the protospacer adjacent motif (PAM) through a process mediated by two catalytic domains in the protein: an HNH domain that cleaves the complementary strand of DNA and a RuvC-like domain that cleaves the non-complementary strand. See Jinke et al., Science 337, 816-821 (2012), which is incorporated by reference in its entirety. Cas9 proteins are known to be present in a number of type II CRISPR systems, including the following, which are reviewed in supplementary information in Makarova et al., Nature Reviews, Microbiology, Vol. 9, June 2011, pp. 467-477: Methanococcus maripaludis strain C7; Corynebacterium diphtheriae; Corynebacterium efficiens strain YS-314; Corynebacterium glutamicum ATCC13032 Kitasato strain; Corynebacterium glutamicum ATCC13032 Bielefeld strain; Corynebacterium glutanicum strain R; Corynebacterium kroppenstedtii strain DSM44385; Mycobacterium abscessus strain ATCC19977; Nocardia farcinica farcinica strain IFM10152; Rhodococcus erythropolis strain PR4; Rhodococcus jostii strain RHA1; Rhodococcus opacus strain B4 uid36573; Acidothermus cellulolyticus strain 11B;Arthrobacter chlorophenolicus strain A6; Kribbella flavida strain DSM17836 uid43465; Thermomonospora curvata strain DSM43183; Bifidobacterium dentium strain Bd1; Bifidobacterium longum strain DJO10A; Slackia heliotrinireducens strain DSM20476; Persephonella marina strain EX H1; Bacteroides fragilis strain NCTC9434; Capnocytophaga ochracea strain DSM7271;Flavobacterium psychrophilum strain JIP0286;Akkermansia muciniphila strain ATCC BAA835;Roseiflexus castenholzii strain DSM13941;Roseiflexus strain RS1;Synechocystis strain PCC6803;Elusimicrobium minutum strain Pei191;Uncultured bacteria Termite group 1 phylotype Rs D17;Fibrobacter succinogenes strain S85;Bacillus cereus cereus ATCC10987 strain; Listeria innocua; Lactobacillus casei; Lactobacillus rhamnosus GG strain; Lactobacillus salivarius UCC118 strain;Streptococcus agalactiae strain A909; Streptococcus agalactiae strain NEM316; (Streptococcus agalactiae strain 2603; Streptococcus dysgalactiae equisimilis strain GGS124; Streptococcus equi zooepidemicus strain MGCS10565; (Streptococcus gallolyticus) strain UCN34 uid46061; Streptococcus gordonii Challis subst. strain CH1; Streptococcus mutans N2025 uid46353;Streptococcus mutans;Streptococcus pyogenes M1 GAS;Streptococcus pyogenes MGAS5005;Streptococcus pyogenes MGAS2096;Streptococcus pyogenes MGAS9429;Streptococcus pyogenes MGAS10270;Streptococcus pyogenes MGAS6180;Streptococcus pyogenes MGAS315;Streptococcus pyogenes SSI-1;Streptococcus pyogenes MGAS10750;Streptococcus pyogenes NZ131;Streptococcus thermophilus thermophiles strain CNRZ1066; Streptococcus thermophilus strain LMD-9; Streptococcus thermophilus strain LMG18311; Clostridium botulinum strain A3 Loch Maree; Clostridium botulinum strain B Eklund 17B; Clostridium botulinum strain Ba4 657; Clostridium botulinum strain F Langeland; Clostridium cellulolyticum strain H10;Finegoldia magna (strain ATCC29328); Eubacterium rectale (strain ATCC33656); Mycoplasma gallisepticum; Mycoplasma mobile (strain 163K); Mycoplasma penetrans; Mycoplasma synoviae (strain 53); Streptobacillus moniliformis (strain DSM12112); Bradyrhizobium (strain BTAil); Nitrobacter hamburgensis (strain X14); Rhodopseudomonas palustris (BisB) 18 strains; Rhodopseudomonas palustris strain BisB5; Parvibaculum lavamentivorans strain DS-1; Dinoroseobacter shibae strain DFL12; Gluconacetobacter diazotrophicus Pal5 strain FAPERJ; Gluconacetobacter diazotrophicus Pal5 strain JGI; Azospirillum B510 strain uid46085; Rhodospirillum rubrum strain ATCC11170; Diaphorobacter TPSY strain uid29975; Verminephrobacter eiseniae strain EF01-2; Neisseria meningitides strain 053442; Neisseria meningitidis strain alpha14; Neisseria meningitidis strain Z2491; Desulfovibrio salexigens strain DSM2638;Campylobacter jejuni doylei strain 269 97;Campylobacter jejuni strain 81116;Campylobacter jejuni;Campylobacter lari strain RM2100;Helicobacter hepaticus;Wolinella succinogenes;Tolumonas auensis strain DSM9187;Pseudoalteromonas atlantica strain T6c;Shewanella pealeana strain ATCC700345;Legionella pneumophila pneumophila strain Paris; Actinobacillus succinogenes strain 130Z; Pasteurella multocida; Francisella tularensis novicida strain U112; Francisella tularensis subsp. holarctica; Francisella tularensis strain FSC198; Francisella tularensis; Francisella tularensis strain WY96-3418; and Treponema denticola strain ATCC35405. Thus, aspects of the present disclosure relate to Cas9 proteins present in type II CRISPR systems.;

[0022] The Cas9 protein is sometimes referred to in the literature by those skilled in the art as Csn1. The sequence of the Cas9 protein from S. pyogenes, which is the subject of the experiments described herein, is shown below. See Deltcheva et al., Nature 471, 602-607 (2011), which is incorporated by reference in its entirety.

[0023] [ka]

[0024] In some embodiments, the RNA-guided DNA-binding proteins include homologs and orthologs of Cas9 that retain the ability to bind to DNA and cleave DNA guided by RNA. In some embodiments, the Cas9 proteins include sequences described for native Cas9 from S. pyogenes and protein sequences that have at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% homology thereto and are DNA-binding proteins, such as RNA-guided DNA-binding proteins.

[0025] According to one embodiment, a modified Cas9-gRNA system is provided that allows site-specific RNA-guided genome cleavage as required and allows genome modification by insertion of exogenous donor nucleic acid. The guide RNA is complementary to a target site or locus on DNA. The guide RNA may be a crRNA-tracrRNA chimera. Cas9 binds to or near the target genomic DNA. One or more guide RNAs bind to or near the target genomic DNA. Cas9 cleaves the target genomic DNA and exogenous donor DNA is inserted into the DNA at the cleavage site.

[0026] Thus, the method relates to the use of guide RNA together with Cas9 protein and exogenous donor nucleic acid to perform multiple insertion of exogenous donor nucleic acid into DNA in Cas9 expressing cells by repeating the insertion of RNA encoding nucleic acid and exogenous donor nucleic acid, expression of RNA, colocalization of RNA, Cas9 and DNA to cleave DNA, and insertion of exogenous donor nucleic acid.The method steps can be repeated as many times as desired to produce any number of DNA modifications.Thus, the method of the present disclosure relates to the editing of targeted genes using Cas9 protein and guide RNA described herein to produce multiple genetic and epigenetic modifications of cells.

[0027] Another aspect of the present disclosure relates to the use of DNA-binding proteins or DNA-binding systems in general for multiple insertions of exogenous donor nucleic acids into DNA, such as genomic DNA, of cells, such as human cells. Those of skill in the art will be able to readily identify exemplary DNA-binding systems based on the present disclosure.

[0028] The cells of the present disclosure include any cell into which foreign nucleic acid can be introduced and expressed as described herein. It should be understood that the basic concepts of the present disclosure described herein are not limited by cell type. The cells of the present disclosure include eukaryotic cells, prokaryotic cells, animal cells, plant cells, fungal cells, archaeal cells, eubacterial cells, and the like. The cells include eukaryotic cells such as yeast cells, plant cells, and animal cells. Specific cells include mammalian cells, such as human cells. Additionally, the cells include any cell in which DNA modification is beneficial or desirable.

[0029] Target nucleic acids include any nucleic acid sequence that the colocalization complex described herein may be useful for either nicking or cleavage. Target nucleic acids include genes. For purposes of this disclosure, DNA, such as double-stranded DNA, may comprise the target nucleic acid, and the colocalization complex may bind to or otherwise colocalize with the DNA at, adjacent to, or near the target nucleic acid, such that the colocalization complex may have a desired effect on the target nucleic acid. Such target nucleic acids may include endogenous (or natural) and exogenous (or foreign) nucleic acids. Based on this disclosure, one skilled in the art can easily identify or design guide RNAs and Cas9 proteins that colocalize with DNA that comprises the target nucleic acid. One skilled in the art can further identify transcriptional regulator proteins or transcriptional regulatory regions that similarly colocalize with DNA that comprises the target nucleic acid. DNA includes genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA. According to certain aspects, materials and methods useful for practicing the present disclosure include those described in Di Carlo, et al., Nucleic Acids Research, 2013, vol. 41, No. 7 4336-4343, which is incorporated by reference in its entirety for all purposes, including exemplary strains and media, plasmid construction, transformation of plasmids, electroporation of transient gRNA cassettes and donor nucleic acids, transformation of Cas9-expressing cells with gRNA plasmids carrying donor DNA, galactose induction of Cas9, identification of CRISPR-Cas targets in the yeast genome, etc.Additional references containing information, materials, and methods useful to one of skill in the art in practicing the present invention include Mali, P., Yang, L., Esvelt, KM, Aach, J., Guell, M., DiCarlo, JE, Norville, JE and Church, GM (2013) RNA-Guided human genome engineering via Cas9. Science, 10.1126fscience. l232033; Storici, F., Durham, CL, Gordenin, DA and Resnick, MA (2003) Chromosomal site-specific double-strand breaks are efficiently targeted for repair by oligonucleotides in yeast. PNAS, 100, 14994-14999; and Jinek, M., Chylinski, K., Fonfara, l., Hauer, M., Doudna, JA and Charpentier, E. (2012) A, each of which is incorporated by reference in its entirety for all purposes. Programmable dual-RNA-Guided DNA endonuclease in adaptive bacterial immunity. Science, 337, 816-821.

[0030] Exogenous nucleic acid (i.e., nucleic acid that is not part of the cell's natural nucleic acid composition) may be introduced into a cell using any method of introduction known to those of skill in the art. Such methods include gene transfer, transduction, viral transduction, microinjection, lipofection, nucleofection, nanoparticle bombardment, transformation, conjugation, and the like. Those of skill in the art will readily understand and apply such methods using readily identifiable literature sources.

[0031] The following examples are provided as representative of the present disclosure, and should not be construed as limiting the scope of the disclosure, as these and other equivalent embodiments will become apparent in light of the present disclosure, the drawings, and the appended claims. EXAMPLES

[0032] Example I A general process for multiple gene editing using CRISPR-Cas9 in yeast Cas9, derived from the CRISPR immune system of Streptococcus pyogenes, is used to promote homologous recombination and select against cells that do not recombine transformed DNA in Saccharomyces cerevisiae. A general method for RNA-guided DNA cleavage using Cas9 is shown in Figure 1. A colocalized complex is formed between Cas9, guide RNA, and target DNA. Cas9 creates a double-stranded break in the target DNA. Donor DNA is then inserted into the DNA by homologous recombination. The donor DNA contains flanking sequences on either side of the cleavage site, as well as a sequence that removes the Cas9 cleavage site. As a result, the donor DNA is integrated into DNA, which may be genomic DNA.

[0033] A general method of high frequency donor DNA recombination by multiple DNA modifications in yeast using Cas9 is described below with reference to FIG. 2. Cells that do not have a native RNA-guided endonuclease Cas9 may be transformed with DNA that allows the cells to express the RNA-guided endonuclease Cas9. Cells that express the RNA-guided endonuclease Cas9 are grown. For the introduction and expression of one or more guide RNAs into cells, a plasmid is created that contains one or more nucleic acids that code for one or more guide RNAs and a selectable marker known to those skilled in the art. As shown in FIG. 2, a pool of plasmids is shown, each of which contains a nucleic acid that codes for a guide RNA that is used to insert different genes, namely gene A, gene B, gene C, gene D, and gene E, into the genomic DNA of a cell. A pool of donor DNA is also provided that contains double-stranded donor DNA of gene A, gene B, gene C, gene D, and gene E.

[0034] The cells are washed and conditioned with lithium acetate. The cells may be further washed, and a pool of exogenous donor nucleic acids, such as DNA cassettes, double-stranded oligonucleotides, and a plasmid containing nucleic acid encoding guide RNA may be mixed with the cells. As shown in Figure 2, the cells are transformed with exogenous donor nucleic acids and plasmids using PEG3350 and lithium acetate.

[0035] As shown in FIG. 2, the cells are selected for one or more guide RNAs using a selection marker. The selected cells express one or more guide RNAs. One or more co-localized complexes of the guide RNA, the RNA-guided endonuclease Cas9, and DNA are formed in the cells. The endonuclease cleaves the DNA and the donor nucleic acid is inserted into the cells by recombination, such as homologous recombination. The cells are then cured of the plasmid, and the above steps are repeated once or more as necessary. Multiple cycles may be performed. Cells that have undergone multiple cycles show a high recombination frequency. Alternatively, the cells are deselected for plasmid maintenance or placed in a medium to select for non-plasmid-bearing cells. The process is then repeated, beginning with the cell growth step. Thus, the method includes repeating cells that have already been modified in the previous cycle, or selecting cells that have not been modified from the previous cycle, and further repeating the unmodified cells to modify the DNA as described herein.

[0036] Example II Detailed repeat protocol (Uracil auxotroph, constitutive Cas9 expression) Grow cells in 5 ml of SC yeast medium or SC+FOA (100 μg / ml) to an optical density of 0.8-1.0. Spin cells at 2250 x g for 3 min and wash once with 10 ml of water. Spin cells and resuspend in 1 ml of 100 mM lithium acetate. Pellet cells and resuspend in 500 μl of 100 mM lithium acetate. Prepare transformation mixture by adding, in that order: 50 μl of cells; a DNA mixture containing 1 nmol of double-stranded oligonucleotide pool, 5 μg of each guide RNA (p426 vector with uracil marker), and water added to the desired final volume of 70 μl; 240 μl of 50% PEG3350; and 36 μl of 1 M lithium acetate. Incubate the mixture at 30 °C for 30 min. Then, vortex the mixture and heat shock the cells by incubating the mixture at 42 °C for 20 min. The cells are then pelleted and the supernatant removed. The cells are plated in 5 ml of SC-uracil to select for gRNA plasmids containing the uracil gene. The cells are allowed to recover for 2 days. After 2 days, 100 μl of cell culture is plated in 5 ml of freshly prepared SC and grown for 12 hours to non-select for plasmid maintenance. 100 μl of SC cultured cells are then plated in 5 ml of SC+FOA (100 μg / mL) medium to select for non-plasmid-carrying cells. This completes one cycle of the process. This process is repeated for the desired number of cycles. The entire process may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, etc.

[0037] Example III Thermotolerance to heat shock in selected mutants Using the methods described herein, thermotolerance to heat shock in selected mutants was demonstrated. Genes that have been shown to increase yeast thermotolerance by knockout or point mutation were targeted by the guide RNA-Cas9 system described herein. Four genes were selected for mutation: UBC1, SCH9, TFS1, and RAS2. SCH9 is a protein kinase that controls osmostress, nutrient, and environmental stress genes. TFS1 inhibits carboxypeptidase Y and Ira2p, inhibits Ras GAP activity, and responds to DNA replication stress. RAS2 is a GTP-binding protein that controls nitrogen starvation and is involved in stress response pathways. For each of SCH9, TFS1, and RAS2, donor DNA was created that contains an allele containing a serine to alanine mutation in the coding region. UBC1-E2 is a ubiquitin-conjugating enzyme. Donor DNA was created that contains a point mutation that removes the phosphorylation site, resulting in thermotolerance.

[0038] Using the methods described herein, genes were targeted using guide RNAs designed to guide Cas9 to cleave at gene loci along with altered double-stranded oligonucleotides. As shown in FIG. 3, allelic replacement was achieved using oligonucleotides targeting four loci responsible for thermotolerance in yeast. Four types of plasmids were created according to the schematic, each incorporating a nucleic acid encoding a guide RNA for one of the genes: UBC1 gRNA plasmid, TFS1 gRNA plasmid, SCH9 gRNA plasmid, and RAS2 gRNA plasmid. Each plasmid had a corresponding double-stranded donor oligonucleotide: ubc1(S97A) double-stranded oligonucleotide, tfs1(tag) double-stranded oligonucleotide, sch9(tag) double-stranded oligonucleotide, and ras(tag) double-stranded oligonucleotide. Yeast was co-transformed with a pool of plasmids and their corresponding double-stranded donor oligonucleotides. Two cycles were performed, and the number of modifications per cell as a function of the percentage of cells in the cell population is shown in FIG. 4. A large number of cells contained one and two modifications after two cycles. One triple mutant could be isolated (data not shown).

[0039] Figure 5A is a table of strains resulting from the methods described herein showing strains transformed with one donor oligonucleotide, two donor oligonucleotides, and three donor oligonucleotides. Figure 5B shows the effect of incubation at 42°C for 3 hours compared to no incubation, and a slight reduction in the number of wild type cells. Figure 5B also shows the effect of incubation at 55°C for 2 hours compared to no incubation. The mutants most resistant to heat shock at 55°C were sch9, sch9 tfs1, and tfs1 ubc1(s97a).

[0040] Figure 6 generally provides graphical information regarding the optimization of multiple oligonucleotide incorporation for two loci. Figure 6A shows the frequency of transformation versus the amount (μg) of each plasmid used for transformation. Figure 6B shows the individual recombination frequency versus the amount (μg) of each plasmid used for transformation. Figure 6C shows the frequency of co-recombination at the can1 and KanMX loci versus the amount (μg) of each plasmid used for transformation.

[0041] Figure 7 generally provides graphical information regarding multiple linear cassette integration at two loci. The graph shows the transformation frequency of the p426 gRNA ADE2+HygR cassette in the leftmost bar, the next bar the transformation frequency of the p426 gRNA CAN1+G418R cassette, and the next three bars the transformation frequency of the p426 gRNA+ADE2 p426 gRNA CAN1+HygR cassette+G418R cassette.

[0042] FIG. 8 generally depicts an analysis of growth rates showing doubling times during logarithmic growth at elevated temperatures for selected mutants. FIG. 8A graphs the fold change in doubling time at 30° C. for wild type and identified mutants. FIG. 8B graphs the fold change in doubling time at 37° C. for wild type and identified mutants. FIG. 8C graphs the fold change in doubling time at 42° C. for wild type and identified mutants inoculated from late stationary phase cultures. FIG. 8D graphs the fold change in doubling time at 42° C. for wild type and identified mutants inoculated from late logarithmic growth cultures. The graphical data shows a shorter doubling time at 37° C. in sch9 tfs1 and tfs1 ubc1(S97A). Graphical data show that doubling times are shortened at 42°C in ras2 tfs1, sch9 ubc1(S97A), tfs1 ubc1(S97A), and ras2 tfs1 ubc1(S97A). Examples of aspects of the present disclosure are given below. <1> 1. A method for producing multiple changes in a target DNA in a cell expressing an enzyme that forms a colocalized complex with an RNA complementary to the target DNA and that site-specifically cleaves the target DNA, comprising: (a) introducing into the cell a first exogenous nucleic acid encoding one or more RNAs that are complementary to a target DNA and that guide the enzyme to the target DNA, wherein the one or more RNAs and the enzyme are components of a co-localized complex to the target DNA; introducing into the cell a second exogenous nucleic acid encoding one or more donor nucleic acid sequences, wherein the one or more RNAs and the one or more donor nucleic acid sequences are expressed, the one or more RNAs and the enzyme co-localize to the target DNA, the enzyme cleaves the target DNA, and the donor nucleic acid is inserted into the target DNA to generate an altered DNA in the cell; and The method comprises repeating step (a) a plurality of times to produce a plurality of alterations in said DNA in said cell. <2> The enzyme is an RNA-guided DNA-binding protein. <1> The method according to <3> wherein the enzyme is Cas9; <1> The method according to <4> the cell is a eukaryotic cell, <1> The method according to <5> The cell is a yeast cell, a plant cell, or an animal cell. <1> The method according to <6> The RNA is about 10 to about 500 nucleotides. <1> The method according to <7> The RNA is about 20 to about 100 nucleotides. <1> The method according to <8> The one or more types of RNA are guide RNAs; <1> The method according to <9> the one or more RNAs are tracrRNA-crRNA fusions; <1> The method according to <10> The DNA is genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA; <1> The method according to <11> the one or more donor nucleic acid sequences are inserted by recombination; <1> The method according to <12> the one or more donor nucleic acid sequences are inserted by homologous recombination; <1> The method according to <13> the one or more RNAs and the one or more donor nucleic acid sequences are present on one or more plasmids; <1> The method according to

Claims

1. A eukaryotic cell expressing a Cas9 protein that forms a colocalized complex with a guide RNA complementary to a target DNA and site-specifically cleaves the target DNA, (a) a plurality of guide RNAs complementary to different sites of DNA in the eukaryotic cell; and (b) a plurality of exogenous donor nucleic acid sequences for insertion into DNA in the eukaryotic cell to modify the DNA; The eukaryotic cell comprising:

2. The eukaryotic cell of claim 1 , wherein the eukaryotic cell comprises multiple exogenous donor nucleic acid sequence insertions into the DNA within the eukaryotic cell.

3. The eukaryotic cell of claim 1 , wherein the eukaryotic cell is a yeast cell, a plant cell, or an animal cell.

4. 2. The eukaryotic cell of claim 1, wherein each guide RNA in the plurality of guide RNAs is 10-500 nucleotides.

5. 2. The eukaryotic cell of claim 1, wherein each guide RNA in the plurality of guide RNAs is 20-100 nucleotides.

6. 2. The eukaryotic cell of claim 1, wherein each guide RNA in the plurality of guide RNAs is a tracrRNA-crRNA fusion.

7. The eukaryotic cell of claim 1 , wherein the DNA is genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA.

8. 2. The eukaryotic cell of claim 1, wherein each guide RNA in the plurality of guide RNAs is present on a plasmid.

9. 2. The eukaryotic cell of claim 1, wherein each guide RNA in the plurality of guide RNAs and each exogenous donor nucleic acid sequence in the plurality of exogenous donor nucleic acid sequences are present on a plasmid.

10. The eukaryotic cell of claim 1 , wherein each exogenous donor nucleic acid sequence in the plurality of exogenous donor nucleic acid sequences comprises a homologous sequence or arm flanking a cleavage site.

11. The eukaryotic cell of claim 1 , wherein each exogenous donor nucleic acid sequence in the plurality of exogenous donor nucleic acid sequences comprises a sequence for removing a cleavage site.

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