Multiple RNA-induced genome editing

Abstract

To iterate or repeat steps of DNA modification on a cell to create a cell having multiple DNA modifications in the cell.SOLUTION: Methods of multiplex genome engineering in a cell using Cas9 is provided which include a cycle of a step of introducing into the cell a first foreign nucleic acid encoding one or more RNAs which are complementary to a target DNA and which guide an enzyme to the target DNA, where the one or more RNAs and the enzyme are constituents of a co-localization complex for the target DNA, and a step of introducing into the cell a second foreign nucleic acid encoding one or more donor nucleic acid sequences, where the cycle is repeated a desired number of times to perform multiplex DNA modification in the cell.SELECTED DRAWING: None

Description

Technical Field

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

[0002] Statement of Government Interest This invention was made with government support under Grant Number DE-FG02-02ER63445 from the U.S. Department of Energy, Grant Number NSF-SynBERC from the National Science Foundation, and Grant Number SA5283-11210 from the National Science Foundation. The United States Government has certain rights in this invention.

Background Art

[0003] The CRISPR-Cas system in bacteria and archaea relies on short-chain guide RNA, which forms a complex with the Cas protein, 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, See 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).In recent years, in vitro rearrangement of the type II CRISPR system in Streptococcus pyogenes has demonstrated that crRNA ("CRISPR RNA") fused with tracrRNA ("trans-activated CRISPR RNA"), which is normally trans-encoded, is sufficient to cause the Cas9 protein to sequence-specifically cleave a target DNA sequence matching that crRNA. Expression of a gRNA homologous to the target site recruits Cas9, leading to degradation of the target DNA. See H. Deveau et al, Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. Journal of Bacteriology 190, 1390 (Feb, 2008). [Overview of the project] [Problems that the invention aims to solve]

[0004] Aspects of this disclosure relate to multiplex modification of DNA in cells, using one or more guide RNAs (ribonucleic acid) to guide a nuclease-active enzyme expressed by a cell, such as a DNA-binding protein with nuclease activity, to a target site on DNA (deoxyribonucleic acid). In this modification, the DNA is cleaved by the enzyme, and an exogenous donor nucleic acid is inserted into this DNA by homologous recombination or the like. Aspects of this disclosure include creating cells with multiple DNA modifications by repeating or iterating the DNA modification steps for each cell. Modifications may include the insertion of exogenous donor nucleic acid. [Means for solving the problem]

[0005] Multiple insertion of exogenous nucleic acids can be achieved in a single step of introducing nucleic acids encoding multiple RNAs and multiple exogenous donor nucleic acids into enzyme-expressing cells, such as by simultaneous transformation. In this step, RNA is expressed, the enzyme is guided to a specific site on the DNA by each of the multiple types of RNA, the DNA is cleaved by the enzyme, and one of the multiple types of exogenous nucleic acids is inserted into this cleavage site. According to this embodiment, many changes or modifications occur in the DNA within the cell in a single cycle.

[0006] Multiple insertions of exogenous nucleic acids can be achieved in cells by a repetitive step or cycle of introducing one or more types of nucleic acids encoding one or more types of RNA, and one or more types of exogenous nucleic acids, into an enzyme-expressing cell. In this step or cycle, the RNA is expressed, the enzyme is guided to a specific site on the DNA, the DNA is cleaved by the enzyme, and the exogenous nucleic acid is inserted into this cleavage site, resulting in a cell having multiple alterations or exogenous DNA insertions in the cell's DNA. In one embodiment, the enzyme-expressing cell may be a cell that naturally expresses the enzyme, or it may be a cell that has been genetically modified to express the enzyme, for example, by introducing a nucleic acid that encodes the enzyme and can be expressed by the cell. Thus, embodiments of the present disclosure include a repetition of the steps of introducing RNA into an enzyme-expressing cell, introducing an exogenous donor nucleic acid into this cell, the RNA being expressed, a colocalization complex of RNA, enzyme, and DNA being formed, the DNA being enzymatically cleaved by the enzyme, and the donor nucleic acid being inserted into this DNA. Repeating or iterating the above steps results in multiple genetic modifications in cells at multiple loci, i.e., cells with multiple genetic changes are obtained.

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

[0008] In one embodiment, the DNA-binding proteins or enzymes included in the scope of this disclosure include proteins that form a complex with a guide RNA, which guides the complex to a double-stranded DNA sequence, where the complex binds to the DNA sequence. In one embodiment, the enzyme may be an RNA-guided DNA-binding protein, such as an RNA-guided DNA-binding protein of a type II CRISPR system that binds to DNA and is guided by RNA. In one embodiment, the RNA-guided DNA-binding protein is the Cas9 protein.

[0009] This aspect of the present disclosure may 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, cells with multiple genetic modifications, such as multiple insertions of exogenous donor DNA, can be created by cleaving multiple sites of double-stranded DNA using a DNA-binding protein-guide RNA complex.

[0010] According to one embodiment, a method is provided for causing multiple changes in target DNA in a cell that expresses an enzyme that forms a co-localization complex with RNA complementary to the target DNA and site-specifically cleaves the target DNA. This method includes the steps of (a) introducing into a cell a first foreign nucleic acid encoding one or more types of RNA complementary to the target DNA and guiding the enzyme to the target DNA, wherein the one or more types of RNA and the enzyme are components of a co-localization complex to the target DNA, wherein the one or more types of RNA and the one or more types of donor nucleic acid sequences are expressed, the one or more types of RNA and the enzyme co-localize to the target DNA, the target DNA is cleaved by the enzyme, the donor nucleic acid is inserted into the target DNA, and altered DNA is generated in the cell, and repeating step (a) multiple times to cause multiple changes in the DNA in the cell.

[0011] In one embodiment, the cell is a eukaryotic cell. In another embodiment, the cell is a yeast cell, a plant cell, or an animal cell. In another embodiment, the cell is a mammalian cell.

[0012] According to one embodiment, RNA consists of approximately 10 to 500 nucleotides. According to another embodiment, RNA consists of approximately 20 to 100 nucleotides.

[0013] According to one embodiment, one or more types of RNA are guide RNAs. According to another embodiment, one or more types of RNA are tracrRNA-crRNA fusions.

[0014] In some embodiments, DNA may be genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA.

[0015] Other features and advantages of specific embodiments of this embodiment will become more readily apparent in the following description of the embodiments and their drawings, and from the claims.

[0016] The above and other features and advantages of this embodiment will be better understood from the following detailed description of exemplary embodiments in conjunction with the accompanying drawings. [Brief explanation of the drawing]

[0017] [Figure 1] This is a schematic diagram of RNA-induced genome cleavage mediated by Cas9. [Figure 2] This is a schematic diagram illustrating multiple genome modification in yeast using Cas9. [Figure 3] This is a schematic diagram illustrating allele substitution using oligonucleotides that target four loci important for heat tolerance in yeast. [Figure 4] This graph shows the number of modifications per cell after one and two cycles. [Figure 5A] This is a table of strains that have mutations. [Figure 5B] This figure shows the heat resistance to heat shock for various strains. [Figure 6A] This figure shows graph data regarding transformation frequency. [Figure 6B] The graph shows the recombination frequency data for each individual. [Figure 6C] This figure shows graph data on the frequency of simultaneous recombination in the can1 and KanMX seating positions. [Figure 7] This figure shows graph data regarding the acquisition of multi-linear cassettes for two seated positions. [Figure 8A] This figure shows graph data regarding the rate of change in doubling time at 30°C. [Figure 8B] This figure shows graph data regarding the rate of change in doubling time at 37°C. [Figure 8C]Fig. 1 is a graph showing the fold change in doubling time at 42°C when cells were seeded from a culture in late stationary phase. [Figure 8D] Fig. 2 is a graph showing the fold change in doubling time at 42°C when cells were seeded from a culture in late logarithmic growth phase. DETAILED DESCRIPTION OF EMBODIMENTS

[0018] Embodiments of the present disclosure are based on the repeated use of exogenous DNA, a nuclease enzyme such as a DNA binding protein, and a guide RNA to co-localize to DNA, digest or cleave DNA, and insert exogenous DNA by homologous recombination or the like. Those skilled in the art will readily appreciate such DNA binding proteins that bind to DNA for a variety of purposes. Such DNA binding proteins may be naturally occurring. DNA binding proteins included within the scope of the present disclosure include those that can be guided by an RNA, referred to herein as a guide RNA. According to this aspect, the guide RNA and the RNA-guided DNA binding protein form a co-localized complex at DNA. Such DNA binding proteins having nuclease activity are known to those skilled in the art, and include naturally occurring DNA binding proteins having nuclease activity such as the Cas9 protein present in, for example, a type II CRISPR system. Such Cas9 proteins and type II CRISPR systems are well established 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 possessing nuclease activity function to form nicks in double-stranded DNA or to cleave double-stranded DNA. Such nuclease activity can arise from a DNA-binding protein having one or more polypeptide sequences exhibiting nuclease activity. Such an exemplary DNA-binding protein may have two distinct nuclease domains, each involved in cleaving a specific strand of double-stranded DNA or forming a nick. Exemplary polypeptide sequences possessing nuclease activity known to those skilled in the art include the McrA-HNH nuclease-associated domain and the RuvC-like nuclease domain. Thus, an exemplary DNA-binding protein is a native protein containing one or more of the McrA-HNH nuclease-associated domain and the RuvC-like nuclease domain.

[0020] An example of a DNA-binding protein is the RNA-induced DNA-binding protein of the type II CRISPR system. Another example of a DNA-binding protein is the Cas9 protein.

[0021] In Streptococcus pyogenes, Cas9 forms a blunt-ended double-strand 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 DNA strand and a RuvC-like domain that cleaves the non-complementary strand. See Jinke et al., Science 337, 816-821 (2012), for the full details, as referenced. The Cas9 protein is known to be present in numerous type II CRISPR systems, including those confirmed in the supplementary information of Makarova et al., Nature Reviews, Microbiology, Vol. 9, June 2011, pp. 467-477. Methanococcus maripaludis C7 strain; Corynebacterium diphtheriae; Corynebacterium efficiens YS-314 strain; Corynebacterium glutamicum ATCC13032 Kitasato strain; Corynebacterium glutamicum ATCC13032 Bielefeld strain; Corynebacterium glutamicum R strain; Corynebacterium kroppenstedtii DSM44385 strain; Mycobacterium abscessus ATCC19977 strain; Nocardia farsinica Rhodococcus farcinica IFM10152 strain; Rhodococcus erythropolis PR4 strain; Rhodococcus jostii RHA1 strain; Rhodococcus opacus B4 uid36573 strain; Acidothermus cellulolyticus 11B strain;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 ochracea) DSM7271 strain; Flavobacterium psychrophilum JIP0286 strain; Akkermansia muciniphila ATCC BAA835 strain; Roseiflexus castenholzii DSM13941 strain; Roseiflexus RS1 strain; Synechocystis PCC6803 strain; Elusimicrobium minutum Pei191 strain; Uncultured bacterium Termite group 1 phylotype Rs D17; Fibrobacter succinogenes S85 strain; Bacillus cereus Lactobacillus 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 strain N2025 uid46353 strain; Streptococcus mutans; Streptococcus pyogenes M1 GAS strain; Streptococcus pyogenes MGAS5005; Streptococcus pyogenes MGAS2096 strain; Streptococcus pyogenes MGAS9429 strain; Streptococcus pyogenes MGAS10270 strain; Streptococcus pyogenes MGAS6180 strain; Streptococcus pyogenes MGAS315 strain; Streptococcus pyogenes SSI-1; Streptococcus pyogenes MGAS10750 strain; Streptococcus pyogenes NZ131 strain; Streptococcus thermophilus *Streptococcus thermophilus* CNRZ1066 strain; *Streptococcus thermophilus* LMD-9 strain; *Streptococcus thermophilus* LMG18311 strain; *Clostridium botulinum* A3 Loch Maree strain; *Clostridium botulinum* B Eklund 17B strain; *Clostridium botulinum* Ba4 657 strain; *Clostridium botulinum* F Langeland strain; *Clostridium cellulolyticum* H10 strain;Finegoldia magna ATCC29328 strain; Eubacterium rectale ATCC33656 strain; Mycoplasma gallisepticum; Mycoplasma mobile 163K strain; Mycoplasma penetrans; Mycoplasma synoviae 53 strain; Streptobacillus moniliformis DSM12112 strain; Bradyrhizobium BTAil strain; Nitrobacter hamburgensis X14 strain; Rhodopseudomonas palustris BisB 18 strains; Rhodopseudomonas palustris BisB5 strain; Parvibaculum lavamentivorans DS-1 strain; Dinoroseobacter shibae DFL12 strain; Gluconacetobacter diazotrophicus Pal5 FAPERJ strain; Gluconacetobacter diazotrophicus Pal5 JGI strain; Azospirillum B510 uid46085 strain; Rhodospirillum rubrum ATCC11170 strain; Diaphorobacter TPSY uid29975 strain; Verminephrobacter eiseniae Neisseria eiseniae) EF01-2 strain; Neisseria meningitides 053442 strain; Neisseria meningitides alpha14 strain; Neisseria meningitides Z2491 strain; Desulfovibrio salexigens DSM2638 strain;Campylobacter jejuni doylei (26997 plants); Campylobacter jejuni (81116 plants); Campylobacter jejuni; Campylobacter lari (RM2100 plants); Helicobacter hepaticus; Wolinella succinogenes; Tolumonas auensis (DSM9187 plants); Pseudoalteromonas atlantica (T6c plant); Shewanella pealeana (ATCC700345 plants); Legionella pneumophila *Pneumophila* (Paris strain); *Actinobacillus succinogenes* (130Z strain); *Pasteurella multocida*; *Francisella tularensis novicida* (U112 strain); *Francisella tularensis holarctica*; *Francisella tularensis* (FSC198 strain); *Francisella tularensis*; *Francisella tularensis* (WY96-3418 strain); and *Treponema denticola* (ATCC35405 strain). Therefore, aspects of this disclosure relate to the Cas9 protein present in the type II CRISPR system.

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

[0023] [ka]

[0024] In one embodiment, RNA-induced DNA-binding proteins include homologs and orthologues of Cas9 that bind to DNA, are guided by RNA, and possess the ability to cleave DNA. In one embodiment, Cas9 proteins include sequences described for natural Cas9 from Streptococcus pyogenes, and protein sequences that have at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% homology to those sequences and are DNA-binding proteins such as RNA-induced DNA-binding proteins.

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

[0026] Therefore, the method relates to a method for multiple insertion of exogenous donor nucleic acids into DNA in Cas9-expressing cells by using guide RNA together with the Cas9 protein and exogenous donor nucleic acid, and repeating the insertion of the RNA-encoding nucleic acid and the exogenous donor nucleic acid, RNA expression, colocalization of RNA, Cas9, and DNA such that the RNA cleaves the DNA, and insertion of the exogenous donor nucleic acid. The method steps may be repeated a desired number of times to produce any number of DNA modifications. Therefore, the method of the present disclosure relates to editing of target genes that result in multiple gene modifications and multiple epigenetic modifications of cells using the Cas9 protein and guide RNA described herein.

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

[0028] The cells relating to this disclosure include any cells that can be introduced and expressed with foreign nucleic acids as described herein. The fundamental concepts of this disclosure as described herein should be understood not to be limited by cell type. Cells relating to this disclosure include eukaryotic cells, prokaryotic cells, animal cells, plant cells, fungal cells, archaeal cells, and bacterial cells. Cells include eukaryotic cells such as yeast cells, plant cells, and animal cells. Specific examples of cells include mammalian cells such as human cells. Furthermore, cells include any cells in which DNA modification is beneficial or desirable.

[0029] Target nucleic acids include any nucleic acid sequence in which the co-localization complex described herein may be useful for either nick formation or cleavage. Examples of target nucleic acids include genes. For the purposes of this disclosure, DNA, such as double-stranded DNA, may contain the target nucleic acid, and the co-localization complex may bind to or otherwise co-localize with the DNA in, adjacent to, or near the target nucleic acid, so that the co-localization complex may exert a desired effect on the target nucleic acid. Such target nucleic acids may include endogenous (or native) nucleic acids and exogenous (or foreign) nucleic acids. Those skilled in the art can readily identify or design guide RNA and Cas9 proteins co-localizing with DNA containing the target nucleic acid based on this disclosure. Those skilled in the art can further identify transcription factor proteins or transcriptional regulatory regions that similarly co-localize with DNA containing the target nucleic acid. DNA may include genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA. In some embodiments, materials and methods useful for carrying out the present disclosure include those described in whole by reference in Di Carlo, et al., Nucleic Acids Research, 2013, vol. 41, No. 7 4336-4343, for any purpose including exemplary strains and media, plasmid construction, plasmid transformation, transient gRNA cassette and donor nucleic acid electroporation, transformation of Cas9-expressing cells with gRNA plasmids containing donor DNA, galactose induction of Cas9, and identification of CRISPR-Cas targets in the yeast genome.Other references containing information, materials, and methods useful to those skilled in the art in carrying out the present invention are, for all purposes, incorporated by reference in their entirety: 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 This is described in "Programmable dual-RNA-Guided DNA endonuclease in adaptive bacterial immunity." Science, 337, 816-821.

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

[0031] The following embodiments are described as representative examples of the present disclosure. These embodiments and other equivalent embodiments become apparent from the present disclosure, drawings, and appended claims, and should not be construed as limiting the scope of the present disclosure. [Examples]

[0032] Example I General process of multiple gene editing using CRISPR-Cas9 in yeast Using Cas9 derived from the CRISPR immune system of Streptococcus pyogenes, homologous recombination is promoted, and cells that do not recombinate with the transformed DNA are selected in Saccharomyces cerevisiae. A general method of RNA-induced DNA cleavage using Cas9 is shown in Figure 1. A colocalization complex is formed between Cas9, guide RNA, and target DNA. Cas9 induces a double-strand break in the target DNA. Next, donor DNA is inserted into the DNA by homologous recombination. The donor DNA contains adjacent sequences on both sides of the cleavage site and a sequence that removes the Cas9 cleavage site. As a result, the donor DNA is incorporated into DNA that may be genomic DNA.

[0033] A general method for high-frequency donor DNA recombination by multiple DNA modification in yeast using Cas9 is described below with reference to Figure 2. Cells lacking the naturally occurring RNA-induced endonuclease Cas9 may be transformed with DNA that enables the expression of RNA-induced endonuclease Cas9 by the cells. Cells expressing RNA-induced endonuclease Cas9 are grown. Plasmids are prepared for the introduction and expression of one or more guide RNAs into the cells, each containing one or more guide RNAs and one or more nucleic acids encoding a selection marker known to those skilled in the art. As shown in Figure 2, a pool of plasmids is shown, each containing nucleic acids encoding guide RNAs used to insert different genes, namely gene A, gene B, gene C, gene D, and gene E, into the genomic DNA of the cells. A pool of donor DNA containing double-stranded donor DNA of genes A, B, C, D, and E is also provided.

[0034] The cells are washed and acclimated with lithium acetate. The cells may be washed again and mixed with a plasmid containing a pool of exogenous donor nucleic acids, such as double-stranded oligonucleotides, and nucleic acids encoding guide RNA, for example, a DNA cassette. The cells are transformed with the exogenous donor nucleic acids and plasmid using PEG3350 and lithium acetate, as shown in Figure 2.

[0035] As shown in Figure 2, cells are selected for one or more types of guide RNA using a selection marker. The selected cells express one or more types of guide RNA. One or more colocalization complexes are formed in the cells with the guide RNA, the RNA-inducible endonuclease Cas9, and DNA. The DNA is cleaved by the endonuclease, and the donor nucleic acid is inserted into the cell by recombination, such as homologous recombination. Next, the plasmid in the cell is removed (cure), and the above steps are repeated once or further for the cell as needed. Multiple cycles may be performed. Cells that have undergone multiple cycles show a high recombination frequency. Alternatively, cells are deselected for plasmid maintenance, or cells are placed in a medium to select cells that do not have the plasmid. Then, the process starting with the cell growth step is repeated. 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 these unmodified cells to perform the DNA modification described herein.

[0036] Example II Detailed iterative protocol (Uracil trophication requirement, constitutive Cas9 expression) cells are grown in 5 ml of SC yeast medium or SC+FOA (100 μg / ml) until the optical density reaches 0.8–1.0. The cells are spun at 2250 × g for 3 minutes and washed once with 10 ml of water. The cells are spun and resuspended in 1 ml of 100 mM lithium acetate. The cells are pelleted and resuspended in 500 μl of 100 mM lithium acetate. A transformation mixture is prepared by adding 50 μl of cells; a DNA mixture containing 1 nmol of double-stranded oligonucleotide pool, each containing 5 μg of guide RNA (p426 vector with uracil marker), with water added to 70 μl to reach the desired final volume; 240 μl of 50% PEG3350; and 36 μl of 1 M lithium acetate in this order. The mixture is incubated at 30°C for 30 minutes. The cells are then subjected to heat shock by vortexing the mixture and incubating the mixture at 42°C for 20 minutes. Next, the cells are pelleted and the supernatant is removed. The cells are seeded in 5 ml of SC-uracil to select the gRNA plasmid containing the uracil gene. The cells are allowed to recover for 2 days. After 2 days, 100 μl of the cell culture is seeded in 5 ml of freshly prepared SC and grown for 12 hours to deselect for plasmid retention. Next, 100 μl of the SC cultured cells are seeded in 5 ml of SC+FOA (100 μg / mL) medium to select cells that do not contain the plasmid. This completes one cycle of the process. This process is repeated for the desired number of cycles. The entire process may consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 cycles, etc.

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

[0038] Using the method described herein, genes were targeted with a guide RNA designed to induce cleavage at Cas9 of the gene locus, along with a double-stranded oligonucleotide that conferred the alteration. Allele substitution was achieved using oligonucleotides targeting four loci responsible for heat tolerance in yeast, as shown in Figure 3. Following the schematic diagram, four plasmids were created, 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 contained a corresponding double-stranded donor oligonucleotide, namely 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 using a pool of plasmids and their corresponding double-stranded donor oligonucleotides. Two cycles were performed, and the number of alterations per cell as a function of the proportion of cells in the cell population is shown in Figure 4. Numerous cells contained one and two alterations after two cycles. We were able to isolate one triple mutant (data not published).

[0039] Figure 5A is a table of strains obtained from the method described herein, showing strains transformed with one donor oligonucleotide, strains transformed with two donor oligonucleotides, and strains transformed with three donor oligonucleotides. Figure 5B shows the effect of incubation at 42°C for 3 hours compared to no incubation, and a slight decrease 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 that were 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 relative to the amount (μg) of each plasmid used for transformation. Figure 6B shows the individual recombination frequencies relative to the amount (μg) of each plasmid used for transformation. Figure 6C shows the frequency of co-recombination at the can1 and KanMX loci relative to the amount (μg) of each plasmid used for transformation.

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

[0042] Figure 8 generally shows an analysis of growth rates, indicating doubling time during logarithmic growth at high temperatures, for selected mutants. Figure 8A graphs the multiplier change in doubling time at 30°C for the wild type and identified mutants. Figure 8B graphs the multiplier change in doubling time at 37°C for the wild type and identified mutants. Figure 8C graphs the multiplier change in doubling time at 42°C for the wild type and mutants identified as seeded from late-stationary-stage cultures. Figure 8D graphs the multiplier change in doubling time at 42°C for the wild type and mutants identified as seeded from late-stage logarithmic growth cultures. The graph data shows that the doubling time is shorter at 37°C in sch9 tfs1 and tfs1 ubc1 (S97A). The graph data shows that the doubling time is shorter at 42°C for ras2 tfs1, sch9 ubc1(S97A), tfs1 ubc1(S97A), and ras2 tfs1 ubc1(S97A). Examples of the aspects related to this disclosure are given below. <1> A method for inducing multiple changes in target DNA in cells that express an enzyme that forms a co-localization complex with RNA complementary to the target DNA and site-specifically cleaves the target DNA, (a) Introducing into the cell a first foreign nucleic acid encoding one or more types of RNA that is complementary to the target DNA and guides the enzyme to the target DNA, wherein the one or more types of RNA and the enzyme are components of a colocalization complex to the target DNA. Introducing a second foreign nucleic acid encoding one or more types of donor nucleic acid sequences into the cells, wherein the sequences of the one or more types of RNA and the one or more types of donor nucleic acids are expressed, the one or more types of RNA and the enzyme co-localize to the target DNA, the target DNA is cleaved by the enzyme, the donor nucleic acid is inserted into the target DNA, and altered DNA is generated in the cells, and A method comprising repeating step (a) multiple times to cause multiple changes in the DNA in the cells. <2> The enzyme is an RNA-induced DNA-binding protein. <1> Methods used. <3> The enzyme is Cas9. <1> Methods used. <4> The aforementioned cells are eukaryotic cells. <1> Methods used. <5> The aforementioned cells are yeast cells, plant cells, or animal cells. <1> Methods used. <6> The RNA in question is approximately 10 to 500 nucleotides long. <1> Methods used. <7> The RNA in question is approximately 20 to 100 nucleotides long. <1> Methods used. <8> The aforementioned one or more types of RNA are guide RNAs. <1> Methods used. <9> The aforementioned one or more types of RNA are tracrRNA-crRNA fusions. <1> Methods used. <10> The DNA in question is genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA. <1> Methods used. <11> The aforementioned one or more types of donor nucleic acid sequences are inserted by recombination. <1> Methods used. <12> The aforementioned one or more types of donor nucleic acid sequences are inserted by homologous recombination. <1> Methods used. <13> The aforementioned one or more types of RNA and the aforementioned one or more types of donor nucleic acid sequences are present on one or more types of plasmids. <1> Methods used.

Claims

1. A cell expressing a Cas9 protein that forms a co-localization complex with a guide RNA complementary to the target DNA and site-specifically cleaves the target DNA, (a) Multiple guide RNAs complementary to different sites of the target DNA within the cell, and (b) Multiple exogenous donor nucleic acid sequences The cells, including the cells.

2. The cell according to claim 1, wherein the cell includes the insertion of a plurality of exogenous donor nucleic acid sequences into the target DNA within the cell.

3. The cell according to claim 1, wherein each of the guide RNAs in the plurality of guide RNAs is 10 to 500 nucleotides.

4. The cell according to claim 1, wherein each of the guide RNAs in the plurality of guide RNAs is 20 to 100 nucleotides.

5. The cell according to claim 1, wherein each of the guide RNAs in the plurality of guide RNAs is a tracrRNA-crRNA fusion.

6. The cell according to claim 1, wherein the target DNA is genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA.

7. The cell according to claim 1, wherein each of the guide RNAs in the plurality of guide RNAs is located on a plasmid.

8. The cell according to claim 1, wherein each of the guide RNAs in the plurality of guide RNAs and each of the exogenous donor nucleic acid sequences in the plurality of exogenous donor nucleic acid sequences are present on the plasmid.

9. The cell according to claim 1, wherein one of the exogenous donor nucleic acid sequences in the plurality of exogenous donor nucleic acid sequences includes a homologous sequence or arm adjacent to the cleavage site.

10. The cell according to claim 1, wherein one of the exogenous donor nucleic acid sequences in the plurality of exogenous donor nucleic acid sequences includes a sequence for removing a cleavage site.

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