KR102664852B1 - Vector system for light-inducible modulating of gene expression and uses thereof - Google Patents

Abstract

Pertaining to a vector system for controlling light-induced gene expression and its use, a kit for transformation of stem cells according to one aspect uses guide RNA (gRNA) for multiple target genes at the same time to transform stem cells using one vector. It is possible to produce stem cells that stably express multiple target genes simultaneously by introducing them into the cell and irradiating light. In addition, according to a method for controlling target gene expression of stem cells according to one aspect, target gene expression of stem cells is controlled by simple manipulation by irradiating or stopping light of a specific wavelength to stem cells transformed using the kit. It can be increased or decreased again. Therefore, the kit for transformation of stem cells, transformed stem cells, and methods for controlling target gene expression of stem cells can be usefully used in basic science research related to target genes and in the field of cell therapy.

Description

Vector system for light-inducible modulating of gene expression and uses thereof}

It relates to a vector system for regulating light-induced gene expression and its use.

Gene editing technology using the CRISPR (Clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated protein) system is widely used in basic science and the medical industry, including gene therapy and the production of new transformed organisms. The Cas protein binds to guide RNA (gRNA) and effectively cuts the target gene, causing DNA double-strand breaks. The cut DNA is largely divided into non-homologous end joining (NHEJ) or homologous recombination. It is repaired through two pathways: recombination (HR), where target-specific mutations and genetic modifications occur, making targeted gene editing possible.

Recently, with technological advances, new systems for regulating genes are being created based on the CRISPR/Cas system. For example, Cas9 mutant (dead Cas9: dCas9) lacking the enzyme activity to cut DNA and gRNA are being used to Technologies that can control the expression of target genes are being developed. In addition, technology is being attempted to control gene expression by irradiation of light by combining the CRISPR/Cas system and optogenetics technology, but such research is minimal in the stem cell field.

Republic of Korea Patent Publication No. 10-2019-0093171

One aspect is to provide a kit for transformation of stem cells.

Another aspect is to provide stem cells transformed using the above transformation kit.

Another aspect is to provide a method for controlling target gene expression in the transformed stem cells.

One aspect includes a first vector comprising polynucleotides each encoding a dead Cas (dCas) protein and a first light-specific binding protein; And a second vector containing a polynucleotide each encoding a guide RNA (gRNA) of a target gene whose expression is to be controlled in stem cells and a second light-specific binding protein, for transformation of stem cells. Kits are provided.

As used herein, the term “dCas” refers to a protein lacking the enzymatic activity to cleave DNA from the Cas (CRISPR-associated protein) protein, “dead Cas”, “nuclease-deficient Cas”, or “nuclease-deactivated Cas”. Can be used interchangeably with others.

The dCas protein may include dCas9 protein. The polynucleotide encoding the dCas9 protein may include the base sequence of SEQ ID NO: 27.

As used herein, the term "light-specific binding protein" refers to a protein that forms a light-induced heterodimer or a protein that forms a light-induced homodimer, which is different from or of the same type by irradiation of light of a specific wavelength. It may refer to a protein that can combine to form a heterodimer or homodimer. For example, the light-specific binding protein may be a light-induced heterodimer forming protein, specifically, CIB1 (cryptochrome-interacting basic helix-loop-helix protein), CIBN (N-terminal fragment of CIB1), CRY2 ( cryptochrome 2), CRY2PHR (N-terminal photolyase homology domain of CRY2), PhyB (phytochrome B), PIF (phytochrome interacting factor), FKF1 (Flavin-binding, Kelch repeat, F-box 1), or GIGANTEA, It is not limited to this.

The first light-specific binding protein may include CIB1 or a fragment thereof, and the fragment of CIB1 may include, for example, CIBN. The polynucleotide encoding the CIBN may include the base sequence of SEQ ID NO: 28.

The second light-specific binding protein may include CRY2 or a fragment thereof, and the fragment of CRY2 may include, for example, CRY2PHR. The polynucleotide encoding CRY2 may include the base sequence of SEQ ID NO: 29.

The first light-specific binding protein and the second light-specific binding protein may bind to each other to form a heterodimer when irradiated with blue light, for example, light having a wavelength of about 440 to 490 nm.

The first vector may further include a promoter operably linked to a polynucleotide encoding the dCas protein and the first light-specific binding protein, respectively. For example, the first vector may further include a promoter selected from the group consisting of the Ef1α promoter, UbC promoter, and CAGG promoter. In one embodiment, the promoter may regulate transcription of a fusion protein of the dCas protein and the first light-specific binding protein.

As used herein, the term “operably linked” may mean that the gene sequence is functionally linked to a promoter sequence that initiates and mediates transcription of a polynucleotide encoding a target protein.

The first vector may include the base sequence of SEQ ID NO: 21.

The second vector may further include a polynucleotide encoding a factor that promotes transcription of the polynucleotide encoding the dCas protein. The factor that promotes transcription may be, for example, VP64 or VP16, but is not limited thereto. The polynucleotide encoding the VP64 may include the base sequence of SEQ ID NO: 30.

The second vector may further include a promoter operably linked to a polynucleotide encoding a factor that promotes transcription of the polynucleotide encoding the second light-specific binding protein and the dCas protein, respectively. For example, the second vector may further include a promoter selected from the group consisting of the Ef1α promoter, UbC promoter, and CAGG promoter. In one embodiment, the promoter may regulate transcription of a fusion protein of a second light-specific binding protein and a transcriptional promotion factor.

The second vector may further include a promoter operably linked to a polynucleotide encoding a gRNA of a target gene whose expression is to be controlled in stem cells. For example, the second vector may further include the mU6 promoter, hU6 promoter, hH1 promoter, and h7SK promoter, but is not limited thereto, and is any promoter that regulates the transcription of the polynucleotide encoding the gRNA of the target gene. may be included.

The second vector contains polynucleotides encoding gRNAs of two or more target genes and different promoters for the gRNAs of each target gene, so that stem cells transformed using the transformation kit are exposed to light irradiation. This may induce overexpression of two or more target genes at the same time.

The second vector may include any one of the base sequences of SEQ ID NOs: 22 to 26.

The first vector and the second vector may be viral vectors or non-viral vectors, for example, viral vectors such as lentivirus, retrovirus, adenovirus, herpesvirus, or avipox. It may be an avipoxvirus vector, but is not limited thereto.

The first vector and the second vector may include at least one selection marker, and the selection marker may be different from each other. In one embodiment, the first vector may contain puromycin (Puro) as a selection marker, and the second vector may contain blasticidin (BSD) as a selection marker.

As used herein, the term “stem cell” refers to an undifferentiated cell that can differentiate into various cells that make up biological tissues, and refers to a cell that is pluripotent or multipotent. You can.

The stem cells may be embryonic stem cells (ESC), adult stem cells, or induced pluripotent stem cells (iPSC). Embryonic stem cells may refer to the inner cell mass extracted from a blastocyst stage embryo just before the fertilized egg implants in the mother's uterus and cultured in vitro. Adult stem cells are undifferentiated cells that exist in extremely small amounts in each tissue of the body and can refer to cells that replace dead or damaged tissues. Induced pluripotent stem cells (iPSC) can refer to cells that induce pluripotency, like embryonic stem cells, by injecting cell differentiation-related genes into differentiated somatic cells and returning them to the cell stage before differentiation. The embryonic stem cells may be, for example, human embryonic stem cells derived from humans.

Additionally, the embryonic stem cells may be somatically cloned embryonic stem cells. As used herein, the term "somatic cell cloned embryonic stem cell" may mean "embryonic stem cell generated by somatic cell nuclear transfer (SCNT) technology. The "somatic cell nuclear transfer" refers to " It can be used interchangeably with "somatic cell nuclear transfer". The somatic cell nuclear transfer technology may refer to a technology for cloning by transplanting the nucleus of a somatic cell after removing the nucleus of an egg. For example, embryonic stem cells cloned from somatic cells are somatic cells. The somatic cell-cloned embryo may be obtained by separating the nucleus of the egg, removing the nucleus of the egg, transplanting the isolated somatic cell nucleus into the egg from which the nucleus has been removed, and culturing the nucleus to differentiate into an embryonic stem cell. Since stem cells have genes from the somatic cell nucleus, the embryonic stem cells cloned from somatic cells may be homozygous for HLA (human leukocyte antigen). , The embryonic stem cells cloned from HLA homozygous somatic cells can be used to develop cell therapeutics with less immune rejection.

There may be two or more target genes whose expression is to be controlled in the stem cells. For example, the target gene may include, but is not limited to, any one or more selected from the group consisting of INS, GCK, PCSK1, and PCSK2, and is not limited to any gene whose expression is to be controlled by irradiation of light in stem cells. May contain genes.

The INS refers to a gene encoding an insulin protein, the GCK refers to a gene encoding a glucokinase protein, and the PCSK1 refers to a gene encoding a PCSK (Proprotein Convertase Subtilisin/Kexin) type 1 protein. And, PCSK2 refers to a gene encoding PCSK type 2 protein.

The polynucleotide encoding the gRNA of the INS may include any one base sequence selected from the group consisting of the base sequences of SEQ ID NOs. 1 to 5 and their respective complementary base sequences, and encodes the gRNA of the GCK. The polynucleotide may include any one base sequence selected from the group consisting of the base sequences of SEQ ID NOs: 6 to 10 and their respective complementary base sequences, and the polynucleotide encoding the gRNA of PCSK1 has SEQ ID NO: It may include any one base sequence selected from the group consisting of base sequences 11 to 15 and complementary base sequences of each of these, and the polynucleotide encoding the gRNA of PCSK2 has the base sequence of SEQ ID NOs. 16 to 20. And it may include any one base sequence selected from the group consisting of each of these complementary base sequences.

Another aspect provides stem cells transformed using the kit for transformation of stem cells.

As used herein, the term “transfection” is also called “transfection” and may mean injecting a nucleic acid into a host cell. In one embodiment, the term “transfection” is used to refer to the injection of a nucleic acid into a host cell using a lentiviral vector. This may mean injecting .

As used herein, the term "transduction" may mean the introduction of foreign DNA into another cell through a viral vector, and in one embodiment, the virus produced by a cell transfected by a lentiviral vector This may mean processing the particles into another host cell so that the target nucleic acid is introduced into the host cell.

As used herein, the term "transformation" may mean changing the genetic properties of an organism by externally given DNA, and includes, for example, both "transfection" and "transduction". It could be a concept.

Methods for transformation using the kit for transformation of stem cells include various conventionally known methods, for example, microinjection, electroporation, particle bombardment, and using sperm. Methods such as sperm-mediated gene transfer, viral infection, and techniques using insulators or transposons can be appropriately selected and applied. In one embodiment, the transformation may be performed using a lentiviral vector. Specifically, after transfecting the lentiviral vector into a primary host cell, the produced viral particles are transferred to a stem cell, which is a secondary host cell. It may be carried out by introducing a trait into .

The transformed stem cells may have increased target gene expression when irradiated with blue light. For example, when light having a wavelength of 440 to 490 nm is irradiated to the transformed stem cells, target gene expression of the stem cells may increase.

The target gene may include, but is not limited to, any one or more selected from the group consisting of INS, GCK, PCSK1, and PCSK2, and may include all genes whose expression is to be controlled by irradiation of light in stem cells. You can.

In one embodiment, the transformed stem cells may simultaneously increase the expression of two or more target genes selected from the group consisting of INS, GCK, PCSK1, and PCSK2 by irradiation of light with a wavelength of 440 to 490 nm. .

When irradiation of light is stopped to stem cells with increased expression of the target gene, the increased expression of the target gene may decrease again.

For example, in the transformed stem cell, in the absence of light having the above wavelength, a fusion protein of the dCas protein and the first light-specific binding protein is present at the target gene site by the dCas protein that recognizes the sequence of the gRNA. When light having the above wavelength is irradiated, the fusion protein of the second photo-specific binding protein and the transcriptional promoter binds to the fusion protein containing the first photospecific binding protein, thereby targeting the target by the action of the transcriptional promoter. Gene expression may be increased. In addition, when irradiation of light with the above wavelength is stopped, the binding between the first and second light-specific binding proteins is released again, preventing the transcription promoting factor from acting on the target gene, thereby increasing the expression of the target gene. This may be decreasing again.

Another aspect provides a method of controlling target gene expression of stem cells, comprising increasing target gene expression of the stem cells by irradiating light having a wavelength of 440 to 490 nm to the transformed stem cells.

The light may be blue and have a wavelength of 440 to 490 nm, but is not limited to the wavelength range as long as it induces binding of the first light-specific binding protein and the second light-specific binding protein. .

The light may be light with pulses, for example, 10 to 90%, 20 to 80%, 30 to 70%, or 40 to 60% of pulses.

The light may be irradiated with a current of 10 to 2000 mA, 100 to 2000 mA, 100 to 1000 mA, or 300 to 1000 mA, or 500 to 800 mA, but the first light-specific binding protein and the second light If light induces binding of a specific binding protein, it is not limited to the above current range.

The light is applied to the transformed stem cells for 6 hours or more, for example, 6 to 120 hours, 10 to 100 hours, 10 to 80 hours, 10 to 50 hours, 20 to 100 hours, 20 to 80 hours, or 20 to 20 hours. The irradiation may be for 50 hours, but the time range is not limited as long as the irradiation is for a time capable of inducing binding between the first light-specific binding protein and the second light-specific binding protein.

The target gene may include, but is not limited to, any one or more selected from the group consisting of INS, GCK, PCSK1, and PCSK2, and may include all genes whose expression is to be controlled by irradiation of light in stem cells. You can.

The method of controlling the target gene expression of the stem cells includes increasing the target gene expression of the stem cells by irradiating light with a wavelength of 440 to 490 nm to the transformed stem cells, and then stopping the irradiation of the light to increase the target gene expression. It may further include a step of reducing target gene expression of the stem cells again.

A kit for transformation of stem cells according to one aspect is to simultaneously introduce gRNAs for multiple target genes into stem cells using one vector to produce stem cells that stably express multiple target genes at the same time by irradiation of light. It can be produced. In addition, according to a method for controlling target gene expression of stem cells according to one aspect, target gene expression of stem cells is controlled by simple manipulation by irradiating or stopping light of a specific wavelength to stem cells transformed using the kit. It can be increased or decreased again. Therefore, the kit for transformation of stem cells, transformed stem cells, and methods for controlling target gene expression of stem cells can be usefully used in basic science research related to target genes and in the field of cell therapy.

Figure 1 is a diagram showing the production process of the pcDNA3.1-int_Ef1α ^P -CibN-dCas9-CibN vector according to one aspect.
Figure 2 is a diagram showing the production process of the Lenti-int_Ef1α ^P -CibN-dCas9-CibN-Puro vector (first vector) according to one aspect.
Figure 3 is a diagram showing the construction process of pmU6-hINS gRNA (A), phU6-hGCK gRNA (B), phH1-hPCSK1 gRNA (C), and ph7SK-hPCSK2 (D) gRNA vectors according to one aspect.
Figure 4 is a diagram showing the production process of the pcDNA3.1-BsmBI-int_Ef1α ^P -Cry2FL-VP64 vector according to one aspect.
Figure 5 is a diagram showing the production process of the Lenti-BsmBI-int_Ef1α ^P -Cry2FL-VP64-BSD vector according to one aspect.
Figure 6 is a diagram showing the production process of the Lenti-4gRNA-int_Ef1α ^P -Cry2FL-VP64-BSD vector (second vector) according to one aspect.
Figure 7 is a diagram showing the results of electrophoresis after cutting the first and second vectors according to one aspect with the restriction enzyme SpeI.
Figure 8 is a graph showing the results of measuring the mRNA expression level of target genes in human embryonic stem cells (hES) by irradiating blue light for 24 hours (A: hINS; B: hGCK; C: hPCSK1; and D :hPCSK2).
Figure 9 is a graph showing the results of measuring the mRNA expression level of target genes by irradiating hES with blue light with a pulse of 50% for 16 or 24 hours (A: hINS; B: hGCK; C: hPCSK1; and D:hPCSK2).
Figure 10 is a graph showing the results of measuring the mRNA expression level of target genes in hES by irradiating blue light for 24 or 48 hours.
Figure 11 is a diagram showing the results of karyotype analysis of transgenic embryonic stem cells that regulate light-induced gene expression according to one aspect.
Figure 12 is a diagram showing the production process of a transformed cell that regulates light-induced gene expression according to one aspect.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are intended to illustrate one or more embodiments and the scope of the present invention is not limited to these examples.

Example 1. Construction of the first vector containing the gene sequence encoding dCas9

1-1. pcDNA3.1-int_Ef1α ^P Construction of -CibN-dCas9-CibN vector

1-1-1. int_Ef1α ^P amplification of

To amplify Ef1α ^P (hereinafter, "int_Ef1α ^P ") containing the intron A region in the lenti-EF1α-dCas9-KRAB-Puro (#99372, Addgene) vector, primers linking SpeI/SacI linkers were prepared (Table 1), After performing Herculase II PCR (agile), partial digestion was performed with restriction enzymes SpeI (New England BioLabs: NEB) and SacI (NEB), and the desired region was isolated using a DNA gel elution kit (FAVORGEN). was obtained.

SpeI tagging oligo sequence (5'->3') SacI tagging oligo sequence (5'->3') ct ACTAGT CTAGGTCTTGAAAGGAGTGG ac GAGCTC ACGTCACGACACCTGAAATG

Using XL10-Gold® Ultracompetent Cells (STRATAGENE), int_Ef1α ^P was introduced into pJET1.2/blunt (Thermo), and after mini prep (GeneAll), int_Ef1α ^P was amplified by cutting with restriction enzymes SpeI and SacI.

1-1-2. Creation of vectors

CMV P of the pcDNA3.1-CMV ^P -CibN-dCas9-CibN (#60553, Addgene) vector was removed by cutting with restriction enzymes SpeI and SacI, and T4 ligase (Thermo) was used to remove the CMV ^P from the vector (#60553, Addgene). Connect int_Ef1α ^P amplified from -1 to create pcDNA3.1-int_Ef1α ^P -CibN-dCas9-CibN A vector was constructed (Figure 1).

1-2. Lenti-int_Ef1α ^P Construction of the -CibN-dCas9-CibN-Puro vector

pcDNA3.1-int_Ef1α ^P -CibN-dCas9-CibN prepared in Example 1-1 To amplify the int_Ef1α ^P -CibN-dCas9-CibN site in the vector, primers linking the XbaI/BsrGI linker were prepared (Table 2), Herculase II PCR was performed, and restriction enzymes After cutting, the desired region was obtained using a DNA gel elution kit.

XbaI tagging oligo sequence (5'->3') BsrGI tagging oligo sequence (5'->3') G TCTAGA AGATATACGCGTTGACATT CG TGTACA AATATAATCCGTTTTCTCC

The obtained int_Ef1α ^P -CibN-dCas9-CibN site was amplified in the same manner as described in Example 1-1-1 above, except that XbaI and BsrGI were used instead of restriction enzymes SpeI and SacI.

After cutting the Lenti-Ef1α-dCas9-KRAB-Puro (#99372, Addgene) lentiviral vector with restriction enzymes NheI (NEB) and BsrGI, the int_Ef1α ^P -CibN-dCas9-CibN site amplified using T4 ligase was By linking, the Lenti-int_Ef1α ^P -CibN-dCas9-CibN-Puro vector was created as the first vector (Figure 2).

Example 2. Construction of a second vector containing a gene sequence encoding gRNA

2-1. Construction of vector containing gene sequence encoding gRNA

In order to insert the gene sequence encoding each gRNA for the target genes hINS, hGCK, hPCSK1 and hPCSK2 genes into each chimeric gRNA site of pmU6-gRNA, phU6-gRNA, phH1-gRNA and ph7SK-gRNA plasmids, as follows: Oligonucleotides were produced together.

Specifically, BbsI at the chimeric gRNA site of pmU6-gRNA (#53187, Addgene), phU6-gRNA (#53188, Addgene), phH1-gRNA (#53186, Addgene), and ph7SK-gRNA (#53189, Addgene) plasmids. The oligonucleotide pairs shown in Table 3 were designed and produced for each target gene, including the sequence of the site cut by a restriction enzyme and each of five gene sequences encoding gRNA. http://crispr-era.stanford.edu/ was used for the design and production of each oligonucleotide sequence.

target
gene set
number Forward oligonucleotide sequence
(5'->3') Reverse oligonucleotide sequence
(5'->3') hINS One TTGTTTG ACCCCAGGCCCTAATGGGCC AAAC GGCCCATTAGGGCCTGGGGT CAA 2 TTGTTTG CTAATGACCCGCTGGTCCTG AAAC CAGGACCAGCGGGTCATTAG CAA 3 TTGTTTG TGAGACTATAAAAGCCAGCGG AAAC CCGCTGGCTTTATAGTCTCA CAA 4 TTGTTTG CTCTGAGACTATAAAAGCCAG AAAC CTGGCTTTATAGTCTCAGAG CAA 5 TTGTTTG GGAAGAGGTGCTGACGACCA AAAC TGGTGCTCAGCACCTCTTCC CAA hGCK One CACCG AGAGTATTTGAGCAGCCGCC AAAC GGCGGCTGCTCAAATACTCT C 2 CACCG GCCCGCACAAGGCTGAGGAA AAAC TTCCTCAGCCTTGTGCGGGC C 3 CACCG TAGCGGTCACTGTAGTGACA AAAC TGTCACTACAGTGACGCTA C 4 CACCG AGTGGGGGATTCTCCTGCCA AAAC TGGCAGGAGAATCCCCACT C 5 CACCG GATCCTCTGCTCATCAGAAC AAAC GTTCTGATGAGCAGAGGATC C hPCSK1 One TCCCA AGCTGCTGGGACTCGCGACC AAAC GGTCGCGAGTCCCAGCAGCT T 2 TCCCA ATCTATCCCGCGACTCTCT AAAC AGAGAGTCGCGGGGATAGAT T 3 TCCCA CGCGACTCTCTAGGGAGCTC AAAC GAGCTCCCTAGAGAGTCGCG T 4 TCCCA CGCGGAGTAGCCTGGGAAGC AAAC GCTTCCCAGGCTACTCCGCG T 5 TCCCA CTGGTAGTTATGTTCAAGT AAAC ACTTGAACATAACTACCAAG T hPCSK2 One CCTCGGCCGCTGGCCTGATTACAGC AAAC GCTGTAATCAGGCCAGCGGC C 2 CCTCG TGCGCTGGCGCCCCCAGAAGG AAAC CCTTCTGGGGGCGCAGCGCA C 3 CCTCG GGCGGGCCCGGGGCGGCCCA AAAC TGGGCCGCCCCGGGCCCGCC C 4 CCTCG CCGCCCTCATTGGCCGCTGG AAAC CCAGCGGCCAATGAGGGCGG C 5 CCTCG GCCCGGGGCGGCCCAGGGCG AAAC CGCCCTGGGCCGCCCCGGGC C

In the sequences in Table 3, the underlined sequence corresponds to the sequence encoding the gRNA for each target gene, and the remaining sequence corresponds to the sequence of the site cut by the BbsI restriction enzyme in the chimeric gRNA site of each plasmid. do.

An equal amount (1:1) of each of the forward and reverse oligonucleotides for each gene in Table 3 was placed in a 1.5 ml tube for experimentation and reacted at 95°C for 5 minutes. Then, the temperature was gradually lowered to room temperature and the complementary Double strands were formed between DNA (complementary DNA: cDNA). After cutting the pmU6-gRNA, phU6-gRNA, phH1-gRNA, and ph7SK-gRNA plasmids with the restriction enzyme BbsI (Thermo scientific), pure plasmids were obtained from the PCR-amplified products using a Gel elution kit. As shown in Figure 3, the DNA double strand of each target gene gRNA was ligated to each plasmid with T4 ligase enzyme to produce pmU6-hINS gRNA, phU6-hGCK gRNA, phH1-hPCSK1 gRNA, and ph7SK-hPCSK2 gRNA plasmid, each set 1. To 5 were produced.

2-2. pcDNA3.1-BsmBI-int_Ef1α ^P -Creation of Cry2FL-VP64 vector

To amplify the BsmBI site in the pLV hUbC-VP64 dCas9 VP64-T2A-GFP (#59791, Addgene) vector, primers linking NruI/MluI linkers were prepared (Table 4), Herculase II PCR was performed, and restriction enzymes were used. After cutting with NruI (NEB) and MluI (NEB), the desired region was obtained using a DNA gel elution kit.

NruI tagging oligo sequence (5'->3') MluI tagging oligo sequence (5'->3') T TCGCGA TTACAGGGACAGCAGAGATCCAG T ACGCGT GAGGCGCCAAAACCCGGCGCGGA

The obtained BsmBI site was amplified in the same manner as described in Example 1-1-1 above, except that NruI and MluI were used instead of restriction enzymes SpeI and SacI.

In Examples 1-1-1 and 1-1-2, instead of the pcDNA3.1-CMV ^P -CibN-dCas9-CibN (#60553) vector, pcDNA3.1-CMV ^P -Cry2FL-VP64 (#60554, Addgene) Using the vector, pcDNA3.1-int_Ef1α ^P -Cry2FL-VP64 vector was constructed. After cutting the vector with restriction enzymes NruI and MluI, the amplified BsmBI site was ligated using T4 ligase to construct the pcDNA3.1-BsmBI-int_Ef1α ^P -Cry2FL-VP64 vector (Figure 4).

2-3. Lenti-BsmBI-int_Ef1α ^P - Construction of the Cry2FL-VP64-BSD vector

Primers ^linked to ^the The desired region was obtained in the same manner as described in Example 1-2, except that .

XbaI tagging oligo sequence (5'->3') BsrGI tagging oligo sequence (5'->3') G TCTAGA TTAGGGTTAGGCGTTTTGC G TGTACA AGAAGCGTAGTCGGGAACG

The obtained BsmBI-int_Ef1α ^P -Cry2FL-VP64 site was amplified in the same manner as described in Example 1-1-1 above, except that XbaI and BsrGI were used instead of restriction enzymes SpeI and SacI.

Lenti-dCas9-KRAB-BSD (#89567, Addgene) lentiviral vector was cut with restriction enzymes NheI and BsrGI, and then the amplified BsmBI-int_Ef1α ^P -Cry2FL-VP64 site was linked using T4 ligase to create Lenti-BsmBI. -int_Ef1α ^P -Cry2FL-VP64-BSD vector was constructed (Figure 5).

2-4. Lenti-4gRNA-int_Ef1α ^P - Construction of the Cry2FL-VP64-BSD vector

The gRNA plasmids having four different promoters prepared in Example 2-1 were combined into sets of the same number, and mixed with the lentiviral vector prepared in Example 2-3 at the composition and dosage shown in Table 6 below. Golden Gate assembly was performed under the reaction conditions in Table 7.

reagent Volume Lenti-BsmBI-int_Ef1αP- ^Cry2FL -VP64-BSD vector 200ng pmU6-hINS gRNA 200ng phU6-hGCK gRNA 200ng phH1-hPCSK1 gRNA 200ng ph7SK-hPCSK2 gRNA 200ng T4 DNA ligase 1ul Restriction enzyme BsmBI (Thermo scientific) 1ul T4 ligase buffer 2ul total 20ul

reaction temperature reaction time 37°C 10 minutes 16°C 15 minutes 37°C 30 minutes 80°C 5 minutes 4°C ∞

Through the above process, sets 1 to 5 of lentiviral vectors (second vectors), which allow the expression of four gRNAs with four different promoters in one vector, were finally created (FIG. 6).

Example 3. Confirmation of normal construction of the first and second vectors

In order to confirm whether the first and second vectors prepared in Examples 1 and 2, respectively, were constructed as intended, electrophoresis was performed.

Specifically, the Lenti-int_Ef1α ^P -CibN-dCas9-CibN-Puro vector (first vector) and the Lenti-4gRNA-int_Ef1α ^P - Cry2FL-VP64-BSD vector (second vector) have one common restriction enzyme, SpeI. Since the recognition sequence was present, each vector was cut with the restriction enzyme SpeI and then electrophoresed.

As a result, as shown in FIG. 7, it was confirmed that bands at positions 9,533 bp and 4,060 bp appeared in common in the second vectors of sets 1 to 5, and bands at positions 12,857 bp and 2,382 bp appeared in the first vector. Confirmed. Through the above results, it can be seen that the sum of the bp sizes of the fragments cut into two pieces from each vector corresponds to the theoretical bp size according to each vector sequence, and that each vector was constructed according to the expected sequence.

Example 4. Confirmation of target gene expression by blue light induction in human somatic cells

4-1. Production of lentivirus

A set of the first vector (Lenti-int_Ef1α ^P -CibN-dCas9-CibN-Puro) prepared in Example 1 and the second vector (Lenti-4gRNA-int_Ef1α ^P -Cry2FL-VP64-BSD) prepared in Example 2. 1 to 5, respectively, using a transfection reagent (Superfect transfection reagent, #301305, Qiagen) together with the lentivirus packaging plasmid psPAX2 (#12260, Addgene) and the lentivirus envelope plasmid pMD2.G (#12259, Addgene). and transfected into 293T cells. After 6 hours, the medium was changed to serum-free expression media, and the expression medium containing the virus was collected at 26, 38, and 50 hours after transfection, and the obtained samples were stored at -80°C. A large volume of virus was concentrated to about 250ul using an ultracentrifugal filter (Amicon Ultra-15 Centrifugal Filter, UFC910024, Merck millipore) and transduction was performed, or aliquoted and stored at -80°C.

4-2. Construction of blue light-mediated human foreskin fibroblasts and human skin fibroblasts overexpressing hINS, hGCK, hPCSK1, and hPCSK2 genes.

Human Foreskin Fibroblast (hFF) and Human Dermal Fibroblast (hDF) were transduced with the lentivirus prepared in Example 4-1, and hINS, hGCK, Cells in which overexpression of hPCSK1 and hPCSK2 genes was induced were produced.

Specifically, the medium of hFF and hDF was changed to a medium to which 10 ug/mL polybrene (TR-1003-G, Merck millipore) was added to increase transduction efficiency, and then prepared in Example 4-1. Approximately 20 to 50 ul of each int_Ef1α ^P -CibN-dCas9-CibN-Puro lentivirus concentrate was added, along with approximately 20 to 50 ul of each of the 4gRNA-int_Ef1α ^P -Cry2FL-VP64-BSD lentivirus sets 1 to 5 concentrates. . Afterwards, after treatment with the selection markers puromycin (Puro) and blasticidin (BSD), cells resistant to all of the above antibiotics were selected as cells into which all of the lentiviruses had been introduced.

4-3. Confirmation of induction of hINS, hGCK, hPCSK1 and hPCSK2 gene expression by blue light irradiation in transduced hFF

Real-Time Quantitative Reverse Transcription PCR (Real-Time Quantitative Reverse Transcription PCR: Real-Time qRT- PCR) was performed to confirm.

Specifically, after irradiating each hFF prepared in Example 4-2 with blue light at 15 mA for 12 hours, the cells were recovered, and total RNA was extracted using a kit (RNeasy Plus Mini Kit, Qiagen). cDNA was synthesized using a TOPscript ^TM RT DryMIX kit (Enzynomics). Using the synthesized cDNA as a template, real-time qRT-PCR was performed using the primers shown in Table 8 below and ^SYBR Green Master Mix (ThermoFisher).

gene Forward 5' → 3' Reverse 5' → 3' hGAPDH GAC CCC TTC ATT GAC CTC AAC TAC TTG ACG GTG CCA TGG AAT TT hINS TCA CTC TTG GTG CAA AGG AA CGG CCC ATA TAA CTG ATG TTC hGCK TAT TCC CGA AGA GCA AAG GA TGT CGT TTG TCT GAG CCT TTT hPCSK1 GCT TAA CAT CAT GAT GGA CCT AGT TTA GGA GAC TCC ATA GCA hPCSK2 TTA GAC CCC TGA TTG CTG CT CCT TGA AGG GGT TGT TGG TA

The mRNA expression level of each gene was measured and normalized based on the mRNA expression level of GAPDH, the relative mRNA expression level of each gene was calculated using the ΔΔCt method, and the mRNA expression level of the target gene was calculated relative to the wild-type hFF control group in which no gene was manipulated. The fold increase in expression level was calculated.

As a result, it was confirmed that overexpression of four target genes was induced in transduced hFF by blue light irradiation.

Example 5. Confirmation of target gene expression by blue light induction in human embryonic stem cells

5-1. Production of lentivirus

The first vector (Lenti-int_Ef1α ^P -CibN-dCas9-CibN-Puro) constructed in Example 1 and the second vector (Lenti-4gRNA-int_Ef1α ^P -Cry2FL-VP64-) constructed in Example 2 were added to 293FT cells. BSD) set 1 was transfected to produce lentivirus.

Specifically, as a transfection reagent, 450 μL of sterile distilled water was added to 12 μg of lentiviral packaging plasmid psPAX2, 10 μg of lentiviral envelope plasmid pMD2.G, 20 μg of first vector, and 20 μg of second vector. Mixed well. Here, 500 μL of 2X BBS solution and 60 μL of 2.5 M CaCl ₂ solution were added and left at room temperature for 20 minutes. 1 mL of the above reagent was added dropwise to a 10 cm culture dish containing 293FT cells, mixed slowly, and cultured for 16 hours. Afterwards, it was replaced with a basic culture medium that did not contain the transfection reagent and cultured for an additional 48 hours. The cell culture medium was recovered, centrifuged at 3,000 rpm for 10 minutes, and the supernatant was filtered through a 0.45 μm filter. A virus extraction reagent (PEG -it Virus Precipitation Solution) was added at 20% by volume of the total solution, mixed, and left at 4°C for 24 hours. Afterwards, the mixture was centrifuged at 4°C and 1,500 g for 30 minutes, and the settled white precipitate of lentivirus particles was released into the medium and used for subsequent transduction.

5-2. Construction of blue light-mediated human embryonic stem cells overexpressing hINS, hGCK, hPCSK1, and hPCSK2 genes

Human embryonic stem cells (hES) were maintained and cultured in mTeSR1 culture medium in Matrigel-coated 4-well culture dishes, and when they grew to about 60 to 80%, the culture medium was replaced with 300 μL/well of new mTeSR1 culture medium. The int_Ef1α ^P -CibN-dCas9-CibN-Puro lentivirus concentrate produced in Example 5-1, the 4gRNA-int_Ef1α ^P -Cry2FL-VP64-BSD lentivirus concentrate, and 8 μg/mL of polybrene were added to each well. Added and incubated for 24 hours. Replaced with a new mTeSR1 culture medium and cultured for an additional 3 days, then treated with selection markers puromycin and blasticidin, cells resistant to all of the above antibiotics were selected as cells into which all of the lentiviruses had been introduced. did.

5-3. Confirmation of induction of hINS, hGCK, hPCSK1 and hPCSK2 gene expression by blue light irradiation in transduced hES

Real-time qRT-PCR was performed to confirm whether expression of the target genes hINS, hGCK, hPCSK1, and hPCSK2 was induced by blue light irradiation in the transduced hES produced in Example 5-2.

Specifically, after irradiating the hES prepared in Example 5-2 with 455 nm blue light at 700 mA, the cells were recovered, using the primers shown in Table 9 below, and the expression level of GAPDH was calculated based on the measured mRNA expression level. qRT-PCR was performed in the same manner as described in Example 4-3, except that the relative mRNA expression level of the target gene was calculated and expressed normalized based on .

gene Forward 5' → 3' Reverse 5' → 3' hGAPDH GGT GGT CTC CTC TGA CTT CAA CA GTG GTC GTT GAG GGC AAT G hINS GGA ACG AGG CTT CTT CTA CAC ACA ATG CCA CGC TTC TGC hGCK CTT CCC TCA GTT TTT CGG TGG TTG ATT CCA GCG AGA AAG GTG hPCSK1 AAG CAA ACC CAA ATC TCA CCT GGC TCA CCA TCA AGC CTG CTC CAT TCT hPCSK2 AAG ATG GCT TTG CAG CAG GAA GGA AGC CAC ATT CAA ATC AAG GCC AGG

As a result, it was confirmed that when blue light was irradiated for 24 hours, the mRNA expression level of each target gene increased in the transduced hES, as shown in Figure 8.

In addition, when blue light was irradiated for 16 or 24 hours and 50% of the light was pulsed, as shown in Figure 9, when blue light was irradiated for 24 hours, the mRNA expression of each target gene in the transduced hES It was confirmed that the amount increased significantly.

In addition, as a result of comparing the mRNA expression level by irradiating blue light for 24 or 48 hours, as shown in Figure 10, when blue light was irradiated to the transduced hES, the mRNA expression level of each target gene increased, and at 24 hours, In comparison, it was confirmed that the expression level further increased when irradiated for 48 hours.

Example 6. Karyotype analysis of human embryonic stem cells overexpressing blue light-mediated target genes

The karyotype of the transduced hES produced in Example 5-2 was analyzed.

Specifically, the cells were cultured in a 4-well plate, 10 μL of colcemid was added per well, and the cells were further cultured for 30 minutes. The cultured cells were washed with a hypotonic solution mixed with 0.8% sodium citrate and 0.075M KCl at a volume ratio of 1:1, and then 500 μL of the hypotonic solution was added per well and incubated for an additional 23 minutes. . Afterwards, a fixation reagent mixed with methanol and acetic acid at a volume ratio of 3:1 was added and centrifuged at 1,800 rpm for 10 minutes. The pellet was collected and resuspended with the above fixation reagent, and centrifuged under the same conditions and resuspended in 3 steps. Repeated more times. The suspended cell solution was dropped onto a glass slide, left in an oven at 80°C for 2 hours, and then Giemsa staining was performed to stain and visualize chromosomes (FIG. 11).

<110> CHA University Industry-Academic Cooperation Foundation Konkuk University Industrial Cooperation Corp <120> Vector system for light-inducible modulating of gene expression and uses it <130> PN131429KR <160> 30 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hINS gRNA 1 <400> 1 acccccaggcc ctaatgggcc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hINS gRNA 2 <400> 2 ctaatgaccc gctggtcctg 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hINS gRNA 3 <400> 3 tgagactata aagccagcgg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hINS gRNA 4 <400> 4 ctctgagact ataaagccag 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hINS gRNA 5 <400> 5 ggaagaggtg ctgacgacca 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hGCK gRNA 1 <400> 6 agagtatttg agcagccgcc 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hGCK gRNA 2 <400> 7 gcccgcacaa ggctgaggaa 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hGCK gRNA 3 <400> 8 tagcggtcac tgtagtgaca 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hGCK gRNA 4 <400> 9 tagcggtcac tgtagtgaca 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hGCK gRNA 5 <400> 10 gatcctctgc tcatcagaac 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hPCSK1 gRNA 1 <400> 11 agctgctggg actcgcgacc 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hPCSK1 gRNA 2 <400> 12 atctatcccc gcgactctct 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hPCSK1 gRNA 3 <400> 13 cgcgactctc tagggagctc 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hPCSK1 gRNA 4 <400> 14 cgcgactctc tagggagctc 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hPCSK1 gRNA 5 <400> 15 cttggtagtt atgttcaagt 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hPCSK2 gRNA 1 <400> 16 gccgctggcc tgattacagc 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hPCSK2 gRNA 2 <400> 17 tgcgctgcgc ccccagaagg 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hPCSK2 gRNA 3 <400> 18 ggcggggcccg gggcggccca 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hPCSK2 gRNA 4 <400> 19 ccgccctcat tggccgctgg 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hPCSK2 gRNA 5 <400> 20 gcccggggcg gcccagggcg 20 <210> 21 <211> 15239 <212> DNA <213> Artificial Sequence <220> <223> Lentivirus vector 1 <400> 21 aagtaagacc accgcacagc aagcggccgc tgatcttcag acctggagga ggagatatga 60 gggacaattg gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag 120 tagcacccac caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag 180 gagctttgtt ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga 240 cgctgacggt acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc 300 tgagggctat tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atca agcagc 360 tccaggcaag aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt 420 ggggttgctc tggaaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta 480 ataaatctct ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta 540 acaattacac aagcttaata cactccttaa ttgaagaatc aaaccag caagaaaaga 600 atgaacaaga attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa 660 caaattggct gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa 720 gaatagtttt tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat 780 cgtttcagac ccacctccca accccgaggg gacccgacag gcccgaagga atagaagaag 840 aaggtggaga gagagacaga gacagatcca ttcgattagt gaacggatcg gcactgcgtg 900 cgccaattct gcagacaaat ggcagtattc atccacaatt ttaaaagaaa aggggggatt 960 ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa 1 020 gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag ggacagcaga 1080 gatccagttt ggttaattag ctagcagata tacgcgttga cattgattat tgactagtct 1140 aggtcttgaa aggagtggga attggctccg gtgcccgtca gtgggcagag cgcacatcgc 1200 ccacagtccc cgagaagttg gggggagggg tcggcaattg aaccggtgcc tagagaaggt 1260 ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg 1320 ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg 1380 ccgccagaac acaggtaagt gccgtgtg tg gttcccgcgg gcctggcctc tttacgggtt 1440 atggcccttg cgtgccttga attacttcca cctggctgca gtacgtgatt cttgatcccg 1500 agcttcgggt tggaagtggg tgggagagtt cgaggccttg cgcttaagga gccccttcgc 1560 ctcgtgcttg agttgaggcc tggcctgggc gctggggccg ccgcgtgcga atctggtggc 1620 accttcgcgc ctgtctcgct gctttcgata agtctctagc catttaaaat ttttgatgac 1680 ctgctgcgac gctttttttc tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca 1740 ctggtatttc ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat 1800 gttcggcgag gcggggcctg cgagcgcggc cacc gagaat cggacggggg tagtctcaag 1860 ctggccggcc tgctctggtg cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg 1920 caaggctggc ccggtcggca ccagttgcgt gagcggaaag atggccgctt cccggccctg 1980 ctgcagggag ctcaaaatgg a ggacgcggc gctcggggaga gcgggcgggt gagtcaccca 2040 cacaaaggaa aagggccttt ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc 2100 gggcgccgtc caggcacctc gattagttct cgagcttttg gagtacgtcg tctttaggtt 2160 ggggggaggg gttttatgcg atggagtttc cccacactga gtgggtggag actgaagtta 2220 ggccagcttg gcacttgatg taattctcct tggaatttgc cctttttgag tttggatctt 2 280 ggttcattct caagcctcag acagtggttc aaagtttttt tcttccattt caggtgtcgt 2340 gacgtgagct ctctggctaa ctagagaacc cactgcttac tggcttatcg aaattaatac 2400 gactcactat agggagaccc aagctggcta gcgccatgga ctacaaagac cat gacggtg 2460 attataaaga tcatgacatc gatataaatg gagctatagg aggtgacctt ttgctcaatt 2520 ttcctgacat gtcggtccta gagcgccaaa gggctcacct caagtacctc aatcccacct 2580 ttgattctcc tctcgccggc ttctttgccg attcttcaat gattaccggc ggcgagatgg 2640 acagctatct ttcgactgcc ggtttgaatc ttccgatgat gtacggtgag acgacggtgg 2700 aagg tgattc aagactctca atttcgccgg aaacgacgct tgggactgga aatttcaaga 2760 aacggaagtt tgatacagag actaaggatt gtaatgagaa gaagaagaag atgacgatga 2820 acagagatga cctagtagaa gaaggagaag aagagaagtc gaaaataaca gagcaaaaca 2880 atgggagcac a aaaagcatc aagaagatga aacacaaagc caagaaagaa gagaacaatt 2940 tctctaatga ttcatctaaa gtgacgaagg aattggagaa aacggattat attcatcacg 3000 attacaagga tgacgatgac aagatggccc ccaagaagaa gaggaaggtg ggccgcggaa 3060 tggataagaa atactcaata ggcttagcta tcggcacaaa tagcgtcgga tgggcggtga 3120 tcactgatga atataaggtt ccgtctaaaa ag ttcaaggt tctgggaaat acagaccgcc 3180 acagtatcaa aaaaaatctt ataggggctc ttttatttga cagtggagag acagcggaag 3240 cgactcgtct caaacggaca gctcgtagaa ggtatacacg tcggaagaat cgtatttgtt 3300 atctacagga gattttttca aatgagatgg cgaaagtaga tgatagtttc tttcatcgac 3360 ttgaagagtc ttttttggtg gaagaagaca agaagcatga acgtcatcct atttttggaa 3420 atatagtaga tgaagttgct tatcatgaga aatatccaac tatctatcat ctgcgaaaaa 3480 aattggtaga ttctactgat aaagcggatt tgcgcttaat ctatttggcc ttagcgcata 3540 tgattaagtt tcgtggtcat tttttgattg aggga gattt aaatcctgat aatagtgatg 3600 tggacaaact atttatccag ttggtacaaa cctacaatca attatttgaa gaaaacccta 3660 ttaacgcaag tggagtagat gctaaagcga ttctttctgc acgattgagt aaatcaagac 3720 gattagaaaa tctcattgct cagctccccg gtga gaagaa aaatggctta tttgggaatc 3780 tcattgcttt gtcattgggt ttgaccccta attttaaatc aaattttgat ttggcagaag 3840 atgctaaatt acagctttca aaagatactt acgatgatga tttagataat ttattggcgc 3900 aaattggaga tcaatatgct gatttgtttt tggcagctaa gaatttatca gatgctattt 3960 tactttcaga tatcctaaga gtaaatactg aaataactaa ggctccccta tcagcttcaa 4020 tgattaaacg ctacgatgaa catcatcaag acttgactct tttaaaagct ttagttcgac 4080 aacaacttcc agaaaagtat aaagaaatct tttttgatca atcaaaaaac ggatatgcag 4140 gttatattga tgggggagct agccaagaag aattttataa atttatcaaa ccaatt ttag 4200 aaaaaatgga tggtactgag gaattattgg tgaaactaaa tcgtgaagat ttgctgcgca 4260 agcaacggac ctttgacaac ggctctattc cccatcaaat tcacttgggt gagctgcatg 4320 ctattttgag aagacaagaa gacttttatc catttttaaa agacaatcgt gagaagattg 4380 aaaaaatctt gacttttcga attccttatt atgttggtcc attggcgcgt gg caatagtc 4440 gttttgcatg gatgactcgg aagtctgaag aaacaattac cccatggaat tttgaagaag 4500 ttgtcgataa aggtgcttca gctcaatcat ttattgaacg catgacaaac tttgataaaa 4560 atcttccaaa tgaaaaagta ctaccaaaac atagtttgct ttatgag tat tttacggttt 4620 ataacgaatt gacaaaggtc aaatatgtta ctgaaggaat gcgaaaacca gcatttcttt 4680 caggtgaaca gaagaaagcc attgttgatt tactcttcaa aacaaatcga aaagtaaccg 4740 ttaagcaatt aaaagaagat tatttcaaaa aaatagaatg ttttgatagt gttgaaattt 4800 caggagttga agatagattt aatgcttcat taggtaccta ccatgatttg ctaaaaatta 4860 ttaaagataa agattttttg gataatgaag aaaatgaaga tatcttagag gatattgttt 4920 taacattgac cttatttgaa gatagggaga tgattgagga aagacttaaa acatatgctc 4980 acctctttga tgaataaggtg atgaaacagc ttaaacgtcg ccgttatact ggttggggac 504 0 gtttgtctcg aaaattgatt aatggtatta gggataagca atctggcaaa acaatattag 5100 attttttgaa atcagatggt tttgccaatc gcaattttat gcagctgatc catgatgata 5160 gtttgacatt taaagaagac attcaaaaag cacaagtgtc tggacaaggc gatagtttac 5220 atgaacatat tgcaaattta gctggtagcc ctgctattaa aaaaggtatt ttacagactg 5280 taaaagttg t tgatgaattg gtcaaagtaa tggggcggca taagccagaa aatatcgtta 5340 ttgaaatggc acgtgaaaat cagacaactc aaaagggcca gaaaaattcg cgagagcgta 5400 tgaaacgaat cgaagaaggt atcaaagaat taggaagtca gattcttaaa gagcatcctg 5460 ttgaaaatac t caattgcaa aatgaaaagc tctatctcta ttatctccaa aatggaagag 5520 acatgtatgt ggaccaagaa ttagatatta atcgtttaag tgattatgat gtcgatgcca 5580 ttgttccaca aagtttcctt aaagacgatt caatagacaa taaggtctta acgcgttctg 5640 ataaaaatcg tggtaaatcg gataacgttc caagtgaaga agtagtcaaa aagatgaaaa 5700 actattggag a caacttcta aacgccaagt taatcactca acgtaagttt gataatttaa 5760 cgaaagctga acgtggaggt ttgagtgaac ttgataaagc tggttttatc aaacgccaat 5820 tggttgaaac tcgccaaatc actaagcatg tggcacaaat tttggatagt cgcatgaata 5880 ctaaatacga tgaaaatgat aaacttattc gagaggttaa agtgattacc ttaaaatcta 5940 aattagtttc tgacttccga aaagatttcc aattctataa agtacgtgag attaacaatt 6000 accatcatgc ccatgatgcg tatctaaatg ccgtcgttgg aactgctttg attaagaaat 6060 atccaaaact tgaatcggag tttgtctatg gtgattataa agtttatgat gttcgtaaaa 6120 tgattgctaa gcaa gaaataggca aagcaaccgc aaaatatttc ttttactcta 6180 atatcatgaa cttcttcaaa acagaaatta cacttgcaaa tggagagatt cgcaaacgcc 6240 ctctaatcga aactaatggg gaaactggag aaattgtctg ggataaaggg cgagattttg 6300 ccacagtgc g caaagtattg tccatgcccc aagtcaatat tgtcaagaaa acagaagtac 6360 agacaggcgg attctccaag gagtcaattt taccaaaaag aaattcggac aagcttattg 6420 ctcgtaaaaa agactgggat ccaaaaaaat atggtggttt tgatagtcca acggtagctt 6480 attcagtcct agtggttgct aaggtggaaa aagggaaatc gaagaagtta aaatccgtta 6540 aagagttact agggatcaca attatggaaa ga agttcctt tgaaaaaaat ccgattgact 6600 ttttagaagc taaaggatat aaggaagtta aaaaagactt aatcattaaa ctacctaaat 6660 atagtctttt tgagttagaa aacggtcgta aacggatgct ggctagtgcc ggagaattac 6720 aaaaaggaaa tgagctggct a atatgtgaa ttttttatat ttagctagtc 6780 attatgaaaa gttgaagggt agtccagaag ataacgaaca aaaacaattg tttgtggagc 6840 agcataagca ttatttagat gagattattg agcaaatcag tgaattttct aagcgtgtta 6900 ttttagcaga tgccaattta gataaagttc ttagtgcata taacaacat agagacaaac 6960 caatacgtga acaagcagaa aatattattc atttatttac gttgacgaat ct tggagctc 7020 ccgctgcttt taaatatttt gatacaacaa ttgatcgtaa acgatatacg tctacaaaag 7080 aagttttaga tgccactctt atccatcaat ccatcactgg tctttatgaa acacgcattg 7140 atttgagtca gctaggaggt gacccaattg ccggatccaa ggctag cccg aaaaagaaac 7200 gcaaagttgg gcgcgccacc atgaatggag ctataggagg tgaccttttg ctcaattttc 7260 ctgacatgtc ggtcctagag cgccaaaggg ctcacctcaa gtacctcaat cccacctttg 7320 attctcctct cgccggcttc tttgccgatt cttcaatgat taccggcggc gagatggaca 7380 gctatctttc gactgccggt ttgaatcttc cgatgatgta gacg acggtggaag 7440 gtgattcaag actctcaatt tcgccggaaa cgacgcttgg gactggaaat ttcaagaaac 7500 ggaagtttga tacagagact aaggattgta atgagaagaa gaagaagatg acgatgaaca 7560 gagatgacct agtagaagaa ggagaagaag agaagtcgaa aataacagag caaaacaatg 7620 ggagcacaaa aagcatcaag aagatgaaac acaaagccaa gaaagaagag aacaatttct 7680 ctaatgattc atctaaagtg acgaaggaat tggagaaaac ggattatatt tgtacaggtt 7740 caggggaggg aagaggttcc ctgctcacat gcggagacgt agaagagaat cccggaccca 7800 tgacctctag agagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg 78 60 ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc 7920 cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc 7980 tcgacatcgg caaggtgtgg gtcgcggacg gccgc ggtggcggtc tggaccacgc 8040 cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga 8100 gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca 8160 aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc 8220 tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtg cccgcct 8280 tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca 8340 ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg 8400 cctgagaatt cgatatcaag cttatcggta atcaacctct ggattacaaa atttgtgaaa 8460 gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa 8520 tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat 8580 cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt 8640 gcactgtgtt tgctgacgca accccactg gttggggcat tgccaccacc tgtcagctcc 87 00 tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc 8760 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg 8820 ggaaatcatc gtcctttcct tggctgtgttg c cacctggatt ctgcgcggga 8880 cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc 8940 tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc 9000 tttgggccgc ctccccgcat cgataccgtc gacctcgaga cctagaaaaa catggagcaa 9060 tcacaagtag caatacagca gctaccaatg ctgattgtgc ctggctagaa gcacaagagg 9120 aggaggaggt gggttttcca gt cacacctc aggtaccttt aagaccaatg acttacaagg 9180 cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact 9240 cccaacgaag acaagatatc cttgatctgt ggatctacca cacacaaggc tacttccctg 9300 attggcagaa ctacacacca tcagatatcc actgaccttt ggatggtgct 9360 acaagctagt accagttgag caagagaagg tagaagaagc caatgaagga gagaacaccc 9420 gcttgttaca ccctgtgagc ctgcatggga tggatgaccc ggagagagaa gtattagagt 9480 ggaggtttga cagccgccta gcatttcatc acatggcccg agagctgcat ccggactgta 9540 ctgggtctct ctggttagac cagatctgag cctggga gct ctctggctaa ctagggaacc 9600 cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt 9660 tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta 9720 gcagggcccg tttaaacccg ct gatcagcc tcgactgtgc cttctagttg ccagccatct 9780 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt 9840 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg 9900 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg 9960 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggct c tagggggtat 10020 ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg 10080 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc 10140 gccacgttcg ccggct ttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga 10200 tttagtgctt tacggcacct cgacccccaaa aaacttgatt agggtgatgg ttcacgtagt 10260 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat 10320 agtggactct tgttccaaac tggaaacaaca ctcaacccta tctcggtcta ttcttttgat 10380 ttataaggga ttttgccgat ttcggcctat tgg ttaaaaa atgagctgat ttaacaaaaaa 10440 tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct 10500 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa 10560 agtcccccagg ctccccagca ggcaga agta tgcaaagcat gcatctcaat tagtcagcaa 10620 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt 10680 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct 10740 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc 10800 tcccgggagc ttgtatatcc attttcggat ctgatcagca tgaca attaatcatc 10860 ggcatagtat atcggcatag tataatacga caaggtgagg aactaaacca tggccaagtt 10920 gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac 10980 cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg tggtccggga 11040 cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca acaccctggc 11100 ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg tcgtgtccac 11160 gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc cgtgggggcg 11220 ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac tt cgtggccg aggagcagga 11280 ctgacacgtg ctacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg 11340 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt 11400 cttcgcccac acttgt ttaattgcagc ttataatggt tacaaataaa gcaatagcat 11460 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact 11520 catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc 11580 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg 11640 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat 11700 tgc gttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg 11760 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct 11820 cactgactcg ctgcgctcgg tcgttcggct gcggc gagcg gtatcagctc actcaaaggc 11880 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg 11940 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg 12000 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg 12060 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac 12120 cctgccgct t accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca 12180 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt 12240 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc 12300 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag 12360 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac 12420 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaaagagt 12480 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 12540 gcagcagatt acgcgcagaa aaa aaggatc tcaagaagat cctttgatct tttctacggg 12600 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa 12660 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat 12720 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc 12780 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat 12840 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc 12900 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc 12960 tgcaacttta tccgcctcca tccagtctat taattgtt gc cgggaagcta gagtaagtag 13020 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg 13080 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg 13140 atcccccatg gcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag 13200 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt 13260 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga 13320 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc 13380 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc 13440 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc 13500 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc 13560 cgcaaaaaag ggaataaggg cgacacggaa atgt tgaata ctcatactct tcctttttca 13620 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat 13680 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt 13740 cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat 13800 gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcg c tgagtagtgc 13860 gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg 13920 cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat 13980 tgattattga ctagttatta atagtaatca attacgggg t cattagttca tagcccatat 14040 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac 14100 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc 14160 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg 14220 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat 14 280 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc 14340 atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt 14400 gactcacggg gatttccaag tctccacccc attgacgttca atgggagttt g ttttggcac 14460 caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc 14520 ggtaggcgtg tacggtggga ggtctata agcagcgcgt tttgcctgta ctgggtctct 14580 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa 14640 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc 1 4700 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc 14760 ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc tctctcgacg caggactcgg 14820 cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgag tac gccaaaaatt 14880 ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg 14940 agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa 15000 ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg 15060 ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca 15120 ggatcagaag aacttagatc attatataat aca gtagcaa ccctctattg tgtgcatcaa 15180aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaa 15239 <210> 22 <211> 13578 <212> DNA <213> Artificial Sequence <220> <223> Lentivirus vector 2 (4gRNA set 1) <400> 22 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg 60 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt 120 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattg c atgaagaatc 180 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac 240 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat 300 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg 360 accccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 420 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 480 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 540 attatgccca gtacatgacc ttcctacttg gcagtacatc tacgtattag 600 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt 660 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc 720 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg 780 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct 840 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 900 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 960 tctggtaact agagatccct cagacccttt tagtcagtgt g gaaaatctc tagcagtggc 1020 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc 1080 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa 1140 ttttgactag cggagggag atgg gtgcga gagcgtcagt attaagcggg 1200 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata 1260 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc 1320 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga 1380 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc 144 0 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca 1500 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg 1560 agggacaatt ggagaagtga attataaa tataaagtag taaaaattga accattagga 1620 gtagcaccca caaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata 1680 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg 1740 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg 1800 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag 1860 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt 1920 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt 1980 aataaatctc tggaacagat ttggaatcac acgacctgga tggga cagagaaatt 2040 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag 2100 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata 2160 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta 2220 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta 2280 tcgtttcaga c ccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa 2340 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt 2400 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat 2460 agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa 2520 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag 2580 agatccagtt tggttaatta gctagcttag ggttaggcgt tttgcgctgc ttcgcgatta 2640 cagggacagc agagatccag tttggttaat taaataactt cgtatagcat acatttatacg 2700 aagttatgat agatccgac g ccgccatctc taggcccgcg ccggccccct cgcacagact 2760 tgtgggagaa gctcggctac tcccctgccc cggttaattt gcatataata tttcctagta 2820 actatagagg cttaatgtgc gataaaagac agataatctg ttctttttaa tactagctac 2880 attttacatg ataggcttgg att tctataa gagatacaaa tactaaatta ttattttaaa 2940 aaacagcaca aaaggaaact caccctaact gtaaagtaat tgtgtgtttt gagactataa 3000 atatcccttg gagaaaagcc ttgtttgacc ccaggcccta atgggccgtt ttagagctag 3060 aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg 3120 tgcttttttt gtacgct cag cggagaaaaa aagcaccgac tcggtgccac tttttcaagt 3180 tgataacgga ctagccttat tttaacttgc tatttctagc tctaaaacgg cggctgctca 3240 aatactctcg gtgtttcgtc ctttccacaa gatatataaa gccaagaaat cgaaatactt 3300 tcaagttacg gtaagcatat gatagtccat tttaaaacat aattttaaaa ctgcaaacta 3360 cccaagaaat tattactttc tacgtcacgt attttgtact aatatctttg tgtttacagt 3420 caaattaatt ccaattatct ctctaacagc cttgtatcgt atatgcaaat atgaaggaat 3480 catgggaaat aggccctctt cctgcccgac cttggatcga tccgcctgaa cgctgacgtc 3540 atcaacccgc tccaagggaat cgcgggcc ca gtgtcactag gcgggaacac ccagcgcgcg 3600 tgcgccctgg caggaagatg gctgtgaggg acaggggagt ggcgccctgc aatatttgca 3660 tgtcgctatg tgttctggga aatcaccata aacgtgaaat gtctttggat ttgggaatct 3720 gtatgagacc actctttccc aagctgctgg gactcgcgac cgttttagag 3780 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag 3840 tcggtgcttt ttttggtcat cagtccgaga aaaaaagcac cgactcggtg ccactttttc 3900 aagttgataa cggactagcc ttattttaac ttgctatttc tagctctaaa acgctgtaat 3960 caggccagcg gccgaggtac ccaagcggcg cacaagctat ataaacctga aggaagtctc 4020 aactttacac ttaggtcaag ttgcttatcg tactagagct tcagcaggaa atttaactaa 4080 aatctaattt aaccagcata gcaaatatca tttattccca aaatgctaaa gtttgagata 4140 aacggacttg atttccggct gttttgacac tatccagaat gcc ttgcaga tgggtggggc 4200 atgctaaata ctgcagacaa ttaattaacc cgtgtcggct ccagatctgg cctccgcgcc 4260 gggttttggc gcctcacgcg ttgacattga ttattgacta gtctaggtct tgaaaggagt 4320 gggaattggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa 4380 gttgggggga ggggtcggca attgaaccgg tgcctagaga a ggtggcgcg gggtaaactg 4440 ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat 4500 aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt 4560 aagtgccg tg tgtggttccc gcgggcctgg cctctttacg ggttatggcc cttgcgtgcc 4620 ttgaattact tccacctggc tgcagtacgt gattcttgat cccgagcttc gggttggaag 4680 tgggtgggag agttcgaggc cttgcgctta aggagcccct tcgcctcgtg cttgagttga 4740 ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg tggcaccttc gcgcctgtct 4800 cgctgctttc gataagtctc tagccattta ga tgacctgctg cgacgctttt 4860 tttctggcaa gatagtcttg taaatgcggg ccaagatctg cacactggta tttcggtttt 4920 tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc acatgttcgg cgaggcgggg 4980 cctgcgagcg cggccaccga gaatcggacg ggggtagtct caagctggcc ggcctgctct 5040 ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg gcggcaaggc tggcccggtc 5100 ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc cctgctgcag ggagctcaaa 5160 atggaggacg cggcgctcgg gagagcgggc gggtgagtca cccacacaaa ggaaaagggc 5220 ctttccgtcc tcagccgtcg cttcatgtga ctccacggag taccgggcgc cgtcca ggca 5280 cctcgattag ttctcgagct tttggagtac gtcgtcttta ggttgggggg aggggtttta 5340 tgcgatggag tttccccaca ctgagtgggt ggagactgaa gttaggccag cttggcactt 5400 gatgtaattc tccttggaat ttgccctttt gga tcttggttca ttctcaagcc 5460 tcagacagtg gttcaaagtt tttttcttcc atttcaggtg tcgtgacgtg agctctctgg 5520 ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag 5580 acccaagctg gctagcgcca tggactacaa agaccatgac ggtgattata aagatcatga 5640 catcgattac aaggatgacg atgacaagat ggcccccaag aagaagagga aggtgggccg 5700 catgaagatg gacaaaaaga ctatagttt g gtttagaaga gacctaagga ttgaggataa 5760 tcctgcatta gcagcagctg ctcacgaagg atctgttttt cctgtcttca tttggtgtcc 5820 tgaagaagaa ggacagtttt atcctggaag agcttcaaga tggtggatga aacaatcact 5880 tgctcactta tctcaatcc t tgaaggctct tggatctgac ctcactttaa tcaaaaccca 5940 caacacgatt tcagcgatct tggattgtat ccgcgttacc ggtgctacaa aagtcgtctt 6000 taaccacctc tatgatcctg tttcgttagt tcgggaccat accgtaaagg agaagctggt 6060 ggaacgtggg atctctgtgc aaagctacaa tggagatcta ttgtatgaac cgtgggagat 6120 atactgcgaa aag ggcaaac cttttacgag tttcaattct tactggaaga aatgcttaga 6180 tatgtcgatt gaatccgtta tgcttcctcc tccttggcgg ttgatgccaa taactgcagc 6240 ggctgaagcg atttgggcgt gttcgattga agaactaggg ctggagaatg aggccgagaa 6300 acc gagcaat gcgttgttaa ctagagcttg gtctccagga tggagcaatg ctgataagtt 6360 actaaatgag ttcatcgaga agcagttgat agattatgca aagaacagca agaaagttgt 6420 tgggaattct acttcactac tttctccgta tctccatttc ggggaaataa gcgtcagaca 6480 cgttttccag tgtgcccgga tgaaacaaat tatatgggca agagataaga acagtgaagg 6540 agaagaaagt gcagatcttt ttcttagggg aatcggttta agagagtatt ctcggtatat 6600 atgtttcaac ttcccgttta ctcacgagca atcgttgttg agtcatcttc ggtttttccc 6660 ttgggatgct gatgttgata agttcaaggc ctggagacaa ggcaggaccg gttatccgtt 6720 gg tggatgcc ggaatgagag agctttgggc taccggatgg atgcataaca gaataagagt 6780 gattgtttca agctttgctg tgaagtttct tctccttcca tggaaatggg gaatgaagta 6840 tttctgggat acacttttgg atgctgattt ggaatgtgac atccttggct ggcagtatat 6900 ctctgggagt atccccgatg gccacgagct tgatcgcttg gacaatcccg cgttacaagg 6960 cgccaaatat gacccagaag gtgagtacat aaggcaat gg cttcccgagc ttgcgagatt 7020 gccaactgaa tggatccatc atccatggga cgctccttta accgtactca aagcttctgg 7080 tgtggaactc ggaacaaact atgcgaaacc cattgtagac atcgacacag ctcgtgagct 7140 actagctaaa gctatttcaa gaacccgtga agc acagatc atgatcggag cagcacctga 7200 tgagattgta gcagatagct tcgaggcctt aggggctaat accattaaag aacctggtct 7260 ttgcccatct gtgtcttcta atgaccaaca agtaccttcg gctgttcgtt acaacgggtc 7320 aaagagagtg aaacctgagg aagaagaaga gagagacatg aagaaatcta ggggattcga 7380 tgaaagggag ttgttttcga ctgctgaatc ttcttcttct tcga gtgtgt ttttcgtttc 7440 gcagtcttgc tcgttggcat cagaagggaa gaatctggaa ggtattcaag attcatctga 7500 tcagattact acaagtttgg gaaaaaatgg ttgcaaacca attgccggat ccaaggctag 7560 cccgaaaaag aaacgcaaag ttgggcgc gc cgacgcgctg gacgatttcg atctcgacat 7620 gctgggttct gatgccctcg atgactttga cctggatatg ttgggaagcg acgcattgga 7680 tgactttgat ctggacatgc tcggctccga tgctctggac gatttcgatc tcgatatgtt 7740 aattaactac ccgtacgacg ttccggacta cgcttcttgt acaggcagtg gagagggcag 7800 aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct ggcccaatgg ccaagccttt 7860 gtctcaagaa gaatccaccc tcattgaaag agcaacggct acaatcaaca gcatccccat 7920 ctctgaagac tacagcgtcg ccagcgcagc tctctctagc gacggccgca tcttcactgg 7980 tgtcaatgta tatcatttta ctgggggacc ctcgtggtgc tgggcactgc 8040 tgctgctgcg gcagctggca acctgacttg tatcgtcgcg atcggaaatg agaacagggg 8100 catcttgagc ccctgcggac ggtgccgaca ggtgcttctc gatctgcatc ctgggatcaa 8160 agccatagtg aaggacagtg atggacagcc gacggcagtt gggattcgtg aattgctgcc 8220 ctctggttat gtgtgggagg gctaagaatt cgatatcaag cttatcggta atcaacctct 828 0 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct 8340 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat 8400 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt gttgt 8460 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat 8520 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc 8580 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga 8640 caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc 8700 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga 8760 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc 8820 tcagacgagt cggatctccc tttgggccgc ctcc ccgcat cgataccgtc gacctcgaga 8880 cctagaaaaa catggagcaa tcacaagtag caatacagca gctaccaatg ctgattgtgc 8940 ctggctagaa gcacaagagg aggaggaggt gggttttcca gtcacacctc aggtaccttt 9000 aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg 9060 actggaaggg ctaattcact cccaacgaag acaagatatc cttgatctgt ggatctacca 9120 cacacaaggc tacttccctg attggcagaa ctacacacca gggccaggga tcagatatcc 9180 actgaccttt ggatggtgct acaagctagt accagttgag caagagaagg tagaagaagc 9240 caatgaagga gagaacaccc gcttgttaca ccctgtgagc ctgcatggga tggatgaccc 9300 ggagagagaa gtattagagt ggaggtttga ca gccgccta gcatttcatc acatggcccg 9360 agagctgcat ccggactgta ctgggtctct ctggttagac cagatctgag cctgggagct 9420 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca 9480 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta 9540 gtcagtgtgg aaaatctcta cccg tttaaacccg ctgatcagcc tcgactgtgc 9600 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag 9660 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta 9720 ggtgt cattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag 9780 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca 9840 gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg 9900 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg 9960 ctttcttccc ttcctttctc gccacg ttcg ccggctttcc ccgtcaagct ctaaatcggg 10020 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt 10080 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt 101 40 tggagtccac gttctttaat agtggactct tgttccaaac tggaaacaaca ctcaacccta 10200 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa 10260 atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg 10320 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta 10380 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat 10440 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac 10500 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga 10560 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag aggct tttttggagg 10620 cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca 10680 cgtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg 10740 aactaaacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc 10800 ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt ggaggacgac 10860 ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg 10920 gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag 10980 tggtcgggagg gaacttccgg gacgcctccg ggccggccat gaccgagatc 11040 ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac 11100 ttcgtggccg aggagcagga ctgacacgtg ctacgagatt tcgattccac cgccgccttc 11160 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc 11220 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttatattgcagc ttataatggt 11280 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct 11340 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct 11400 agctagagct tggcgtaatc atggtcatag ctgtttcct g tgtgaaattg ttatccgctc 11460 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 11520 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 11580 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 11640 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 11700 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 11760 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 11820 gcgtttttcc ataggctccg cccccctgac gagcatca ca aaaatcgacg ctcaagtcag 11880 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 11940 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 12000 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 12060 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 12120 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 12180 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 12240 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 12300 gttaccttcg gaaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 12360 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 12420 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 12480 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 12540 tttaaatcaa tctaaagtat atatgagtaa acttgg tctg acagttacca atgcttaatc 12600 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 12660 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 12720 ccgcgagacc cacg ctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 12780 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 12840 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 12900 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 12960 cgatcaaggc gagttacatg atcccc catg ttgtgcaaaa aagcggttag ctccttcggt 13020 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 13080 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 13140 tcaaccaagt cat tctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 13200 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaaacgt 13260 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 13320 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 13380 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 13440 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 13500 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 13560cgaaaagtgc cacctgac 13578 <210> 23 <211> 13578 <212> DNA <213> Artificial Sequence <220> <223> Lentivirus vector 2 (4gRNA set 2) <400> 23 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg 60 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt 120 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattg c atgaagaatc 180 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac 240 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat 300 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg 360 accccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 420 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 480 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 540 attatgccca gtacatgacc ttcctacttg gcagtacatc tacgtattag 600 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt 660 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc 720 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg 780 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct 840 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 900 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 960 tctggtaact agagatccct cagacccttt tagtcagtgt g gaaaatctc tagcagtggc 1020 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc 1080 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa 1140 ttttgactag cggagggag atgg gtgcga gagcgtcagt attaagcggg 1200 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata 1260 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc 1320 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga 1380 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc 144 0 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca 1500 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg 1560 agggacaatt ggagaagtga attataaa tataaagtag taaaaattga accattagga 1620 gtagcaccca caaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata 1680 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg 1740 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg 1800 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag 1860 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt 1920 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt 1980 aataaatctc tggaacagat ttggaatcac acgacctgga tggga cagagaaatt 2040 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag 2100 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata 2160 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta 2220 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta 2280 tcgtttcaga c ccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa 2340 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt 2400 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat 2460 agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa 2520 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag 2580 agatccagtt tggttaatta gctagcttag ggttaggcgt tttgcgctgc ttcgcgatta 2640 cagggacagc agagatccag tttggttaat taaataactt cgtatagcat acatttatacg 2700 aagttatgat agatccgac g ccgccatctc taggcccgcg ccggccccct cgcacagact 2760 tgtgggagaa gctcggctac tcccctgccc cggttaattt gcatataata tttcctagta 2820 actatagagg cttaatgtgc gataaaagac agataatctg ttctttttaa tactagctac 2880 attttacatg ataggcttgg att tctataa gagatacaaa tactaaatta ttattttaaa 2940 aaacagcaca aaaggaaact caccctaact gtaaagtaat tgtgtgtttt gagactataa 3000 atatcccttg gagaaaagcc ttgtttgcta atgacccgct ggtcctggtt ttagagctag 3060 aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg 3120 tgcttttttt gtac gctcag cggagaaaaa aagcaccgac tcggtgccac tttttcaagt 3180 tgataacgga ctagccttat tttaacttgc tatttctagc tctaaaactt cctcagcctt 3240 gtgcgggccg gtgtttcgtc ctttccacaa gatatataaa gccaagaaat cgaaatactt 3300 t caagttacg gtaagcatat gatagtccat tttaaaacat aattttaaaa ctgcaaacta 3360 cccaagaaat tattactttc tacgtcacgt attttgtact aatatctttg tgtttacagt 3420 caaattaatt ccaattatct ctctaacagc cttgtatcgt atatgcaaat atgaaggaat 3480 catgggaaat aggccctctt cctgcccgac cttggatcga tccgcctgaa cgctgacgtc 3540 atcaacccgc tccaagggaat cgcgggcc ca gtgtcactag gcgggaacac ccagcgcgcg 3600 tgcgccctgg caggaagatg gctgtgaggg acaggggagt ggcgccctgc aatatttgca 3660 tgtcgctatg tgttctggga aatcaccata aacgtgaaat gtctttggat ttgggaatct 3720 gtatgagacc actctttccc aatctatccc cgcgactctc tgttttagag 3780 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag 3840 tcggtgcttt ttttggtcat cagtccgaga aaaaaagcac cgactcggtg ccactttttc 3900 aagttgataa cggactagcc ttattttaac ttgctatttc tagctctaaa acccttctgg 3960 gggcgcagcg cacgaggtac ccaagcggcg cacaagct at ataaacctga aggaagtctc 4020 aactttacac ttaggtcaag ttgcttatcg tactagagct tcagcaggaa atttaactaa 4080 aatctaattt aaccagcata gcaaatatca tttatattccca aaatgctaaa gtttgagata 4140 aacggacttg atttccggct gttttgacac tatccagaat g ccttgcaga tgggtggggc 4200 atgctaaata ctgcagacaa ttaattaacc cgtgtcggct ccagatctgg cctccgcgcc 4260 gggttttggc gcctcacgcg ttgacattga ttattgacta gtctaggtct tgaaaggagt 4320 gggaattggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa 4380 gttgggggga ggggtcggca attgaaccgg tgcctagaga a ggtggcgcg gggtaaactg 4440 ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat 4500 aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt 4560 aagtgccg tg tgtggttccc gcgggcctgg cctctttacg ggttatggcc cttgcgtgcc 4620 ttgaattact tccacctggc tgcagtacgt gattcttgat cccgagcttc gggttggaag 4680 tgggtgggag agttcgaggc cttgcgctta aggagcccct tcgcctcgtg cttgagttga 4740 ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg tggcaccttc gcgcctgtct 4800 cgctgctttc gataagtctc tagccattta ga tgacctgctg cgacgctttt 4860 tttctggcaa gatagtcttg taaatgcggg ccaagatctg cacactggta tttcggtttt 4920 tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc acatgttcgg cgaggcgggg 4980 cctgcgagcg cggccaccga gaatcggacg ggggtagtct caagctggcc ggcctgctct 5040 ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg gcggcaaggc tggcccggtc 5100 ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc cctgctgcag ggagctcaaa 5160 atggaggacg cggcgctcgg gagagcgggc gggtgagtca cccacacaaa ggaaaagggc 5220 ctttccgtcc tcagccgtcg cttcatgtga ctccacggag taccgggcgc cgtcca ggca 5280 cctcgattag ttctcgagct tttggagtac gtcgtcttta ggttgggggg aggggtttta 5340 tgcgatggag tttccccaca ctgagtgggt ggagactgaa gttaggccag cttggcactt 5400 gatgtaattc tccttggaat ttgccctttt gga tcttggttca ttctcaagcc 5460 tcagacagtg gttcaaagtt tttttcttcc atttcaggtg tcgtgacgtg agctctctgg 5520 ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag 5580 acccaagctg gctagcgcca tggactacaa agaccatgac ggtgattata aagatcatga 5640 catcgattac aaggatgacg atgacaagat ggcccccaag aagaagagga aggtgggccg 5700 catgaagatg gacaaaaaga ctatagttt g gtttagaaga gacctaagga ttgaggataa 5760 tcctgcatta gcagcagctg ctcacgaagg atctgttttt cctgtcttca tttggtgtcc 5820 tgaagaagaa ggacagtttt atcctggaag agcttcaaga tggtggatga aacaatcact 5880 tgctcactta tctcaatcc t tgaaggctct tggatctgac ctcactttaa tcaaaaccca 5940 caacacgatt tcagcgatct tggattgtat ccgcgttacc ggtgctacaa aagtcgtctt 6000 taaccacctc tatgatcctg tttcgttagt tcgggaccat accgtaaagg agaagctggt 6060 ggaacgtggg atctctgtgc aaagctacaa tggagatcta ttgtatgaac cgtgggagat 6120 atactgcgaa aag ggcaaac cttttacgag tttcaattct tactggaaga aatgcttaga 6180 tatgtcgatt gaatccgtta tgcttcctcc tccttggcgg ttgatgccaa taactgcagc 6240 ggctgaagcg atttgggcgt gttcgattga agaactaggg ctggagaatg aggccgagaa 6300 acc gagcaat gcgttgttaa ctagagcttg gtctccagga tggagcaatg ctgataagtt 6360 actaaatgag ttcatcgaga agcagttgat agattatgca aagaacagca agaaagttgt 6420 tgggaattct acttcactac tttctccgta tctccatttc ggggaaataa gcgtcagaca 6480 cgttttccag tgtgcccgga tgaaacaaat tatatgggca agagataaga acagtgaagg 6540 agaagaaagt gcagatcttt ttcttagggg aatcggttta agagagtatt ctcggtatat 6600 atgtttcaac ttcccgttta ctcacgagca atcgttgttg agtcatcttc ggtttttccc 6660 ttgggatgct gatgttgata agttcaaggc ctggagacaa ggcaggaccg gttatccgtt 6720 gg tggatgcc ggaatgagag agctttgggc taccggatgg atgcataaca gaataagagt 6780 gattgtttca agctttgctg tgaagtttct tctccttcca tggaaatggg gaatgaagta 6840 tttctgggat acacttttgg atgctgattt ggaatgtgac atccttggct ggcagtatat 6900 ctctgggagt atccccgatg gccacgagct tgatcgcttg gacaatcccg cgttacaagg 6960 cgccaaatat gacccagaag gtgagtacat aaggcaat gg cttcccgagc ttgcgagatt 7020 gccaactgaa tggatccatc atccatggga cgctccttta accgtactca aagcttctgg 7080 tgtggaactc ggaacaaact atgcgaaacc cattgtagac atcgacacag ctcgtgagct 7140 actagctaaa gctatttcaa gaacccgtga agc acagatc atgatcggag cagcacctga 7200 tgagattgta gcagatagct tcgaggcctt aggggctaat accattaaag aacctggtct 7260 ttgcccatct gtgtcttcta atgaccaaca agtaccttcg gctgttcgtt acaacgggtc 7320 aaagagagtg aaacctgagg aagaagaaga gagagacatg aagaaatcta ggggattcga 7380 tgaaagggag ttgttttcga ctgctgaatc ttcttcttct tcga gtgtgt ttttcgtttc 7440 gcagtcttgc tcgttggcat cagaagggaa gaatctggaa ggtattcaag attcatctga 7500 tcagattact acaagtttgg gaaaaaatgg ttgcaaacca attgccggat ccaaggctag 7560 cccgaaaaag aaacgcaaag ttgggcgc gc cgacgcgctg gacgatttcg atctcgacat 7620 gctgggttct gatgccctcg atgactttga cctggatatg ttgggaagcg acgcattgga 7680 tgactttgat ctggacatgc tcggctccga tgctctggac gatttcgatc tcgatatgtt 7740 aattaactac ccgtacgacg ttccggacta cgcttcttgt acaggcagtg gagagggcag 7800 aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct ggcccaatgg ccaagccttt 7860 gtctcaagaa gaatccaccc tcattgaaag agcaacggct acaatcaaca gcatccccat 7920 ctctgaagac tacagcgtcg ccagcgcagc tctctctagc gacggccgca tcttcactgg 7980 tgtcaatgta tatcatttta ctgggggacc ctcgtggtgc tgggcactgc 8040 tgctgctgcg gcagctggca acctgacttg tatcgtcgcg atcggaaatg agaacagggg 8100 catcttgagc ccctgcggac ggtgccgaca ggtgcttctc gatctgcatc ctgggatcaa 8160 agccatagtg aaggacagtg atggacagcc gacggcagtt gggattcgtg aattgctgcc 8220 ctctggttat gtgtgggagg gctaagaatt cgatatcaag cttatcggta atcaacctct 828 0 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct 8340 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat 8400 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt gttgt 8460 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat 8520 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc 8580 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga 8640 caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc 8700 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga 8760 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc 8820 tcagacgagt cggatctccc tttgggccgc ctcc ccgcat cgataccgtc gacctcgaga 8880 cctagaaaaa catggagcaa tcacaagtag caatacagca gctaccaatg ctgattgtgc 8940 ctggctagaa gcacaagagg aggaggaggt gggttttcca gtcacacctc aggtaccttt 9000 aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg 9060 actggaaggg ctaattcact cccaacgaag acaagatatc cttgatctgt ggatctacca 9120 cacacaaggc tacttccctg attggcagaa ctacacacca gggccaggga tcagatatcc 9180 actgaccttt ggatggtgct acaagctagt accagttgag caagagaagg tagaagaagc 9240 caatgaagga gagaacaccc gcttgttaca ccctgtgagc ctgcatggga tggatgaccc 9300 ggagagagaa gtattagagt ggaggtttga ca gccgccta gcatttcatc acatggcccg 9360 agagctgcat ccggactgta ctgggtctct ctggttagac cagatctgag cctgggagct 9420 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca 9480 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta 9540 gtcagtgtgg aaaatctcta cccg tttaaacccg ctgatcagcc tcgactgtgc 9600 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag 9660 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta 9720 ggtgt cattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag 9780 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca 9840 gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg 9900 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg 9960 ctttcttccc ttcctttctc gccacg ttcg ccggctttcc ccgtcaagct ctaaatcggg 10020 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt 10080 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt 101 40 tggagtccac gttctttaat agtggactct tgttccaaac tggaaacaaca ctcaacccta 10200 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa 10260 atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg 10320 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta 10380 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat 10440 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac 10500 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga 10560 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag aggct tttttggagg 10620 cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca 10680 cgtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg 10740 aactaaacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc 10800 ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt ggaggacgac 10860 ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg 10920 gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag 10980 tggtcgggagg gaacttccgg gacgcctccg ggccggccat gaccgagatc 11040 ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac 11100 ttcgtggccg aggagcagga ctgacacgtg ctacgagatt tcgattccac cgccgccttc 11160 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc 11220 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttatattgcagc ttataatggt 11280 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct 11340 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct 11400 agctagagct tggcgtaatc atggtcatag ctgtttcct g tgtgaaattg ttatccgctc 11460 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 11520 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 11580 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 11640 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 11700 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 11760 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 11820 gcgtttttcc ataggctccg cccccctgac gagcatca ca aaaatcgacg ctcaagtcag 11880 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 11940 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 12000 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 12060 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 12120 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 12180 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 12240 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 12300 gttaccttcg gaaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 12360 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 12420 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 12480 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 12540 tttaaatcaa tctaaagtat atatgagtaa acttgg tctg acagttacca atgcttaatc 12600 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 12660 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 12720 ccgcgagacc cacg ctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 12780 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 12840 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 12900 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 12960 cgatcaaggc gagttacatg atcccc catg ttgtgcaaaa aagcggttag ctccttcggt 13020 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 13080 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 13140 tcaaccaagt cat tctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 13200 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaaacgt 13260 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 13320 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 13380 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 13440 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 13500 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 13560cgaaaagtgc cacctgac 13578 <210> 24 <211> 13578 <212> DNA <213> Artificial Sequence <220> <223> Lentivirus vector 2 (4gRNA set 3) <400> 24 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg 60 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt 120 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattg c atgaagaatc 180 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac 240 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat 300 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg 360 accccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 420 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 480 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 540 attatgccca gtacatgacc ttcctacttg gcagtacatc tacgtattag 600 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt 660 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc 720 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg 780 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct 840 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 900 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 960 tctggtaact agagatccct cagacccttt tagtcagtgt g gaaaatctc tagcagtggc 1020 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc 1080 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa 1140 ttttgactag cggagggag atgg gtgcga gagcgtcagt attaagcggg 1200 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata 1260 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc 1320 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga 1380 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc 144 0 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca 1500 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg 1560 agggacaatt ggagaagtga attataaa tataaagtag taaaaattga accattagga 1620 gtagcaccca caaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata 1680 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg 1740 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg 1800 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag 1860 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt 1920 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt 1980 aataaatctc tggaacagat ttggaatcac acgacctgga tggga cagagaaatt 2040 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag 2100 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata 2160 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta 2220 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta 2280 tcgtttcaga c ccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa 2340 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt 2400 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat 2460 agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa 2520 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag 2580 agatccagtt tggttaatta gctagcttag ggttaggcgt tttgcgctgc ttcgcgatta 2640 cagggacagc agagatccag tttggttaat taaataactt cgtatagcat acatttatacg 2700 aagttatgat agatccgac g ccgccatctc taggcccgcg ccggccccct cgcacagact 2760 tgtgggagaa gctcggctac tcccctgccc cggttaattt gcatataata tttcctagta 2820 actatagagg cttaatgtgc gataaaagac agataatctg ttctttttaa tactagctac 2880 attttacatg ataggcttgg att tctataa gagatacaaa tactaaatta ttattttaaa 2940 aaacagcaca aaaggaaact caccctaact gtaaagtaat tgtgtgtttt gagactataa 3000 atatcccttg gagaaaagcc ttgtttgtga gactataaag ccagcgggtt ttagagctag 3060 aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg 3120 tgcttttttt gtacg ctcag cggagaaaaa aagcaccgac tcggtgccac tttttcaagt 3180 tgataacgga ctagccttat tttaacttgc tatttctagc tctaaaactg tcactacagt 3240 gaccgctacg gtgtttcgtc ctttccacaa gatatataaa gccaagaaat cgaaatactt 3300 cg gtaagcatat gatagtccat tttaaaacat aattttaaaa ctgcaaacta 3360 cccaagaaat tattactttc tacgtcacgt attttgtact aatatctttg tgtttacagt 3420 caaattaatt ccaattatct ctctaacagc cttgtatcgt atatgcaaat atgaaggaat 3480 catgggaaat aggccctctt cctgcccgac cttggatcga tccgcctgaa cgctgacgtc 3540 atcaacccgc tccaagggaat cgcgggcc ca gtgtcactag gcgggaacac ccagcgcgcg 3600 tgcgccctgg caggaagatg gctgtgaggg acaggggagt ggcgccctgc aatatttgca 3660 tgtcgctatg tgttctggga aatcaccata aacgtgaaat gtctttggat ttgggaatct 3720 gtatgagacc actctttccc acgcgactct ctagggagct cgttttagag 3780 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag 3840 tcggtgcttt ttttggtcat cagtccgaga aaaaaagcac cgactcggtg ccactttttc 3900 aagttgataa cggactagcc ttattttaac ttgctatttc tagctctaaa actgggccgc 3960 cccgggcccg cccgaggtac ccaagcggcg cacaagctat ataaacctga aggaagtctc 4020 aactttacac ttaggtcaag ttgcttatcg tactagagct tcagcaggaa atttaactaa 4080 aatctaattt aaccagcata gcaaatatca tttattccca aaatgctaaa gtttgagata 4140 aacggacttg atttccggct gttttgacac tatccagaat gcc ttgcaga tgggtggggc 4200 atgctaaata ctgcagacaa ttaattaacc cgtgtcggct ccagatctgg cctccgcgcc 4260 gggttttggc gcctcacgcg ttgacattga ttattgacta gtctaggtct tgaaaggagt 4320 gggaattggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa 4380 gttgggggga ggggtcggca attgaaccgg tgcctagaga a ggtggcgcg gggtaaactg 4440 ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat 4500 aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt 4560 aagtgccg tg tgtggttccc gcgggcctgg cctctttacg ggttatggcc cttgcgtgcc 4620 ttgaattact tccacctggc tgcagtacgt gattcttgat cccgagcttc gggttggaag 4680 tgggtgggag agttcgaggc cttgcgctta aggagcccct tcgcctcgtg cttgagttga 4740 ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg tggcaccttc gcgcctgtct 4800 cgctgctttc gataagtctc tagccattta ga tgacctgctg cgacgctttt 4860 tttctggcaa gatagtcttg taaatgcggg ccaagatctg cacactggta tttcggtttt 4920 tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc acatgttcgg cgaggcgggg 4980 cctgcgagcg cggccaccga gaatcggacg ggggtagtct caagctggcc ggcctgctct 5040 ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg gcggcaaggc tggcccggtc 5100 ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc cctgctgcag ggagctcaaa 5160 atggaggacg cggcgctcgg gagagcgggc gggtgagtca cccacacaaa ggaaaagggc 5220 ctttccgtcc tcagccgtcg cttcatgtga ctccacggag taccgggcgc cgtcca ggca 5280 cctcgattag ttctcgagct tttggagtac gtcgtcttta ggttgggggg aggggtttta 5340 tgcgatggag tttccccaca ctgagtgggt ggagactgaa gttaggccag cttggcactt 5400 gatgtaattc tccttggaat ttgccctttt gga tcttggttca ttctcaagcc 5460 tcagacagtg gttcaaagtt tttttcttcc atttcaggtg tcgtgacgtg agctctctgg 5520 ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag 5580 acccaagctg gctagcgcca tggactacaa agaccatgac ggtgattata aagatcatga 5640 catcgattac aaggatgacg atgacaagat ggcccccaag aagaagagga aggtgggccg 5700 catgaagatg gacaaaaaga ctatagttt g gtttagaaga gacctaagga ttgaggataa 5760 tcctgcatta gcagcagctg ctcacgaagg atctgttttt cctgtcttca tttggtgtcc 5820 tgaagaagaa ggacagtttt atcctggaag agcttcaaga tggtggatga aacaatcact 5880 tgctcactta tctcaatcc t tgaaggctct tggatctgac ctcactttaa tcaaaaccca 5940 caacacgatt tcagcgatct tggattgtat ccgcgttacc ggtgctacaa aagtcgtctt 6000 taaccacctc tatgatcctg tttcgttagt tcgggaccat accgtaaagg agaagctggt 6060 ggaacgtggg atctctgtgc aaagctacaa tggagatcta ttgtatgaac cgtgggagat 6120 atactgcgaa aag ggcaaac cttttacgag tttcaattct tactggaaga aatgcttaga 6180 tatgtcgatt gaatccgtta tgcttcctcc tccttggcgg ttgatgccaa taactgcagc 6240 ggctgaagcg atttgggcgt gttcgattga agaactaggg ctggagaatg aggccgagaa 6300 acc gagcaat gcgttgttaa ctagagcttg gtctccagga tggagcaatg ctgataagtt 6360 actaaatgag ttcatcgaga agcagttgat agattatgca aagaacagca agaaagttgt 6420 tgggaattct acttcactac tttctccgta tctccatttc ggggaaataa gcgtcagaca 6480 cgttttccag tgtgcccgga tgaaacaaat tatatgggca agagataaga acagtgaagg 6540 agaagaaagt gcagatcttt ttcttagggg aatcggttta agagagtatt ctcggtatat 6600 atgtttcaac ttcccgttta ctcacgagca atcgttgttg agtcatcttc ggtttttccc 6660 ttgggatgct gatgttgata agttcaaggc ctggagacaa ggcaggaccg gttatccgtt 6720 gg tggatgcc ggaatgagag agctttgggc taccggatgg atgcataaca gaataagagt 6780 gattgtttca agctttgctg tgaagtttct tctccttcca tggaaatggg gaatgaagta 6840 tttctgggat acacttttgg atgctgattt ggaatgtgac atccttggct ggcagtatat 6900 ctctgggagt atccccgatg gccacgagct tgatcgcttg gacaatcccg cgttacaagg 6960 cgccaaatat gacccagaag gtgagtacat aaggcaat gg cttcccgagc ttgcgagatt 7020 gccaactgaa tggatccatc atccatggga cgctccttta accgtactca aagcttctgg 7080 tgtggaactc ggaacaaact atgcgaaacc cattgtagac atcgacacag ctcgtgagct 7140 actagctaaa gctatttcaa gaacccgtga agc acagatc atgatcggag cagcacctga 7200 tgagattgta gcagatagct tcgaggcctt aggggctaat accattaaag aacctggtct 7260 ttgcccatct gtgtcttcta atgaccaaca agtaccttcg gctgttcgtt acaacgggtc 7320 aaagagagtg aaacctgagg aagaagaaga gagagacatg aagaaatcta ggggattcga 7380 tgaaagggag ttgttttcga ctgctgaatc ttcttcttct tcga gtgtgt ttttcgtttc 7440 gcagtcttgc tcgttggcat cagaagggaa gaatctggaa ggtattcaag attcatctga 7500 tcagattact acaagtttgg gaaaaaatgg ttgcaaacca attgccggat ccaaggctag 7560 cccgaaaaag aaacgcaaag ttgggcgc gc cgacgcgctg gacgatttcg atctcgacat 7620 gctgggttct gatgccctcg atgactttga cctggatatg ttgggaagcg acgcattgga 7680 tgactttgat ctggacatgc tcggctccga tgctctggac gatttcgatc tcgatatgtt 7740 aattaactac ccgtacgacg ttccggacta cgcttcttgt acaggcagtg gagagggcag 7800 aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct ggcccaatgg ccaagccttt 7860 gtctcaagaa gaatccaccc tcattgaaag agcaacggct acaatcaaca gcatccccat 7920 ctctgaagac tacagcgtcg ccagcgcagc tctctctagc gacggccgca tcttcactgg 7980 tgtcaatgta tatcatttta ctgggggacc ctcgtggtgc tgggcactgc 8040 tgctgctgcg gcagctggca acctgacttg tatcgtcgcg atcggaaatg agaacagggg 8100 catcttgagc ccctgcggac ggtgccgaca ggtgcttctc gatctgcatc ctgggatcaa 8160 agccatagtg aaggacagtg atggacagcc gacggcagtt gggattcgtg aattgctgcc 8220 ctctggttat gtgtgggagg gctaagaatt cgatatcaag cttatcggta atcaacctct 828 0 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct 8340 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat 8400 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt gttgt 8460 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat 8520 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc 8580 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga 8640 caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc 8700 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga 8760 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc 8820 tcagacgagt cggatctccc tttgggccgc ctcc ccgcat cgataccgtc gacctcgaga 8880 cctagaaaaa catggagcaa tcacaagtag caatacagca gctaccaatg ctgattgtgc 8940 ctggctagaa gcacaagagg aggaggaggt gggttttcca gtcacacctc aggtaccttt 9000 aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg 9060 actggaaggg ctaattcact cccaacgaag acaagatatc cttgatctgt ggatctacca 9120 cacacaaggc tacttccctg attggcagaa ctacacacca gggccaggga tcagatatcc 9180 actgaccttt ggatggtgct acaagctagt accagttgag caagagaagg tagaagaagc 9240 caatgaagga gagaacaccc gcttgttaca ccctgtgagc ctgcatggga tggatgaccc 9300 ggagagagaa gtattagagt ggaggtttga ca gccgccta gcatttcatc acatggcccg 9360 agagctgcat ccggactgta ctgggtctct ctggttagac cagatctgag cctgggagct 9420 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca 9480 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta 9540 gtcagtgtgg aaaatctcta cccg tttaaacccg ctgatcagcc tcgactgtgc 9600 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag 9660 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta 9720 ggtgt cattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag 9780 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca 9840 gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg 9900 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg 9960 ctttcttccc ttcctttctc gccacg ttcg ccggctttcc ccgtcaagct ctaaatcggg 10020 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt 10080 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt 101 40 tggagtccac gttctttaat agtggactct tgttccaaac tggaaacaaca ctcaacccta 10200 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa 10260 atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg 10320 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta 10380 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat 10440 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac 10500 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga 10560 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag aggct tttttggagg 10620 cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca 10680 cgtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg 10740 aactaaacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc 10800 ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt ggaggacgac 10860 ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg 10920 gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag 10980 tggtcggagg gaacttccgg gacgcctccg ggccggccat gaccgagatc 11040 ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac 11100 ttcgtggccg aggagcagga ctgacacgtg ctacgagatt tcgattccac cgccgccttc 11160 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc 11220 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttatattgcagc ttataatggt 11280 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct 11340 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct 11400 agctagagct tggcgtaatc atggtcatag ctgtttcct g tgtgaaattg ttatccgctc 11460 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 11520 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 11580 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 11640 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 11700 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 11760 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 11820 gcgtttttcc ataggctccg cccccctgac gagcatca ca aaaatcgacg ctcaagtcag 11880 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 11940 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 12000 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 12060 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 12120 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 12180 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 12240 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 12300 gttaccttcg gaaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 12360 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 12420 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 12480 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 12540 tttaaatcaa tctaaagtat atatgagtaa acttgg tctg acagttacca atgcttaatc 12600 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 12660 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 12720 ccgcgagacc cacg ctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 12780 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 12840 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 12900 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 12960 cgatcaaggc gagttacatg atcccc catg ttgtgcaaaa aagcggttag ctccttcggt 13020 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 13080 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 13140 tcaaccaagt cat tctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 13200 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaaacgt 13260 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 13320 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 13380 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 13440 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 13500 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 13560cgaaaagtgc cacctgac 13578 <210> 25 <211> 13578 <212> DNA <213> Artificial Sequence <220> <223> Lentivirus vector 2 (4gRNA set 4) <400> 25 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg 60 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt 120 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattg c atgaagaatc 180 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac 240 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat 300 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg 360 accccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 420 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 480 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 540 attatgccca gtacatgacc ttcctacttg gcagtacatc tacgtattag 600 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt 660 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc 720 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg 780 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct 840 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 900 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 960 tctggtaact agagatccct cagacccttt tagtcagtgt g gaaaatctc tagcagtggc 1020 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc 1080 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa 1140 ttttgactag cggagggag atgg gtgcga gagcgtcagt attaagcggg 1200 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata 1260 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc 1320 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga 1380 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc 144 0 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca 1500 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg 1560 agggacaatt ggagaagtga attataaa tataaagtag taaaaattga accattagga 1620 gtagcaccca caaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata 1680 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg 1740 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg 1800 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag 1860 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt 1920 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt 1980 aataaatctc tggaacagat ttggaatcac acgacctgga tggga cagagaaatt 2040 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag 2100 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata 2160 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta 2220 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta 2280 tcgtttcaga c ccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa 2340 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt 2400 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat 2460 agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa 2520 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag 2580 agatccagtt tggttaatta gctagcttag ggttaggcgt tttgcgctgc ttcgcgatta 2640 cagggacagc agagatccag tttggttaat taaataactt cgtatagcat acatttatacg 2700 aagttatgat agatccgac g ccgccatctc taggcccgcg ccggccccct cgcacagact 2760 tgtgggagaa gctcggctac tcccctgccc cggttaattt gcatataata tttcctagta 2820 actatagagg cttaatgtgc gataaaagac agataatctg ttctttttaa tactagctac 2880 attttacatg ataggcttgg att tctataa gagatacaaa tactaaatta ttattttaaa 2940 aaacagcaca aaaggaaact caccctaact gtaaagtaat tgtgtgtttt gagactataa 3000 atatcccttg gagaaaagcc ttgtttgctc tgagactata aagccaggtt ttagagctag 3060 aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg 3120 tgcttttttt gtacgct cag cggagaaaaa aagcaccgac tcggtgccac tttttcaagt 3180 tgataacgga ctagccttat tttaacttgc tatttctagc tctaaaactg gcaggagaat 3240 cccccactcg gtgtttcgtc ctttccacaa gatatataaa gccaagaaat cgaaatactt 3300 tcaagttacg g taagcatat gatagtccat tttaaaacat aattttaaaa ctgcaaacta 3360 cccaagaaat tattactttc tacgtcacgt attttgtact aatatctttg tgtttacagt 3420 caaattaatt ccaattatct ctctaacagc cttgtatcgt atatgcaaat atgaaggaat 3480 catgggaaat aggccctctt cctgcccgac cttggatcga tccgcctgaa cgctgacgtc 3540 atcaacccgc tccaagggaat cgcgggcc ca gtgtcactag gcgggaacac ccagcgcgcg 3600 tgcgccctgg caggaagatg gctgtgaggg acaggggagt ggcgccctgc aatatttgca 3660 tgtcgctatg tgttctggga aatcaccata aacgtgaaat gtctttggat ttgggaatct 3720 gtatgagacc actctttccc acgcggagta gcctgggaag cgttttagag 3780 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag 3840 tcggtgcttt ttttggtcat cagtccgaga aaaaaagcac cgactcggtg ccactttttc 3900 aagttgataa cggactagcc ttattttaac ttgctatttc tagctctaaa acccagcggc 3960 caatgagggc ggcgaggtac ccaagcggcg cacaagct at ataaacctga aggaagtctc 4020 aactttacac ttaggtcaag ttgcttatcg tactagagct tcagcaggaa atttaactaa 4080 aatctaattt aaccagcata gcaaatatca tttatattccca aaatgctaaa gtttgagata 4140 aacggacttg atttccggct gttttgacac tatccagaat g ccttgcaga tgggtggggc 4200 atgctaaata ctgcagacaa ttaattaacc cgtgtcggct ccagatctgg cctccgcgcc 4260 gggttttggc gcctcacgcg ttgacattga ttattgacta gtctaggtct tgaaaggagt 4320 gggaattggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa 4380 gttgggggga ggggtcggca attgaaccgg tgcctagaga a ggtggcgcg gggtaaactg 4440 ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat 4500 aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt 4560 aagtgccg tg tgtggttccc gcgggcctgg cctctttacg ggttatggcc cttgcgtgcc 4620 ttgaattact tccacctggc tgcagtacgt gattcttgat cccgagcttc gggttggaag 4680 tgggtgggag agttcgaggc cttgcgctta aggagcccct tcgcctcgtg cttgagttga 4740 ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg tggcaccttc gcgcctgtct 4800 cgctgctttc gataagtctc tagccattta ga tgacctgctg cgacgctttt 4860 tttctggcaa gatagtcttg taaatgcggg ccaagatctg cacactggta tttcggtttt 4920 tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc acatgttcgg cgaggcgggg 4980 cctgcgagcg cggccaccga gaatcggacg ggggtagtct caagctggcc ggcctgctct 5040 ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg gcggcaaggc tggcccggtc 5100 ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc cctgctgcag ggagctcaaa 5160 atggaggacg cggcgctcgg gagagcgggc gggtgagtca cccacacaaa ggaaaagggc 5220 ctttccgtcc tcagccgtcg cttcatgtga ctccacggag taccgggcgc cgtcca ggca 5280 cctcgattag ttctcgagct tttggagtac gtcgtcttta ggttgggggg aggggtttta 5340 tgcgatggag tttccccaca ctgagtgggt ggagactgaa gttaggccag cttggcactt 5400 gatgtaattc tccttggaat ttgccctttt gga tcttggttca ttctcaagcc 5460 tcagacagtg gttcaaagtt tttttcttcc atttcaggtg tcgtgacgtg agctctctgg 5520 ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag 5580 acccaagctg gctagcgcca tggactacaa agaccatgac ggtgattata aagatcatga 5640 catcgattac aaggatgacg atgacaagat ggcccccaag aagaagagga aggtgggccg 5700 catgaagatg gacaaaaaga ctatagttt g gtttagaaga gacctaagga ttgaggataa 5760 tcctgcatta gcagcagctg ctcacgaagg atctgttttt cctgtcttca tttggtgtcc 5820 tgaagaagaa ggacagtttt atcctggaag agcttcaaga tggtggatga aacaatcact 5880 tgctcactta tctcaatcc t tgaaggctct tggatctgac ctcactttaa tcaaaaccca 5940 caacacgatt tcagcgatct tggattgtat ccgcgttacc ggtgctacaa aagtcgtctt 6000 taaccacctc tatgatcctg tttcgttagt tcgggaccat accgtaaagg agaagctggt 6060 ggaacgtggg atctctgtgc aaagctacaa tggagatcta ttgtatgaac cgtgggagat 6120 atactgcgaa aag ggcaaac cttttacgag tttcaattct tactggaaga aatgcttaga 6180 tatgtcgatt gaatccgtta tgcttcctcc tccttggcgg ttgatgccaa taactgcagc 6240 ggctgaagcg atttgggcgt gttcgattga agaactaggg ctggagaatg aggccgagaa 6300 acc gagcaat gcgttgttaa ctagagcttg gtctccagga tggagcaatg ctgataagtt 6360 actaaatgag ttcatcgaga agcagttgat agattatgca aagaacagca agaaagttgt 6420 tgggaattct acttcactac tttctccgta tctccatttc ggggaaataa gcgtcagaca 6480 cgttttccag tgtgcccgga tgaaacaaat tatatgggca agagataaga acagtgaagg 6540 agaagaaagt gcagatcttt ttcttagggg aatcggttta agagagtatt ctcggtatat 6600 atgtttcaac ttcccgttta ctcacgagca atcgttgttg agtcatcttc ggtttttccc 6660 ttgggatgct gatgttgata agttcaaggc ctggagacaa ggcaggaccg gttatccgtt 6720 gg tggatgcc ggaatgagag agctttgggc taccggatgg atgcataaca gaataagagt 6780 gattgtttca agctttgctg tgaagtttct tctccttcca tggaaatggg gaatgaagta 6840 tttctgggat acacttttgg atgctgattt ggaatgtgac atccttggct ggcagtatat 6900 ctctgggagt atccccgatg gccacgagct tgatcgcttg gacaatcccg cgttacaagg 6960 cgccaaatat gacccagaag gtgagtacat aaggcaat gg cttcccgagc ttgcgagatt 7020 gccaactgaa tggatccatc atccatggga cgctccttta accgtactca aagcttctgg 7080 tgtggaactc ggaacaaact atgcgaaacc cattgtagac atcgacacag ctcgtgagct 7140 actagctaaa gctatttcaa gaacccgtga agc acagatc atgatcggag cagcacctga 7200 tgagattgta gcagatagct tcgaggcctt aggggctaat accattaaag aacctggtct 7260 ttgcccatct gtgtcttcta atgaccaaca agtaccttcg gctgttcgtt acaacgggtc 7320 aaagagagtg aaacctgagg aagaagaaga gagagacatg aagaaatcta ggggattcga 7380 tgaaagggag ttgttttcga ctgctgaatc ttcttcttct tcga gtgtgt ttttcgtttc 7440 gcagtcttgc tcgttggcat cagaagggaa gaatctggaa ggtattcaag attcatctga 7500 tcagattact acaagtttgg gaaaaaatgg ttgcaaacca attgccggat ccaaggctag 7560 cccgaaaaag aaacgcaaag ttgggcgc gc cgacgcgctg gacgatttcg atctcgacat 7620 gctgggttct gatgccctcg atgactttga cctggatatg ttgggaagcg acgcattgga 7680 tgactttgat ctggacatgc tcggctccga tgctctggac gatttcgatc tcgatatgtt 7740 aattaactac ccgtacgacg ttccggacta cgcttcttgt acaggcagtg gagagggcag 7800 aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct ggcccaatgg ccaagccttt 7860 gtctcaagaa gaatccaccc tcattgaaag agcaacggct acaatcaaca gcatccccat 7920 ctctgaagac tacagcgtcg ccagcgcagc tctctctagc gacggccgca tcttcactgg 7980 tgtcaatgta tatcatttta ctgggggacc ctcgtggtgc tgggcactgc 8040 tgctgctgcg gcagctggca acctgacttg tatcgtcgcg atcggaaatg agaacagggg 8100 catcttgagc ccctgcggac ggtgccgaca ggtgcttctc gatctgcatc ctgggatcaa 8160 agccatagtg aaggacagtg atggacagcc gacggcagtt gggattcgtg aattgctgcc 8220 ctctggttat gtgtgggagg gctaagaatt cgatatcaag cttatcggta atcaacctct 828 0 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct 8340 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat 8400 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt gttgt 8460 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat 8520 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc 8580 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga 8640 caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc 8700 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga 8760 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc 8820 tcagacgagt cggatctccc tttgggccgc ctcc ccgcat cgataccgtc gacctcgaga 8880 cctagaaaaa catggagcaa tcacaagtag caatacagca gctaccaatg ctgattgtgc 8940 ctggctagaa gcacaagagg aggaggaggt gggttttcca gtcacacctc aggtaccttt 9000 aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg 9060 actggaaggg ctaattcact cccaacgaag acaagatatc cttgatctgt ggatctacca 9120 cacacaaggc tacttccctg attggcagaa ctacacacca gggccaggga tcagatatcc 9180 actgaccttt ggatggtgct acaagctagt accagttgag caagagaagg tagaagaagc 9240 caatgaagga gagaacaccc gcttgttaca ccctgtgagc ctgcatggga tggatgaccc 9300 ggagagagaa gtattagagt ggaggtttga ca gccgccta gcatttcatc acatggcccg 9360 agagctgcat ccggactgta ctgggtctct ctggttagac cagatctgag cctgggagct 9420 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca 9480 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta 9540 gtcagtgtgg aaaatctcta cccg tttaaacccg ctgatcagcc tcgactgtgc 9600 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag 9660 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta 9720 ggtgt cattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag 9780 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca 9840 gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg 9900 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg 9960 ctttcttccc ttcctttctc gccacg ttcg ccggctttcc ccgtcaagct ctaaatcggg 10020 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt 10080 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt 101 40 tggagtccac gttctttaat agtggactct tgttccaaac tggaaacaaca ctcaacccta 10200 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa 10260 atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg 10320 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta 10380 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat 10440 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac 10500 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga 10560 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag aggct tttttggagg 10620 cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca 10680 cgtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg 10740 aactaaacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc 10800 ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt ggaggacgac 10860 ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg 10920 gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag 10980 tggtcggagg gaacttccgg gacgcctccg ggccggccat gaccgagatc 11040 ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac 11100 ttcgtggccg aggagcagga ctgacacgtg ctacgagatt tcgattccac cgccgccttc 11160 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc 11220 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttatattgcagc ttataatggt 11280 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct 11340 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct 11400 agctagagct tggcgtaatc atggtcatag ctgtttcct g tgtgaaattg ttatccgctc 11460 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 11520 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 11580 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 11640 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 11700 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 11760 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 11820 gcgtttttcc ataggctccg cccccctgac gagcatca ca aaaatcgacg ctcaagtcag 11880 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 11940 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 12000 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 12060 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 12120 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 12180 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 12240 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 12300 gttaccttcg gaaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 12360 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 12420 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 12480 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 12540 tttaaatcaa tctaaagtat atatgagtaa acttgg tctg acagttacca atgcttaatc 12600 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 12660 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 12720 ccgcgagacc cacg ctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 12780 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 12840 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 12900 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 12960 cgatcaaggc gagttacatg atcccc catg ttgtgcaaaa aagcggttag ctccttcggt 13020 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 13080 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 13140 tcaaccaagt cat tctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 13200 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaaacgt 13260 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 13320 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 13380 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 13440 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 13500 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 13560cgaaaagtgc cacctgac 13578 <210> 26 <211> 13578 <212> DNA <213> Artificial Sequence <220> <223> Lentivirus vector 2 (4gRNA set 5) <400> 26 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg 60 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt 120 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattg c atgaagaatc 180 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac 240 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat 300 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg 360 accccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 420 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 480 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 540 attatgccca gtacatgacc ttcctacttg gcagtacatc tacgtattag 600 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt 660 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc 720 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg 780 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct 840 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 900 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 960 tctggtaact agagatccct cagacccttt tagtcagtgt g gaaaatctc tagcagtggc 1020 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc 1080 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa 1140 ttttgactag cggagggag atgg gtgcga gagcgtcagt attaagcggg 1200 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata 1260 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc 1320 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga 1380 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc 144 0 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca 1500 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg 1560 agggacaatt ggagaagtga attataaa tataaagtag taaaaattga accattagga 1620 gtagcaccca caaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata 1680 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg 1740 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg 1800 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag 1860 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt 1920 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt 1980 aataaatctc tggaacagat ttggaatcac acgacctgga tggga cagagaaatt 2040 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag 2100 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata 2160 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta 2220 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta 2280 tcgtttcaga c ccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa 2340 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt 2400 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat 2460 agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa 2520 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag 2580 agatccagtt tggttaatta gctagcttag ggttaggcgt tttgcgctgc ttcgcgatta 2640 cagggacagc agagatccag tttggttaat taaataactt cgtatagcat acatttatacg 2700 aagttatgat agatccgac g ccgccatctc taggcccgcg ccggccccct cgcacagact 2760 tgtgggagaa gctcggctac tcccctgccc cggttaattt gcatataata tttcctagta 2820 actatagagg cttaatgtgc gataaaagac agataatctg ttctttttaa tactagctac 2880 attttacatg ataggcttgg att tctataa gagatacaaa tactaaatta ttattttaaa 2940 aaacagcaca aaaggaaact caccctaact gtaaagtaat tgtgtgtttt gagactataa 3000 atatcccttg gagaaaagcc ttgtttggga agaggtgctg acgaccagtt ttagagctag 3060 aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg 3120 tgcttttttt gtacg ctcag cggagaaaaa aagcaccgac tcggtgccac tttttcaagt 3180 tgataacgga ctagccttat tttaacttgc tatttctagc tctaaaacgt tctgatgagc 3240 agaggatccg gtgtttcgtc ctttccacaa gatatataaa gccaagaaat cgaaatactt 3300 ttacg gtaagcatat gatagtccat tttaaaacat aattttaaaa ctgcaaacta 3360 cccaagaaat tattactttc tacgtcacgt attttgtact aatatctttg tgtttacagt 3420 caaattaatt ccaattatct ctctaacagc cttgtatcgt atatgcaaat atgaaggaat 3480 catgggaaat aggccctctt cctgcccgac cttggatcga tccgcctgaa cgctgacgtc 3540 atcaacccgc tccaagggaat cgcgggcc ca gtgtcactag gcgggaacac ccagcgcgcg 3600 tgcgccctgg caggaagatg gctgtgaggg acaggggagt ggcgccctgc aatatttgca 3660 tgtcgctatg tgttctggga aatcaccata aacgtgaaat gtctttggat ttgggaatct 3720 gtatgagacc actctttccc acttggtagt tatgttcaag tgttttagag 3780 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag 3840 tcggtgcttt ttttggtcat cagtccgaga aaaaaagcac cgactcggtg ccactttttc 3900 aagttgataa cggactagcc ttattttaac ttgctatttc tagctctaaa accgccctgg 3960 gccgccccgg gccgaggtac ccaagcggcg cacaagctat at aaacctga aggaagtctc 4020 aactttacac ttaggtcaag ttgcttatcg tactagagct tcagcaggaa atttaactaa 4080 aatctaattt aaccagcata gcaaatatca tttattccca aaatgctaaa gtttgagata 4140 aacggacttg atttccggct gttttgacac tatccagaat gcct tgcaga tgggtggggc 4200 atgctaaata ctgcagacaa ttaattaacc cgtgtcggct ccagatctgg cctccgcgcc 4260 gggttttggc gcctcacgcg ttgacattga ttattgacta gtctaggtct tgaaaggagt 4320 gggaattggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa 4380 gttgggggga ggggtcggca attgaaccgg tgcctagaga a ggtggcgcg gggtaaactg 4440 ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat 4500 aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt 4560 aagtgccg tg tgtggttccc gcgggcctgg cctctttacg ggttatggcc cttgcgtgcc 4620 ttgaattact tccacctggc tgcagtacgt gattcttgat cccgagcttc gggttggaag 4680 tgggtgggag agttcgaggc cttgcgctta aggagcccct tcgcctcgtg cttgagttga 4740 ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg tggcaccttc gcgcctgtct 4800 cgctgctttc gataagtctc tagccattta ga tgacctgctg cgacgctttt 4860 tttctggcaa gatagtcttg taaatgcggg ccaagatctg cacactggta tttcggtttt 4920 tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc acatgttcgg cgaggcgggg 4980 cctgcgagcg cggccaccga gaatcggacg ggggtagtct caagctggcc ggcctgctct 5040 ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg gcggcaaggc tggcccggtc 5100 ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc cctgctgcag ggagctcaaa 5160 atggaggacg cggcgctcgg gagagcgggc gggtgagtca cccacacaaa ggaaaagggc 5220 ctttccgtcc tcagccgtcg cttcatgtga ctccacggag taccgggcgc cgtcca ggca 5280 cctcgattag ttctcgagct tttggagtac gtcgtcttta ggttgggggg aggggtttta 5340 tgcgatggag tttccccaca ctgagtgggt ggagactgaa gttaggccag cttggcactt 5400 gatgtaattc tccttggaat ttgccctttt gga tcttggttca ttctcaagcc 5460 tcagacagtg gttcaaagtt tttttcttcc atttcaggtg tcgtgacgtg agctctctgg 5520 ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag 5580 acccaagctg gctagcgcca tggactacaa agaccatgac ggtgattata aagatcatga 5640 catcgattac aaggatgacg atgacaagat ggcccccaag aagaagagga aggtgggccg 5700 catgaagatg gacaaaaaga ctatagttt g gtttagaaga gacctaagga ttgaggataa 5760 tcctgcatta gcagcagctg ctcacgaagg atctgttttt cctgtcttca tttggtgtcc 5820 tgaagaagaa ggacagtttt atcctggaag agcttcaaga tggtggatga aacaatcact 5880 tgctcactta tctcaatcc t tgaaggctct tggatctgac ctcactttaa tcaaaaccca 5940 caacacgatt tcagcgatct tggattgtat ccgcgttacc ggtgctacaa aagtcgtctt 6000 taaccacctc tatgatcctg tttcgttagt tcgggaccat accgtaaagg agaagctggt 6060 ggaacgtggg atctctgtgc aaagctacaa tggagatcta ttgtatgaac cgtgggagat 6120 atactgcgaa aag ggcaaac cttttacgag tttcaattct tactggaaga aatgcttaga 6180 tatgtcgatt gaatccgtta tgcttcctcc tccttggcgg ttgatgccaa taactgcagc 6240 ggctgaagcg atttgggcgt gttcgattga agaactaggg ctggagaatg aggccgagaa 6300 acc gagcaat gcgttgttaa ctagagcttg gtctccagga tggagcaatg ctgataagtt 6360 actaaatgag ttcatcgaga agcagttgat agattatgca aagaacagca agaaagttgt 6420 tgggaattct acttcactac tttctccgta tctccatttc ggggaaataa gcgtcagaca 6480 cgttttccag tgtgcccgga tgaaacaaat tatatgggca agagataaga acagtgaagg 6540 agaagaaagt gcagatcttt ttcttagggg aatcggttta agagagtatt ctcggtatat 6600 atgtttcaac ttcccgttta ctcacgagca atcgttgttg agtcatcttc ggtttttccc 6660 ttgggatgct gatgttgata agttcaaggc ctggagacaa ggcaggaccg gttatccgtt 6720 gg tggatgcc ggaatgagag agctttgggc taccggatgg atgcataaca gaataagagt 6780 gattgtttca agctttgctg tgaagtttct tctccttcca tggaaatggg gaatgaagta 6840 tttctgggat acacttttgg atgctgattt ggaatgtgac atccttggct ggcagtatat 6900 ctctgggagt atccccgatg gccacgagct tgatcgcttg gacaatcccg cgttacaagg 6960 cgccaaatat gacccagaag gtgagtacat aaggcaat gg cttcccgagc ttgcgagatt 7020 gccaactgaa tggatccatc atccatggga cgctccttta accgtactca aagcttctgg 7080 tgtggaactc ggaacaaact atgcgaaacc cattgtagac atcgacacag ctcgtgagct 7140 actagctaaa gctatttcaa gaacccgtga agc acagatc atgatcggag cagcacctga 7200 tgagattgta gcagatagct tcgaggcctt aggggctaat accattaaag aacctggtct 7260 ttgcccatct gtgtcttcta atgaccaaca agtaccttcg gctgttcgtt acaacgggtc 7320 aaagagagtg aaacctgagg aagaagaaga gagagacatg aagaaatcta ggggattcga 7380 tgaaagggag ttgttttcga ctgctgaatc ttcttcttct tcga gtgtgt ttttcgtttc 7440 gcagtcttgc tcgttggcat cagaagggaa gaatctggaa ggtattcaag attcatctga 7500 tcagattact acaagtttgg gaaaaaatgg ttgcaaacca attgccggat ccaaggctag 7560 cccgaaaaag aaacgcaaag ttgggcgc gc cgacgcgctg gacgatttcg atctcgacat 7620 gctgggttct gatgccctcg atgactttga cctggatatg ttgggaagcg acgcattgga 7680 tgactttgat ctggacatgc tcggctccga tgctctggac gatttcgatc tcgatatgtt 7740 aattaactac ccgtacgacg ttccggacta cgcttcttgt acaggcagtg gagagggcag 7800 aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct ggcccaatgg ccaagccttt 7860 gtctcaagaa gaatccaccc tcattgaaag agcaacggct acaatcaaca gcatccccat 7920 ctctgaagac tacagcgtcg ccagcgcagc tctctctagc gacggccgca tcttcactgg 7980 tgtcaatgta tatcatttta ctgggggacc ctcgtggtgc tgggcactgc 8040 tgctgctgcg gcagctggca acctgacttg tatcgtcgcg atcggaaatg agaacagggg 8100 catcttgagc ccctgcggac ggtgccgaca ggtgcttctc gatctgcatc ctgggatcaa 8160 agccatagtg aaggacagtg atggacagcc gacggcagtt gggattcgtg aattgctgcc 8220 ctctggttat gtgtgggagg gctaagaatt cgatatcaag cttatcggta atcaacctct 828 0 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct 8340 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat 8400 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt gttgt 8460 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat 8520 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc 8580 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga 8640 caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc 8700 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga 8760 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc 8820 tcagacgagt cggatctccc tttgggccgc ctcc ccgcat cgataccgtc gacctcgaga 8880 cctagaaaaa catggagcaa tcacaagtag caatacagca gctaccaatg ctgattgtgc 8940 ctggctagaa gcacaagagg aggaggaggt gggttttcca gtcacacctc aggtaccttt 9000 aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg 9060 actggaaggg ctaattcact cccaacgaag acaagatatc cttgatctgt ggatctacca 9120 cacacaaggc tacttccctg attggcagaa ctacacacca gggccaggga tcagatatcc 9180 actgaccttt ggatggtgct acaagctagt accagttgag caagagaagg tagaagaagc 9240 caatgaagga gagaacaccc gcttgttaca ccctgtgagc ctgcatggga tggatgaccc 9300 ggagagagaa gtattagagt ggaggtttga ca gccgccta gcatttcatc acatggcccg 9360 agagctgcat ccggactgta ctgggtctct ctggttagac cagatctgag cctgggagct 9420 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca 9480 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta 9540 gtcagtgtgg aaaatctcta cccg tttaaacccg ctgatcagcc tcgactgtgc 9600 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag 9660 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta 9720 ggtgt cattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag 9780 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca 9840 gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg 9900 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg 9960 ctttcttccc ttcctttctc gccacg ttcg ccggctttcc ccgtcaagct ctaaatcggg 10020 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt 10080 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt 101 40 tggagtccac gttctttaat agtggactct tgttccaaac tggaaacaaca ctcaacccta 10200 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa 10260 atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg 10320 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta 10380 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat 10440 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac 10500 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga 10560 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag aggct tttttggagg 10620 cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca 10680 cgtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg 10740 aactaaacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc 10800 ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt ggaggacgac 10860 ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg 10920 gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag 10980 tggtcggagg gaacttccgg gacgcctccg ggccggccat gaccgagatc 11040 ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac 11100 ttcgtggccg aggagcagga ctgacacgtg ctacgagatt tcgattccac cgccgccttc 11160 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc 11220 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttatattgcagc ttataatggt 11280 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct 11340 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct 11400 agctagagct tggcgtaatc atggtcatag ctgtttcct g tgtgaaattg ttatccgctc 11460 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 11520 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 11580 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 11640 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 11700 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 11760 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 11820 gcgtttttcc ataggctccg cccccctgac gagcatca ca aaaatcgacg ctcaagtcag 11880 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 11940 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 12000 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 12060 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 12120 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 12180 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 12240 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 12300 gttaccttcg gaaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 12360 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 12420 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 12480 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 12540 tttaaatcaa tctaaagtat atatgagtaa acttgg tctg acagttacca atgcttaatc 12600 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 12660 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 12720 ccgcgagacc cacg ctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 12780 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 12840 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 12900 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 12960 cgatcaaggc gagttacatg atcccc catg ttgtgcaaaa aagcggttag ctccttcggt 13020 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 13080 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 13140 tcaaccaagt cat tctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 13200 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaaacgt 13260 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 13320 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 13380 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 13440 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 13500 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 13560cgaaaagtgc cacctgac 13578 <210> 27 <211> 4104 <212> DNA <213> Artificial Sequence <220> <223>dCas9 <400> 27 atggataaga aatactcaat aggcttagct atcggcacaa atagcgtcgg atgggcggtg 60 atcactgatg aatataaggt tccgtctaaa aagttcaagg ttctgggaaa tacagaccgc 120 cacagtatca aaaaaaatct tataggggct cttttatttg acagtggaga gacagcggaa 180 gcgactcgtc tcaaacggac agctcgtaga aggtatacac gtcggaagaa tcgtatttgt 240 tatctacagg agattttttc aaatgagatg gcgaaagtag atgatagttt ctttcatcga 300 cttgaagagt cttttttggt ggaagaagac aagaagcatg aacgtcatcc tatttttgga 360 aatatagtag atgaagttgc ttatcatgag aaatatccaa ctatctatca tctgcgaaaa 420 aaattggtag attctactga taaagcggat ttgcgcttaa tctatttggc cttagcgcat 480 atgattaagt ttcgtggtca ttttttgatt gagggagatt taaatcctga taatagtgat 540 gtggacaaac tatttatcca gttggtacaa acctacaatc aattatttga agaaaaccct 600 attaacgcaa gtggagtaga tgctaaagcg attctttctg cacgattgag taaatcaaga 660 cgattagaaa atctcattgc tcagctcccc ggtgagaaga aaaatggctt atttgggaat 720 ctcattgctt tgtcattggg tttgacccct aattttaaat caaattttga tttggcagaa 780 gatgctaaat tacagctttc aaaagatact tacgatgatg atttagataa tttatggcg 840 caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc agatgctatt 900 ttactttcag atatcctaag agtaaatact gaaataacta aggctcccct atcagcttca 960 atgattaaac gctacgatga acatcatcaa gacttgactc ttttaaaagc tttagttcga 1020 caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa cggatatgca 1080 ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa accaatttta 1140 gaaaaaatgg atggtactga ggaattattg gtgaaactaa atcgtgaaga tttgctgcgc 1200 aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg tgagctgcat 1260 gctattttga gaagacaaga agacttttat ccatttttaa aagacaatcg tgagaagatt 1320 gaaaaaatct tgacttttcg aattccttat tatgttggtc cattggcgcg tggcaatagt 1380 cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa ttttgaagaa 1440 gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa ctttgataaa 1500 aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta ttttacggtt 1560 tataacgaat tgacaaaggt caaatatgtt actgaaggaa tgcgaaaacc agcatttctt 1620 tcaggtgaac agaagaaagc cattgttgat ttactcttca aaaacaaatcg aaaagtaacc 1680 gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttgatag tgttgaaatt 1740 tcaggagttg aagatagatt taatgcttca ttaggtacct accatgattt gctaaaaatt 1800 attaaagata aagatttttt ggataatgaa gaaaatgaag atatcttaga ggatattgtt 1860 ttaacattga ccttattga agatagggag atgattgagg aaagacttaa aacatatgct 1920 cacctctttg atgataaggt gatgaaacag cttaaacgtc gccgttatac tggttgggga 1980 cgtttgtctc gaaaattgat taatggtatt agggataagc aatctggcaa aacaatatta 2040 gattttttga aatcagatgg ttttgccaat cgcaatttta tgcagctgat ccatgatgat 2100 agtttgacat ttaaagaaga cattcaaaaa gcacaagtgt ctggacaagg cgatagttta 2160 catgaacata ttgcaaattt agctggtagc cctgctatta aaaaaggtat tttacagact 2220 gtaaaagttg ttgatgaatt ggtcaaagta atggggcggc ataagccaga aaatatcgtt 2280 attgaaatgg cacgtgaaaa tcagacaact caaaagggcc agaaaaattc gcgagagcgt 2340 atgaaacgaa tcgaagaagg tatcaaagaa ttaggaagtc agattcttaa agagcatcct 2400 gttgaaaata ctcaattgca aaatgaaaag ctctatctct attatctcca aaatggaaga 2460 gacatgtatg tggaccaaga attagatatt aatcgtttaa gtgattatga tgtcgatgcc 2520 attgttccac aaagtttcct taaagacgat tcaatagaca ataaggtctt aacgcgttct 2580 gataaaaatc gtggtaaatc ggataacgtt ccaagtgaag aagtagtcaa aaagatgaaa 2640 aactattgga gacaacttct aaacgccaag ttaatcactc aacgtaagtt tgataattta 2700 acgaaagctg aacgtggagg tttgagtgaa cttgataaag ctggttttat caaacgccaa 2760 ttggttgaaa ctcgccaaat cactaagcat gtggcacaaa ttttggatag tcgcatgaat 2820 actaaatacg atgaaaatga taaacttatt cgagaggtta aagtgattac cttaaaatct 2880 aaattagttt ctgacttccg aaaagatttc caattctata aagtacgtga gattaacaat 2940 taccatcatg cccatgatgc gtatctaaat gccgtcgttg gaactgcttt gattaagaaa 3000 tatccaaaac ttgaatcgga gtttgtctat ggtgattata aagtttatga tgttcgtaaa 3060 atgattgcta agtctgagca agaaataggc aaagcaaccg caaaatattt cttttactct 3120 aatatcatga acttcttcaa aacagaaatt acacttgcaa atggagagat tcgcaaacgc 3180 cctctaatcg aaactaatgg ggaaactgga gaaattgtct gggataaagg gcgagatttt 3240 gccacagtgc gcaaagtatt gtccatgccc caagtcaata ttgtcaagaa aacagaagta 3300 cagacaggcg gattctccaa ggagtcaatt ttaccaaaaa gaaattcgga caagcttatt 3360 gctcgtaaaa aagactggga tccaaaaaaaa tatggtggtt ttgatagtcc aacggtagct 3420 tattcagtcc tagtggttgc taaggtggaa aaagggaaat cgaagaagtt aaaatccgtt 3480 aaagagttac tagggatcac aattatggaa agaagttcct ttgaaaaaaa tccgattgac 3540 tttttagaag ctaaaggata taaggaagtt aaaaaagact taatcattaa actacctaaa 3600 tatagtcttt ttgagttaga aaacggtcgt aaacggatgc tggctagtgc cggagaatta 3660 caaaaaggaa atgagctggc tctgccaagc aaatatgtga attttttata tttagctagt 3720 cattatgaaa agttgaaggg tagtccagaa gataacgaac aaaaacaatt gtttgtggag 3780 cagcataagc attattaga tgagattatt gagcaaatca gtgaattttc taagcgtgtt 3840 attttagcag atgccaattt agataaagtt cttagtgcat ataacaaaca tagagacaaa 3900 ccaatacgtg aacaagcaga aaatattatt catttattta cgttgacgaa tcttggagct 3960 cccgctgctt ttaaatattt tgatacaaca attgatcgta aacgatatac gtctacaaaa 4020 gaagttttag atgccactct tatccatcaa tccatcactg gtctttatga aacacgcatt 4080 gatttgagtc agctaggagg tgac 4104 <210> 28 <211> 510 <212> DNA <213> Artificial Sequence <220> <223> CIBN <400> 28 atgaatggag ctataggagg tgaccttttg ctcaattttc ctgacatgtc ggtcctagag 60 cgccaaaggg ctcacctcaa gtacctcaat cccacctttg attctcctct cgccggcttc 120 tttgccgatt cttcaatgat taccggcggc gagatggaca gctatctttc gactgccggt 180 ttgaatcttc cgatgatgta cggtgagacg acggtggaag gtgattcaag actctcaatt 240 tcgccggaaa cgacgcttgg gactggaaat ttcaagaaac ggaagtttga tacagagact 300 aaggattgta atgagaagaa gaagaagatg acgatgaaca gagatgacct agtagaagaa 360 ggagaagaag agaagtcgaa aataacagag caaaacaatg ggagcacaaa aagcatcaag 420 aagatgaaac acaaagccaa gaaagaagag aacaatttct ctaatgattc atctaaagtg 480 acgaaggaat tggagaaaac ggattatatt 510 <210> 29 <211> 1836 <212> DNA <213> Artificial Sequence <220> <223> CRY2FL <400> 29 atgaagatgg acaaaaagac tatagtttgg tttagaagag acctaaggat tgaggataat 60 cctgcattag cagcagctgc tcacgaagga tctgtttttc ctgtcttcat ttggtgtcct 120 gaagaagaag gacagtttta tcctggaaga gcttcaagat ggtggatgaa acaatcactt 180 gctcacttat ctcaatcctt gaaggctctt ggatctgacc tcactttaat caaaacccac 240 aacacgattt cagcgatctt ggattgtatc cgcgttaccg gtgctacaaa agtcgtcttt 300 aaccacctct atgatcctgt ttcgttagtt cgggaccata ccgtaaagga gaagctggtg 360 gaacgtggga tctctgtgca aagctacaat ggagatctat tgtatgaacc gtgggagata 420 tactgcgaaa agggcaaacc ttttacgagt ttcaattctt actggaagaa atgcttagat 480 atgtcgattg aatccgttat gcttcctcct ccttggcggt tgatgccaat aactgcagcg 540 gctgaagcga tttgggcgtg ttcgattgaa gaactagggc tggagaatga ggccgagaaa 600 ccgagcaatg cgttgttaac tagagcttgg tctccaggat ggagcaatgc tgataagtta 660 ctaaatgagt tcatcgagaa gcagttgata gattatgcaa agaacagcaa gaaagttgtt 720 gggaattcta cttcactact ttctccgtat ctccatttcg gggaaataag cgtcagacac 780 gttttccagt gtgcccggat gaaacaaatt atatgggcaa gagataagaa cagtgaagga 840 gaagaaagtg cagatctttt tcttagggga atcggtttaa gagagtattc tcggtatata 900 tgtttcaact tcccgtttac tcacgagcaa tcgttgttga gtcatcttcg gtttttccct 960 tgggatgctg atgttgataa gttcaaggcc tggagacaag gcaggaccgg ttatccgttg 1020 gtggatgccg gaatgagaga gctttgggct accggatgga tgcataacag aataagagtg 1080 attgtttcaa gctttgctgt gaagtttctt ctccttccat ggaaatgggg aatgaagtat 1140 ttctgggata cacttttgga tgctgatttg gaatgtgaca tccttggctg gcagtatatc 1200 tctgggagta tccccgatgg ccacgagctt gatcgcttgg acaatcccgc gttacaaggc 1260 gccaaatatg accgaagg tgagtacata aggcaatggc ttcccgagct tgcgagattg 1320 ccaactgaat ggatccatca tccatgggac gctcctttaa ccgtactcaa agcttctggt 1380 gtggaactcg gaacaaacta tgcgaaaccc attgtagaca tcgacacagc tcgtgagcta 1440 ctagctaaag ctatttcaag aacccgtgaa gcacagatca tgatcggagc agcacctgat 1500 gagattgtag cagatagctt cgaggcctta ggggctaata ccattaaaga acctggtctt 1560 tgcccatctg tgtcttctaa tgaccaacaa gtaccttcgg ctgttcgtta caacgggtca 1620 aagagagtga aacctgagga agaagaagag agagacatga agaaatctag gggattcgat 1680 gaaagggagt tgttttcgac tgctgaatct tcttcttctt cgagtgtgtt tttcgtttcg 1740 cagtcttgct cgttggcatc agaagggaag aatctggaag gtattcaaga ttcatctgat 1800 cagattacta caagtttggg aaaaaatggt tgcaaa 1836 <210> 30 <211> 150 <212> DNA <213> Artificial Sequence <220> <223> VP64 <400>30 gacgcgctgg acgatttcga tctcgacatg ctgggttctg atgccctcga tgactttgac 60 ctggatatgt tgggaagcga cgcattggat gactttgatc tggacatgct cggctccgat 120 gctctggacg atttcgatct cgatatgtta 150

Claims (19)

A first vector comprising polynucleotides each encoding a dead Cas9 (dCas9) protein and a cryptochrome-interacting basic helix-loop-helix protein (CIB1) protein; and
Guide RNA (gRNA) of the target genes of INS, GCK, PCSK1, and PCSK2 whose expression in stem cells is to be regulated, mU6 promoter, hU6 promoter, hH1 promoter, and h7SK promoter for the gRNA of each target gene, and CRY2 ( A kit for transformation of stem cells, comprising a second vector containing polynucleotides each encoding a cryptochrome 2) protein,
A kit for transformation of stem cells, wherein the first vector contains the base sequence of SEQ ID NO: 21, and the second vector contains any one of the base sequences of SEQ ID NOs: 22 to 26. delete delete delete The kit for transformation of stem cells according to claim 1, wherein the first vector further includes a promoter selected from the group consisting of the Ef1α promoter, the UbC promoter, and the CAGG promoter. delete The kit for transformation of stem cells according to claim 1, wherein the second vector further includes a promoter selected from the group consisting of the Ef1α promoter, the UbC promoter, and the CAGG promoter. delete The method of claim 1, wherein the stem cells are embryonic stem cells (ES), adult stem cells, or induced pluripotent stem cells (iPSC). Kit for. The kit for transformation of stem cells according to claim 9, wherein the embryonic stem cells are somatic cell cloned embryonic stem cells. delete The method of claim 1, wherein the polynucleotide encoding the gRNA of the INS includes any one base sequence selected from the group consisting of the base sequences of SEQ ID NOs: 1 to 5 and their respective complementary base sequences;
The polynucleotide encoding the gRNA of GCK includes any one base sequence selected from the group consisting of the base sequences of SEQ ID NOs: 6 to 10 and their respective complementary base sequences;
The polynucleotide encoding the gRNA of PCSK1 includes any one base sequence selected from the group consisting of the base sequences of SEQ ID NOs: 11 to 15 and their respective complementary base sequences; and
A kit for transformation of stem cells, wherein the polynucleotide encoding the gRNA of PCSK2 includes any one base sequence selected from the group consisting of the base sequences of SEQ ID NOs: 16 to 20 and their respective complementary base sequences. . Stem cells transformed using the kit for transformation of stem cells according to any one of claims 1, 5, 7, 9, 10, and 12. The method of claim 13, wherein when light having a wavelength of 440 to 490 nm is irradiated to the transformed stem cells, INS, GCK, PCSK1, and PCSK2 gene expression of the stem cells increases. delete The method of claim 14, wherein when irradiation of light is stopped to stem cells in which the expression of the two or more target genes is increased, the increased expression of the INS, GCK, PCSK1, and PCSK2 genes decreases again. A method of regulating target gene expression in stem cells comprising simultaneously increasing the INS, GCK, PCSK1, and PCSK2 gene expression of the stem cells by irradiating the transformed stem cells of claim 13 with light having a wavelength of 440 to 490 nm. . delete The method of claim 17, wherein the light is light having a pulse of 30 to 70%.