CN110511286B - RNA base editing molecule - Google Patents

Abstract

The invention relates to the field of biotechnology, in particular to a fusion protein which can be an RNA base editing molecule. The invention provides a fusion protein, which comprises a dCas13Rx fragment, wherein an ADAR2DD fragment is inserted into a loop3 site of the dCas13Rx fragment in a chimeric way. The invention realizes accurate RNA positioning capacity by utilizing the protein dCasRx capable of specifically targeting RNA in the CRISPR system, embeds unbiased RNA double-stranded deaminase ADAR into the dCasRx on the premise of not changing the targeting activity of the dCasRx, not only blocks the random off-target activity of the ADAR on double-stranded RNA, but also realizes the RNA base editing purpose of a specific site under the mediation of the dCasRx, not only has higher base editing efficiency, but also depends on the specificity of CasRx more, and only needs shorter gRNA for guidance, so the accuracy is higher, and the protein dCasRx has the advantage of adult treatment because the volume is small, can be packaged into AAV.

Description

RNA base editing molecule

Technical Field

The invention relates to the field of biotechnology, in particular to a fusion protein which can be an RNA base editing molecule.

Background

With the progress of RNA base editing technology, RNA-dependent Adenosine Deaminase (ADAR) has been able to carry out highly efficient adenine (a) to hypoxanthine (I) mutations at the site of interest (Woolf et al, 1995); however, over-expressed ADAR causes severe off-target of transcriptome RNA (valley cillo-Viejo et al, 2018), thus limiting the application of ADAR-based RNA editing techniques. Therefore, it is still a difficult point to realize precise, efficient and low off-target base editing by using the CasRx protein mediated ADAR of the target RNA in the CRISPR system.

Single-base editing can BE realized at the DNA and RNA level, but the currently reported DNA base editing tools ABE and BE3 depend on a CRISPR/Cas9 system and have PAM limitation, so that all sites cannot BE covered (Rees and Liu, 2018). The RNA base editing system has no PAM dependency, has a wider editing range, and can recode genetic information at the RNA level, resulting in changes in protein function and RNA processing. In the case of compensatory responses that may be caused by DNA mutations, RNA editing techniques can compensate for this gap. Secondly, modulation of the relative abundance of alternatively spliced transcripts can be optimally achieved by manipulation at the RNA level (Konermann et al, 2018). Importantly, changes in RNA levels are reversible and therefore safer than DNA editing.

RNA site-directed base editing techniques were first reported in 1995 (Woolf et al, 1995). Various RNA base editing techniques developed at present use guide RNA to guide ADAR deaminase to a target site to achieve site-directed editing (egglington et al, 2011). However, these methods have various limitations and cannot achieve high efficiency, accuracy and convenience at the same time. For example, in 2018, Science reports that efficient RNA site-directed editing is achieved by using Cas13b protein capable of specifically targeting RNA in CRISPR system to mediate ADAR, but its off-target effect is severe, and the optimized V2 version reduces off-target effect but also greatly affects editing efficiency (Cox et al, 2017). And Cas13b requires a longer (40-50nt) guideRNA, which has inherent disadvantages.

The current RNA base editing technology is divided into two major categories, one is to edit a target site by using ADAR expressed in cells under the mediation of chemically modified gRNA, but not all cells express ADAR, and the background expression amount of ADAR in different cells is different, which will affect the editing efficiency (Merkle et al, 2019). Another category requires overexpression of ADAR, but direct overexpression of ADAR can cause severe transcriptome-level off-target (egglington et al, 2011).

Disclosure of Invention

In view of the above-described drawbacks of the prior art, it is an object of the present invention to provide a base editing molecule for solving the problems of the prior art.

To achieve the above and other related objects, the present invention provides a fusion protein comprising dCas13Rx fragment, wherein ADAR2DD fragment is inserted into the loop3 site of dCas13Rx fragment.

In some embodiments of the invention, the fusion protein comprises, in order from N-terminus to C-terminus, a first dCas13Rx fragment, an ADAR2DD fragment, and a second dCas13Rx fragment.

In some embodiments of the invention, the amino acid sequence of the first dCas13Rx fragment comprises:

a) an amino acid sequence shown as SEQ ID NO. 1; or the like, or, alternatively,

b) an amino acid sequence having a sequence similarity of 80% or more to SEQ ID No.1 and having the function of the amino acid sequence defined in a), preferably having dCasRx targeting activity;

and/or the amino acid sequence of the second dCas13Rx fragment comprises:

e) an amino acid sequence shown as SEQ ID NO. 3; or the like, or, alternatively,

f) an amino acid sequence having a sequence similarity of 80% or more to SEQ ID NO.3 and having the function of the amino acid sequence defined in e), preferably having dCasRx targeting activity; .

In some embodiments of the invention, the amino acid sequence of the ADAR2DD fragment comprises:

c) an amino acid sequence shown as SEQ ID NO. 2; or the like, or, alternatively,

d) an amino acid sequence having a sequence similarity of 80% or more to SEQ ID NO.2 and having the function of the amino acid sequence defined in c); preferably having adenosine deaminase activity.

In some embodiments of the invention, the fusion protein further comprises an NLS fragment, preferably, the N-terminus of the first dCas13Rx fragment, between the first dCas13Rx fragment and ADAR2DD fragment, between the ADAR2DD fragment and the second dCas13Rx fragment, and the C-terminus of the second dCas13Rx fragment are provided with the NLS fragment, preferably, the NLS fragment comprises an amino acid sequence as shown in SEQ ID No. 4.

In some embodiments of the invention, the amino acid sequence of the fusion protein comprises the amino acid sequence shown as SEQ ID No. 5.

In another aspect, the present invention provides an isolated polynucleotide encoding the fusion protein described above.

In another aspect, the invention provides a construct comprising the isolated polynucleotide described above.

In another aspect, the present invention provides an expression system comprising the above-described construct or a polynucleotide having an exogenous sequence integrated into its genome.

In some embodiments of the invention, the host cell of the expression system is selected from eukaryotic cells or prokaryotic cells, preferably from mouse cells, more preferably from mouse brain neuroma cells, more preferably from N2a cells.

In another aspect, the present invention provides the use of the fusion protein described above, the isolated polynucleotide described above, the construct described above or the expression system described above in gene editing.

In some embodiments of the invention, the use is in particular in gene editing in eukaryotes.

In another aspect, the invention provides a base editing system, which includes the fusion protein, and the base editing system further includes sgRNA.

In another aspect, the present invention provides a gene editing method, including: the gene is edited by the above-mentioned fusion protein or the above-mentioned base editing system.

Drawings

FIG. 1 is a schematic diagram of Vx, wherein the upper diagram is a schematic diagram of a Vx carrier, and the lower diagram is a schematic diagram of a Vx structure.

FIG. 2 is a fluorescent reporter system design.

FIG. 3 is a graph of the position of A within the deep-seq sequencing range.

FIG. 4 shows FACS results of efficiency of Vx editing on the reporting system.

FIG. 5 shows the fluorescent results of efficiency of Vx editing on a reporter system under guidance of sgRNA of 30nt length

FIG. 6 is the deep-seq sequencing analysis of Vx on a reporter system (heat map).

FIG. 7 is an edit of Vx flanking non-target A on the reporting system.

FIG. 8a shows the results of the fluorescence ratios of efficiencies compiled by Vx on the reporter system under different lengths of sgRNA guidance.

FIG. 8b is the sequencing result of efficiency of Vx editing on the reporter system under guidance of different length sgRNAs.

FIG. 9 shows the result of dep-seq sequencing analysis of Vx at target site editing A45 guided by sgRNAs of different lengths.

FIG. 10a shows the fluorescence results of the editing efficiency of Vx under sgRNA guidance of 22nt length of different mismatches C.

FIG. 10b shows the result of deep-seq sequencing analysis of target site A45 with Vx guided by sgRNA of 22nt length of different mismatches C.

FIG. 11 shows the sequencing analysis of deep-seq for Vx targeting different bases on both sides of A.

FIG. 12a is the fluorescence scale results of the efficiency of Vx editing at the reporter system targeting different sites.

FIG. 12b is the sequencing analysis result of the edit efficiency deep-seq of Vx in targeting A in different reporting systems.

FIG. 13 shows the result of editing on endogenous transcripts

FIG. 14 shows the whole transcriptome off-target for V1, V2, and Vx at 50nt sgRNA guide.

Detailed Description

In order to make the objects, technical solutions and advantageous technical effects of the present invention more clear, the present invention is further described in detail with reference to the following embodiments. It should be understood that the embodiments described in this specification are only for the purpose of explaining the present invention and are not intended to limit the present invention.

For the sake of brevity, only some numerical ranges are explicitly disclosed herein. However, any lower limit may be combined with any upper limit to form ranges not explicitly recited; and any lower limit may be combined with any other lower limit to form a range not explicitly recited, and any upper limit may be combined with any other upper limit to form a range not explicitly recited. Also, although not explicitly recited, each point or individual numerical value between the endpoints of a range is encompassed within that range. Thus, each point or individual value can form a range not explicitly recited as its own lower or upper limit in combination with any other point or individual value or in combination with other lower or upper limits.

In the description herein, it is to be noted that, unless otherwise specified, "above" and "below" are inclusive, and "one or more" of "plural" means two or more.

The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The following description more particularly exemplifies illustrative embodiments. At various points throughout this application, guidance is provided through a list of embodiments that can be used in various combinations. In each instance, the list is merely a representative group and should not be construed as exhaustive.

The present inventors have conducted extensive exploratory studies and provided a fusion protein, which is a novel base editing molecule, and which can include a dCas13Rx fragment and an ADAR2DD fragment, can achieve efficient editing of a > I in a sgRNA target region, and has higher efficiency and specificity, thereby completing the present invention.

The invention provides a fusion protein, which comprises a dCas13Rx fragment, wherein an ADAR2DD fragment is inserted into a loop3 site of the dCas13Rx fragment in a chimeric mode. The specific position of the loop3 site of the dCas13Rx fragment is known to those skilled in the art (DOI: https:// doi.org/10.1016/j.cell.2018.09.001), for example, the position can be the position between 558Asn and 587Met of dCas13Rx fragment, the 559-586 amino acid fragment is replaced by an ADAR2DD fragment, the ADAR2DD fragment is chimeric between dCasRx to reduce the off-target effect of ADAR, and base editing is performed at the target site under the guide of sgRNA, so that the base editing efficiency of the target site can be increased.

In the fusion protein provided by the invention, the loop3 site of the dCas13Rx fragment is inserted with the ADAR2DD fragment in a chimeric way, so that the dCas13Rx fragment can comprise a first dCas13Rx fragment positioned at the N-terminal of the ADAR2DD fragment and a second dCas13Rx fragment positioned at the C-terminal of the ADAR2DD fragment, and the fusion protein sequentially comprises the first dCas13Rx fragment, the ADAR2DD fragment and the second dCas13Rx fragment from the N-terminal to the C-terminal.

In the fusion protein provided by the present invention, the amino acid sequence of the first dCas13Rx fragment may include: a) an amino acid sequence shown as SEQ ID NO. 1; or b) an amino acid sequence having a sequence similarity of 80% or more to SEQ ID NO.1 and having the function of the amino acid sequence defined in a). Specifically, the amino acid sequence in b) specifically refers to: the amino acid sequence shown as SEQ ID No.1 is obtained by substituting, deleting or adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2 or 3) amino acids, or by adding one or more (specifically, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1, 2, or 3) amino acids to the N-terminus and/or C-terminus, and has the function of the polypeptide fragment with the amino acid shown as SEQ ID No.1, for example, it may be that the first dCas13Rx fragment still has the targeting activity of dCasRx after being complexed with the second dCas13Rx fragment, and more particularly may be capable of targeting RNA under the direction of an appropriate gRNA. The amino acid sequence in b) may have more than 80%, 85%, 90%, 93%, 95%, 97%, or 99% similarity to SEQ ID No. 1.

IEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARLEKIVEGDSIRSVNEGEAFSAEMADKNAGYKIGNAKFSHPKGYAVVANNPLYTGPVQQDMLGLKETLEKRYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDKDIIGFGKFSTVYTYDEFKDPEHHRAAFNNNDKLINAIKAQYDEFDNFLDNPRLGYFGQAFFSKEGRNYIINYGNECYDILALLSGLAHWVVANNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITNELTNSFSKNSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDRKDMSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFVINLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAGRDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAFFKDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTN(SEQ ID NO.1)

In the fusion protein provided by the present invention, the amino acid sequence of the second dCas13Rx fragment may include: e) an amino acid sequence shown as SEQ ID NO. 3; or, f) an amino acid sequence having a sequence similarity of 80% or more to SEQ ID NO.3 and having the function of the amino acid sequence defined in e). Specifically, the amino acid sequence in f) specifically refers to: the amino acid sequence shown as SEQ ID No.3 is obtained by substituting, deleting or adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2 or 3) amino acids, or by adding one or more (specifically, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1, 2, or 3) amino acids to the N-terminus and/or C-terminus, and has the function of the polypeptide fragment having the amino acid shown in SEQ ID No.3, for example, it may be that the first dCas13Rx fragment still has the targeting activity of dCasRx after being complexed with the second dCas13Rx fragment, and more particularly may be capable of targeting RNA under the direction of an appropriate gRNA. The amino acid sequence in f) may have more than 80%, 85%, 90%, 93%, 95%, 97%, or 99% similarity to SEQ ID No. 3.

MRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQKKQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGRENAEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEEKGFSSVTKLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEKKAEEFTRQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFANKAVALEVARYVHAYINDIAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYNDRLLKLLCVPFGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS(SEQ ID NO.3)

In the fusion protein provided by the present invention, the ADAR2DD fragment generally refers to the deamidase domain of ADAR, and the amino acid sequence of the ADAR2DD fragment may include: c) an amino acid sequence shown as SEQ ID NO. 2; or d) an amino acid sequence having a sequence similarity of 80% or more to SEQ ID NO.2 and having the function of the amino acid sequence defined in c). Specifically, the amino acid sequence in d) specifically refers to: the amino acid sequence shown as SEQ ID No.2 is obtained by substituting, deleting or adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2 or 3) amino acids, or by adding one or more (specifically, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, or 1 to 3, 1, 2, or 3) amino acids to the N-terminus and/or C-terminus, and has the function of the polypeptide fragment with the amino acid shown as SEQ ID No.2, for example, may have adenosine deaminase activity, and more specifically may be a function of editing adenine (A) into hypoxanthine (I). The amino acid sequence in d) may have more than 80%, 85%, 90%, 93%, 95%, 97%, or 99% similarity to SEQ ID No. 2.

QLHLPQVLADAVSRLVLGKFGDLTDNFSSPHARRKVLAGVVMTTGTDVKDAKVISVSTGTKCINGEYMSDRGLALNDCHAEIISRRSLLRFLYTQLELYLNNKDDQKRSIFQKSERGGFRLKENVQFHLYISTSPCGDARIFSPHEPILEEPADRHPNRKARGQLRTKIESGQGTIPVRSNASIQTWDGVLQGERLLTMSCSDKIARWNVVGIQGSLLSIFVEPIYFSSIILGSLYHGDHLSRAMYQRISNIEDLPPLYTLNKPLLSGISNAEARQPGKAPNFSVNWTVGDSAIEVINATTGKDELGRASRLCKHALYCRWMRVHGKVPSHLLRSKITKPNVYHESKLAAKEYQAAKARLFTAFIKAGLGAWVEKPTEQDQFSLT(SEQ ID NO.2)

In the fusion protein provided by the invention, the substitution, deletion or addition can be conservative amino acid substitution. The "conservative amino acid substitution" may specifically refer to the case where an amino acid residue is substituted with another amino acid residue having a similar side chain. Families of amino acid residues with similar side chains should be known to those skilled in the art and may be, for example, families including, but not limited to, basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). More specifically, conservative amino acid substitutions may include, but are not limited to, the particulars listed in the following table, where the numbers in table 1 (amino acid similarity matrix) indicate the degree of similarity between two amino acids, and where the numbers are greater than or equal to 0, they are considered conservative amino acid substitutions, and table 2 is an exemplary scheme of conservative amino acid substitutions.

TABLE 1

	C	G	P	S	A	T	D	E	N	Q	H	K	R	V	M	I	L	F	Y	W
																					W	-8	-7	-6	-2	-6	-5	-7	-7	-4	-5	-3	-3	2	-6	-4	-5	-2	0	0	17
Y	0	-5	-5	-3	-3	-3	-4	-4	-2	-4	0	-4	-5	-2	-2	-1	-1	7	10
																					F	-4	-5	-5	-3	-4	-3	-6	-5	-4	-5	-2	-5	-4	-1	0	1	2	9
L	-6	-4	-3	-3	-2	-2	-4	-3	-3	-2	-2	-3	-3	2	4	2	6
																					I	-2	-3	-2	-1	-1	0	-2	-2	-2	-2	-2	-2	-2	4	2	5
M	-5	-3	-2	-2	-1	-1	-3	-2	0	-1	-2	0	0	2	6
																					V	-2	-1	-1	-1	0	0	-2	-2	-2	-2	-2	-2	-2	4
R	-4	-3	0	0	-2	-1	-1	-1	0	1	2	3	6
																					K	-5	-2	-1	0	-1	0	0	0	1	1	0	5
H	-3	-2	0	-1	-1	-1	1	1	2	3	6
																					Q	-5	-1	0	-1	0	-1	2	2	1	4
N	-4	0	-1	1	0	0	2	1	2
																					E	-5	0	-1	0	0	0	3	4
D	-5	1	-1	0	0	0	4
																					T	-2	0	0	1	1	3
A	-2	1	1	1	2
																					S	0	1	1	1
P	-3	-1	6
																					G	-3	5
C	12

TABLE 2

In the fusion protein provided by the invention, the fusion protein can also comprise an NLS (nuclear localization sequence) fragment. The NLS fragment may be located N-terminal to the first dCas13Rx fragment, between the first dCas13Rx fragment and the ADAR2DD fragment, between the ADAR2DD fragment and the second dCas13Rx fragment, or C-terminal to the second dCas13Rx fragment. The NLS fragment can comprise an amino acid sequence shown as SEQ ID NO. 4.

KRTADGSEFESPKKKRKV(SEQ ID NO.4)

In a specific embodiment of the present invention, the amino acid sequence of the fusion protein comprises the amino acid sequence shown as SEQ ID NO. 5.

MKRTADGSEFESPKKKRKVGSIEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARLEKIVEGDSIRSVNEGEAFSAEMADKNAGYKIGNAKFSHPKGYAVVANNPLYTGPVQQDMLGLKETLEKRYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDKDIIGFGKFSTVYTYDEFKDPEHHRAAFNNNDKLINAIKAQYDEFDNFLDNPRLGYFGQAFFSKEGRNYIINYGNECYDILALLSGLAHWVVANNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITNELTNSFSKNSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDRKDMSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFVINLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAGRDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAFFKDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTNGSKRTADGSEFESPKKKRKVGSQLHLPQVLADAVSRLVLGKFGDLTDNFSSPHARRKVLAGVVMTTGTDVKDAKVISVSTGTKCINGEYMSDRGLALNDCHAEIISRRSLLRFLYTQLELYLNNKDDQKRSIFQKSERGGFRLKENVQFHLYISTSPCGDARIFSPHEPILEEPADRHPNRKARGQLRTKIESGQGTIPVRSNASIQTWDGVLQGERLLTMSCSDKIARWNVVGIQGSLLSIFVEPIYFSSIILGSLYHGDHLSRAMYQRISNIEDLPPLYTLNKPLLSGISNAEARQPGKAPNFSVNWTVGDSAIEVINATTGKDELGRASRLCKHALYCRWMRVHGKVPSHLLRSKITKPNVYHESKLAAKEYQAAKARLFTAFIKAGLGAWVEKPTEQDQFSLTGSKRTADGSEFESPKKKRKVGSMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQKKQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGRENAEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEEKGFSSVTKLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEKKAEEFTRQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFANKAVALEVARYVHAYINDIAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYNDRLLKLLCVPFGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNSGSKRTADGSEFESPKKKRKV(SEQ ID NO.5)

In a second aspect, the present invention provides an isolated polynucleotide encoding a fusion protein provided by the first aspect of the present invention.

In a third aspect, the invention provides a construct comprising an isolated polynucleotide provided in the second aspect of the invention. The construct can be generally constructed by inserting the isolated polynucleotide into a suitable expression vector, which can be selected by one of skill in the art, for example, including but not limited to bacterial expression vectors, fungal expression vectors, animal expression vectors (e.g., insect, drosophila, nematode, fish, zebrafish, mammalian, mouse, rat, rabbit, pig, monkey, human, etc.), plant expression vectors, and the like.

In a fourth aspect, the invention provides an expression system comprising a construct or genome provided by the third aspect of the invention and integrated therein an exogenous isolated polynucleotide provided by the second aspect of the invention. The expression system can be a host cell that can express the fusion protein as described above, which can cooperate with the sgRNA such that the fusion protein can be targeted to the target region, enabling base editing of the target region. In another embodiment of the present invention, the host cell may be a eukaryotic cell or a prokaryotic cell, more specifically a mouse cell, a human cell, etc., more specifically a mouse brain neuroma cell, a human kidney epithelial cell line, etc., more specifically N2a cell, 293T, etc.

In a fifth aspect, the present invention provides the use of the fusion protein provided in the first aspect of the present invention, or the isolated polynucleotide provided in the second aspect of the present invention, or the construct provided in the third aspect of the present invention, or the expression system provided in the fourth aspect of the present invention in gene editing, preferably in gene expression of eukaryotes, particularly metazoan, particularly, metazoan, including, but not limited to, mice, etc. The use may specifically be, but is not limited to, repair of a mutation in the gene from G to A, restoration of translation of an inactive protein resulting in a stop codon from a single base mutation, alteration of an RNA clip, and the like. In one embodiment of the present invention, the Gene to be edited may be PRKN (Gene ID: 5071), ADGRV1(Gene ID: 84059), AHI1(Gene ID: 54806), APC (Gene ID: 324), COL3A1(Gene ID: 1281), DNAH5(Gene ID: 7), MECP2(Gene ID: 4204), MYBPC3(Gene ID: 4607), BRCA1(Gene ID: BMPR 2659), BMPR2(Gene ID: BRCA 1672), or the like.

In a sixth aspect, the invention provides a base editing system, including the fusion protein provided in the first aspect, the base editing system further including sgRNA. One skilled in the art can select an appropriate sgRNA targeting the target region based on the target editing region of the gene. For example, the sequence of the sgRNA can be at least partially complementary to the target region, so that it can cooperate with the fusion protein to localize the fusion protein to the target region, and allow base editing of the target region, which can be in particular adenine deamination, i.e. the editing of adenine (a) to hypoxanthine (I). The base editing system provided by the invention can realize editing under a shorter sgRNA system, and still has excellent editing activity under the condition that the sgRNA has a certain mismatching distance, for example, under the condition that the length of a nucleotide sequence of the sgRNA is not more than 25nt, good editing activity is still realized at each mismatching distance (for example, 3-18 nt), and further, for example, under the condition that the length of the nucleotide sequence of the sgRNA is not more than 22nt, the mismatching distance below 18nt still has editing activity, and the editing efficiency can reach about 35% -40% at the mismatching distance of 7 nt-9 nt. In a specific embodiment of the invention, the sgRNA can target genes such as PRKN, ADGRV1, AHI1, APC 22, COL3a1, DNAH5, MECP2, MYBPC3, BRCA1, BMPR2, and the like.

The seventh aspect of the present invention provides a base editing method comprising: the gene editing is performed by the fusion protein provided by the first aspect of the present invention or the base editing system provided by the sixth aspect of the present invention. For example, the gene editing method may include: culturing the expression system provided by the fourth aspect of the present invention under appropriate conditions to express the fusion protein, which can base-edit the target region in the presence of the sgRNA targeting the target region to which it is mated. Methods for providing conditions under which the sgRNA exists should be known to those skilled in the art, and for example, an expression system capable of expressing the sgRNA, which may be a host cell including an expression vector containing a polynucleotide encoding the sgRNA or a host cell having the polynucleotide encoding the sgRNA integrated in a chromosome, may be cultured under appropriate conditions. In one embodiment of the invention, the gene editing is in vitro gene editing.

The fusion protein provided by the invention has the advantages that the CasRx in the CRISPR system based on the fusion protein can target RNA under the guide of RNA, and the CasRx has only 954 amino acids, is the smallest one of the types VI-A, VI-B and VI-C of the current CRISPR system, and has great value for realizing adult treatment by packaging AAV. In addition, the CasRx protein can still retain activity after deleting some amino acid fragments, so that the ADAR is inserted into the CasRx protein, and the chimeric expression dCasRx (1-558) -ADAR-dCasRx (587-966) protein still has the RNA targeting activity of CasRx and deamination of the ADAR mutation target site. Through the CasRx which is a CasRx protein capable of targeting RNA in a CRISPR system, ADAR is fused in the middle position of a dCas13Rx protein, the fusion protein is guided by a gRNA, the target site location is realized by utilizing the precise targeting function of dCas13Rx on the RNA, and the deamination mutation of bases A to I is carried out on the ADAR wrapped in the middle of the CasRx. In addition, the inventor finds that dCasRx wrapped at two ends of the ADAR protein can block the combination of the ADAR to non-target RNA, so that the non-target off-target effect is reduced, and the aim of not only targeting RNA but also reducing RNA base editing off-target is achieved by virtue of a CRISPR system.

Compared with a base editing method for directly over-expressing ADAR, the specificity of a new generation base editing system depending on CRISPR is higher; the gRNA is directly expressed by plasmid, does not need chemical synthesis, and has lower cost and more flexibility. Compared with the V1 version of dCas13b-ADAR system (Cox et al, 2017), the fusion protein provided by the invention (dCasRx-ADAR RNA (Vx) base editing system) is low off-target, the Vx system editing efficiency is higher compared to previous editing systems, and Vx is more dependent on sgRNA with DR sequence, suggesting that targeting is achieved by relying on dCasRx. In addition, V1 and V2 can be guided by sgrnas without DR, which indicates that the grnas mainly form a double-stranded structure with target RNA to recruit ADAR to perform editing, but not dCas13b, so that the fusion protein and the corresponding base editing system provided by the present invention can edit a target site under the guidance of shorter grnas (22 nt).

In conclusion, the invention realizes accurate RNA positioning capacity by utilizing the protein dCasRx capable of specifically targeting RNA in the CRISPR system, and embeds unbiased RNA double-strand deaminase ADAR into the dCasRx on the premise of not changing the targeting activity of dCasRx, thereby not only blocking the random off-target activity of the ADAR on double-strand RNA, but also realizing the purpose of RNA base editing of a specific site under the mediation of dCasRx, having higher base editing efficiency, being more dependent on the specificity of CasRx, and only needing shorter gRNA guide, so that the accuracy is higher, and the protein dCasRx has the advantage of adult treatment because the volume is small, and can be packaged into AAV. In addition, compared with a DNA base editing tool, the editing system provided by the invention is not limited by PAM, has wider editing sites, and does not cause off-target at the DNA level, thereby being safer.

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

It is to be understood that the processing equipment or apparatus not specifically identified in the following examples is conventional in the art.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORY MANUAL, Second edition, Cold Spring Harbor LABORATORY Press, 1989 and Third edition, 2001; ausubel et al, Current PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.

Example 1

Construction of Vx System plasmid:

pC0053-CMV-dPspCas13b-GS-ADAR2DD(E488Q)-delta-984-109(Addgene plasmid # 103869; http:// n2 t.net/addge: 103869; RRID: Addgene _103869) was gifted to the kytoppeak, and plasmid dCas13Rx was codon-optimized for synthesis in genshrit (GenScript). 4184F (CCGATGGATCGAGTTCGAATCCCCTAAAAAGAAAAGAGGTGGGATCCCAGCTGCATTTACCG) (SEQ ID NO.6) and 4184R (TCTTCTTGGGGCTCTCAAATTCGCTGCCGTCAGCAGTCCGTTTCATGGTGGCAAGCTTAAGTTTAAACGCTA) (SEQ ID NO.7) primers were diluted to 10. mu.M and the ADAR2DD (E488Q) fragment was amplified from pC0053 template using the Novozae Hi-Fi kit (Vazyme, p501-d 2). 4183F (AGAGAGCCCCAAGAAGAGAGGAAAGTCGGATCCATCGAGAAGAAGCTTCGCCAA) (SEQ ID NO.8) and 4183R (GGGGATTCGAACTCGGATCCATCGGGCGGTGCGCTTAGAACCGGAGTTGCCGCTCACCT) (SEQ ID NO.9) primers were diluted to 10. mu.M and the dCas13Rx fragment was amplified from synthetic dCas13Rx plasmid template using the Novozae Hi-Fi enzyme kit (Vazyme, p501-d 2). After the PCR amplification product was purified and recovered by AxyPrep PCR Clean-up Kit (Axygen, AP-PCR-500G), the above-mentioned two fragments were recombined with Vazyme Clon express Ultra One Step Cloning Kit (C115-01). The recombinant product was incubated at 37 ℃ for 30min, then transformed and plated, and Sanger sequenced to obtain the correct plasmid, CMV BPNLS-dCasRx-BPNLS-ADAR2 _DD (E488Q) sequence: gacggatcgggagatctcccgatcccctatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctccctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatctgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcgaaattaatacgactcactatagggagacccaagctggcTAGCGTTTAAACTTAAGCTTGCCACCATGAAACGGACTGCTGACGGCAGCGAATTTGAGAGCCCCAAGAAGAAGAGGAAAGTCGGATCCATCGAGAAGAAGAAGAGCTTCGCCAAGGGCATGGGAGTGAAGAGCACCCTGGTGTCCGGCTCTAAGGTGTACATGACCACATTTGCTGAGGGAAGCGACGCCAGGCTGGAGAAGATCGTGGAGGGCGATAGCATCAGATCCGTGAACGAGGGAGAGGCTTTCAGCGCCGAGATGGCTGACAAGAACGCTGGCTACAAGATCGGAAACGCCAAGTTTTCCCACCCAAAGGGCTACGCCGTGGTGGCTAACAACCCACTGTACACCGGACCAGTGCAGCAGGACATGCTGGGACTGAAGGAGACACTGGAGAAGAGGTACTTCGGCGAGTCCGCCGACGGAAACGATAACATCTGCATCCAGGTCATCCACAACATCCTGGATATCGAGAAGATCCTGGCTGAGTACATCACAAACGCCGCTTACGCCGTGAACAACATCTCCGGCCTGGACAAGGATATCATCGGCTTCGGAAAGTTTTCTACCGTGTACACATACGACGAGTTCAAGGATCCAGAGCACCACCGGGCCGCTTTTAACAACAACGACAAGCTGATCAACGCCATCAAGGCTCAGTACGACGAGTTCGATAACTTTCTGGATAACCCCAGGCTGGGCTACTTCGGACAGGCTTTCTTTTCTAAGGAGGGCAGAAACTACATCATCAACTACGGAAACGAGTGTTACGACATCCTGGCCCTGCTGAGCGGACTGGCCCACTGGGTGGTGGCCAACAACGAGGAGGAGTCTCGGATCAGCCGCACCTGGCTGTACAACCTGGACAAGAACCTGGATAACGAGTACATCTCCACACTGAACTACCTGTACGACAGGATCACCAACGAGCTGACAAACAGCTTCTCCAAGAACTCTGCCGCTAACGTGAACTACATCGCTGAGACCCTGGGCATCAACCCAGCTGAGTTCGCTGAGCAGTACTTCAGATTTTCCATCATGAAGGAGCAGAAGAACCTGGGCTTCAACATCACAAAGCTGAGAGAAGTGATGCTGGACAGAAAGGATATGTCCGAGATCAGGAAGAACCACAAGGTGTTCGATTCTATCAGAACCAAGGTGTACACAATGATGGACTTTGTGATCTACAGGTACTACATCGAGGAGGATGCCAAGGTGGCCGCTGCCAACAAGAGCCTGCCCGACAACGAGAAGTCTCTGAGCGAGAAGGATATCTTCGTGATCAACCTGAGAGGCTCCTTTAACGACGATCAGAAGGACGCTCTGTACTACGATGAGGCCAACAGGATCTGGAGAAAGCTGGAGAACATCATGCACAACATCAAGGAGTTCCGGGGAAACAAGACCCGCGAGTACAAGAAGAAGGACGCTCCAAGGCTGCCTAGGATCCTGCCTGCTGGAAGGGACGTGAGCGCCTTCAGCAAGCTGATGTACGCCCTGACAATGTTTCTGGACGGAAAGGAGATCAACGATCTGCTGACCACACTGATCAACAAGTTCGACAACATCCAGTCTTTTCTGAAAGTGATGCCTCTGATCGGCGTGAACGCTAAGTTCGTGGAGGAGTACGCCTTCTTTAAGGACAGCGCCAAGATCGCTGATGAGCTGCGGCTGATCAAGTCCTTTGCCAGGATGGGAGAGCCAATCGCTGACGCTAGGAGAGCTATGTACATCGATGCCATCCGGATCCTGGGAACCAACCTGTCTTACGACGAGCTGAAGGCTCTGGCCGACACCTTCAGCCTGGATGAGAACGGCAACAAGCTGAAGAAGGGCAAGCACGGAATGCGCAACTTCATCATCAACAACGTGATCAGCAACAAGCGGTTTCACTACCTGATCAGATACGGCGACCCAGCTCACCTGCACGAGATCGCTAAGAACGAGGCCGTGGTGAAGTTCGTGCTGGGACGGATCGCCGATATCCAGAAGAAGCAGGGCCAGAACGGAAAGAACCAGATCGACCGCTACTACGAGACCTGCATCGGCAAGGATAAGGGAAAGTCCGTGTCTGAGAAGGTGGACGCTCTGACCAAGATCATCACAGGCATGAACTACGACCAGTTCGATAAGAAGAGATCTGTGATCGAGGACACCGGAAGGGAGAACGCCGAGAGAGAGAAGTTTAAGAAGATCATCAGCCTGTACCTGACAGTGATCTACCACATCCTGAAGAACATCGTGAACATCAACGCTAGATACGTGATCGGCTTCCACTGCGTGGAGCGCGATGCCCAGCTGTACAAGGAGAAGGGATACGACATCAACCTGAAGAAGCTGGAGGAGAAGGGCTTTAGCTCCGTGACCAAGCTGTGCGCTGGAATCGACGAGACAGCCCCCGACAAGAGGAAGGATGTGGAGAAGGAGATGGCCGAGAGAGCTAAGGAGAGCATCGACTCCCTGGAGTCTGCTAACCCTAAGCTGTACGCCAACTACATCAAGTACTCCGATGAGAAGAAGGCCGAGGAGTTCACCAGGCAGATCAACAGAGAGAAGGCCAAGACCGCTCTGAACGCCTACCTGAGGAACACAAAGTGGAACGTGATCATCCGGGAGGACCTGCTGCGCATCGATAACAAGACCTGTACACTGTTCGCTAACAAGGCTGTGGCCCTGGAGGTGGCTCGCTACGTGCACGCCTACATCAACGACATCGCCGAGGTGAACTCCTACTTTCAGCTGTACCACTACATCATGCAGAGGATCATCATGAACGAGAGATACGAGAAGTCTAGCGGCAAGGTGTCTGAGTACTTCGACGCCGTGAACGATGAGAAGAAGTACAACGATAGACTGCTGAAGCTGCTGTGCGTGCCTTTCGGATACTGTATCCCACGGTTTAAGAACCTGAGCATCGAGGCCCTGTTCGACCGCAACGAGGCTGCCAAGTTTGATAAGGAGAAGAAGAAGGTGAGCGGCAACTCCGGTTCTAAGCGCACCGCCGATGGATCCGAGTTCGAATCCCCTAAAAAGAAAAGAAAGGTGGGATCCCAGCTGCATTTACCGcaggttttagctgacgctgtctcacgcctggtcctgggtaagtttggtgacctgaccgacaacttctcctcccctcacgctcgcagaaaagtgctggctggagtcgtcatgacaacaggcacagatgttaaagatgccaaggtgataagtgtttctacaggaacaaaatgtattaatggtgaatacatgagtgatcgtggccttgcattaaatgactgccatgcagaaataatatctcggagatccttgctcagatttctttatacacaacttgagctttacttaaataacaaagatgatcaaaaaagatccatctttcagaaatcagagcgaggggggtttaggctgaaggagaatgtccagtttcatctgtacatcagcacctctccctgtggagatgccagaatcttctcaccacatgagccaatcctggaagaaccagcagatagacacccaaatcgtaaagcaagaggacagctacggaccaaaatagagtctggtCaggggacgattccagtgcgctccaatgcgagcatccaaacgtgggacggggtgctgcaaggggagcggctgctcaccatgtcctgcagtgacaagattgcacgctggaacgtggtgggcatccagggatcActgctcagcattttcgtggagcccatttacttctcgagcatcatcctgggcagcctttaccacggggaccacctttccagggccatgtaccagcggatctccaacatagaggacctgccacctctctacaccctcaacaagcctttgctcagtggcatcagcaatgcagaagcacggcagccagggaaggcccccaacttcagtgtcaactggacggtaggcgactccgctattgaggtcatcaacgccacgactgggaaggatgagctgggccgcgcgtcccgcctgtgtaagcacgcgttgtactgtcgctggatgcgtgtgcacggcaaggttccctcccacttactacgctccaagattaccaagcccaacgtgtaccatgagtccaagctggcggcaaaggagtaccaggccgccaaggcgcgtctgttcacagccttcatcaaggcggggctgggggcctgggtggagaagcccaccgagcaggaccagttctcactcacgTAAgcggccgctcgagtctagagggcccgtttaaacccgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagctggggctctagggggtatccccacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattaattctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctctgcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagctcccgggagcttgtatatccattttcggatctgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgagcgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccaccccaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgtataccgtcgacctctagctagagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtc(9558bp)。(SEQ ID NO.10)

With CMV BPNLS-dCasRx-BPNLS-ADAR2 _DD (E488Q) plasmid as template, 4366F (AGGATGACGATGACAAGTAAGCCGCTCGAGCCTA) (SEQ ID NO.11) and 4366R (AGAGCCCCCGCCGCCTCCGTTGGTTCCCAGGATCCGGATG) (SEQ ID NO.12) primer PCR vector fragment 1. With CMV BPNLS-dCasRx-BPNLS-ADAR2 _DD (E488Q) plasmid as template, 4367F (GGAGGCGGGGGGGCTCTGGATCCCAGCTGCATTTACCGCAGGTTTT) (SEQ ID NO.13) and 4367R (GCTTCCGCCCCCCCTCCAGAGCCCGTGAGTGAACTGGTCCTGCTCG) (SEQ ID NO.14) primer PCR vector fragment 2. With CMV BPNLS-dCasRx-BPNLS-ADAR2 _DD (E488Q) plasmid as template, 4368F (GGAGGGGGCGGAAGCATGCAGGCAACTTCATCATCATCAACAACGTGATCAGCAA) (SEQ ID NO.15) and 4368R (CTTGTCATCGTCATCTGTATCATGATCTTTATAATCACCGTCATGTCTT) (SEQ ID NO.16) primer PCR vector fragment 3. The three PCR amplification products were purified and recovered by AXYPREP PCRCLEAN-UP Kit (AXYGEN, AP-PCR-500G), and the two fragments were recombined with Vazyme Clon express Ultra One Step Cloning Kit (C115-01). The recombinant product was incubated at 37 ℃ for 30min before transformation and plating, and Sanger sequencing gave the correct plasmid CMV-BPNLS-dCasRx (1I-558G) -ADAR-dCasRx (587M-966S) -BPNLS with the following sequence: gacggatcgggagatctcccgatcccctatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctccctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatctgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcgaaattaatacgactcactatagggagacccaagctggcTAGCGTTTAAACTTAAGCTTGCCACCATGAAACGGACTGCTGACGGCAGCGAATTTGAGAGCCCCAAGAAGAAGAGGAAAGTCGGATCCATCGAGAAGAAGAAGAGCTTCGCCAAGGGCATGGGAGTGAAGAGCACCCTGGTGTCCGGCTCTAAGGTGTACATGACCACATTTGCTGAGGGAAGCGACGCCAGGCTGGAGAAGATCGTGGAGGGCGATAGCATCAGATCCGTGAACGAGGGAGAGGCTTTCAGCGCCGAGATGGCTGACAAGAACGCTGGCTACAAGATCGGAAACGCCAAGTTTTCCCACCCAAAGGGCTACGCCGTGGTGGCTAACAACCCACTGTACACCGGACCAGTGCAGCAGGACATGCTGGGACTGAAGGAGACACTGGAGAAGAGGTACTTCGGCGAGTCCGCCGACGGAAACGATAACATCTGCATCCAGGTCATCCACAACATCCTGGATATCGAGAAGATCCTGGCTGAGTACATCACAAACGCCGCTTACGCCGTGAACAACATCTCCGGCCTGGACAAGGATATCATCGGCTTCGGAAAGTTTTCTACCGTGTACACATACGACGAGTTCAAGGATCCAGAGCACCACCGGGCCGCTTTTAACAACAACGACAAGCTGATCAACGCCATCAAGGCTCAGTACGACGAGTTCGATAACTTTCTGGATAACCCCAGGCTGGGCTACTTCGGACAGGCTTTCTTTTCTAAGGAGGGCAGAAACTACATCATCAACTACGGAAACGAGTGTTACGACATCCTGGCCCTGCTGAGCGGACTGGCCCACTGGGTGGTGGCCAACAACGAGGAGGAGTCTCGGATCAGCCGCACCTGGCTGTACAACCTGGACAAGAACCTGGATAACGAGTACATCTCCACACTGAACTACCTGTACGACAGGATCACCAACGAGCTGACAAACAGCTTCTCCAAGAACTCTGCCGCTAACGTGAACTACATCGCTGAGACCCTGGGCATCAACCCAGCTGAGTTCGCTGAGCAGTACTTCAGATTTTCCATCATGAAGGAGCAGAAGAACCTGGGCTTCAACATCACAAAGCTGAGAGAAGTGATGCTGGACAGAAAGGATATGTCCGAGATCAGGAAGAACCACAAGGTGTTCGATTCTATCAGAACCAAGGTGTACACAATGATGGACTTTGTGATCTACAGGTACTACATCGAGGAGGATGCCAAGGTGGCCGCTGCCAACAAGAGCCTGCCCGACAACGAGAAGTCTCTGAGCGAGAAGGATATCTTCGTGATCAACCTGAGAGGCTCCTTTAACGACGATCAGAAGGACGCTCTGTACTACGATGAGGCCAACAGGATCTGGAGAAAGCTGGAGAACATCATGCACAACATCAAGGAGTTCCGGGGAAACAAGACCCGCGAGTACAAGAAGAAGGACGCTCCAAGGCTGCCTAGGATCCTGCCTGCTGGAAGGGACGTGAGCGCCTTCAGCAAGCTGATGTACGCCCTGACAATGTTTCTGGACGGAAAGGAGATCAACGATCTGCTGACCACACTGATCAACAAGTTCGACAACATCCAGTCTTTTCTGAAAGTGATGCCTCTGATCGGCGTGAACGCTAAGTTCGTGGAGGAGTACGCCTTCTTTAAGGACAGCGCCAAGATCGCTGATGAGCTGCGGCTGATCAAGTCCTTTGCCAGGATGGGAGAGCCAATCGCTGACGCTAGGAGAGCTATGTACATCGATGCCATCCGGATCCTGGGAACCAACGGAGGCGGGGGCTCTGGATCCCAGCTGCATTTACCGCAGGTTTtagctgacgctgtctcacgcctggtcctgggtaagtttggtgacctgaccgacaacttctcctcccctcacgctcgcagaaaagtgctggctggagtcgtcatgacaacaggcacagatgttaaagatgccaaggtgataagtgtttctacaggaacaaaatgtattaatggtgaatacatgagtgatcgtggccttgcattaaatgactgccatgcagaaataatatctcggagatccttgctcagatttctttatacacaacttgagctttacttaaataacaaagatgatcaaaaaagatccatctttcagaaatcagagcgaggggggtttaggctgaaggagaatgtccagtttcatctgtacatcagcacctctccctgtggagatgccagaatcttctcaccacatgagccaatcctggaagaaccagcagatagacacccaaatcgtaaagcaagaggacagctacggaccaaaatagagtctggtCaggggacgattccagtgcgctccaatgcgagcatccaaacgtgggacggggtgctgcaaggggagcggctgctcaccatgtcctgcagtgacaagattgcacgctggaacgtggtgggcatccagggatcActgctcagcattttcgtggagcccatttacttctcgagcatcatcctgggcagcctttaccacggggaccacctttccagggccatgtaccagcggatctccaacatagaggacctgccacctctctacaccctcaacaagcctttgctcagtggcatcagcaatgcagaagcacggcagccagggaaggcccccaacttcagtgtcaactggacggtaggcgactccgctattgaggtcatcaacgccacgactgggaaggatgagctgggccgcgcgtcccgcctgtgtaagcacgcgttgtactgtcgctggatgcgtgtgcacggcaaggttccctcccacttactacgctccaagattaccaagcccaacgtgtaccatgagtccaagctggcggcaaaggagtaccaggccgccaaggcgcgtctgttcacagccttcatcaaggcggggctgggggcctgggtggagaagcccaccgagcaggaccagttctcactcacgGGCTCTGGAGGGGGCGGAAGCATGCGCAACTTCATCATCAACAACGTGATCAGCAACAAGCGGTTTCACTACCTGATCAGATACGGCGACCCAGCTCACCTGCACGAGATCGCTAAGAACGAGGCCGTGGTGAAGTTCGTGCTGGGACGGATCGCCGATATCCAGAAGAAGCAGGGCCAGAACGGAAAGAACCAGATCGACCGCTACTACGAGACCTGCATCGGCAAGGATAAGGGAAAGTCCGTGTCTGAGAAGGTGGACGCTCTGACCAAGATCATCACAGGCATGAACTACGACCAGTTCGATAAGAAGAGATCTGTGATCGAGGACACCGGAAGGGAGAACGCCGAGAGAGAGAAGTTTAAGAAGATCATCAGCCTGTACCTGACAGTGATCTACCACATCCTGAAGAACATCGTGAACATCAACGCTAGATACGTGATCGGCTTCCACTGCGTGGAGCGCGATGCCCAGCTGTACAAGGAGAAGGGATACGACATCAACCTGAAGAAGCTGGAGGAGAAGGGCTTTAGCTCCGTGACCAAGCTGTGCGCTGGAATCGACGAGACAGCCCCCGACAAGAGGAAGGATGTGGAGAAGGAGATGGCCGAGAGAGCTAAGGAGAGCATCGACTCCCTGGAGTCTGCTAACCCTAAGCTGTACGCCAACTACATCAAGTACTCCGATGAGAAGAAGGCCGAGGAGTTCACCAGGCAGATCAACAGAGAGAAGGCCAAGACCGCTCTGAACGCCTACCTGAGGAACACAAAGTGGAACGTGATCATCCGGGAGGACCTGCTGCGCATCGATAACAAGACCTGTACACTGTTCGCTAACAAGGCTGTGGCCCTGGAGGTGGCTCGCTACGTGCACGCCTACATCAACGACATCGCCGAGGTGAACTCCTACTTTCAGCTGTACCACTACATCATGCAGAGGATCATCATGAACGAGAGATACGAGAAGTCTAGCGGCAAGGTGTCTGAGTACTTCGACGCCGTGAACGATGAGAAGAAGTACAACGATAGACTGCTGAAGCTGCTGTGCGTGCCTTTCGGATACTGTATCCCACGGTTTAAGAACCTGAGCATCGAGGCCCTGTTCGACCGCAACGAGGCTGCCAAGTTTGATAAGGAGAAGAAGAAGGTGAGCGGCAACTCCGGTTCTAAGCGCACCGCCGATGGATCCGAGTTCGAATCCCCTAAAAAGAAAAGAAAGGTGGGATCCGACTACAaagaccatgacggtgattataaagatcatgatatcgattacaAGGATGACGATGACAAGTAAGCGGCCGCTCGAGCCTAgagggcccgtttaaacccgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagctggggctctagggggtatccccacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattaattctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctctgcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagctcccgggagcttgtatatccattttcggatctgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgagcgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccaccccaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgtataccgtcgacctctagctagagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtc(9582bp)。(SEQ ID NO.17)

4369R (TTGGTTCCCAGGATCGGATGGCATCGATGTACAT) (SEQ ID NO.18) and 4369F (GACGATGACAAGTAAGCGGCCGCTCGAGCCTAGGGC) (SEQ ID NO.19) primers were diluted to 10. mu.M and fragment 4 was amplified from CMV-BPNLS-dCasRx (1I-558G) -ADAR-dCasRx (587M-966S) -BPNLS template using the Novozam Hi-Fi enzyme kit (Vazyme, p501-d 2). 4370F (GGATCCGAGTTTCGAATCCCCTAAAAAGAAAAGGTGGGATCCCAGCTGCATTTACCG) (SEQ ID NO.20) and 4370R (CTCTCAAACTCAGATCCATCCGCAGTTTTTTGGATCCGTGAGTGAACTGGTCCTGC) (SEQ ID NO.21) primers were diluted to 10. mu.M and fragment 5 was amplified from CMV-BPNLS-dCasRx (1I-558G) -ADAR-dCasRx (587M-966S) -BPNLS template using the Novozam high fidelity enzyme kit (Vazyme, p501-d 2). 4371F (CCGGATCCTGGGAACCAACGGTTTCTAAGCGCACCGCCGATGGATCCGAGTTCGAATCCCC) (SEQ ID NO.22) and 4371R (GGATGGATCTGATGAGTTTGAGGTCCAAAAAAGAGGAGGAAGGTCGGTTCTATGCGCAACTT) (SEQ ID NO.23) primers were diluted to 10. mu.M and fragment 6 was amplified using the Novozam high fidelity enzyme kit (Vazyme, p501-d2) using amplified fragment 5 as a template. 4372F (TTCTATGCGCAACTTCATCAACACGTGATCAGC) (SEQ ID NO.24) and 4372R (TAGGCTCGAGCGGCCGCTTACTTGTCATCGTCGTCAT) (SEQ ID NO.25) primers were diluted to 10. mu.M and fragment 7 was amplified from CMV-BPNLS-dCasRx (1I-558G) -ADAR-dCasRx (587M-966S) -BPS template using the Norrespect Hi-Fi enzyme kit (Vazyme, p501-d 2).

The three PCR amplification products 4, 6, and 7 were purified and recovered by AxyPrep PCR Clean-up Kit (Axygen, AP-PCR-500G), and then recombined with Vazyme Clonexpress Ultra One Step Cloning Kit (C115-01). The recombinant product was incubated at 37 ℃ for 30min before transformation and plating, and Sanger sequencing gave the correct Vx plasmid CMV-BPNLS-dCasRx (1I-558G) -BPNLS-ADAR-BPNLS-dCasRx (587M-966S) -BPNLS sequence: gacggatcgggagatctcccgatcccctatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctccctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatctgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcgaaattaatacgactcactatagggagacccaagctggcTAGCGTTTAAACTTAAGCTTGCCACCATGAAACGGACTGCTGACGGCAGCGAATTTGAGAGCCCCAAGAAGAAGAGGAAAGTCGGATCCATCGAGAAGAAGAAGAGCTTCGCCAAGGGCATGGGAGTGAAGAGCACCCTGGTGTCCGGCTCTAAGGTGTACATGACCACATTTGCTGAGGGAAGCGACGCCAGGCTGGAGAAGATCGTGGAGGGCGATAGCATCAGATCCGTGAACGAGGGAGAGGCTTTCAGCGCCGAGATGGCTGACAAGAACGCTGGCTACAAGATCGGAAACGCCAAGTTTTCCCACCCAAAGGGCTACGCCGTGGTGGCTAACAACCCACTGTACACCGGACCAGTGCAGCAGGACATGCTGGGACTGAAGGAGACACTGGAGAAGAGGTACTTCGGCGAGTCCGCCGACGGAAACGATAACATCTGCATCCAGGTCATCCACAACATCCTGGATATCGAGAAGATCCTGGCTGAGTACATCACAAACGCCGCTTACGCCGTGAACAACATCTCCGGCCTGGACAAGGATATCATCGGCTTCGGAAAGTTTTCTACCGTGTACACATACGACGAGTTCAAGGATCCAGAGCACCACCGGGCCGCTTTTAACAACAACGACAAGCTGATCAACGCCATCAAGGCTCAGTACGACGAGTTCGATAACTTTCTGGATAACCCCAGGCTGGGCTACTTCGGACAGGCTTTCTTTTCTAAGGAGGGCAGAAACTACATCATCAACTACGGAAACGAGTGTTACGACATCCTGGCCCTGCTGAGCGGACTGGCCCACTGGGTGGTGGCCAACAACGAGGAGGAGTCTCGGATCAGCCGCACCTGGCTGTACAACCTGGACAAGAACCTGGATAACGAGTACATCTCCACACTGAACTACCTGTACGACAGGATCACCAACGAGCTGACAAACAGCTTCTCCAAGAACTCTGCCGCTAACGTGAACTACATCGCTGAGACCCTGGGCATCAACCCAGCTGAGTTCGCTGAGCAGTACTTCAGATTTTCCATCATGAAGGAGCAGAAGAACCTGGGCTTCAACATCACAAAGCTGAGAGAAGTGATGCTGGACAGAAAGGATATGTCCGAGATCAGGAAGAACCACAAGGTGTTCGATTCTATCAGAACCAAGGTGTACACAATGATGGACTTTGTGATCTACAGGTACTACATCGAGGAGGATGCCAAGGTGGCCGCTGCCAACAAGAGCCTGCCCGACAACGAGAAGTCTCTGAGCGAGAAGGATATCTTCGTGATCAACCTGAGAGGCTCCTTTAACGACGATCAGAAGGACGCTCTGTACTACGATGAGGCCAACAGGATCTGGAGAAAGCTGGAGAACATCATGCACAACATCAAGGAGTTCCGGGGAAACAAGACCCGCGAGTACAAGAAGAAGGACGCTCCAAGGCTGCCTAGGATCCTGCCTGCTGGAAGGGACGTGAGCGCCTTCAGCAAGCTGATGTACGCCCTGACAATGTTTCTGGACGGAAAGGAGATCAACGATCTGCTGACCACACTGATCAACAAGTTCGACAACATCCAGTCTTTTCTGAAAGTGATGCCTCTGATCGGCGTGAACGCTAAGTTCGTGGAGGAGTACGCCTTCTTTAAGGACAGCGCCAAGATCGCTGATGAGCTGCGGCTGATCAAGTCCTTTGCCAGGATGGGAGAGCCAATCGCTGACGCTAGGAGAGCTATGTACATCGATGCCATCCGGATCCTGGGAACCAACGGTTCTAAGCGCACCGCCGATGGATCCGAGTTCGAATCCCCTAAAAAGAAAAGAAAGGTGGGATCCCAGCTGCATTTACCGcaggttttagctgacgctgtctcacgcctggtcctgggtaagtttggtgacctgaccgacaacttctcctcccctcacgctcgcagaaaagtgctggctggagtcgtcatgacaacaggcacagatgttaaagatgccaaggtgataagtgtttctacaggaacaaaatgtattaatggtgaatacatgagtgatcgtggccttgcattaaatgactgccatgcagaaataatatctcggagatccttgctcagatttctttatacacaacttgagctttacttaaataacaaagatgatcaaaaaagatccatctttcagaaatcagagcgaggggggtttaggctgaaggagaatgtccagtttcatctgtacatcagcacctctccctgtggagatgccagaatcttctcaccacatgagccaatcctggaagaaccagcagatagacacccaaatcgtaaagcaagaggacagctacggaccaaaatagagtctggtCaggggacgattccagtgcgctccaatgcgagcatccaaacgtgggacggggtgctgcaaggggagcggctgctcaccatgtcctgcagtgacaagattgcacgctggaacgtggtgggcatccagggatcActgctcagcattttcgtggagcccatttacttctcgagcatcatcctgggcagcctttaccacggggaccacctttccagggccatgtaccagcggatctccaacatagaggacctgccacctctctacaccctcaacaagcctttgctcagtggcatcagcaatgcagaagcacggcagccagggaaggcccccaacttcagtgtcaactggacggtaggcgactccgctattgaggtcatcaacgccacgactgggaaggatgagctgggccgcgcgtcccgcctgtgtaagcacgcgttgtactgtcgctggatgcgtgtgcacggcaaggttccctcccacttactacgctccaagattaccaagcccaacgtgtaccatgagtccaagctggcggcaaaggagtaccaggccgccaaggcgcgtctgttcacagccttcatcaaggcggggctgggggcctgggtggagaagcccaccgagcaggacCAGTTCTCACTCACGGGATCCAAAAGAACTGCGGATGGATCTGAGTTTGAGAGTCCAAAAAAGAAGAGGAAGGTCGGTTCTATGCGCAACTTCATCATCAACAACGTGATCAGCAACAAGCGGTTTCACTACCTGATCAGATACGGCGACCCAGCTCACCTGCACGAGATCGCTAAGAACGAGGCCGTGGTGAAGTTCGTGCTGGGACGGATCGCCGATATCCAGAAGAAGCAGGGCCAGAACGGAAAGAACCAGATCGACCGCTACTACGAGACCTGCATCGGCAAGGATAAGGGAAAGTCCGTGTCTGAGAAGGTGGACGCTCTGACCAAGATCATCACAGGCATGAACTACGACCAGTTCGATAAGAAGAGATCTGTGATCGAGGACACCGGAAGGGAGAACGCCGAGAGAGAGAAGTTTAAGAAGATCATCAGCCTGTACCTGACAGTGATCTACCACATCCTGAAGAACATCGTGAACATCAACGCTAGATACGTGATCGGCTTCCACTGCGTGGAGCGCGATGCCCAGCTGTACAAGGAGAAGGGATACGACATCAACCTGAAGAAGCTGGAGGAGAAGGGCTTTAGCTCCGTGACCAAGCTGTGCGCTGGAATCGACGAGACAGCCCCCGACAAGAGGAAGGATGTGGAGAAGGAGATGGCCGAGAGAGCTAAGGAGAGCATCGACTCCCTGGAGTCTGCTAACCCTAAGCTGTACGCCAACTACATCAAGTACTCCGATGAGAAGAAGGCCGAGGAGTTCACCAGGCAGATCAACAGAGAGAAGGCCAAGACCGCTCTGAACGCCTACCTGAGGAACACAAAGTGGAACGTGATCATCCGGGAGGACCTGCTGCGCATCGATAACAAGACCTGTACACTGTTCGCTAACAAGGCTGTGGCCCTGGAGGTGGCTCGCTACGTGCACGCCTACATCAACGACATCGCCGAGGTGAACTCCTACTTTCAGCTGTACCACTACATCATGCAGAGGATCATCATGAACGAGAGATACGAGAAGTCTAGCGGCAAGGTGTCTGAGTACTTCGACGCCGTGAACGATGAGAAGAAGTACAACGATAGACTGCTGAAGCTGCTGTGCGTGCCTTTCGGATACTGTATCCCACGGTTTAAGAACCTGAGCATCGAGGCCCTGTTCGACCGCAACGAGGCTGCCAAGTTTGATAAGGAGAAGAAGAAGGTGAGCGGCAACTCCGGTTCTAAGCGCACCGCCGATGGATCCGAGTTCGAATCCCCTAAAAAGAAAAGAAAGGTGGGATCCGACTACAaagaccatgacggtgattataaagatcatgatatcgattacaAGGATGACGATGACAAGTAAGCGGCCGCTCGAGCCTAGAGGGCCCgtttaaacccgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagctggggctctagggggtatccccacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattaattctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctctgcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagctcccgggagcttgtatatccattttcggatctgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgagcgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccaccccaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgtataccgtcgacctctagctagagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtc (9672 bp). (SEQ ID NO.26)

The U6-CasRx crRNA-BspQI-BspQI-CasRx crRNA plasmid was synthesized in Kinsley with the sequence: tgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgCTGCAATGATACCGCGAGATCCACGCTCACCGGCTCCAGATTTATCAGCAAtaaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagcccaagctaccatgataagtaagtaatattaaggtacgggaggtacttggagcggccgcaataaaatatctttattttcattacatctgtgtgttggttttttgtgtgaatcgatagtactaacatacgctctccatcaaaacaaaacgaaacaaaacaaactagcaaaataggctgtccccagtgcaagtgcaggtgccagaacatttctctatcgataggtaccgattagtgaacggatctcgacggtatcgatcacgagactagcctcgagcggccgcccccttcaccgagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggCTTTATATATCTTGTGGAAAGGACgaaacaccGCAAGTAAACCCCTACCAACTGGTCGGGGTTTGAAACAGAAGAGCCTCGAGGCTCTTCTCAAGTAAACCCCTACCAACTGGTCGGGGTTTGAAACGAAGACTTTTTTTTTCGCTTCCTCGCTCACTgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcAAAGGCGGTAATACGGTCCTCGAGACAAATGGCAGTAttcatccacaattttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttcgggtttattacagggacagcagagatccactttggccgcggctcgagggggttggggttgcgccttttccaaggcagccctgggtttgcgcagggacgcggctgctctgggcgtggttccgggaaacgcagcggcgccgaccctgggactcgcacattcttcacgtccgttcgcagcgtcacccggatcttcgccgctacccttgtgggccccccggcgacgcttcctgctccgcccctaagtcgggaaggttccttgcggttcgcggcgtgccggacgtgacaaacggaagccgcacgtctcactagtaccctcgcagacggacagcgccagggagcaatggcagcgcgccgaccgcgatgggctgtggccaatagcggctgctcagcagggcgcgccgagagcagcggccgggaaggggcggtgcgggaggcggggtgtggggcggtagtgtgggccctgttcctgcccgcgcggtgttccgcattctgcaagcctccggagcgcacgtcggcagtcggctccctcgttgaccgaatcaccgacctctctccccagggggatccaccGGAGCTTACCatgaccgagtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgtacgcaccctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccgccacatcgagcgggtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacggcgccgcggtggcggtctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcggcccgcgcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggcgtctcgcccgaccaccagggcaagggtctgggcagcgccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggaAacctccgcgccccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctggtgcatgacccgcaagcccggtgcctgaCGCCCGCCCCACGACCCGCAGCGCCCGACCGAAAGGAGCGCACGACCCCATGCATCGgtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttctagagtcggggcggccggccgcttcgagcagacatgataagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagttaacaacaacaattgcattcattttatgtttcaggttcagggggaggtgtgggaggttttttaaAGCAAGTAAAACCTCTACAAATGTGGTCGCTTCCTCGCTCACTgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatg (4875bp), (SEQ ID NO.27) respectively designs 18-60 base complementary paired upstream and downstream primers according to the target site sequence, and dissolves to 100 μ M with sterilized water. After annealing, the recombinant plasmid was ligated to a U6-CasRx crRNA-BspQI-BspQI-CasRx crRNA vector to construct a target-specific sgRNA. The primer sequences of sgrnas are as follows:

4582B_ADGRV1 TTGctgagggtccccagtgtttctggatgacatcatGgatcagcccagctgtc(SEQ ID NO.28)

4582T_ADGRV1 AACgacagctgggctgatcCatgatgtcatccagaaacactggggaccctcag(SEQ ID NO.29)

4583B_AHI1 TTGttacaggatgctatgattccatgatacggatatGgaaagttgagatgaga(SEQ ID NO.30)

4583T_AHI1 AACtctcatctcaactttcCatatccgtatcatggaatcatagcatcctgtaa(SEQ ID NO.31)

4584B_APC TTGtggaacagatacgcgcttactgtgaaacctgttGggagtggcaggaagct(SEQ ID NO.32)

4584T_APC AACagcttcctgccactccCaacaggtttcacagtaagcgcgtatctgttcca(SEQ ID NO.33)

4585B_BMPR2 TTGtatgcaagtatttaagtctccacacaagtgactGggtaagctcttgccgt(SEQ ID NO.34)

4585T_BMPR2 AACacggcaagagcttaccCagtcacttgtgtggagacttaaatacttgcata(SEQ ID NO.35)

4586B_BRCA1 TTGgatgtgtgaggcacctgtggtgacccgagagtgGgtgttggacagtgtag(SEQ ID NO.36)

4586T_BRCA1 AACctacactgtccaacacCcactctcgggtcaccacaggtgcctcacacatc(SEQ ID NO.37)

4587B_COL3A1 TTGtctgccatcctgaactcaagagtggagaatactGggttgaccctaaccaa(SEQ ID NO.38)

4587T_COL3A1 AACttggttagggtcaaccCagtattctccactcttgagttcaggatggcaga(SEQ ID NO.39)

4588B_DNAH5 TTGtcatcaaggaaccaaatgatctgttaaagctgtGgaagcatgagtgtaaa(SEQ ID NO.40)

4588T_DNAH5 AACtttacactcatgcttcCacagctttaacagatcatttggttccttgatga(SEQ ID NO.41)

4589B_MECP2 TTGccatgtatgatgaccccaccctgcctgaaggctGgacacggaagcttaag(SEQ ID NO.42)

4589T_MECP2 AACcttaagcttccgtgtcCagccttcaggcagggtggggtcatcatacatgg(SEQ ID NO.43)

4590B_MYBPC3 TTGagccaccccaggatgtcggcaacacggagctctGggggtacacagtgcag(SEQ ID NO.44)

4590T_MYBPC3 AACctgcactgtgtaccccCagagctccgtgttgccgacatcctggggtggct(SEQ ID NO.45)

4591B_PRKN TTGggctcgagtggtgctggaactgtggctgcgagtGgaaccgcgtctgcatg(SEQ ID NO.46)

4591T_PRKN AACcatgcagacgcggttcCactcgcagccacagttccagcaccactcgagcc(SEQ ID NO.47)

4591B_PRKN_60 TTGccccagtgcaggctcgagtggtgctggaactgtggctgcgagtGgaaccgcgtctgcatg(SEQ ID NO.48)

4591T_PRKN_60 AACcatgcagacgcggttcCactcgcagccacagttccagcaccactcgagcctgcactgggg(SEQ ID NO.49)

4591B_PRKN_40 TTGgtgctggaactgtggctgcgagtGgaaccgcgtctgcatg(SEQ ID NO.50)

4591T_PRKN_40 AACcatgcagacgcggttcCactcgcagccacagttccagcac(SEQ ID NO.51)

4591B_PRKN_30 TTGtgtggctgcgagtGgaaccgcgtctgcatg(SEQ ID NO.52)

4591T_PRKN_30 AACcatgcagacgcggttcCactcgcagccaca(SEQ ID NO.53)

4591B_PRKN_25 TTGgtggctgcgagtGgaaccgcgtctg(SEQ ID NO.54)

4591T_PRKN_25 AACcagacgcggttcCactcgcagccac(SEQ ID NO.55)

4591B_PRKN_22 TTGgctgcgagtGgaaccgcgtctg(SEQ ID NO.56)

4591T_PRKN_22 AACcagacgcggttcCactcgcagc(SEQ ID NO.57)

4591B_PRKN_18 TTGcgagtGgaaccgcgtctg(SEQ ID NO.58)

4591T_PRKN_18 AACcagacgcggttcCactcg(SEQ ID NO.59)

the U6-CasRx crRNA-BspQI-CasRx crRNA plasmid was cleaved with BspQI (NEB, R0712L) to give a linearized sgRNA vector. And (3) recovering the enzyme digestion product by using an AxyPrep DNA gel recovery kit (Axygen, AP-GX-250G) as a tapping gel to obtain a linear carrier. 20ng of the linearized vector and 1 μ l of the annealed product were ligated by Solution I (Takara, 6022), incubated at 16 ℃ for 1 hour, and then transformed into a plate, and subjected to Sanger sequencing to obtain the correct target-specific sgRNA.

Example 2

Base editing efficiency comparison of Vx system on reporter system:

n2a cells were transfected using the Vx system described above as follows:

1) n2a cells (from ATCC) were recovered and cultured in 10cm dishes (Corning,430167) in DMEM (HyClone, SH30243.01) mixed with 10% fetal bovine serum (HyClone, SV 30087). The culture temperature was 37 ℃ and the carbon dioxide concentration was 5%. After multiple passages when the cell density was 80%, cells were plated into 24-well plates.

2) When the cell concentration is 80%, the cell state is recovered to the optimum state by changing the culture medium with 10% serum DMEM and culturing for 2 hours. The amount of plasmid transfected per well was 300ng Vx plasmid, 200ng sgRNA plasmid, 20ng reporter plasmid, respectively. The plasmid was mixed in 50. mu.l of Opti-MEM (Gibco,11058021) medium. Systems V1 and V2 were used as positive control groups, the preparation methods of the systems V1 and V2 were the same as the Vx system, and the references to plasmid information: d.b.t.cox et al, Science 10.1126/science.aaq0180(2017).

3) Mu.l of Lipofectamine 2000 transfection reagent (Thermo,11668019) was mixed into 50. mu.l of Opti-MEM medium and allowed to stand for 5 minutes.

4) The plasmid-mixed Opti-MEM was added to the plasmid-mixed Opti-MEM mixed with Lipofectamine 2000, gently whipped, mixed well, and allowed to stand for 20 minutes.

5) Opti-MEM mixed with plasmid and Lipofectamine 2000 was added to 24-well plates, respectively.

6) 6 hours after transfection, the cells were replaced with 10% FBS in DMEM.

7) 12 hours after transfection, a drug kill treatment was performed with Puromycin (Invivogen, nt-pr-1) at a final concentration of 2 ng/ml.

8) Cells were harvested 48 hours after transfection, and RNA was extracted by TRIzol-chloroform method. 500ng of Total RNA was reverse transcribed using the Novozan reverse transcription kit HiScript II Q RT SuperMix kit (Vazyme, R223-01).

9) PCR primers (CGCCGAGGGCCGCCACTCCAC (SEQ ID NO.60) and ACGCTGAACTTGTGGCCGTTT) (SEQ ID NO.61) were designed and synthesized, and diluted to 10. mu.M with water. The target site fragment was PCR amplified using Novozam high fidelity enzyme kit (Vazyme, p501-d 2). Recovering PCR product sample by using AxyPrep DNA gel recovery kit (Axygen, AP-GX-250G) as tapping gel to remove non-specific band; sending to deep-seq sequencing analysis.

The Vx system has higher editing efficiency and accuracy under sgRNA with the length of 30 nt:

cas13d family member CasRx is known to have several loops on the outer protein surface that can be removed without affecting the protein function. We found that insertion of ADAR2DD (E488Q) into such a loop results in a base editing tool capable of targeting RNA, abbreviated repair (Vx, fig. 1), as detailed below.

To compare Vx, V1, and V2, we used a target sequence known to be editable by V1. A specific target sequence is G with clinical relevance>A mutated human PRKN mRNA fragment (GAGCAGGCTCGTTGGGAAGCAGCCTCCAAAGAAACCATCAAGAAAACCACCAAGCCCTGTCCCCGCTGCCATGTACCAGTGGAAAAAAATGGAGGCTGCATGCACATGAAGTGTCCGCAGCCCCAGTGCAGGCTCGAGTGGTGCTGGAACTGTGGCTGCGAGTAGAACCGCGTCTGCATGGGGGACCACTGGTTCGACGTG) (SEQ ID NO.62) which generates a premature stop codon (UAG, W55X; FIG. 2), additionally targeting the mutation status of the remaining A on the mRNA may reflect the accuracy of the editing tool (FIG. 3). To more intuitively examine the editing efficiency of target sites A to I, we constructed a GFP-based reporterA particle carrying a mutated fragment inserted upstream of GFP, wherein the stop codon prevents GFP expression until a is mutated to I. On the other hand, the reporter plasmid constitutively expresses mCherry, which serves as a standardized control for the recovery of GFP expression. The grnas for all three REPAIR versions share a 30nt complementary region, but the positions and sequences of the crrnas differ. Finally, control grnas lacking Direct Region (DR) were used to analyze the role of dCas in editing; this control is critical because the ADAR2 _DD Has the potential ability to recognize and edit any double-stranded RNA independently of dCas. This control was not set in previous studies with V1 and V2. Therefore our data can more closely compare and describe the performance of the three systems V1, V2 and Vx.

To evaluate the editing activity of REPAIR, we co-transfected the reporter plasmid and V1/V2/Vx and the respective gRNA plasmids into N2a cells (the corresponding primers used are shown below in 30nt-mismatch17C up and 30nt-mismatch17C down), and analyzed the cells for fluorescent expression after 48 hours. Consistent with the expectation, V1 induced GFP expression more efficiently than V2, but Vx was more efficient than V1 (fig. 5). sgRNA lacking DR affected induction of GFP from Vx groups, but GFP signal from V1 and V2 was largely independent of dCas13b (fig. 5).

We next quantified GFP induction efficiency using FACS (fig. 4). We transfected cells with a plasmid that had repaired W55X as a positive control. GFP was constitutively co-expressed with mCherry in transfected cells, and the Mean Fluorescence Intensity (MFI) of the two proteins was similar (3222 vs 2551, ratio approximately equal to 1; FIG. 4, line 1, middle). GFP could not be detected in cells transfected with the plasmid of the W55X reporter system alone (fig. 4, line 1, right), but three editing systems, V1, V2 and Vx, were all able to induce GFP expression, and we used the GFP/mCherry ratio for evaluation of editing efficiency (fig. 5). In the presence of grnas with DR at 30nt length, the efficiency of editing was Vx > V1> V2, whereas DR-independent GFP induction was similar for the three editing systems (fig. 5).

We then used deep-seq to directly quantify the editing efficiency of the targeted adenosine (A45) and the flanking non-targeted A within the 224-bp PRKN mRNA fragment (53 in total). The efficiencies of all 54 a's are shown in a heat map (fig. 6), and the efficiencies of a45 and the other 53 wings off target are bar chart and violin chart, respectively (fig. 7). For a45, Vx was indeed the most effective in the presence of DR (efficiency 67%, whereas V1 and V2 were 40% and 5%, respectively), whereas DR independent editing was comparable in three-bit editing (V1, V2 and Vx 14%, 7%, 10%, respectively), consistent with FACS analysis results. Of the 53 non-target a, the editing efficiency of all three editing systems differed greatly (fig. 7). For V1, these efficiencies ranged from 0.01% to 20% (median 0.4%) in the presence of DR. Importantly, Vx is more specific with a median of 0.08%, which is the same as V2 (median of 0.08%). DR deletion did not affect off-target effects (fig. 7).

Overall, the data for the reporting system indicates that Vx is superior to both V1 and V2 in efficiency and accuracy.

Effect of sgRNA length and mismatch distance on Vx editing:

the above-described reporter system uses sgrnas of 30 nt. Next, the sgRNA length was changed from 18 to 60nt, and primers used for constructing the sgRNA were as follows:

18nt-mismatch17C up：AACcatgcagacgcggttcCa；(SEQ ID NO.63)

18nt-mismatch17C down：TTGtGgaaccgcgtctgcatg；(SEQ ID NO.64)

22nt-mismatch17C up：AACcatgcagacgcggttcCactcg；(SEQ ID NO.65)

22nt-mismatch17C down：TTGcgagtGgaaccgcgtctgcatg；(SEQ ID NO.66)

25nt-mismatch17C up：AACcatgcagacgcggttcCactcgcag；(SEQ ID NO.67)

25nt-mismatch17C down：TTGctgcgagtGgaaccgcgtctgcatg；(SEQ ID NO.68)

30nt-mismatch17C up：AACcatgcagacgcggttcCactcgcagccaca；(SEQ ID NO.69)

30nt-mismatch17C down：TTGtgtggctgcgagtGgaaccgcgtctgcatg；(SEQ ID NO.70)

40nt-mismatch17C up：AACcatgcagacgcggttcCactcgcagccacagttccagcac；(SEQ ID NO.71)

40nt-mismatch17C down：TTGgtgctggaactgtggctgcgagtGgaaccgcgtctgcatg；(SEQ ID NO.72)

50nt-mismatch17C up：AACcatgcagacgcggttcCactcgcagccacagttccagcaccactcgagcc；(SEQ ID NO.73)

50nt-mismatch17C down：TTGggctcgagtggtgctggaactgtggctgcgagtGgaaccgcgtctgcatg。(SEQ ID NO.74)

and determining the influence on the efficiencies of V1, V2 and Vx (FIG. 8a, FIG. 8 b).

The editing efficiency of a45 was first examined using FACS and deep-seq, with reference to the above examination method of editing efficiency for sgrnas of 30nt length. First, V1 and V2 required sgrnas of 40nt in length to obtain optimal activity, but sgrnas as short as 25nt were sufficient to support maximum editing activity of Vx, consistent with Cas13b and CasRx attributes, respectively. But shorter sgRNA lengths can reduce off-target of ADAR (fig. 8a, fig. 8 b). Second, Vx is more efficient than V1 (especially at 22nt and 25nt, where Vx is about 3 times more active than V1) at each length tested in the presence of grnas carrying DR. Third, DR deletion significantly reduced the editing efficiency of sgrnas of all lengths of Vx, demonstrating the key role of dCasRx in editing. DR deletion also compromised the editing efficiency of V2 at sgrnas of 40nt length, but the editing efficiency of sgrnas of 50nt length was independent of the presence or absence of DR, where long RNA duplexes were known to be sufficient to recruit ADAR directly (fig. 8a, fig. 8 b).

Further described here is the off-target editing of a45 flanking a, with Vx off-target effect much weaker than V1 but comparable to V2 regardless of DR status and all sgRNA lengths tested (fig. 9).

Finally, it was determined how the mismatch C distance affected Vx (fig. 10a, fig. 10 b). Primers used for construction of sgrnas were as follows:

22nt-mismatch3C up：AACtcCactcgcagccacagttcca；(SEQ ID NO.75)

22nt-mismatch3C down：TTGtggaactgtggctgcgagtGga；(SEQ ID NO.76)

22nt-mismatch5C up：AACgttcCactcgcagccacagttcc；(SEQ ID NO.77)

22nt-mismatch5C down：TTGggaactgtggctgcgagtGgaac；(SEQ ID NO.78)

22nt-mismatch6C up：AACggttcCactcgcagccacagtt；(SEQ ID NO.79)

22nt-mismatch6C down：TTGaactgtggctgcgagtGgaacc；(SEQ ID NO.80)

22nt-mismatch7C up：AACcggttcCactcgcagccacagt；(SEQ ID NO.81)

22nt-mismatch7C down：TTGactgtggctgcgagtGgaaccg；(SEQ ID NO.82)

22nt-mismatch8C up：AACgcggttcCactcgcagccacag；(SEQ ID NO.83)

22nt-mismatch8C down：TTGctgtggctgcgagtGgaaccgc；(SEQ ID NO.84)

22nt-mismatch9C up：AACcgcggttcCactcgcagccaca；(SEQ ID NO.85)

22nt-mismatch9C down：TTGtgtggctgcgagtGgaaccgcg；(SEQ ID NO.86)

22nt-mismatch10C up：AACacgcggttcCactcgcagccac；(SEQ ID NO.87)

22nt-mismatch10C down：TTGgtggctgcgagtGgaaccgcgt；(SEQ ID NO.88)

22nt-mismatch11C up：AACgacgcggttcCactcgcagcca；(SEQ ID NO.89)

22nt-mismatch11C down：TTGtggctgcgagtGgaaccgcgtc；(SEQ ID NO.90)

22nt-mismatch12C up：AACagacgcggttcCactcgcagcc；(SEQ ID NO.91)

22nt-mismatch12C down：TTGggctgcgagtGgaaccgcgtct；(SEQ ID NO.92)

22nt-mismatch13C up：AACcagacgcggttcCactcgcagc；(SEQ ID NO.93)

22nt-mismatch13C down：TTGgctgcgagtGgaaccgcgtctg；(SEQ ID NO.94)

22nt-mismatch14C up：AACgcagacgcggttcCactcgcag；(SEQ ID NO.95)

22nt-mismatch14C down：TTGctgcgagtGgaaccgcgtctgc；(SEQ ID NO.96)

22nt-mismatch15C up：AACtgcagacgcggttcCactcgca；(SEQ ID NO.97)

22nt-mismatch15C down：TTGtgcgagtGgaaccgcgtctgca；(SEQ ID NO.98)

22nt-mismatch18C up：AACccatgcagacgcggttcCactc；(SEQ ID NO.99)

22nt-mismatch18C down：TTGgagtGgaaccgcgtctgcatgg；(SEQ ID NO.100)

in the case of sgrnas of 22nt length, mismatches of 6-15nt were efficiently edited by > 40% with the exception of 8nt and 11nt (20% and 30%, respectively; fig. 10 b). The sgRNA of 18nt still had editing activity, although the efficiency at only a few mismatch distances, i.e. at 7nt and 9nt, was close to 40% (fig. 10 b).

It can be seen that Vx can achieve optimal editing activity even with various mismatch distances in short 25 nt-length sgrnas.

Reflection of ADAR2 using sgRNA of 22nt length _DD Base bias properties on both sides of edit a:

codon bias for Vx was explored by testing all possible combinations of 5 'and 3' nucleotides targeting the a flank. The sgRNA length is 22nt, and sgRNA primers were constructed as follows:

22nt-TAT mismatch12C up：AACagacgcggttaCactcgcagcc；(SEQ ID NO.101)

22nt-TAT mismatch12C down：TTGggctgcgagtGtaaccgcgtct；(SEQ ID NO.102)

22nt-AAT mismatch12C up：AACagacgcggtttCactcgcagcc；(SEQ ID NO.103)

22nt-AAT mismatch12C down：TTGggctgcgagtGaaaccgcgtct；(SEQ ID NO.104)

22nt-CAT mismatch12C up：AACagacgcggttgCactcgcagcc；(SEQ ID NO.105)

22nt-CAT mismatch12C down：TTGggctgcgagtGcaaccgcgtct；(SEQ ID NO.106)

22nt-AAA mismatch12C up：AACagacgcggtttCtctcgcagcc；(SEQ ID NO.107)

22nt-AAA mismatch12C down：TTGggctgcgagaGaaaccgcgtct；(SEQ ID NO.108)

22nt-AAT mismatch12C up：AACagacgcggttaCtctcgcagcc；(SEQ ID NO.109)

22nt-AAT mismatch12C down：TTGggctgcgagtGaaaccgcgtct；(SEQ ID NO.110)

22nt-CAA mismatch12C up：AACagacgcggttgCtctcgcagc；(SEQ ID NO.111)

22nt-CAA mismatch12C down：TTGggctgcgagaGcaaccgcgtct；(SEQ ID NO.112)

22nt-GAA mismatch12C up：AACagacgcggttcCtctcgcagc；(SEQ ID NO.113)

22nt-GAA mismatch12C down：TTGggctgcgagaGgaaccgcgtct；(SEQ ID NO.114)

22nt-AAC mismatch12C up：AACagacgcggtttCgctcgcagc；(SEQ ID NO.115)

22nt-AAC mismatch12C down：TTGggctgcgagcGaaaccgcgtct；(SEQ ID NO.116)

22nt-TAC mismatch12C up：AACagacgcggttaCgctcgcagc；(SEQ ID NO.117)

22nt-TAC mismatch12C down：TTGggctgcgagcGtaaccgcgtct；(SEQ ID NO.118)

22nt-CAC mismatch12C up：AACagacgcggttgCgctcgcagc；(SEQ ID NO.119)

22nt-CAC mismatch12C down：TTGggctgcgagcGcaaccgcgtct；(SEQ ID NO.120)

22nt-GAC mismatch12C up：AACagacgcggttcCgctcgcagc；(SEQ ID NO.121)

22nt-GAC mismatch12C down：TTGggctgcgagcGgaaccgcgtct；(SEQ ID NO.122)

22nt-AAG mismatch12C up：AACagacgcggtttCcctcgcagc；(SEQ ID NO.123)

22nt-AAG mismatch12C down：TTGggctgcgaggGaaaccgcgtct；(SEQ ID NO.124)

22nt-TAG mismatch12C up：AACagacgcggttaCcctcgcagc；(SEQ ID NO.125)

22nt-TAG mismatch12C down：TTGggctgcgaggGtaaccgcgtct；(SEQ ID NO.126)

22nt-CAG mismatch12C up：AACagacgcggttgCcctcgcagc；(SEQ ID NO.127)

22nt-CAG mismatch12C down：TTGggctgcgaggGcaaccgcgtct；(SEQ ID NO.128)

22nt-GAG mismatch12C up：AACagacgcggttcCcctcgcagc；(SEQ ID NO.129)

22nt-GAG mismatch12C down：TTGggctgcgaggGgaaccgcgtct；(SEQ ID NO.130)

even in the case of grnas only 22nt long, Vx can edit all 16 triplets (9-40% efficiency), except for 4 codons beginning with G, which are slightly less efficient (< 8%; fig. 11), consistent with the known ADAR2DD properties.

Other disease-associated G > a mutations were corrected using sgrnas of 22nt length:

it has been demonstrated that Vx can repair PRKN mutations with high efficiency and precision, further extending this analysis to 7 more disease-associated G > a mutations. The sgRNA length is 22nt, and sgRNA primers were constructed as follows:

ADGRV1 22-mismatch12C up：AACctgggctgatcCatgatgtcat(SEQ ID NO.131)

ADGRV1 22-mismatch12C down：TTGatgacatcatGgatcagcccag(SEQ ID NO.132)

AHI1 22-mismatch12 up：AACtctcaactttcCatatccgtat(SEQ ID NO.133)

AHI1 22-mismatch12 down：TTGatacggatatGgaaagttgaga(SEQ ID NO.134)

APC 22-mismatch12 up：AACcctgccactccCaacaggtttc(SEQ ID NO.135)

APC 22-mismatch12 down：TTGgaaacctgttGggagtggcagg(SEQ ID NO.136)

COL3A1 22-mismatch12 up：AACtagggtcaaccCagtattctcc(SEQ ID NO.137)

COL3A1 22-mismatch12 down：TTGggagaatactGggttgacccta(SEQ ID NO.138)

DNAH5 22-mismatch12 up：AACactcatgcttcCacagctttaa(SEQ ID NO.139)

DNAH5 22-mismatch12 down：TTGttaaagctgtGgaagcatgagt(SEQ ID NO.140)

MECP2 22-mismatch12 up：AACgcttccgtgtcCagccttcagg(SEQ ID NO.141)

MECP2 22-mismatch12 down：TTGcctgaaggctGgacacggaagc(SEQ ID NO.142)

MYBPC3 22-mismatch12 up：AACctgtgtaccccCagagctccgt(SEQ ID NO.143)

MYBPC3 22-mismatch12 down：TTGacggagctctGggggtacacag(SEQ ID NO.144)

vx can edit all 7 genes even with a suboptimal gRNA of 22nt length with an efficiency of 12-53% (fig. 12a, fig. 12b), better than the editing efficiency of V1 at sgrnas of 50nt length (20-28%) (fig. 12a, fig. 12 b).

Editing and off-target effects on endogenous transcripts:

to test the efficiency of Vx editing on endogenous transcripts, transcripts that targeted successful editing of previous V1 and V2 (KRAS and PPIB) were selected. However, Kras mRNA was very low expressed in N2a cells, and Ppib expression level was relatively high. V1 is known to edit two sites on human PPIB mRNA with an efficiency of about 40% and 8%, respectively, compared to 10% -15% and 5% for V2. The exact editing site on the human transcript is missing from the mouse transcript, so the targeting sequences for sgrnas for the two nearby sites are as follows:

ppib1 up:AACtttgtaaatcaaagtaCaccttgactgtga；(SEQ ID NO.145)

ppib1 down:TTGtcacagtcaaggtGtactttgatttacaaa；(SEQ ID NO.146)

ppib2 up:AACcaaagacgactcgtccCacagattcatctc；(SEQ ID NO.147)

ppib2 down:TTGgagatgaatctgtGggacgagtcgtctttg；(SEQ ID NO.148)

the PCR primers were as follows:

Ppib forward:TCTTCCTTTTGCTGCCCGGACCCTC(SEQ ID NO.149)

Ppib reverse:ACATCCATGCCCTCTAGAACTTTGCCGAA(SEQ ID NO.150)

sanger sequencing primers:

Ppib sequencing:GCCGAAAACCACATGCTTGCC(SEQ ID NO.151)

as shown by Sanger sequencing (fig. 13), V1 achieved 33% -39% editing at two sites, compared to about 22% for V2. Importantly, Vx editing site 1 is more efficient (51% versus 39%) than V1 (31% versus 33%) and 2.

Three systems were further tested on GusB transcripts, the targeting sequences of sgrnas were as follows:

gusb1 up:AACtcccaagtagaaatacCagtgtcaggaagt；(SEQ ID NO.152)

gusb1 down:TTGacttcctgacactGgtatttctacttggga；(SEQ ID NO.153)

gusb2 up:AACgctttgatgtcacttcCaaagtccccttgt；(SEQ ID NO.154)

gusb2 down:TTG acaaggggactttGgaagtgacatcaaagc；(SEQ ID NO.155)

the PCR primers were as follows:

gusb forward:AGCTCATCTGGAATTTCGCCGACT(SEQ ID NO.156)

gusb reverse:AAGGCACTGTAAGTTATTATCCCCACGGAA(SEQ ID NO.157)

sanger sequencing primers:

gusb sequencing:GAATTTCGCCGACTTCATGACGA(SEQ ID NO.158)

its human counterpart can be edited by SNAP-ADAR. At both test sites, the efficiency of Vx (37% and 35%) was comparable to V1 (35% and 41%), but significantly higher than V2 (17% and 18%). Notably, at position 1, Vx edits an adenosine near a weak target (14%, whereas V1 is 22%). Finally, Vx was also effective (60%) for Actb mRNA. The results were reproducible in three independent experiments (fig. 13). sgRNA target sequences are as follows:

actb up:AACtctcctgctcgaagtcCagagcaacatag；(SEQ ID NO.159)

actb down:TTGctatgttgctctGgacttcgagcaggaga；(SEQ ID NO.160)

the PCR primers were as follows:

actb forward:ACATCCGTAAAGACCTCTATGCCAACAC；(SEQ ID NO.161)

actb reverse:CCAGGGAGACCAAAGCCTTCATACATCA；(SEQ ID NO.162)

sanger sequencing primers:

actb sequencing:CCGACAGGATGCAGAAGGAGA；(SEQ ID NO.163)

whole transcriptome random off-target conditions:

whole transcriptome random off-target events are important for occasional weak off-target effects near the target RNA as described above. V1 showed high off-target rates, 24606 off-target sites, whereas V2 was only 144 (fig. 14). Notably, Vx is close to the V2 off-target site (205 sites). As expected, DR deletion did not significantly affect off-target editing (V1, V2 and Vx 18292,182 and 132, respectively; FIG. 14).

It can be seen that Vx has similar RNA targeting activity to V1 and has the advantage of low off-target as V2, demonstrating that the Vx version is currently the best RNA base editing system based on CRISPR systems.

In conclusion, the present invention effectively overcomes various disadvantages of the prior art and has high industrial utilization value.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Sequence listing

<110> Shanghai science and technology university

<120> an RNA base-editing molecule

<160> 163

<170> SIPOSequenceListing 1.0

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Tyr Tyr Ile Glu Glu Asp Ala Lys Val Ala Ala Ala Asn Lys Ser Leu

370 375 380

Pro Asp Asn Glu Lys Ser Leu Ser Glu Lys Asp Ile Phe Val Ile Asn

385 390 395 400

Leu Arg Gly Ser Phe Asn Asp Asp Gln Lys Asp Ala Leu Tyr Tyr Asp

405 410 415

Glu Ala Asn Arg Ile Trp Arg Lys Leu Glu Asn Ile Met His Asn Ile

420 425 430

Lys Glu Phe Arg Gly Asn Lys Thr Arg Glu Tyr Lys Lys Lys Asp Ala

435 440 445

Pro Arg Leu Pro Arg Ile Leu Pro Ala Gly Arg Asp Val Ser Ala Phe

450 455 460

Ser Lys Leu Met Tyr Ala Leu Thr Met Phe Leu Asp Gly Lys Glu Ile

465 470 475 480

Asn Asp Leu Leu Thr Thr Leu Ile Asn Lys Phe Asp Asn Ile Gln Ser

485 490 495

Phe Leu Lys Val Met Pro Leu Ile Gly Val Asn Ala Lys Phe Val Glu

500 505 510

Glu Tyr Ala Phe Phe Lys Asp Ser Ala Lys Ile Ala Asp Glu Leu Arg

515 520 525

Leu Ile Lys Ser Phe Ala Arg Met Gly Glu Pro Ile Ala Asp Ala Arg

530 535 540

Arg Ala Met Tyr Ile Asp Ala Ile Arg Ile Leu Gly Thr Asn

545 550 555

<210> 2

<211> 385

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Gln Leu His Leu Pro Gln Val Leu Ala Asp Ala Val Ser Arg Leu Val

1 5 10 15

Leu Gly Lys Phe Gly Asp Leu Thr Asp Asn Phe Ser Ser Pro His Ala

20 25 30

Arg Arg Lys Val Leu Ala Gly Val Val Met Thr Thr Gly Thr Asp Val

35 40 45

Lys Asp Ala Lys Val Ile Ser Val Ser Thr Gly Thr Lys Cys Ile Asn

50 55 60

Gly Glu Tyr Met Ser Asp Arg Gly Leu Ala Leu Asn Asp Cys His Ala

65 70 75 80

Glu Ile Ile Ser Arg Arg Ser Leu Leu Arg Phe Leu Tyr Thr Gln Leu

85 90 95

Glu Leu Tyr Leu Asn Asn Lys Asp Asp Gln Lys Arg Ser Ile Phe Gln

100 105 110

Lys Ser Glu Arg Gly Gly Phe Arg Leu Lys Glu Asn Val Gln Phe His

115 120 125

Leu Tyr Ile Ser Thr Ser Pro Cys Gly Asp Ala Arg Ile Phe Ser Pro

130 135 140

His Glu Pro Ile Leu Glu Glu Pro Ala Asp Arg His Pro Asn Arg Lys

145 150 155 160

Ala Arg Gly Gln Leu Arg Thr Lys Ile Glu Ser Gly Gln Gly Thr Ile

165 170 175

Pro Val Arg Ser Asn Ala Ser Ile Gln Thr Trp Asp Gly Val Leu Gln

180 185 190

Gly Glu Arg Leu Leu Thr Met Ser Cys Ser Asp Lys Ile Ala Arg Trp

195 200 205

Asn Val Val Gly Ile Gln Gly Ser Leu Leu Ser Ile Phe Val Glu Pro

210 215 220

Ile Tyr Phe Ser Ser Ile Ile Leu Gly Ser Leu Tyr His Gly Asp His

225 230 235 240

Leu Ser Arg Ala Met Tyr Gln Arg Ile Ser Asn Ile Glu Asp Leu Pro

245 250 255

Pro Leu Tyr Thr Leu Asn Lys Pro Leu Leu Ser Gly Ile Ser Asn Ala

260 265 270

Glu Ala Arg Gln Pro Gly Lys Ala Pro Asn Phe Ser Val Asn Trp Thr

275 280 285

Val Gly Asp Ser Ala Ile Glu Val Ile Asn Ala Thr Thr Gly Lys Asp

290 295 300

Glu Leu Gly Arg Ala Ser Arg Leu Cys Lys His Ala Leu Tyr Cys Arg

305 310 315 320

Trp Met Arg Val His Gly Lys Val Pro Ser His Leu Leu Arg Ser Lys

325 330 335

Ile Thr Lys Pro Asn Val Tyr His Glu Ser Lys Leu Ala Ala Lys Glu

340 345 350

Tyr Gln Ala Ala Lys Ala Arg Leu Phe Thr Ala Phe Ile Lys Ala Gly

355 360 365

Leu Gly Ala Trp Val Glu Lys Pro Thr Glu Gln Asp Gln Phe Ser Leu

370 375 380

Thr

385

<210> 3

<211> 380

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Arg Asn Phe Ile Ile Asn Asn Val Ile Ser Asn Lys Arg Phe His

1 5 10 15

Tyr Leu Ile Arg Tyr Gly Asp Pro Ala His Leu His Glu Ile Ala Lys

20 25 30

Asn Glu Ala Val Val Lys Phe Val Leu Gly Arg Ile Ala Asp Ile Gln

35 40 45

Lys Lys Gln Gly Gln Asn Gly Lys Asn Gln Ile Asp Arg Tyr Tyr Glu

50 55 60

Thr Cys Ile Gly Lys Asp Lys Gly Lys Ser Val Ser Glu Lys Val Asp

65 70 75 80

Ala Leu Thr Lys Ile Ile Thr Gly Met Asn Tyr Asp Gln Phe Asp Lys

85 90 95

Lys Arg Ser Val Ile Glu Asp Thr Gly Arg Glu Asn Ala Glu Arg Glu

100 105 110

Lys Phe Lys Lys Ile Ile Ser Leu Tyr Leu Thr Val Ile Tyr His Ile

115 120 125

Leu Lys Asn Ile Val Asn Ile Asn Ala Arg Tyr Val Ile Gly Phe His

130 135 140

Cys Val Glu Arg Asp Ala Gln Leu Tyr Lys Glu Lys Gly Tyr Asp Ile

145 150 155 160

Asn Leu Lys Lys Leu Glu Glu Lys Gly Phe Ser Ser Val Thr Lys Leu

165 170 175

Cys Ala Gly Ile Asp Glu Thr Ala Pro Asp Lys Arg Lys Asp Val Glu

180 185 190

Lys Glu Met Ala Glu Arg Ala Lys Glu Ser Ile Asp Ser Leu Glu Ser

195 200 205

Ala Asn Pro Lys Leu Tyr Ala Asn Tyr Ile Lys Tyr Ser Asp Glu Lys

210 215 220

Lys Ala Glu Glu Phe Thr Arg Gln Ile Asn Arg Glu Lys Ala Lys Thr

225 230 235 240

Ala Leu Asn Ala Tyr Leu Arg Asn Thr Lys Trp Asn Val Ile Ile Arg

245 250 255

Glu Asp Leu Leu Arg Ile Asp Asn Lys Thr Cys Thr Leu Phe Ala Asn

260 265 270

Lys Ala Val Ala Leu Glu Val Ala Arg Tyr Val His Ala Tyr Ile Asn

275 280 285

Asp Ile Ala Glu Val Asn Ser Tyr Phe Gln Leu Tyr His Tyr Ile Met

290 295 300

Gln Arg Ile Ile Met Asn Glu Arg Tyr Glu Lys Ser Ser Gly Lys Val

305 310 315 320

Ser Glu Tyr Phe Asp Ala Val Asn Asp Glu Lys Lys Tyr Asn Asp Arg

325 330 335

Leu Leu Lys Leu Leu Cys Val Pro Phe Gly Tyr Cys Ile Pro Arg Phe

340 345 350

Lys Asn Leu Ser Ile Glu Ala Leu Phe Asp Arg Asn Glu Ala Ala Lys

355 360 365

Phe Asp Lys Glu Lys Lys Lys Val Ser Gly Asn Ser

370 375 380

<210> 4

<211> 18

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys Arg

1 5 10 15

Lys Val

<210> 5

<211> 1408

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys

1 5 10 15

Arg Lys Val Gly Ser Ile Glu Lys Lys Lys Ser Phe Ala Lys Gly Met

20 25 30

Gly Val Lys Ser Thr Leu Val Ser Gly Ser Lys Val Tyr Met Thr Thr

35 40 45

Phe Ala Glu Gly Ser Asp Ala Arg Leu Glu Lys Ile Val Glu Gly Asp

50 55 60

Ser Ile Arg Ser Val Asn Glu Gly Glu Ala Phe Ser Ala Glu Met Ala

65 70 75 80

Asp Lys Asn Ala Gly Tyr Lys Ile Gly Asn Ala Lys Phe Ser His Pro

85 90 95

Lys Gly Tyr Ala Val Val Ala Asn Asn Pro Leu Tyr Thr Gly Pro Val

100 105 110

Gln Gln Asp Met Leu Gly Leu Lys Glu Thr Leu Glu Lys Arg Tyr Phe

115 120 125

Gly Glu Ser Ala Asp Gly Asn Asp Asn Ile Cys Ile Gln Val Ile His

130 135 140

Asn Ile Leu Asp Ile Glu Lys Ile Leu Ala Glu Tyr Ile Thr Asn Ala

145 150 155 160

Ala Tyr Ala Val Asn Asn Ile Ser Gly Leu Asp Lys Asp Ile Ile Gly

165 170 175

Phe Gly Lys Phe Ser Thr Val Tyr Thr Tyr Asp Glu Phe Lys Asp Pro

180 185 190

Glu His His Arg Ala Ala Phe Asn Asn Asn Asp Lys Leu Ile Asn Ala

195 200 205

Ile Lys Ala Gln Tyr Asp Glu Phe Asp Asn Phe Leu Asp Asn Pro Arg

210 215 220

Leu Gly Tyr Phe Gly Gln Ala Phe Phe Ser Lys Glu Gly Arg Asn Tyr

225 230 235 240

Ile Ile Asn Tyr Gly Asn Glu Cys Tyr Asp Ile Leu Ala Leu Leu Ser

245 250 255

Gly Leu Ala His Trp Val Val Ala Asn Asn Glu Glu Glu Ser Arg Ile

260 265 270

Ser Arg Thr Trp Leu Tyr Asn Leu Asp Lys Asn Leu Asp Asn Glu Tyr

275 280 285

Ile Ser Thr Leu Asn Tyr Leu Tyr Asp Arg Ile Thr Asn Glu Leu Thr

290 295 300

Asn Ser Phe Ser Lys Asn Ser Ala Ala Asn Val Asn Tyr Ile Ala Glu

305 310 315 320

Thr Leu Gly Ile Asn Pro Ala Glu Phe Ala Glu Gln Tyr Phe Arg Phe

325 330 335

Ser Ile Met Lys Glu Gln Lys Asn Leu Gly Phe Asn Ile Thr Lys Leu

340 345 350

Arg Glu Val Met Leu Asp Arg Lys Asp Met Ser Glu Ile Arg Lys Asn

355 360 365

His Lys Val Phe Asp Ser Ile Arg Thr Lys Val Tyr Thr Met Met Asp

370 375 380

Phe Val Ile Tyr Arg Tyr Tyr Ile Glu Glu Asp Ala Lys Val Ala Ala

385 390 395 400

Ala Asn Lys Ser Leu Pro Asp Asn Glu Lys Ser Leu Ser Glu Lys Asp

405 410 415

Ile Phe Val Ile Asn Leu Arg Gly Ser Phe Asn Asp Asp Gln Lys Asp

420 425 430

Ala Leu Tyr Tyr Asp Glu Ala Asn Arg Ile Trp Arg Lys Leu Glu Asn

435 440 445

Ile Met His Asn Ile Lys Glu Phe Arg Gly Asn Lys Thr Arg Glu Tyr

450 455 460

Lys Lys Lys Asp Ala Pro Arg Leu Pro Arg Ile Leu Pro Ala Gly Arg

465 470 475 480

Asp Val Ser Ala Phe Ser Lys Leu Met Tyr Ala Leu Thr Met Phe Leu

485 490 495

Asp Gly Lys Glu Ile Asn Asp Leu Leu Thr Thr Leu Ile Asn Lys Phe

500 505 510

Asp Asn Ile Gln Ser Phe Leu Lys Val Met Pro Leu Ile Gly Val Asn

515 520 525

Ala Lys Phe Val Glu Glu Tyr Ala Phe Phe Lys Asp Ser Ala Lys Ile

530 535 540

Ala Asp Glu Leu Arg Leu Ile Lys Ser Phe Ala Arg Met Gly Glu Pro

545 550 555 560

Ile Ala Asp Ala Arg Arg Ala Met Tyr Ile Asp Ala Ile Arg Ile Leu

565 570 575

Gly Thr Asn Gly Ser Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser

580 585 590

Pro Lys Lys Lys Arg Lys Val Gly Ser Gln Leu His Leu Pro Gln Val

595 600 605

Leu Ala Asp Ala Val Ser Arg Leu Val Leu Gly Lys Phe Gly Asp Leu

610 615 620

Thr Asp Asn Phe Ser Ser Pro His Ala Arg Arg Lys Val Leu Ala Gly

625 630 635 640

Val Val Met Thr Thr Gly Thr Asp Val Lys Asp Ala Lys Val Ile Ser

645 650 655

Val Ser Thr Gly Thr Lys Cys Ile Asn Gly Glu Tyr Met Ser Asp Arg

660 665 670

Gly Leu Ala Leu Asn Asp Cys His Ala Glu Ile Ile Ser Arg Arg Ser

675 680 685

Leu Leu Arg Phe Leu Tyr Thr Gln Leu Glu Leu Tyr Leu Asn Asn Lys

690 695 700

Asp Asp Gln Lys Arg Ser Ile Phe Gln Lys Ser Glu Arg Gly Gly Phe

705 710 715 720

Arg Leu Lys Glu Asn Val Gln Phe His Leu Tyr Ile Ser Thr Ser Pro

725 730 735

Cys Gly Asp Ala Arg Ile Phe Ser Pro His Glu Pro Ile Leu Glu Glu

740 745 750

Pro Ala Asp Arg His Pro Asn Arg Lys Ala Arg Gly Gln Leu Arg Thr

755 760 765

Lys Ile Glu Ser Gly Gln Gly Thr Ile Pro Val Arg Ser Asn Ala Ser

770 775 780

Ile Gln Thr Trp Asp Gly Val Leu Gln Gly Glu Arg Leu Leu Thr Met

785 790 795 800

Ser Cys Ser Asp Lys Ile Ala Arg Trp Asn Val Val Gly Ile Gln Gly

805 810 815

Ser Leu Leu Ser Ile Phe Val Glu Pro Ile Tyr Phe Ser Ser Ile Ile

820 825 830

Leu Gly Ser Leu Tyr His Gly Asp His Leu Ser Arg Ala Met Tyr Gln

835 840 845

Arg Ile Ser Asn Ile Glu Asp Leu Pro Pro Leu Tyr Thr Leu Asn Lys

850 855 860

Pro Leu Leu Ser Gly Ile Ser Asn Ala Glu Ala Arg Gln Pro Gly Lys

865 870 875 880

Ala Pro Asn Phe Ser Val Asn Trp Thr Val Gly Asp Ser Ala Ile Glu

885 890 895

Val Ile Asn Ala Thr Thr Gly Lys Asp Glu Leu Gly Arg Ala Ser Arg

900 905 910

Leu Cys Lys His Ala Leu Tyr Cys Arg Trp Met Arg Val His Gly Lys

915 920 925

Val Pro Ser His Leu Leu Arg Ser Lys Ile Thr Lys Pro Asn Val Tyr

930 935 940

His Glu Ser Lys Leu Ala Ala Lys Glu Tyr Gln Ala Ala Lys Ala Arg

945 950 955 960

Leu Phe Thr Ala Phe Ile Lys Ala Gly Leu Gly Ala Trp Val Glu Lys

965 970 975

Pro Thr Glu Gln Asp Gln Phe Ser Leu Thr Gly Ser Lys Arg Thr Ala

980 985 990

Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys Arg Lys Val Gly Ser

995 1000 1005

Met Arg Asn Phe Ile Ile Asn Asn Val Ile Ser Asn Lys Arg Phe His

1010 1015 1020

Tyr Leu Ile Arg Tyr Gly Asp Pro Ala His Leu His Glu Ile Ala Lys

1025 1030 1035 1040

Asn Glu Ala Val Val Lys Phe Val Leu Gly Arg Ile Ala Asp Ile Gln

1045 1050 1055

Lys Lys Gln Gly Gln Asn Gly Lys Asn Gln Ile Asp Arg Tyr Tyr Glu

1060 1065 1070

Thr Cys Ile Gly Lys Asp Lys Gly Lys Ser Val Ser Glu Lys Val Asp

1075 1080 1085

Ala Leu Thr Lys Ile Ile Thr Gly Met Asn Tyr Asp Gln Phe Asp Lys

1090 1095 1100

Lys Arg Ser Val Ile Glu Asp Thr Gly Arg Glu Asn Ala Glu Arg Glu

1105 1110 1115 1120

Lys Phe Lys Lys Ile Ile Ser Leu Tyr Leu Thr Val Ile Tyr His Ile

1125 1130 1135

Leu Lys Asn Ile Val Asn Ile Asn Ala Arg Tyr Val Ile Gly Phe His

1140 1145 1150

Cys Val Glu Arg Asp Ala Gln Leu Tyr Lys Glu Lys Gly Tyr Asp Ile

1155 1160 1165

Asn Leu Lys Lys Leu Glu Glu Lys Gly Phe Ser Ser Val Thr Lys Leu

1170 1175 1180

Cys Ala Gly Ile Asp Glu Thr Ala Pro Asp Lys Arg Lys Asp Val Glu

1185 1190 1195 1200

Lys Glu Met Ala Glu Arg Ala Lys Glu Ser Ile Asp Ser Leu Glu Ser

1205 1210 1215

Ala Asn Pro Lys Leu Tyr Ala Asn Tyr Ile Lys Tyr Ser Asp Glu Lys

1220 1225 1230

Lys Ala Glu Glu Phe Thr Arg Gln Ile Asn Arg Glu Lys Ala Lys Thr

1235 1240 1245

Ala Leu Asn Ala Tyr Leu Arg Asn Thr Lys Trp Asn Val Ile Ile Arg

1250 1255 1260

Glu Asp Leu Leu Arg Ile Asp Asn Lys Thr Cys Thr Leu Phe Ala Asn

1265 1270 1275 1280

Lys Ala Val Ala Leu Glu Val Ala Arg Tyr Val His Ala Tyr Ile Asn

1285 1290 1295

Asp Ile Ala Glu Val Asn Ser Tyr Phe Gln Leu Tyr His Tyr Ile Met

1300 1305 1310

Gln Arg Ile Ile Met Asn Glu Arg Tyr Glu Lys Ser Ser Gly Lys Val

1315 1320 1325

Ser Glu Tyr Phe Asp Ala Val Asn Asp Glu Lys Lys Tyr Asn Asp Arg

1330 1335 1340

Leu Leu Lys Leu Leu Cys Val Pro Phe Gly Tyr Cys Ile Pro Arg Phe

1345 1350 1355 1360

Lys Asn Leu Ser Ile Glu Ala Leu Phe Asp Arg Asn Glu Ala Ala Lys

1365 1370 1375

Phe Asp Lys Glu Lys Lys Lys Val Ser Gly Asn Ser Gly Ser Lys Arg

1380 1385 1390

Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys Arg Lys Val

1395 1400 1405

<210> 6

<211> 65

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ccgatggatc cgagttcgaa tcccctaaaa agaaaagaaa ggtgggatcc cagctgcatt 60

taccg 65

<210> 7

<211> 72

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

tcttcttggg gctctcaaat tcgctgccgt cagcagtccg tttcatggtg gcaagcttaa 60

gtttaaacgc ta 72

<210> 8

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

agagccccaa gaagaagagg aaagtcggat ccatcgagaa gaagaagagc ttcgccaa 58

<210> 9

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ggggattcga actcggatcc atcggcggtg cgcttagaac cggagttgcc gctcacct 58

<210> 10

<211> 9558

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60

ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120

cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180

ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240

gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300

tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360

cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420

attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480

atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540

atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600

tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660

actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720

aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780

gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840

ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900

gtttaaactt aagcttgcca ccatgaaacg gactgctgac ggcagcgaat ttgagagccc 960

caagaagaag aggaaagtcg gatccatcga gaagaagaag agcttcgcca agggcatggg 1020

agtgaagagc accctggtgt ccggctctaa ggtgtacatg accacatttg ctgagggaag 1080

cgacgccagg ctggagaaga tcgtggaggg cgatagcatc agatccgtga acgagggaga 1140

ggctttcagc gccgagatgg ctgacaagaa cgctggctac aagatcggaa acgccaagtt 1200

ttcccaccca aagggctacg ccgtggtggc taacaaccca ctgtacaccg gaccagtgca 1260

gcaggacatg ctgggactga aggagacact ggagaagagg tacttcggcg agtccgccga 1320

cggaaacgat aacatctgca tccaggtcat ccacaacatc ctggatatcg agaagatcct 1380

ggctgagtac atcacaaacg ccgcttacgc cgtgaacaac atctccggcc tggacaagga 1440

tatcatcggc ttcggaaagt tttctaccgt gtacacatac gacgagttca aggatccaga 1500

gcaccaccgg gccgctttta acaacaacga caagctgatc aacgccatca aggctcagta 1560

cgacgagttc gataactttc tggataaccc caggctgggc tacttcggac aggctttctt 1620

ttctaaggag ggcagaaact acatcatcaa ctacggaaac gagtgttacg acatcctggc 1680

cctgctgagc ggactggccc actgggtggt ggccaacaac gaggaggagt ctcggatcag 1740

ccgcacctgg ctgtacaacc tggacaagaa cctggataac gagtacatct ccacactgaa 1800

ctacctgtac gacaggatca ccaacgagct gacaaacagc ttctccaaga actctgccgc 1860

taacgtgaac tacatcgctg agaccctggg catcaaccca gctgagttcg ctgagcagta 1920

cttcagattt tccatcatga aggagcagaa gaacctgggc ttcaacatca caaagctgag 1980

agaagtgatg ctggacagaa aggatatgtc cgagatcagg aagaaccaca aggtgttcga 2040

ttctatcaga accaaggtgt acacaatgat ggactttgtg atctacaggt actacatcga 2100

ggaggatgcc aaggtggccg ctgccaacaa gagcctgccc gacaacgaga agtctctgag 2160

cgagaaggat atcttcgtga tcaacctgag aggctccttt aacgacgatc agaaggacgc 2220

tctgtactac gatgaggcca acaggatctg gagaaagctg gagaacatca tgcacaacat 2280

caaggagttc cggggaaaca agacccgcga gtacaagaag aaggacgctc caaggctgcc 2340

taggatcctg cctgctggaa gggacgtgag cgccttcagc aagctgatgt acgccctgac 2400

aatgtttctg gacggaaagg agatcaacga tctgctgacc acactgatca acaagttcga 2460

caacatccag tcttttctga aagtgatgcc tctgatcggc gtgaacgcta agttcgtgga 2520

ggagtacgcc ttctttaagg acagcgccaa gatcgctgat gagctgcggc tgatcaagtc 2580

ctttgccagg atgggagagc caatcgctga cgctaggaga gctatgtaca tcgatgccat 2640

ccggatcctg ggaaccaacc tgtcttacga cgagctgaag gctctggccg acaccttcag 2700

cctggatgag aacggcaaca agctgaagaa gggcaagcac ggaatgcgca acttcatcat 2760

caacaacgtg atcagcaaca agcggtttca ctacctgatc agatacggcg acccagctca 2820

cctgcacgag atcgctaaga acgaggccgt ggtgaagttc gtgctgggac ggatcgccga 2880

tatccagaag aagcagggcc agaacggaaa gaaccagatc gaccgctact acgagacctg 2940

catcggcaag gataagggaa agtccgtgtc tgagaaggtg gacgctctga ccaagatcat 3000

cacaggcatg aactacgacc agttcgataa gaagagatct gtgatcgagg acaccggaag 3060

ggagaacgcc gagagagaga agtttaagaa gatcatcagc ctgtacctga cagtgatcta 3120

ccacatcctg aagaacatcg tgaacatcaa cgctagatac gtgatcggct tccactgcgt 3180

ggagcgcgat gcccagctgt acaaggagaa gggatacgac atcaacctga agaagctgga 3240

ggagaagggc tttagctccg tgaccaagct gtgcgctgga atcgacgaga cagcccccga 3300

caagaggaag gatgtggaga aggagatggc cgagagagct aaggagagca tcgactccct 3360

ggagtctgct aaccctaagc tgtacgccaa ctacatcaag tactccgatg agaagaaggc 3420

cgaggagttc accaggcaga tcaacagaga gaaggccaag accgctctga acgcctacct 3480

gaggaacaca aagtggaacg tgatcatccg ggaggacctg ctgcgcatcg ataacaagac 3540

ctgtacactg ttcgctaaca aggctgtggc cctggaggtg gctcgctacg tgcacgccta 3600

catcaacgac atcgccgagg tgaactccta ctttcagctg taccactaca tcatgcagag 3660

gatcatcatg aacgagagat acgagaagtc tagcggcaag gtgtctgagt acttcgacgc 3720

cgtgaacgat gagaagaagt acaacgatag actgctgaag ctgctgtgcg tgcctttcgg 3780

atactgtatc ccacggttta agaacctgag catcgaggcc ctgttcgacc gcaacgaggc 3840

tgccaagttt gataaggaga agaagaaggt gagcggcaac tccggttcta agcgcaccgc 3900

cgatggatcc gagttcgaat cccctaaaaa gaaaagaaag gtgggatccc agctgcattt 3960

accgcaggtt ttagctgacg ctgtctcacg cctggtcctg ggtaagtttg gtgacctgac 4020

cgacaacttc tcctcccctc acgctcgcag aaaagtgctg gctggagtcg tcatgacaac 4080

aggcacagat gttaaagatg ccaaggtgat aagtgtttct acaggaacaa aatgtattaa 4140

tggtgaatac atgagtgatc gtggccttgc attaaatgac tgccatgcag aaataatatc 4200

tcggagatcc ttgctcagat ttctttatac acaacttgag ctttacttaa ataacaaaga 4260

tgatcaaaaa agatccatct ttcagaaatc agagcgaggg gggtttaggc tgaaggagaa 4320

tgtccagttt catctgtaca tcagcacctc tccctgtgga gatgccagaa tcttctcacc 4380

acatgagcca atcctggaag aaccagcaga tagacaccca aatcgtaaag caagaggaca 4440

gctacggacc aaaatagagt ctggtcaggg gacgattcca gtgcgctcca atgcgagcat 4500

ccaaacgtgg gacggggtgc tgcaagggga gcggctgctc accatgtcct gcagtgacaa 4560

gattgcacgc tggaacgtgg tgggcatcca gggatcactg ctcagcattt tcgtggagcc 4620

catttacttc tcgagcatca tcctgggcag cctttaccac ggggaccacc tttccagggc 4680

catgtaccag cggatctcca acatagagga cctgccacct ctctacaccc tcaacaagcc 4740

tttgctcagt ggcatcagca atgcagaagc acggcagcca gggaaggccc ccaacttcag 4800

tgtcaactgg acggtaggcg actccgctat tgaggtcatc aacgccacga ctgggaagga 4860

tgagctgggc cgcgcgtccc gcctgtgtaa gcacgcgttg tactgtcgct ggatgcgtgt 4920

gcacggcaag gttccctccc acttactacg ctccaagatt accaagccca acgtgtacca 4980

tgagtccaag ctggcggcaa aggagtacca ggccgccaag gcgcgtctgt tcacagcctt 5040

catcaaggcg gggctggggg cctgggtgga gaagcccacc gagcaggacc agttctcact 5100

cacgtaagcg gccgctcgag tctagagggc ccgtttaaac ccgctgatca gcctcgactg 5160

tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg 5220

aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga 5280

gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg 5340

aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa 5400

ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg 5460

gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt 5520

tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 5580

gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg 5640

attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga 5700

cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc 5760

ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa 5820

aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt 5880

agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa 5940

ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag 6000

catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct 6060

aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc 6120

agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg 6180

aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca 6240

agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc 6300

ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc 6360

tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga 6420

cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac 6480

gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct 6540

gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa 6600

agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc 6660

attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct 6720

tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc 6780

caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg 6840

cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct 6900

gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct 6960

tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca 7020

gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa 7080

atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc 7140

tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc 7200

ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt 7260

tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct 7320

agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct 7380

agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc 7440

acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 7500

gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 7560

tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 7620

cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 7680

gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 7740

aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 7800

gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 7860

aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 7920

gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 7980

ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 8040

cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 8100

ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 8160

actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 8220

tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 8280

gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 8340

ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 8400

ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 8460

gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 8520

aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 8580

gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 8640

gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 8700

cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 8760

gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 8820

gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 8880

ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 8940

tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 9000

ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 9060

cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 9120

accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 9180

cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 9240

tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 9300

cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 9360

acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 9420

atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 9480

tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 9540

aaagtgccac ctgacgtc 9558

<210> 11

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

aggatgacga tgacaagtaa gcggccgctc gagccta 37

<210> 12

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

agagcccccg cctccgttgg ttcccaggat ccggatg 37

<210> 13

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

ggaggcgggg gctctggatc ccagctgcat ttaccgcagg ttt 43

<210> 14

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gcttccgccc cctccagagc ccgtgagtga gaactggtcc tgctcg 46

<210> 15

<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

ggagggggcg gaagcatgcg caacttcatc atcaacaacg tgatcagcaa 50

<210> 16

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

cttgtcatcg tcatccttgt aatcgatatc atgatcttta taatcaccgt catggtctt 59

<210> 17

<211> 9582

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60

ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120

cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180

ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240

gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300

tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360

cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420

attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480

atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540

atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600

tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660

actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720

aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780

gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840

ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900

gtttaaactt aagcttgcca ccatgaaacg gactgctgac ggcagcgaat ttgagagccc 960

caagaagaag aggaaagtcg gatccatcga gaagaagaag agcttcgcca agggcatggg 1020

agtgaagagc accctggtgt ccggctctaa ggtgtacatg accacatttg ctgagggaag 1080

cgacgccagg ctggagaaga tcgtggaggg cgatagcatc agatccgtga acgagggaga 1140

ggctttcagc gccgagatgg ctgacaagaa cgctggctac aagatcggaa acgccaagtt 1200

ttcccaccca aagggctacg ccgtggtggc taacaaccca ctgtacaccg gaccagtgca 1260

gcaggacatg ctgggactga aggagacact ggagaagagg tacttcggcg agtccgccga 1320

cggaaacgat aacatctgca tccaggtcat ccacaacatc ctggatatcg agaagatcct 1380

ggctgagtac atcacaaacg ccgcttacgc cgtgaacaac atctccggcc tggacaagga 1440

tatcatcggc ttcggaaagt tttctaccgt gtacacatac gacgagttca aggatccaga 1500

gcaccaccgg gccgctttta acaacaacga caagctgatc aacgccatca aggctcagta 1560

cgacgagttc gataactttc tggataaccc caggctgggc tacttcggac aggctttctt 1620

ttctaaggag ggcagaaact acatcatcaa ctacggaaac gagtgttacg acatcctggc 1680

cctgctgagc ggactggccc actgggtggt ggccaacaac gaggaggagt ctcggatcag 1740

ccgcacctgg ctgtacaacc tggacaagaa cctggataac gagtacatct ccacactgaa 1800

ctacctgtac gacaggatca ccaacgagct gacaaacagc ttctccaaga actctgccgc 1860

taacgtgaac tacatcgctg agaccctggg catcaaccca gctgagttcg ctgagcagta 1920

cttcagattt tccatcatga aggagcagaa gaacctgggc ttcaacatca caaagctgag 1980

agaagtgatg ctggacagaa aggatatgtc cgagatcagg aagaaccaca aggtgttcga 2040

ttctatcaga accaaggtgt acacaatgat ggactttgtg atctacaggt actacatcga 2100

ggaggatgcc aaggtggccg ctgccaacaa gagcctgccc gacaacgaga agtctctgag 2160

cgagaaggat atcttcgtga tcaacctgag aggctccttt aacgacgatc agaaggacgc 2220

tctgtactac gatgaggcca acaggatctg gagaaagctg gagaacatca tgcacaacat 2280

caaggagttc cggggaaaca agacccgcga gtacaagaag aaggacgctc caaggctgcc 2340

taggatcctg cctgctggaa gggacgtgag cgccttcagc aagctgatgt acgccctgac 2400

aatgtttctg gacggaaagg agatcaacga tctgctgacc acactgatca acaagttcga 2460

caacatccag tcttttctga aagtgatgcc tctgatcggc gtgaacgcta agttcgtgga 2520

ggagtacgcc ttctttaagg acagcgccaa gatcgctgat gagctgcggc tgatcaagtc 2580

ctttgccagg atgggagagc caatcgctga cgctaggaga gctatgtaca tcgatgccat 2640

ccggatcctg ggaaccaacg gaggcggggg ctctggatcc cagctgcatt taccgcaggt 2700

tttagctgac gctgtctcac gcctggtcct gggtaagttt ggtgacctga ccgacaactt 2760

ctcctcccct cacgctcgca gaaaagtgct ggctggagtc gtcatgacaa caggcacaga 2820

tgttaaagat gccaaggtga taagtgtttc tacaggaaca aaatgtatta atggtgaata 2880

catgagtgat cgtggccttg cattaaatga ctgccatgca gaaataatat ctcggagatc 2940

cttgctcaga tttctttata cacaacttga gctttactta aataacaaag atgatcaaaa 3000

aagatccatc tttcagaaat cagagcgagg ggggtttagg ctgaaggaga atgtccagtt 3060

tcatctgtac atcagcacct ctccctgtgg agatgccaga atcttctcac cacatgagcc 3120

aatcctggaa gaaccagcag atagacaccc aaatcgtaaa gcaagaggac agctacggac 3180

caaaatagag tctggtcagg ggacgattcc agtgcgctcc aatgcgagca tccaaacgtg 3240

ggacggggtg ctgcaagggg agcggctgct caccatgtcc tgcagtgaca agattgcacg 3300

ctggaacgtg gtgggcatcc agggatcact gctcagcatt ttcgtggagc ccatttactt 3360

ctcgagcatc atcctgggca gcctttacca cggggaccac ctttccaggg ccatgtacca 3420

gcggatctcc aacatagagg acctgccacc tctctacacc ctcaacaagc ctttgctcag 3480

tggcatcagc aatgcagaag cacggcagcc agggaaggcc cccaacttca gtgtcaactg 3540

gacggtaggc gactccgcta ttgaggtcat caacgccacg actgggaagg atgagctggg 3600

ccgcgcgtcc cgcctgtgta agcacgcgtt gtactgtcgc tggatgcgtg tgcacggcaa 3660

ggttccctcc cacttactac gctccaagat taccaagccc aacgtgtacc atgagtccaa 3720

gctggcggca aaggagtacc aggccgccaa ggcgcgtctg ttcacagcct tcatcaaggc 3780

ggggctgggg gcctgggtgg agaagcccac cgagcaggac cagttctcac tcacgggctc 3840

tggagggggc ggaagcatgc gcaacttcat catcaacaac gtgatcagca acaagcggtt 3900

tcactacctg atcagatacg gcgacccagc tcacctgcac gagatcgcta agaacgaggc 3960

cgtggtgaag ttcgtgctgg gacggatcgc cgatatccag aagaagcagg gccagaacgg 4020

aaagaaccag atcgaccgct actacgagac ctgcatcggc aaggataagg gaaagtccgt 4080

gtctgagaag gtggacgctc tgaccaagat catcacaggc atgaactacg accagttcga 4140

taagaagaga tctgtgatcg aggacaccgg aagggagaac gccgagagag agaagtttaa 4200

gaagatcatc agcctgtacc tgacagtgat ctaccacatc ctgaagaaca tcgtgaacat 4260

caacgctaga tacgtgatcg gcttccactg cgtggagcgc gatgcccagc tgtacaagga 4320

gaagggatac gacatcaacc tgaagaagct ggaggagaag ggctttagct ccgtgaccaa 4380

gctgtgcgct ggaatcgacg agacagcccc cgacaagagg aaggatgtgg agaaggagat 4440

ggccgagaga gctaaggaga gcatcgactc cctggagtct gctaacccta agctgtacgc 4500

caactacatc aagtactccg atgagaagaa ggccgaggag ttcaccaggc agatcaacag 4560

agagaaggcc aagaccgctc tgaacgccta cctgaggaac acaaagtgga acgtgatcat 4620

ccgggaggac ctgctgcgca tcgataacaa gacctgtaca ctgttcgcta acaaggctgt 4680

ggccctggag gtggctcgct acgtgcacgc ctacatcaac gacatcgccg aggtgaactc 4740

ctactttcag ctgtaccact acatcatgca gaggatcatc atgaacgaga gatacgagaa 4800

gtctagcggc aaggtgtctg agtacttcga cgccgtgaac gatgagaaga agtacaacga 4860

tagactgctg aagctgctgt gcgtgccttt cggatactgt atcccacggt ttaagaacct 4920

gagcatcgag gccctgttcg accgcaacga ggctgccaag tttgataagg agaagaagaa 4980

ggtgagcggc aactccggtt ctaagcgcac cgccgatgga tccgagttcg aatcccctaa 5040

aaagaaaaga aaggtgggat ccgactacaa agaccatgac ggtgattata aagatcatga 5100

tatcgattac aaggatgacg atgacaagta agcggccgct cgagcctaga gggcccgttt 5160

aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct 5220

cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg 5280

aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc 5340

aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct 5400

ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct 5460

gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg 5520

ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg 5580

gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac 5640

ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct 5700

gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt 5760

tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt 5820

tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt 5880

aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag 5940

aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc 6000

cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc 6060

cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg 6120

ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca 6180

gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg 6240

tatatccatt ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca 6300

agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg 6360

ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg 6420

cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc 6480

agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt 6540

cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc 6600

atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca 6660

tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc 6720

acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg 6780

gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct 6840

cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc 6900

tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc 6960

tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta 7020

cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt 7080

ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga 7140

gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac 7200

gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac 7260

ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat 7320

aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat 7380

catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt 7440

cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag 7500

tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg 7560

cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg 7620

gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc 7680

tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc 7740

acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg 7800

aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat 7860

cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 7920

gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 7980

tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg 8040

tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt 8100

cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 8160

gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 8220

ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt 8280

ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc 8340

ggcaaacaaa ccaccgctgg tagcggtttt tttgtttgca agcagcagat tacgcgcaga 8400

aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 8460

gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 8520

cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 8580

gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 8640

tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct 8700

ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca 8760

ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 8820

atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg 8880

cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct 8940

tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa 9000

aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 9060

tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc 9120

ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 9180

agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa 9240

gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg 9300

agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc 9360

accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg 9420

gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat 9480

cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 9540

ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tc 9582

<210> 18

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

ttggttccca ggatccggat ggcatcgatg tacat 35

<210> 19

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

gacgatgaca agtaagcggc cgctcgagcc tagagggc 38

<210> 20

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

ggatccgagt tcgaatcccc taaaaagaaa agaaaggtgg gatcccagct gcatttaccg 60

<210> 21

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

ctctcaaact cagatccatc cgcagttctt ttggatcccg tgagtgagaa ctggtcctgc 60

<210> 22

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

ccggatcctg ggaaccaacg gttctaagcg caccgccgat ggatccgagt tcgaatcccc 60

<210> 23

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

ggatggatct gagtttgaga gtccaaaaaa gaagaggaag gtcggttcta tgcgcaactt 60

<210> 24

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

ttctatgcgc aacttcatca tcaacaacgt gatcagc 37

<210> 25

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

taggctcgag cggccgctta cttgtcatcg tcat 34

<210> 26

<211> 9672

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60

ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120

cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180

ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240

gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300

tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360

cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420

attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480

atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540

atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600

tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660

actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720

aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780

gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840

ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900

gtttaaactt aagcttgcca ccatgaaacg gactgctgac ggcagcgaat ttgagagccc 960

caagaagaag aggaaagtcg gatccatcga gaagaagaag agcttcgcca agggcatggg 1020

agtgaagagc accctggtgt ccggctctaa ggtgtacatg accacatttg ctgagggaag 1080

cgacgccagg ctggagaaga tcgtggaggg cgatagcatc agatccgtga acgagggaga 1140

ggctttcagc gccgagatgg ctgacaagaa cgctggctac aagatcggaa acgccaagtt 1200

ttcccaccca aagggctacg ccgtggtggc taacaaccca ctgtacaccg gaccagtgca 1260

gcaggacatg ctgggactga aggagacact ggagaagagg tacttcggcg agtccgccga 1320

cggaaacgat aacatctgca tccaggtcat ccacaacatc ctggatatcg agaagatcct 1380

ggctgagtac atcacaaacg ccgcttacgc cgtgaacaac atctccggcc tggacaagga 1440

tatcatcggc ttcggaaagt tttctaccgt gtacacatac gacgagttca aggatccaga 1500

gcaccaccgg gccgctttta acaacaacga caagctgatc aacgccatca aggctcagta 1560

cgacgagttc gataactttc tggataaccc caggctgggc tacttcggac aggctttctt 1620

ttctaaggag ggcagaaact acatcatcaa ctacggaaac gagtgttacg acatcctggc 1680

cctgctgagc ggactggccc actgggtggt ggccaacaac gaggaggagt ctcggatcag 1740

ccgcacctgg ctgtacaacc tggacaagaa cctggataac gagtacatct ccacactgaa 1800

ctacctgtac gacaggatca ccaacgagct gacaaacagc ttctccaaga actctgccgc 1860

taacgtgaac tacatcgctg agaccctggg catcaaccca gctgagttcg ctgagcagta 1920

cttcagattt tccatcatga aggagcagaa gaacctgggc ttcaacatca caaagctgag 1980

agaagtgatg ctggacagaa aggatatgtc cgagatcagg aagaaccaca aggtgttcga 2040

ttctatcaga accaaggtgt acacaatgat ggactttgtg atctacaggt actacatcga 2100

ggaggatgcc aaggtggccg ctgccaacaa gagcctgccc gacaacgaga agtctctgag 2160

cgagaaggat atcttcgtga tcaacctgag aggctccttt aacgacgatc agaaggacgc 2220

tctgtactac gatgaggcca acaggatctg gagaaagctg gagaacatca tgcacaacat 2280

caaggagttc cggggaaaca agacccgcga gtacaagaag aaggacgctc caaggctgcc 2340

taggatcctg cctgctggaa gggacgtgag cgccttcagc aagctgatgt acgccctgac 2400

aatgtttctg gacggaaagg agatcaacga tctgctgacc acactgatca acaagttcga 2460

caacatccag tcttttctga aagtgatgcc tctgatcggc gtgaacgcta agttcgtgga 2520

ggagtacgcc ttctttaagg acagcgccaa gatcgctgat gagctgcggc tgatcaagtc 2580

ctttgccagg atgggagagc caatcgctga cgctaggaga gctatgtaca tcgatgccat 2640

ccggatcctg ggaaccaacg gttctaagcg caccgccgat ggatccgagt tcgaatcccc 2700

taaaaagaaa agaaaggtgg gatcccagct gcatttaccg caggttttag ctgacgctgt 2760

ctcacgcctg gtcctgggta agtttggtga cctgaccgac aacttctcct cccctcacgc 2820

tcgcagaaaa gtgctggctg gagtcgtcat gacaacaggc acagatgtta aagatgccaa 2880

ggtgataagt gtttctacag gaacaaaatg tattaatggt gaatacatga gtgatcgtgg 2940

ccttgcatta aatgactgcc atgcagaaat aatatctcgg agatccttgc tcagatttct 3000

ttatacacaa cttgagcttt acttaaataa caaagatgat caaaaaagat ccatctttca 3060

gaaatcagag cgaggggggt ttaggctgaa ggagaatgtc cagtttcatc tgtacatcag 3120

cacctctccc tgtggagatg ccagaatctt ctcaccacat gagccaatcc tggaagaacc 3180

agcagataga cacccaaatc gtaaagcaag aggacagcta cggaccaaaa tagagtctgg 3240

tcaggggacg attccagtgc gctccaatgc gagcatccaa acgtgggacg gggtgctgca 3300

aggggagcgg ctgctcacca tgtcctgcag tgacaagatt gcacgctgga acgtggtggg 3360

catccaggga tcactgctca gcattttcgt ggagcccatt tacttctcga gcatcatcct 3420

gggcagcctt taccacgggg accacctttc cagggccatg taccagcgga tctccaacat 3480

agaggacctg ccacctctct acaccctcaa caagcctttg ctcagtggca tcagcaatgc 3540

agaagcacgg cagccaggga aggcccccaa cttcagtgtc aactggacgg taggcgactc 3600

cgctattgag gtcatcaacg ccacgactgg gaaggatgag ctgggccgcg cgtcccgcct 3660

gtgtaagcac gcgttgtact gtcgctggat gcgtgtgcac ggcaaggttc cctcccactt 3720

actacgctcc aagattacca agcccaacgt gtaccatgag tccaagctgg cggcaaagga 3780

gtaccaggcc gccaaggcgc gtctgttcac agccttcatc aaggcggggc tgggggcctg 3840

ggtggagaag cccaccgagc aggaccagtt ctcactcacg ggatccaaaa gaactgcgga 3900

tggatctgag tttgagagtc caaaaaagaa gaggaaggtc ggttctatgc gcaacttcat 3960

catcaacaac gtgatcagca acaagcggtt tcactacctg atcagatacg gcgacccagc 4020

tcacctgcac gagatcgcta agaacgaggc cgtggtgaag ttcgtgctgg gacggatcgc 4080

cgatatccag aagaagcagg gccagaacgg aaagaaccag atcgaccgct actacgagac 4140

ctgcatcggc aaggataagg gaaagtccgt gtctgagaag gtggacgctc tgaccaagat 4200

catcacaggc atgaactacg accagttcga taagaagaga tctgtgatcg aggacaccgg 4260

aagggagaac gccgagagag agaagtttaa gaagatcatc agcctgtacc tgacagtgat 4320

ctaccacatc ctgaagaaca tcgtgaacat caacgctaga tacgtgatcg gcttccactg 4380

cgtggagcgc gatgcccagc tgtacaagga gaagggatac gacatcaacc tgaagaagct 4440

ggaggagaag ggctttagct ccgtgaccaa gctgtgcgct ggaatcgacg agacagcccc 4500

cgacaagagg aaggatgtgg agaaggagat ggccgagaga gctaaggaga gcatcgactc 4560

cctggagtct gctaacccta agctgtacgc caactacatc aagtactccg atgagaagaa 4620

ggccgaggag ttcaccaggc agatcaacag agagaaggcc aagaccgctc tgaacgccta 4680

cctgaggaac acaaagtgga acgtgatcat ccgggaggac ctgctgcgca tcgataacaa 4740

gacctgtaca ctgttcgcta acaaggctgt ggccctggag gtggctcgct acgtgcacgc 4800

ctacatcaac gacatcgccg aggtgaactc ctactttcag ctgtaccact acatcatgca 4860

gaggatcatc atgaacgaga gatacgagaa gtctagcggc aaggtgtctg agtacttcga 4920

cgccgtgaac gatgagaaga agtacaacga tagactgctg aagctgctgt gcgtgccttt 4980

cggatactgt atcccacggt ttaagaacct gagcatcgag gccctgttcg accgcaacga 5040

ggctgccaag tttgataagg agaagaagaa ggtgagcggc aactccggtt ctaagcgcac 5100

cgccgatgga tccgagttcg aatcccctaa aaagaaaaga aaggtgggat ccgactacaa 5160

agaccatgac ggtgattata aagatcatga tatcgattac aaggatgacg atgacaagta 5220

agcggccgct cgagcctaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt 5280

ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg 5340

ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt 5400

gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca 5460

atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct 5520

ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg 5580

tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt 5640

tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc 5700

tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg 5760

gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg 5820

agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct 5880

cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg 5940

agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg 6000

tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc 6060

agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca 6120

tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc 6180

gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc 6240

cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct 6300

aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac 6360

aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc 6420

ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc 6480

cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc 6540

cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg 6600

cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt 6660

gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc 6720

catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga 6780

ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga 6840

tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct 6900

caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc 6960

gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt 7020

ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg 7080

cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat 7140

cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc 7200

gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa 7260

aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat 7320

ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa 7380

taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt 7440

ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag 7500

agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt 7560

ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc 7620

taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc 7680

cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct 7740

tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca 7800

gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac 7860

atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 7920

ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 7980

cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 8040

tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 8100

gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 8160

aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 8220

tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 8280

aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 8340

aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc 8400

ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtttt 8460

tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 8520

ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 8580

agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 8640

atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 8700

cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 8760

ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac 8820

ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc 8880

agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct 8940

agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc 9000

gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg 9060

cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc 9120

gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat 9180

tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag 9240

tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat 9300

aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 9360

cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca 9420

cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga 9480

aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc 9540

ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 9600

tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 9660

ccacctgacg tc 9672

<210> 27

<211> 4875

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 60

cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 120

aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 180

cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 240

gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 300

ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 360

cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 420

aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 480

tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc 540

ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 600

tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 660

ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 720

agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 780

atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 840

cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 900

ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagat 960

ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc 1020

agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct 1080

agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc 1140

gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg 1200

cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc 1260

gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat 1320

tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag 1380

tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat 1440

aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 1500

cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca 1560

cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga 1620

aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc 1680

ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 1740

tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 1800

ccacctgacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc 1860

gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt 1920

ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc 1980

cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt 2040

agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt 2100

aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt 2160

gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa 2220

aaatttaacg cgaattttaa caaaatatta acgcttacaa tttgccattc gccattcagg 2280

ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagcccaag 2340

ctaccatgat aagtaagtaa tattaaggta cgggaggtac ttggagcggc cgcaataaaa 2400

tatctttatt ttcattacat ctgtgtgttg gttttttgtg tgaatcgata gtactaacat 2460

acgctctcca tcaaaacaaa acgaaacaaa acaaactagc aaaataggct gtccccagtg 2520

caagtgcagg tgccagaaca tttctctatc gataggtacc gattagtgaa cggatctcga 2580

cggtatcgat cacgagacta gcctcgagcg gccgccccct tcaccgaggg cctatttccc 2640

atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tggaattaat 2700

ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt 2760

gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact 2820

tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgcaagt 2880

aaacccctac caactggtcg gggtttgaaa cagaagagcc tcgaggctct tctcaagtaa 2940

acccctacca actggtcggg gtttgaaacg aagacttttt ttttcgcttc ctcgctcact 3000

gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta 3060

atacggtcct cgagacaaat ggcagtattc atccacaatt ttaaaagaaa aggggggatt 3120

ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa 3180

gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag ggacagcaga 3240

gatccacttt ggccgcggct cgagggggtt ggggttgcgc cttttccaag gcagccctgg 3300

gtttgcgcag ggacgcggct gctctgggcg tggttccggg aaacgcagcg gcgccgaccc 3360

tgggactcgc acattcttca cgtccgttcg cagcgtcacc cggatcttcg ccgctaccct 3420

tgtgggcccc ccggcgacgc ttcctgctcc gcccctaagt cgggaaggtt ccttgcggtt 3480

cgcggcgtgc cggacgtgac aaacggaagc cgcacgtctc actagtaccc tcgcagacgg 3540

acagcgccag ggagcaatgg cagcgcgccg accgcgatgg gctgtggcca atagcggctg 3600

ctcagcaggg cgcgccgaga gcagcggccg ggaaggggcg gtgcgggagg cggggtgtgg 3660

ggcggtagtg tgggccctgt tcctgcccgc gcggtgttcc gcattctgca agcctccgga 3720

gcgcacgtcg gcagtcggct ccctcgttga ccgaatcacc gacctctctc cccaggggga 3780

tccaccggag cttaccatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga 3840

cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca 3900

caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac 3960

gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt 4020

ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat 4080

ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc 4140

gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca 4200

gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg 4260

ggtgcccgcc ttcctggaaa cctccgcgcc ccgcaacctc cccttctacg agcggctcgg 4320

cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg 4380

caagcccggt gcctgacgcc cgccccacga cccgcagcgc ccgaccgaaa ggagcgcacg 4440

accccatgca tcggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca 4500

ctttctagag tcggggcggc cggccgcttc gagcagacat gataagatac attgatgagt 4560

ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg 4620

ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca 4680

ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc 4740

tctacaaatg tggtcgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 4800

cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 4860

gcaggaaaga acatg 4875

<210> 28

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

ttgctgaggg tccccagtgt ttctggatga catcatggat cagcccagct gtc 53

<210> 29

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

aacgacagct gggctgatcc atgatgtcat ccagaaacac tggggaccct cag 53

<210> 30

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

ttgttacagg atgctatgat tccatgatac ggatatggaa agttgagatg aga 53

<210> 31

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

aactctcatc tcaactttcc atatccgtat catggaatca tagcatcctg taa 53

<210> 32

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

ttgtggaaca gatacgcgct tactgtgaaa cctgttggga gtggcaggaa gct 53

<210> 33

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

aacagcttcc tgccactccc aacaggtttc acagtaagcg cgtatctgtt cca 53

<210> 34

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

ttgtatgcaa gtatttaagt ctccacacaa gtgactgggt aagctcttgc cgt 53

<210> 35

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

aacacggcaa gagcttaccc agtcacttgt gtggagactt aaatacttgc ata 53

<210> 36

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

ttggatgtgt gaggcacctg tggtgacccg agagtgggtg ttggacagtg tag 53

<210> 37

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

aacctacact gtccaacacc cactctcggg tcaccacagg tgcctcacac atc 53

<210> 38

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

ttgtctgcca tcctgaactc aagagtggag aatactgggt tgaccctaac caa 53

<210> 39

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

aacttggtta gggtcaaccc agtattctcc actcttgagt tcaggatggc aga 53

<210> 40

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

ttgtcatcaa ggaaccaaat gatctgttaa agctgtggaa gcatgagtgt aaa 53

<210> 41

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 41

aactttacac tcatgcttcc acagctttaa cagatcattt ggttccttga tga 53

<210> 42

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 42

ttgccatgta tgatgacccc accctgcctg aaggctggac acggaagctt aag 53

<210> 43

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 43

aaccttaagc ttccgtgtcc agccttcagg cagggtgggg tcatcataca tgg 53

<210> 44

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 44

ttgagccacc ccaggatgtc ggcaacacgg agctctgggg gtacacagtg cag 53

<210> 45

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 45

aacctgcact gtgtaccccc agagctccgt gttgccgaca tcctggggtg gct 53

<210> 46

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 46

ttgggctcga gtggtgctgg aactgtggct gcgagtggaa ccgcgtctgc atg 53

<210> 47

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 47

aaccatgcag acgcggttcc actcgcagcc acagttccag caccactcga gcc 53

<210> 48

<211> 63

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 48

ttgccccagt gcaggctcga gtggtgctgg aactgtggct gcgagtggaa ccgcgtctgc 60

atg 63

<210> 49

<211> 63

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 49

aaccatgcag acgcggttcc actcgcagcc acagttccag caccactcga gcctgcactg 60

ggg 63

<210> 50

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 50

ttggtgctgg aactgtggct gcgagtggaa ccgcgtctgc atg 43

<210> 51

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 51

aaccatgcag acgcggttcc actcgcagcc acagttccag cac 43

<210> 52

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 52

ttgtgtggct gcgagtggaa ccgcgtctgc atg 33

<210> 53

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 53

aaccatgcag acgcggttcc actcgcagcc aca 33

<210> 54

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 54

ttggtggctg cgagtggaac cgcgtctg 28

<210> 55

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 55

aaccagacgc ggttccactc gcagccac 28

<210> 56

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 56

ttggctgcga gtggaaccgc gtctg 25

<210> 57

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 57

aaccagacgc ggttccactc gcagc 25

<210> 58

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 58

ttgcgagtgg aaccgcgtct g 21

<210> 59

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 59

aaccagacgc ggttccactc g 21

<210> 60

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 60

cgccgagggc cgccactcca c 21

<210> 61

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 61

acgctgaact tgtggccgtt t 21

<210> 62

<211> 201

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 62

gagcaggctc gttgggaagc agcctccaaa gaaaccatca agaaaaccac caagccctgt 60

ccccgctgcc atgtaccagt ggaaaaaaat ggaggctgca tgcacatgaa gtgtccgcag 120

ccccagtgca ggctcgagtg gtgctggaac tgtggctgcg agtagaaccg cgtctgcatg 180

ggggaccact ggttcgacgt g 201

<210> 63

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 63

aaccatgcag acgcggttcc a 21

<210> 64

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 64

ttgtggaacc gcgtctgcat g 21

<210> 65

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 65

aaccatgcag acgcggttcc actcg 25

<210> 66

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 66

ttgcgagtgg aaccgcgtct gcatg 25

<210> 67

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 67

aaccatgcag acgcggttcc actcgcag 28

<210> 68

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 68

ttgctgcgag tggaaccgcg tctgcatg 28

<210> 69

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 69

aaccatgcag acgcggttcc actcgcagcc aca 33

<210> 70

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 70

ttgtgtggct gcgagtggaa ccgcgtctgc atg 33

<210> 71

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 71

aaccatgcag acgcggttcc actcgcagcc acagttccag cac 43

<210> 72

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 72

ttggtgctgg aactgtggct gcgagtggaa ccgcgtctgc atg 43

<210> 73

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 73

aaccatgcag acgcggttcc actcgcagcc acagttccag caccactcga gcc 53

<210> 74

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 74

ttgggctcga gtggtgctgg aactgtggct gcgagtggaa ccgcgtctgc atg 53

<210> 75

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 75

aactccactc gcagccacag ttcca 25

<210> 76

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 76

ttgtggaact gtggctgcga gtgg 24

<210> 77

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 77

aacgttccac tcgcagccac agttcc 26

<210> 78

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 78

ttgggaactg tggctgcgag tggaac 26

<210> 79

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 79

aacggttcca ctcgcagcca cagtt 25

<210> 80

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 80

ttgaactgtg gctgcgagtg gaacc 25

<210> 81

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 81

aaccggttcc actcgcagcc acagt 25

<210> 82

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 82

ttgactgtgg ctgcgagtgg aaccg 25

<210> 83

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 83

aacgcggttc cactcgcagc cacag 25

<210> 84

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 84

ttgctgtggc tgcgagtgga accgc 25

<210> 85

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 85

aaccgcggtt ccactcgcag ccaca 25

<210> 86

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 86

ttgtgtggct gcgagtggaa ccgcg 25

<210> 87

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 87

aacacgcggt tccactcgca gccac 25

<210> 88

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 88

ttggtggctg cgagtggaac cgcgt 25

<210> 89

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 89

aacgacgcgg ttccactcgc agcca 25

<210> 90

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 90

ttgtggctgc gagtggaacc gcgtc 25

<210> 91

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 91

aacagacgcg gttccactcg cagcc 25

<210> 92

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 92

ttgggctgcg agtggaaccg cgtct 25

<210> 93

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 93

aaccagacgc ggttccactc gcagc 25

<210> 94

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 94

ttggctgcga gtggaaccgc gtctg 25

<210> 95

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 95

aacgcagacg cggttccact cgcag 25

<210> 96

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 96

ttgctgcgag tggaaccgcg tctgc 25

<210> 97

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 97

aactgcagac gcggttccac tcgca 25

<210> 98

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 98

ttgtgcgagt ggaaccgcgt ctgca 25

<210> 99

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 99

aacccatgca gacgcggttc cactc 25

<210> 100

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 100

ttggagtgga accgcgtctg catgg 25

<210> 101

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 101

aacagacgcg gttacactcg cagcc 25

<210> 102

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 102

ttgggctgcg agtgtaaccg cgtct 25

<210> 103

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 103

aacagacgcg gtttcactcg cagcc 25

<210> 104

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 104

ttgggctgcg agtgaaaccg cgtct 25

<210> 105

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 105

aacagacgcg gttgcactcg cagcc 25

<210> 106

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 106

ttgggctgcg agtgcaaccg cgtct 25

<210> 107

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 107

aacagacgcg gtttctctcg cagcc 25

<210> 108

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 108

ttgggctgcg agagaaaccg cgtct 25

<210> 109

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 109

aacagacgcg gttactctcg cagcc 25

<210> 110

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 110

ttgggctgcg agtgaaaccg cgtct 25

<210> 111

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 111

aacagacgcg gttgctctcg cagc 24

<210> 112

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 112

ttgggctgcg agagcaaccg cgtct 25

<210> 113

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 113

aacagacgcg gttcctctcg cagc 24

<210> 114

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 114

ttgggctgcg agaggaaccg cgtct 25

<210> 115

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 115

aacagacgcg gtttcgctcg cagc 24

<210> 116

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 116

ttgggctgcg agcgaaaccg cgtct 25

<210> 117

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 117

aacagacgcg gttacgctcg cagc 24

<210> 118

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 118

ttgggctgcg agcgtaaccg cgtct 25

<210> 119

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 119

aacagacgcg gttgcgctcg cagc 24

<210> 120

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 120

ttgggctgcg agcgcaaccg cgtct 25

<210> 121

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 121

aacagacgcg gttccgctcg cagc 24

<210> 122

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 122

ttgggctgcg agcggaaccg cgtct 25

<210> 123

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 123

aacagacgcg gtttccctcg cagc 24

<210> 124

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 124

ttgggctgcg agggaaaccg cgtct 25

<210> 125

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 125

aacagacgcg gttaccctcg cagc 24

<210> 126

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 126

ttgggctgcg agggtaaccg cgtct 25

<210> 127

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 127

aacagacgcg gttgccctcg cagc 24

<210> 128

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 128

ttgggctgcg agggcaaccg cgtct 25

<210> 129

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 129

aacagacgcg gttcccctcg cagc 24

<210> 130

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 130

ttgggctgcg aggggaaccg cgtct 25

<210> 131

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 131

aacctgggct gatccatgat gtcat 25

<210> 132

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 132

ttgatgacat catggatcag cccag 25

<210> 133

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 133

aactctcaac tttccatatc cgtat 25

<210> 134

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 134

ttgatacgga tatggaaagt tgaga 25

<210> 135

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 135

aaccctgcca ctcccaacag gtttc 25

<210> 136

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 136

ttggaaacct gttgggagtg gcagg 25

<210> 137

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 137

aactagggtc aacccagtat tctcc 25

<210> 138

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 138

ttgggagaat actgggttga cccta 25

<210> 139

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 139

aacactcatg cttccacagc tttaa 25

<210> 140

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 140

ttgttaaagc tgtggaagca tgagt 25

<210> 141

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 141

aacgcttccg tgtccagcct tcagg 25

<210> 142

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 142

ttgcctgaag gctggacacg gaagc 25

<210> 143

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 143

aacctgtgta cccccagagc tccgt 25

<210> 144

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 144

ttgacggagc tctgggggta cacag 25

<210> 145

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 145

aactttgtaa atcaaagtac accttgactg tga 33

<210> 146

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 146

ttgtcacagt caaggtgtac tttgatttac aaa 33

<210> 147

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 147

aaccaaagac gactcgtccc acagattcat ctc 33

<210> 148

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 148

ttggagatga atctgtggga cgagtcgtct ttg 33

<210> 149

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 149

tcttcctttt gctgcccgga ccctc 25

<210> 150

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 150

acatccatgc cctctagaac tttgccgaa 29

<210> 151

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 151

gccgaaaacc acatgcttgc c 21

<210> 152

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 152

aactcccaag tagaaatacc agtgtcagga agt 33

<210> 153

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 153

ttgacttcct gacactggta tttctacttg gga 33

<210> 154

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 154

aacgctttga tgtcacttcc aaagtcccct tgt 33

<210> 155

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 155

ttgacaaggg gactttggaa gtgacatcaa agc 33

<210> 156

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 156

agctcatctg gaatttcgcc gact 24

<210> 157

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 157

aaggcactgt aagttattat ccccacggaa 30

<210> 158

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 158

gaatttcgcc gacttcatga cga 23

<210> 159

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 159

aactctcctg ctcgaagtcc agagcaacat ag 32

<210> 160

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 160

ttgctatgtt gctctggact tcgagcagga ga 32

<210> 161

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 161

acatccgtaa agacctctat gccaacac 28

<210> 162

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 162

ccagggagac caaagccttc atacatca 28

<210> 163

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 163

ccgacaggat gcagaaggag a 21

Claims (12)

1. A fusion protein has an amino acid sequence shown as SEQ ID number 5.

2. An isolated polynucleotide encoding the fusion protein of claim 1.

3. A construct comprising the isolated polynucleotide of claim 2.

4. An expression system comprising the construct or genome of claim 3 having integrated therein an exogenous polynucleotide of claim 2.

5. The expression system of claim 4, wherein the host cell of the expression system is selected from the group consisting of eukaryotic cells and prokaryotic cells.

6. The expression system of claim 5, wherein the host cell is selected from a mouse cell.

7. The expression system of claim 6, wherein the host cell is selected from mouse brain neuroma cells.

8. The expression system of claim 7, wherein the host cell is selected from the group consisting of N2a cells.

9. Use of the fusion protein of claim 1, the isolated polynucleotide of claim 2, the construct of claim 3 or the expression system of any one of claims 4 to 8 for gene editing for non-disease diagnosis and treatment purposes.

10. Use according to claim 9, in particular in gene editing in eukaryotes.

11. A base editing system comprising the fusion protein of claim 1, the base editing system further comprising a sgRNA.

12. A method of gene editing comprising: gene editing is performed by the fusion protein according to claim 1 or the base editing system according to claim 11.

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