Patent
Right

Date
Case number

February 25, 20202019 (Gyo-Ke) 10010

Court

Intellectual
PropertyHigh
Court,First
Division- In Article 29-2 of the Patent Act, the "invention" stated in the description, etc. ofthe earlier application means an invention which is understood from a matter statedin the description, etc. of the earlier application and from a matter equivalent tothat stated in the description, etc. of the earlier application, in which the "matter
equivalent to that stated" means a matter which can be derived from the statedmatter by taking common general technical knowledge as of filing into
consideration.
- A case in which, with regard to a patent application concerning an invention titled"ENGINEERING OF SYSTEMS, METHODS AND OPTIMIZED GUIDECOMPOSITIONS FOR SEQUENCE MANIPULATION", there is a statement in
the description, etc. of the earlier application to the extent that a person ordinarily
skilled in the art can understand that the invention of the earlier application isshown in the description, etc. of the earlier application and a person ordinarily
skilled in the art is enabled to work the invention shown in the earlier application,and therefore, it is determined that the invention of the present application is
identical with the invention of the earlier application and is not allowed to be
granted a patent.
Case type: Rescission of Appeal Decision of Refusal
Result: Dismissed
References: Article 29, paragraph (1), item (iii), and Article 29-2 of the Patent Act
Related rights, etc.: Appeal against Examiner's Decision of Refusal No. 2017-13795,
Patent Application No. 2016-117740
Summary of the Judgment
1.

The present case is a lawsuit for rescission of an appeal decision of refusal inwhich the examiner made a decision of refusal with regard to the Plaintiffs' patentapplication concerning an invention titled "ENGINEERING OF SYSTEMS,METHODS AND OPTIMIZED GUIDE COMPOSITIONS FOR SEQUENCEMANIPULATION", against which Plaintiffs filed an appeal, and then the decision of
the JPO dismissing the request for an appeal against the examiner's decision of refusal
was made, and thus Plaintiffs sought the rescission of the appeal decision of refusal.
Reasons for rescission are [i] Erroneous determination of Article 29-2 of the
Patent Act on the basis of Cited Invention 1 (Reason 1 for Rescission) and [ii]Erroneous determination of an inventive step on the basis of Cited Invention 2(Reason 2 for Rescission).
2.

The present judgment, in summary, held with regard to Reason 1 for Rescissionⅰas follows and dismissed the Plaintiff's claim.
(1) It is found that the invention of the present application is identical with Cited
Invention 1.
It should be deemed that Cited Document 1 discloses not only formally but also
substantially that "a guide RNA directs a type II Cas 9 protein to a targeted site inchromosomal sequence in eukaryotic cells where the type II Cas 9 protein introduces
a double-stranded break of chromosomal DNA in the targeted site, and the doublestranded break is repaired by a DNA repair process such that the chromosomal
sequence is modified."

It can be understood that the vector system of Cited

Invention 1 is also disclosed as a vector system which includes the above function.
(2) A. An object of Article 29-2 of the Patent Act .. is as follows.

An invention

stated in the description, etc. of an earlier application, even if the invention is notstated in the Scope of Claims, is laid open to the public in a publication, etc. of theearlier application.

Thus, even if a later application is filed before the publication,etc. of the earlier application, when the invention of the later application is identic alwith the invention of the earlier application, no new technology is laid open to the
public in the publication, etc. of the later application.

Therefore, granting a patent tosuch invention is improper from the viewpoint of the patent system, which intends toprotect an invention as a reward for laying a new invention open to the public.
The "invention" stated in the description, etc. of the earlier application in this
Article is construed as an invention which is understood from a matter stated in thedescription, etc. of the earlier application and from a matter equivalent to that statedin the description, etc. of the earlier application.

The "matter equivalent to th atstated" means a matter which can be derived from the stated matter by taking common
general technical knowledge as of filing into consideration.
Thus, even if there is no particular statement in the description, etc. of the earlier
application, the invention of the earlier application can be found by taking common
general technical knowledge into consideration in order to understand the invention of
the earlier application.

On the other hand, in case where an invention lacks

concreteness or where technical content of an invention is insufficiently disclosed
even taking common general technical knowledge of a person ordinarily skilled in theart into consideration, such invention does not fall under the "invention" mentioned
above, and does not have an effect of excluding the later application stipulated in this
Article.

In addition, it should be deemed that the degree of disclosure of technicalcontent required here should be sufficient if the technical content is disclosed to theextent that a person ordinarily skilled in the art can understand that the invention ofⅱ

the earlier application is shown in the description, etc. of the earlier application andthe person ordinarily skilled in the art is enabled to work the invention shown in theearlier application.
B. In the present case, with regard to the above, Examples 1 to 3 in Cited
Invention 1 disclose in detail a method for producing each vector of (i) to (iii) in
Cited Invention 1, and Example 4 clearly discloses a concrete test method for
confirming whether a donor sequence (GFP gene) is incorporated in or near thetargeted sequence.
In addition, .. from the experimental results of Example 4, it can be understoodthat a combination of RNA-guided endonuclease comprising a nuclear localization
signal, guide RNA, and donor polynucleotides is incorporated into eukaryotic cell s
and that double-stranded break and repair occur in the targeted site.

The

experimental results of Example 5 are not to be interpreted as a bar to the above
understanding.
Further, a vector system comprising the above vectors (i) to (iii) has the technicalmeans necessary for appropriate transcription, translation, nuclear translocation, etc.
in eukaryotic cells, and the technical means necessary for appropriate modification of
the targeted sequence in eukaryotic cells.

Thus, it can be understood that even if thevector system is used, such vector system has functions of cleavage of targeted
sequences and modification of targeted sequences in eukaryotic cells .. .
From the above, it can be deemed that there is a disclosure in Cited Document 1 tothe extent that a person ordinarily skilled in the art can understand that the inventionof the earlier application is shown in the description, etc. of the earlier application anda person ordinarily skilled in the art is enabled to work the invention shown in theearlier application.

Therefore, it is proper to find that in Cited Document 1, atechnology was laid open to the public to the extent that the technology was sufficient
to exclude the later application, in which the technology includes a portion of thefunction such that "a guide RNA directs a type II Cas 9 protein to a targeted site inchromosomal sequence in eukaryotic cells where the type II Cas 9 protein introduces
a double-stranded break of chromosomal DNA in the targeted site, and the doublestranded break is repaired by a DNA repair process such that the chromosomal
sequence is modified."ⅲ

Judgment rendered on February 25, 20202019 (Gyo-Ke) 10010 A case of seeking rescission of the JPO decision
Date of conclusion of oral argument: December 18, 2019
Judgment
Plaintiff:

The Broad Institute Inc.

Plaintiff:

Massachusetts Institute of Technology

Plaintiffs:

President and Fellows of Harvard College

Defendant: Commissioner of the Japan Patent Office

Main Text
1. The Plaintiffs' claim shall be dismissed.
2. The court costs shall be borne by the Plaintiffs.
3. An additional period of 30 days shall be set for the Plaintiffs to file a final
appeal against this judgment and a petition for acceptance of the final appeal.
Facts and ReasonsNo. 1 Claim
The decision rendered by the Japan Patent Office on September 14, 2018 forthe case of Appeal against Examiner's Decision of Refusal No. 2017-13795 shall be
rescinded.
No. 2 Outline of the Case
1. History of Procedures in the Japan Patent Office(1) The Plaintiffs filed a patent application with regard to an invention titled"ENGINEERINGOFSYSTEMS,METHODS,AND

OPTIMIZEDGUIDECOMPOSITIONS FOR SEQUENCE MANIPULATION" on June 14, 2016 (Patent
Application No. 2016-117740, Division of Patent Application No. 2015-547573(Priority claimed: December 12, 2012, United States of America), Publication Date:
1September 15, 2016, Exhibit Ko 9).
(2) With regard to this patent application, the Plaintiffs received an Examiner's
decision of refusal on April 28, 2017 (Exhibit Ko 14).

Accordingly, the Plaintiffs

filed a request for an appeal against the Examiner's decision of refusal on September
15, 2017 (Exhibit Ko 15).

The Japan Patent Office examined the above request as

the case of Appeal against Examiner's Decision of Refusal No. 2017-13795.
(3) On September 14, 2018, the Japan Patent Office rendered a decision thatthe request for the appeal shall be dismissed, as stated in the written decision (a copy)
as shown in Attachment (hereinafter referred to as "the decision of the present case").
The certified copy of the decision of this case was served on the Plaintiffs on October
1, 2018.
(4) The Plaintiffs instituted an action for seeking a rescission of the decision of
the present case on January 29, 2019.
2. Statement of the Scope of Claims
The statement of Claim 1 in the Scope of Claims to which the decision is
directed is as follows (Exhibit Ko 12; hereinafter, the invention stated in the above
Claim 1 is referred to as "the Present Invention"; the description according to thisapplication (Exhibit Ko 9) and the drawings attached thereto are collectively referred
to as "the description of the present application").

Note that "/" in the text below

indicates a line break in the original text (the same shall apply hereinafter).
[Claim 1]
An engineered, non-naturally occurring clustered regularly interspaced shortpalindromic repeats (CRISPR) - CRISPR-associated (Cas) (CRISPR-Cas) vector
system, / comprising one or more vectors comprising: / a) a first regulatory element
operably linked to a nucleotide sequence encoding a CRISPR-Cas systempolynucleotide sequence comprising a guide sequence, tracrRNA, and a tracr matesequence, wherein the guide sequence hybridizes with one or more targeted sequencesin polynucleotide loci in a eukaryotic cell, / b) a second regulatory element operably
linked to a nucleotide sequence encoding a type II Cas9 protein, and / c) a
recombination template, / wherein components (a), (b), and (c) are located on thesame or different vectors of the system, wherein the system further comprises one ormore nuclear localization signals (there can be more than one) (NLS (there can be
more than one)) to be expressed together with the nucleotide sequence encoding theCas9 protein, / whereby the guide sequence targets the one or more polynucleotideloci in the eukaryotic cell and the Cas9 protein cleaves the one or more polynucleotide
loci, whereby the sequence of the one or more polynucleotide loci is modified.
2

3. Summary of Reasons of the Decision of the Present Case
(1) Reasons of the decision of the present case are as stated in the written
decision (a copy) in the Attachment.

In summary, [i] the Present Invention is

identical with the invention as disclosed in Cited Document 1 of the ea rlier
application mentioned below (hereinafter referred to as "Cited Invention 1"), and thus
falls under Article 29-2 of the Patent Act, and [ii] the Present Invention could have
easily been made by a person ordinarily skilled in the art on the basis of the invention
as disclosed in Cited Document 2 mentioned below (hereinafter referred to as "Cited
Invention 2") and the well-known art before the priority date of the present
application (December 12, 2012), and thus falls under Article 29, paragraph (2) of the
Patent Act.

For the foregoing reasons, a patent shall not be granted for the Present

Invention.
a. Cited Document 1: PCT/US2013/073307 (International Publication No.
WO2014/089290, Filing date: December 5, 2013 (Priority claimed: December 6,2012), Publication date: June 12, 2014, Exhibit Ko 1-1)
b. Cited Document 2: "A Programmable Dual-RNA - Guided DNA
Endonuclease in Adaptive Bacterial Immunity" (Science, Aug 2012, Vol. 337, pp.
816-821, Exhibit Ko 2-1) and "Supplementary Materials" (Exhibit Ko 2-2) (Online
publication: June 28, 2012).
(2) The decision of the present case found as follows, with regard to Cited
Invention 1.
A vector system, comprising / (i) a vector comprising a promoter control
sequence operably linked to a nucleic acid encoding at least one type II Cas9 protein
comprising at least one nuclear localization signal, / (ii) a vector comprising a
promoter control sequence operably linked to DNA encoding at least one guide RNA
comprising a first region at the 5' end that is complementary to a targeted site in achromosomal sequence in a eukaryotic cell, a second internal region that forms a stem
loop structure, and a third 3' region that remains essentially single-stranded; / and (iii)a vector comprising at least one donor polynucleotide, / wherein the guide RNA
directs a type II Cas9 protein to a targeted site in the chromosomal sequence in a
eukaryotic cell, where the type II Cas9 protein introduces a chromosomal DNA
double-stranded break in the targeted site, and the double-stranded break is repairedby a DNA repair process such that the chromosomal sequence is modified.
(3) The decision of the present case found as follows, with regard to Cited
Invention 2 and the comparison between the Present Invention and Cited Invention 2.
a. Findings on Cited Invention 2
3

An engineered, non-naturally occurring clustered regularly interspaced shortpalindromic repeats (CRISPR) - CRISPR-associated (Cas) (CRISPR-Cas) system, /comprising: / a) a chimeric RNA comprising a target recognition sequence at the 5'
end followed by a hairpin structure retaining base-pairing interactions that occur
between tracrRNA and crRNA, wherein the target recognition sequence hybridizes
with a targeted sequence in a buffer solution; and / b) a type II Cas9 protein, /whereby the Cas9 protein cleaves the targeted sequence.
b. Common Feature between the Present Invention and Cited Invention 2
An engineered, non-naturally occurring clustered regularly interspaced shortpalindromic repeats (CRISPR) - CRISPR-associated (Cas) (CRISPR-Cas) system, /comprising: / a) a CRISPR-Cas system polynucleotide comprising a guide sequence,
tracrRNA, and a tracr mate sequence, wherein the guide sequence hybridizes with atargeted sequence; and / b) a type II Cas9 protein, / whereby the Cas9 protein cleaves
the targeted sequence.
c. Different Features between the Present Invention and Cited Invention 2(Different Feature 1)
In the Present Invention, a guide sequence "hybridizes with one or moretargeted sequences in polynucleotide loci in a eukaryotic cell" and the system "further
comprises one or more nuclear localization signals (there can be more than one) (NLS
(there can be more than one)) to be expressed together with the nucleotide sequence
encoding the Cas9 protein", whereby the guide sequence targets "the one or morepolynucleotide loci in the eukaryotic cell" and the Cas9 protein cleaves this.

In

contrast, in Cited Invention 2, a targeted sequence is present in a buffer solution.
Further, a Cas9 protein does not comprise a nuclear localization signal.
(Different Feature 2)
The Present Invention comprises a "recombination template", wherein"sequence of the one or more polynucleotide loci is modified."

In contrast, in Cited

Invention 2 does not comprise a recombination template, a targeted sequence is only
cleaved.
(Different Feature 3)
The Present Invention is a CRISPR-Cas "vector" system, comprising: "one ormore vectors" comprising: "a first regulatory element operably linked to a nucleotide
sequence encoding" the CRISPR-Cas system polynucleotide sequence; and "a second
regulatory element operably linked to a nucleotide sequence encoding" a type II Cas9
protein; "wherein components (a), (b), and (c) are located on the same or different
vectors of the system".

In contrast, Cited Invention 2 is a CRISPR -Cas system using4

a "chimeric RNA" and a "Cas9 protein".
4. Reasons for Rescission(1) Erroneous Determinations of Article 29-2 of the Patent Act Based on Cited
Invention 1 (Reason 1 for Rescission)(2) Erroneous Determinations of Inventive Step Based on Cited Invention 2(Reason 2 for Rescission)(omitted)
No. 4 Judgment of This Court
1. Present Invention(1) Statement of the Description of the Present Application
The description of the present application is stated as follows (Exhibit Ko 9).
a. Technical Field[0003] The present invention generally relates to systems, methods, andcompositions used for the control of gene expression involving sequence targeting,such as genome perturbation or gene-editing, that may use vector systems related toClustered

Regularly

Interspaced

Short

Palindromic

Repeats(CRISPR)

andcomponents thereof.
b. Background Art[0005] Precise genome targeting technologies are needed to enable systematic
reverse engineering of causal genetic variations as well as to advance synthetic
biology, biotechnological, and medical applications.

Although genome-editing

techniques such as designer zinc fingers, transcription activator-like effectors (TALE),or homing meganucleases are available , there remains a need for new genome
engineering technologies that are affordable, easy to set up, scalable, and amenable totargeting multiple positions within the eukaryotic genome.
c. Problem to be Solved by the Invention[0007] In the CRISPR/Cas or the CRISPR-Cas system, a single Cas enzyme
can be programmed by a short RNA molecule to recognize a specific DNA target; inother words, the Cas enzyme can be recruited to a specific DNA target using said
short RNA molecule.
d. Means for Solving the Problem[0008] In some embodiments, the CRISPR enzyme is a type II CRISPR system
enzyme.

In some embodiments, the CRISPR enzyme is a Cas9 enzyme.

In someembodiments, the Cas9 enzyme is S. pneumoniae, S. pyogenes, or S. thermophilus5Cas9, and may include mutated Cas9 derived from these organisms.
In some embodiments, the CRISPR enzyme is codon-optimized for expression
in a eukaryotic cell.
In some embodiments, the guide sequence is at least 15, 16, 17, 18, 19, 20, 25
nucleotides in length, or between 10 to 30, or between 15 to 25, or between 15 to 20
nucleotides in length.
In general, and throughout this description, the term "vector" refers to a nucleic
acid molecule capable of transporting another nucleic acid to which it has been linked.
One type of vector is a "plasmid," which refers to a circular double-stranded
DNA loop into which additional DNA segments can be inserted, such as by standard
molecular cloning techniques.

Another type of vector is a viral vector, wherein

virally-derived DNA or RNA sequences are present in the vector for packaging into avirus (..).
Common expression vectors of utility in recombinant DNA techniques areoften in the form of plasmids.
[0010] The term "regulatory element" is intended to include promoters,
enhancers, internal ribosomal entry sites (IRES), and other expression control
elements (e.g. transcription termination signals, such as polyadenylation signals andpoly-U sequences).
In some embodiments, a vector comprises one or more pol III promoter s (..),
one or more pol II promoters (..), one or more pol I promoters (..), or combinations
thereof.

Examples of pol III promoters include, but are not limited to, U6 and H1

promoters.

Examples of pol II promoters include, but are not limited to, theretroviral Rous sarcoma virus (RSV) LTR promoter (..), the cytomegalovirus (CMV)
promoter.

Also encompassed by the term "regulatory element" are enhancer

elements, such as WPRE; CMV enhancers; the R-U5' segment in LTR of HTLV-1;
and SV40 enhancer.

A vector can be introduced into host cells to thereby produce

transcripts, proteins, or peptides, including fusion proteins or peptides, encoded by
nucleic acids as described herein.
[0012] In one aspect, the present invention provides a vector comprising a
regulatory element operably linked to an enzyme-coding sequence encoding a
CRISPR enzyme comprising one or more nuclear localization sequences.
[0023] In aspects of the present invention, the modification comprises anengineered secondary structure.

For example, the modification can com prise areduction in a region of hybridization between a tracr mate sequence and a tracr
sequence.

For example, the modification also may comprise fusing a tracr mate6sequence and a tracr sequence through an artificial loop.

The modification may

comprise a tracr sequence having a length between 40 and 120 bp.
In certain embodiments, a length of tracrRNA includes at least nucleotides 1 to
67 and in some embodiments at least nucleotides 1 to 85 of a wild type tracrRNA.
The modification may comprise sequence optimization.
Sequence optimization may be combined with the reduction in a region of
hybridization between a tracr mate sequence and a tracr sequence; for example, areduction in a length of a tracr sequence.
[0025] In an aspect, the present invention provides a CRISPR-Cas system or a
CRISPR enzyme system wherein the modification comprises adding a polyT
terminator sequence.
[0035] In some aspects of the present invention, a length of tracrRNA required
in a construct of the present invention, e.g., a chimeric construct, need not necessarily
be fixed, and in some aspects of the present invention it can be between 40 and 120 bp,
and in some aspects of the present invention up to a full length of the tracr, e.g., insome aspects of the present invention, until 3' end of tracr as punctuated by atranscription termination signal in a bacterial genome.

In certain embodiments, alength of tracrRNA includes at least nucleotides 1 to 67 and in some embodiments atleast nucleotides 1 to 85 of a wild type tracrRNA.

In some embodiments, at least

nucleotides corresponding to nucleotides 1 to 67 or 1 to 85 of wild type S. pyogenes
Cas9 tracrRNA may be used.
As to the presence of polyT terminator sequences in tracr + tracr matetranscript, e.g., a polyT terminator (TTTTT or more T), in some aspects of the present
invention, it is advantageous that such be added to an end of the transcript, whether it
is in two RNA (tracr and tracr mate) or single guide RNA form.

Concerning loops

and hairpins in tracr and tracr mate transcripts, in some aspects of the present
invention, it is advantageous that a minimum of two hairpins be present in the
chimeric guide RNA.

A first hairpin can be the hairpin formed by complementation

between the tracr and tracr mate (direct repeat) sequence.

A second hairpin can be atthe 3' end of the tracrRNA sequence, and this can provide a secondary structure forinteraction with Cas9.

Additional hairpins may be added to the 3' of the guide RNA,e.g., in some aspects of the present invention, to increase stability of the guide RNA.
e. Mode for Carrying Out the Invention[0065] In general, "CRISPR system" refers collectively to transcripts and otherelements involved in the expression of or directing the activity of CRISPR -associated("Cas") genes, including sequences encoding a Cas gene, a tracr (trans-activating
7

CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr matesequence (encompassing a "direct repeat" and a tracrRNA-processed partial direct
repeat in the context of an endogenous CRISPR system), a guide sequence (alsoreferred to as a "spacer" in the context of an endogenous CRISPR system), or othersequences and transcripts from a CRISPR locus.
In some embodiments, one or more elements of a CRISPR system are derived
from a particular organism comprising an endogenous CRISPR system, such as
Streptococcus pyogenes.

In general, a CRISPR system is characterized by elementsthat promote the formation of a CRISPR complex at the site of a target ed sequence(also referred to as a protospacer in the context of an endogenous CRISPR system).
In the context of formation of a CRISPR complex, "targeted sequence" refers to asequence to which a guide sequence is designed to have complementarity, wherehybridization between a targeted sequence and a guide sequence promotes theformation of a CRISPR complex.
A sequence or template that may be used for recombination into the targeted
locus comprising the targeted sequences is referred to as an "editing template" or"editing polynucleotide" or "editing sequence".

In aspects of the present invention,

an exogenous template polynucleotide may be referred to as an editing template.

In

an aspect of the present invention, the recombination is homologous recombination.
[0066] Without wishing to be bound by theory, the tracr sequence, which may
comprise or consist of all or a portion of a wild-type tracr sequence (e.g. about or
more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wild -type
tracr sequence), may also form part of a CRISPR complex, such as by hybridization
along at least a portion of the tracr sequence to all or a portion of a tracr matesequence that is operably linked to the guide sequence.
[0071] In some embodiments, a vector encodes a CRISPR enzyme comprising
one or more nuclear localization sequences (NLS), such as about or more than about 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs.
Non-limiting examples of NLSs include the NLS of the SV40 virus large Tantigen, having the amino acid sequence PKKKRKV.
[0072] In general, the one or more NLSs are of sufficient strength to drive
accumulation of the CRISPR enzyme in a detectable amount in the nucleus of aeukaryotic cell.
[0076] In general, a tracr mate sequence includes any sequence that has
sufficient complementarity with a tracr sequence to promote one or more of: (1)excision of a guide sequence flanked by tracr mate sequences in a cell containing the8corresponding tracr sequence; and (2) formation of a CRISPR complex (the CRISPR
complex comprises the tracr mate sequence hybridized to the tracr sequence) at atargeted sequence.
In some embodiments, the tracr sequence is about or more than about 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides inlength.

In some embodiments, the tracr sequence and tracr mate sequence arecontained within a single transcript, such that hybridization between the two produces
a transcript having a secondary structure, such as a hairpin.

Preferred loop forming

sequences for use in hairpin structures are four nucleotides in length, and most
preferably have the sequence GAAA.

However, longer or shorter loop sequences

may be used, as may be alternative sequences.

The sequences preferably include anucleotide triplet (for example, AAA), and an additional nucleotide (for example C orG).

Examples of loop forming sequences include CAAA and AAAG.

In anembodiment of the present invention, the transcript or transcribed polynucleotide
sequence has at least two hairpins.
three, four, or five hairpins.

In preferred embodiments, the transcript has two,

In a further embodiment of the present invention, thetranscript has at most five hairpins.

In some embodiments, the single transcript

further includes a transcription termination sequence; preferably this is a polyT
sequence, such as six T nucleotides.

An example illustration of such a hairpin

structure is provided in the lower portion of Figure 8B (as shown in Attachment 1),
where the portion of the sequence 5' of the final "N" and upstream of the loop
corresponds to the tracr mate sequence, and the portion of the sequence 3' of the loop
corresponds to the tracr sequence. (In the original text, "Fig. 13B" is shown.
However, from the context of the preceding and following text, this is recognized as
Fig. 8B.)
f. Examples(a) Example 1: CRISPR Complex Activity in the Nucleus of a Eukaryotic Cell
[0146] To improve expression of CRISPR components in mammalian cells, twogenes of Cas9 (SpCas9) and RNase III (SpRNase III) from the SF370 locus 1 ofStreptococcus pyogenes (S. pyogenes) were codon-optimized.

To facilitate nuclear

localization, a nuclear localization signal (NLS) was included at the amino (N) - orcarboxyl (C)-termini of both SpCas9 and SpRNase III (Figure 2B).
A version of SpCas9 with an NLS attached to both N- and C-termini (2×NLSSpCas9) was also generated.
Whereas the C-terminal NLS was sufficient to target SpRNase III to the
nucleus, attachment of a single copy of these particular NLSs to either the N - or C9

terminus of SpCas9 was unable to achieve adequate nuclear localization in this system.
In

this

example,the

C-terminal

NLS

was

that

of

nucleoplasmin(KRPAATKKAGQAKKKK), and the C-terminal NLS was that of the SV40 large Tantigen (PKKKRKV).

Of the versions of SpCas9 tested, only 2×NLS-SpCas9

exhibited nuclear localization (Figure 2B).
(b) Example 4: Evaluation of Multiple Chimeric crRNA-tracrRNA Hybrids[0165] This example states results obtained for chimeric RNAs (chiRNAs;
comprising a guide sequence, a tracr mate sequence, and a tracr sequence in a single
transcript) having tracr sequences that incorporate different lengths of wild -type
tracrRNA sequence.
Cas9 is driven by the CBh promoter and the chimeric RNA is driven by a U6
promoter.
The guide and tracr sequences are separated by the tracr mate sequence
GUUUUAGAGCUA followed by the loop sequence GAAA.

Results of

SURVEYOR assays for Cas9-mediated indels at the human EMX1 and PVALB lociare illustrated in Figures 18b and 18c, respectively.
ChiRNAs are indicated by their "+n" designation, and crRNA refers to a hybrid
RNA where guide and tracr sequences are expressed as separate transcripts.
Quantification of these results, performed in triplicate, is illustrated byhistograms in Figures 11a and 11b, corresponding to Figures 10b and 10c (as shownin Attachment 1), respectively ("N.D." indicates no indels detected).
[0171] Initially, three sites within the EMX1 locus in human HEK293FT cells
were targeted.

Efficiency of genome modification of each chiRNA was assessedusing the SURVEYOR nuclease assay, which detects mutations resulting from DNA
double-strand breaks (DSB) and their subsequent repair by the non-homologous end
joining (NHEJ) DNA damage repair pathway.

Constructs designated chiRNA(+n)

indicate that up to the +n nucleotide of wild-type tracrRNA is included in the
chimeric RNA construct, with values of 48, 54, 67, and 85 used for n.

Chimeric

RNAs containing longer fragments of wild-type tracrRNA (chiRNA(+67) andchiRNA(+85)) mediated DNA cleavage at all three EMX1 targeted sites, with
chiRNA(+85) in particular demonstrating significantly higher levels of DNA cleavage
than the corresponding crRNA/tracrRNA hybrids that expressed guide and tracr
sequences in separate transcripts (Figures 10b and 10a).

Two sites in the PVALB

locus that yielded no detectable cleavage using the hybrid system (guide sequence andtracr sequence expressed as separate transcripts) were also targeted using chiRNAs.
chiRNA(+67) and chiRNA(+85) were able to mediate significant cleavage at the two
10

PVALB protospacers (Figures 10c and 10b).
[0172] For all five targets in the EMX1 and PVALB loci, a consistent increasein efficiency of genome modification with increasing tracr sequence length wasobserved.

Without wishing to be bound by any theory, the secondary structure

formed by the 3' end of the tracrRNA may play a role in enhancing the rate of
CRISPR complex formation.

An illustration of predicted secondary structures foreach of the chimeric RNAs used in this example is provided in Figure 21.

BecausechiRNAs with longer tracr sequences were able to cleave targets that were not cleaved
by native CRISPR crRNA/tracrRNA hybrids, it is possible that chimeric RNA may beloaded onto Cas9 more efficiently than its native hybrid counterpart.
[0173] Figures 11 (as shown in Attachment 1) and 21 illustrate exemplary
bicistronic expression vectors for expression of chimeric RNA including up to the +85
nucleotides of wild-type tracrRNA sequence, and SpCas9 with nuclear localization
sequences.

SpCas9 is expressed from a CBh promoter and terminated with the bGH

polyA signal (bGHpA).

The expanded sequence illustrated immediately below theschematic corresponds to the region surrounding the guide sequence insertion site,
and includes, from 5' to 3', 3'-portion of the U6 promoter (first shaded region), BbsI
cleavage sites (arrows), partial direct repeat (tracr mate sequence GTTTTAGAGCTA,
underlined), loop sequence GAAA, and +85 tracr sequence (underlined sequence
following loop sequence).

An exemplary guide sequence insert is illustrated below

the guide sequence insertion site, with nucleotides of the guide sequence for aselected target represented by "N".
(2) Features of the Present Invention
The Present Invention relates to genome-editing techniques using a CRISPRCas vector.
In the Present Invention, a Cas enzyme (a type II Cas9 protein) is allowed torecognize a specific DNA target (a polynucleotide locus) in a eukaryotic cell by ashort RNA molecule (a CRISPR-Cas system polynucleotide sequence comprising a
guide sequence, a tracrRNA sequence, and a tracr mate sequence), and the Cas
enzyme cleaves the DNA target, whereby the DNA target is modified.
The Present Invention has the following constituent features; that is, a) a first
regulatory element operably linked to a nucleotide sequence encoding the CRISPRCas system polynucleotide sequence, b) a second regulatory element operably linked
to a nucleotide sequence encoding the type II Cas9 protein, and c) a recombination
template are located on one or more of the same or different vectors of the system,
and comprise a nuclear localization signal to be expressed together with the11nucleotide sequence encoding the Cas9 protein.
2. Reason 1 for Rescission (Erroneous Determinations of Article 29-2 of the
Patent Act Based on of Cited Invention 1)(1) Statement of Cited Document 1 (Exhibit Ko 1-1. The paragraph number isthat of National Publication of International Patent Application No. 2016-502840
which is a national publication (Exhibit Ko 1-2).

Paragraph [0151] is from thedescription on which the priority claim is based with regard to Cited Document 1(Exhibit Ko 105)).
a. Technical Field[0001] The present disclosure relates to targeted genome modification.

In

particular, the disclosure relates to RNA-guided endonucleases or fusion proteinscomprising CRISPR/Cas-like protein and methods of using said proteins to modify or
regulate targeted chromosomal sequences.
b. Background Art[0002] Targeted genome modification is a powerful tool for genetic
manipulation of eukaryotic cells, embryos, and animals.

Current methods rely on theuse of engineered nuclease enzymes, such as, for example, zinc finger nucleases(ZFN) or transcription activator-like effector nucleases (TALEN).

Each newgenomic target, however, requires the design of a new ZFN or TALEN comprising anovel sequence-specific DNA-binding module.

Thus, preparation of these custom

designed nucleases tends to be costly and time-consuming.

Moreover, thespecificities of ZFN and TALEN are such that they can mediate off-target (..)
cleavages.
[0003] Thus, there is a need for a targeted genome modification technologythat does not require the design of a new nuclease for each new targeted genomic
location .. , there is a need for a technology with increased specificity with few or no
off-target effects.
c. Summary of the Invention[0004] Among the various aspects of the present disclosure is the provision of
an isolated RNA-guided endonuclease, wherein the endonuclease comprises at least
one nuclear localization signal, at least one nuclease domain, and at least one domainthat interacts with a guide RNA to target the endonuclease to a specific nucleotide
sequence for cleavage.
In other embodiments, a nucleic acid sequence encoding the RNA-guided
endonuclease can be operably linked to a promoter control sequence, and optionally,
can be part of a vector.

In other embodiments, a vector comprising a sequence12

encoding the RNA-guided endonuclease, which can be operably linked to a promoter
control sequence, can also comprise a sequence encoding a guide RNA, which can be
operably linked to a promoter control sequence.
[Claim 13] A method for modifying a chromosomal sequence in a eukaryotic
cell or embryo, / the method comprising: / a) introducing into the eukaryotic cell orembryo (i) at least one RNA-guided endonuclease comprising at least one nuclear
localization signal or nucleic acid encoding at least one RNA-guided endonuclease
comprising at least one nuclear localization signal, (ii) at least one guide RNA or
DNA encoding at least one guide RNA, and, optionally, (iii) at least one donor
polynucleotide; and / b) culturing the eukaryotic cell or embryo such that each guideRNA directs an RNA-guided endonuclease to a targeted site in the chromosomal
sequence where the RNA-guided endonuclease introduces a double-stranded break in
the targeted site, and the double-stranded break is repaired by a DNA repair process
such that the chromosomal sequence is modified.
[Claim 14] The method of claim 13, wherein the RNA-guided endonuclease isderived from a Cas9 protein.
[Claim 15] The method of claim 13 or claim 14, wherein the nucleic acidencoding the RNA-guided endonuclease is mRNA.
[Claim 16] The method of claim 13 or claim 14, wherein the nucleic acidencoding the RNA-guided endonuclease is DNA.
[Claim 17] The method of claim 16, wherein the DNA is part of a vector thatfurther comprises a sequence encoding the guide RNA.
[0005] Another aspect of the present invention encompasses a method formodifying a chromosomal sequence in a eukaryotic cell or embryo.

The method

comprises introducing into a eukaryotic cell or embryo (i) at least one RNA -guided
endonuclease comprising at least one nuclear localization signal or nucleic acidencoding at least one RNA-guided endonuclease as defined herein, (ii) at least one
guide RNA or DNA encoding at least one guide RNA, and, optionally, (iii) at least
one donor polynucleotide comprising a donor sequence.

The method furth er

comprises culturing the cell or embryo such that each guide RNA directs a RNA guided endonuclease to a targeted site in the chromosomal sequence where the RNA guided endonuclease introduces a double-stranded break in the targeted site, and the
double-stranded break is repaired by a DNA repair process such that the chromosomal
sequence is modified.

In one embodiment, the RNA-guided endonuclease can bederived from a Cas9 protein.

In another embodiment, the nucleic acid encoding theRNA-guided endonuclease introduced into the cell or embryo can be mRNA.
13

In a

further embodiment, the nucleic acid encoding the RNA-guided endonuclease
introduced into the cell or embryo can be DNA.

In a further embodiment, the DNA

encoding the RNA-guided endonuclease can be part of a vector that further comprises
a sequence encoding the guide RNA.
d. Mode for Carrying Out the Invention[0014] In one embodiment, the RNA-guided endonuclease is derived from atype II CRISPR/Cas system.

In a specific embodiment, the RNA-guided

endonuclease is derived from a Cas9 protein.
[0022] In optional embodiments, the RNA-guided endonuclease may be part ofa protein-RNA complex comprising a guide RNA.

The guide RNA interacts with theRNA-guided endonuclease to direct the endonuclease to a specific targeted site (aspecific protospacer sequence at the 5' end of the guide RNA base pairs).
[0060] In embodiments in which the optional donor polynucleotide is present,
the donor sequence in the donor polynucleotide can be exchanged with or inte grated
into the chromosomal sequence during repair of the double-stranded breaks. ,For
example, in embodiments in which the donor sequence is flanked by upstream anddownstream sequences having substantial sequence identity with upstream anddownstream sequences, respectively, of the targeted sites in the chromosomal
sequence, the donor sequence can be exchanged with or integrated into thechromosomal sequence at the targeted site during repair mediated by homologydirected repair process.
(a) RNA-Guided Endonuclease[0063] The method comprises introducing into a cell or embryo at least one
RNA-guided endonuclease comprising at least one nuclear localization signal , or
nucleic acid encoding at least one RNA-guided endonuclease comprising at least one
nuclear localization signal.
[0064] In some embodiments, the RNA-guided endonuclease can be introduced
into the cell or embryo as an isolated protein.

In other embodiments, the RNA -guided endonuclease can be introduced into the cell or embryo as an mRNA molecule.
In still other embodiments, the RNA-guided endonuclease can be introduced into thecell or embryo as a DNA molecule.

The DNA sequence can be linear, or the DNA

sequence can be part of a vector.
(b) Guide RNA[0066] The method also comprises introducing into a cell or embryo at least
one guide RNA or DNA encoding at least one guide RNA.

A guide RNA interacts

with the RNA-guided endonuclease to direct the endonuclease to a specific target ed
14

site, at which site the 5' end of the guide RNA base pairs with a specific protospacer
sequence in the chromosomal sequence.
[0067] Each guide RNA comprises three regions: a first region at the 5' endthat is complementary to the targeted site in the chromosomal sequence, a second
internal region that forms a stem loop structure, and a third 3' region that remains
essentially single-stranded.

.. The first region of each guide RNA is different suchthat each guide RNA guides a fusion protein to a specific targeted site.

.. The second

and third regions of each guide RNA can be the same in all guide RNAs.
[0068] The first region of the guide RNA is complementary to a sequence (i.e.,a protospacer sequence) at the targeted site in the chromosomal sequence such that thefirst region of the guide RNA can base pair with the targeted site.

In an exemplary

embodiment, the first region of the guide RNA is about 19, 20, or 21 nucleotides inlength.
[0069] The guide RNA also comprises a second region that forms a secondary
structure.

In some embodiments, the secondary structure comprises a stem (orhairpin) and a loop.

For example, the loop can range from about 3 to about 10

nucleotides in length, and the stem can range from about 6 to about 20 base pairs inlength.

Thus, the overall length of the second region can range from about 16 toabout 60 nucleotides in length.

In an exemplary embodiment, the loop is abo ut 4

nucleotides in length and the stem comprises about 12 base pairs.
[0070] The guide RNA also comprises a third region at the 3' end that remains
essentially single-stranded.

Thus, the third region has no complementarity to any

chromosomal sequence in the cell of interest and has no complementarity to the restof the guide RNA.

The length of the third region can vary.

region is about 4 or more nucleotides in length.

In general, the third

For example, the length of the third

region ranges from about 5 to about 60 nucleotides in length.
[0071] The combined length of the second and third regions of the guide RNA
can range from about 30 to about 120 nucleotides in length.

In one aspect, thecombined length of the second and third regions of the guide RNA ranges from about
70 to about 100 nucleotides in length.
[0072] In some embodiments, the guide RNA comprises a single moleculecomprising all three regions.
two separate molecules.

In other embodiments, the guide RNA can comprise

The first RNA molecule can comprise the first region of theguide RNA and one half of the "stem" of the second region of the guide RNA.

The

second RNA molecule can comprise the other half of the "stem" of the second regionof the guide RNA and the third region of the guide RNA.
15

Thus, in this embodiment,the first and second RNA molecules each contain a sequence of nucleotides that arecomplementary to one another.

For example, in one embodiment, the first andsecond RNA molecules each comprise a sequence (about 6 to about 20 nucleotides)that base pairs to the other sequence to form a functional guide RNA.
[0073] The guide RNA can be introduced into the cell or embryo as an RNA
molecule.
[0074] In other embodiments, the guide RNA can be introduced into the cell orembryo as a DNA molecule.

In such cases, the DNA encoding the guide RNA canbe operably linked to a promoter control sequence for expression of the guide RNA in
the cell or embryo of interest.

In exemplary embodiments, the RNA encoding asequence is linked to a mouse or human U6 promoter.
[0075] In some embodiments, the DNA sequence encoding the guide RNA canbe part of a vector.

Suitable vectors include plasmid vectors and viral vectors.

[0076] In embodiments in which both the RNA-guided endonuclease and theguide RNA are introduced into the cell as DNA molecules, each can be part of aseparate molecule or both can be part of the same molecule.
(c) Targeted Site
[0077] An RNA-guided endonuclease in conjunction with a guide RNA isdirected to a targeted site in the chromosomal sequence, wherein the RNA-guided
endonuclease introduces a double-stranded break in the chromosomal sequence.
(d) Optional Donor Polynucleotide[0085] Donor polynucleotides comprising the upstream and downstream
sequences with sequence similarity to the targeted chromosomal sequence can belinear or circular.

In embodiments in which the donor polynucleotide is circular, it

can be part of a vector.

For example, the vector can be a plasmid vector.

[0088] Typically, the donor polynucleotide will be DNA.
single-strandedor

double-stranded

and/orlinearor

The DNA may becircular.

The

donor

polynucleotide may be a DNA plasmid, a bacterial artificial chromosome (BAC), a
yeast artificial chromosome (YAC), a viral vector, a linear piece of DNA, a PCR
fragment, a naked nucleic acid, or a nucleic acid complexed with a delivery vehicle
such as a liposome or poloxamer.

In certain embodiments, the donor polynucleotide

comprising the donor sequence can be part of a plasmid vector.

In any of thes esituations, the donor polynucleotide comprising the donor sequence can further
comprise at least one additional sequence.
(e) Example 1: Modification of Cas9 Gene for Mammalian Expression[0138] A Cas9 gene from Streptococcus pyogenes strain MGAS15252
16

(Accession number YP_005388840.1) was optimized with Homo sapiens codon
preference to enhance its translation in mammalian cells.

The Cas9 gene was also

modified by adding a nuclear localization signal PKKKRKV (SEQ ID NO:1) at the C
terminus for targeting the protein into the nuclei of mammalian cells.
[0140] The modified Cas9 DNA sequence was placed under the control of acytomegalovirus (CMV) promoter for constituent expression in mammalian cells.
The modified Cas9 DNA sequence was also placed under the control T7 promoter forin vitro mRNA synthesis with T7 RNA polymerase.
(f) Example 2: Targeting Cas9[0141] The adeno-associated virus integration site 1 (AAVS1) locus was used
as a target for Cas9-mediated human genome modification.

The human AAVS1

locus is located in intron 1 (4427 bp) of protein phosphatase 1, regulatory subunit 12C(PPP1R12C).
[0143] Cas9 guide RNAs were designed for targeting the human AAVS1 locus.
A 42 nucleotide RNA (referred to herein as a "crRNA" sequence) comprising (5' to 3')
a target recognition sequence (i.e., a sequence complementary to the non-coding
strand of the targeted sequence) and a protospacer sequence; a 85 nucleotide RNA(referred to herein as a "tracrRNA" sequence) comprising 5' sequence withcomplementarity to the 3' sequence of the crRNA and additional hairpin sequence;
and a chimeric RNA comprising nucleotides 1-32 of the crRNA, a GAAA loop, andnucleotides 19-45 of the tracrRNA were prepared.

The chimeric RNA coding

sequence was also placed under the control of human U6 promoter for in vivotranscription in human cells.
[0144] [Table 8] (As shown in Attachment 2)(g) Example 3: Preparation of Donor Polynucleotide to Monitor Genome
Modification[0145] Targeted integration of a GFP protein into the N terminus of PPP1R12C
was used to monitor Cas9-mediated genome modification.

To mediate integration byhomologous recombination, a donor polynucleotide was prepared.

The AAVS1-GFP

DNA donor contained a 5' (1185 bp) AAVS1 locus homologous arm, an RNA splicing
receptor, a turbo GFP coding sequence, a 3' transcription terminator, and a 3' (1217bp) AAVS1 locus homologous arm.
[0147] Targeted gene integration will result in a fusion protein between thefirst 107 amino acids of the PPP1R12C and the turbo GFP.

The expected fusion

protein contains the first 107 amino acid residues of PPP1R12C (highlighted in grey)
from RNA splicing between the first exon of PPP1R12C and the engineered splice17

receptor (..).
(h) Example 4: Cas9-Mediated Targeted Integration[0149] Transfection was performed on human K562 cells.

The K562 cell line

was obtained from American Type Culture Collection (ATCC) and grown in Iscove's
Modified Dulbecco's Medium, supplemented with 10% FBS and 2 mM L-glutamine.
Cultures were split one day before transfection (at approximately 0.5 million cells permL).

Cells were transfected with Nucleofector Solution V (Lonza) on a

Nucleofector (Lonza) with the T-016 program.

Transfection treatments are detailedin Table 7.
[0150] [Table 7] (As shown in Attachment 2)[0151] Fluorescence-activated cell sorting (FACS) was performed 4 days after
transfection.

FACS data are presented in Figure 4.

The percent GFP detected ineach of the four experimental treatments (A to D) was greater than in the control
treatments (E, F) (Exhibit Ko 105).
(i) Example 5: PCR Confirmation of Targeted Integration[0152] Genomic DNA was extracted from transfected cells with GenElute
Mammalian Genomic DNA Miniprep Kit (Sigma) 12 days after transfection.
Genomic DNA was then PCR amplified with a forward primer located outside the 5'
homologous arm of the AAVS1-GFP plasmid donor and a reverse primer located atthe5'region

of

the

GFP.

The

forwardprimerwas

5'-

CCACTCTGTGCTGACCACTCT-3' (SEQ ID NO:18) and the reverse primer was 5'GCGGCACTCGATCTCCA-3' (SEQ ID NO:19).
the junction PCR was 1388 bp.

The expected fragment size from

The amplification was carried out with JumpStart

Taq ReadyMix (Sigma), using the following cycling conditions: 98°C for 2 minutes
for initial denaturation; 35 cycles of 98°C for 15 seconds, 62°C for 30 seconds, and72°C for 1 minute and 30 seconds; and a final extension at 72°C for 5 minutes.

PCR

products were resolved on 1% agarose gel.
[0153] Cells transfected with 10 μg of Cas9 mRNA transcribed with an AntiReverse Cap Analog (ARCA), 0.3 nmol of pre-annealed crRNA-tracrRNA duplex,
and 10 μg of AAVS1-GFP plasmid DNA displayed a PCR product of the expected
size (see lane A of Figure 5).
(2) Summary of the Disclosure of Cited Document 1 and Cited Invention 1
Found by the Decision of the Present Casea. The main purpose of Cited Document 1 is to provide an RNA-guided
endonuclease directed by a guide RNA as genome-editing techniques.

According tothis, Cited Document 1 discloses a technology that does not require a design of a new18

nuclease for a targeted genomic location, like the conventional genome editing
techniques such as ZFN and TALEN, and that also has increased specificity ([0001] to[0004]).
b. Concretely, Cited Document 1 discloses that (1) an RNA-guided
endonuclease (Cas9 protein) comprising a nuclear localization signal is introduced
into a eukaryotic cell, (2) a guide RNA directs an RNA-guided endonuclease to atargeted site, and (3) in a eukaryotic cell, an RNA-guided endonuclease introduces adouble-stranded break in a targeted site and the chromosomal sequence is modified bya donor DNA ([Claim 13], [Claim 14], [0005]).
Among these, as (1) a method of introducing an RNA-guided endonuclease intoa eukaryotic cell, Cited Document 1 discloses [i] a method of introducing a protein,[ii] a method of introducing mRNA encoding a protein, and [iii] a method wherein
DNA encoding a protein is made to be part of a vector to introduce the part of thevector ([Claim 15] to [Claim 17], [0005], [0064]).
In addition, as (2) a method of introducing a guide RNA into a eukaryotic cell,
Cited Document 1 discloses [i] a method of introducing a single molecule (single stranded chimeric RNA), [ii] a method of introducing two separate molecules (double stranded crRNA-tracrRNA) ([0072], Example 2, Example 4), and [iii] a method of
introducing DNA encoding the RNA in the above [i] or [ii] (single-stranded or
double-stranded) wherein the DNA is made to be part of a vector to introduce the partof the vector ([0074], [0075]).
Further, with regard to (3) a donor DNA, Cited Document 1 discloses that thedonor DNA is made to be part of a vector to introduce into a eukaryotic cell ([0085],[0088]).
Methods of preparing the above (1) to (3) are disclosed in Examples 1 to 3.
c. In Examples 4 and 5, with regard to Treatment A ((1)[ii], (2)[ii]), Treatments
B and C ((1)[i], (2)[i]) and Treatment D ((1)[iii], (2)[iii]) in which the introducing
methods were changed from (1) and (2), Fluorescence-Activated Cell Sorting (FACS)(Example 4) and PCR test (Example 5) were used to confirm whether or not a
chromosomal sequence is modified in a eukaryotic cell, as compared to control
treatment groups (E, F).

In this regard, among Treatments A to D, none ofTreatments A to C is a vector system.

Only Treatment D is a vector system.

d. The decision of the present case found Cited Invention 1 from Cited
Document 1, as mentioned in No. 2, 3(2) above.
(3) Findings on Cited Invention 1
a. Cited Document 1 discloses an invention related to targeted genome-editing
19([0001]).

With regard to the invention, Cited Document 1 discloses theconfiguration comprising introducing into a eukaryotic cell or embryo at least one
RNA-guided endonuclease comprising at least one nuclear localization signal, or a
nucleic acid encoding at least one RNA-guided endonuclease comprising at least one
nuclear localization signal ([Claim 13], [0005]), among which the RNA-guided
endonuclease is derived from a Cas9 protein ([Claim 14], [0005]), wherein the nucleic
acid encoding the RNA-guided endonuclease is DNA, and the DNA is part of a vector
which further comprises a sequence encoding a guide RNA ([Claim 16], [Claim 17],[0075]).

Cited Document 1 also discloses the configuration that the nucleic acidsequence encoding the RNA-guided endonuclease can be operably linked to apromoter control sequence, and is part of a vector ([0004]).
Thus, Cited Document 1 discloses "a vector comprising a promoter control
sequence operably linked to a nucleic acid encoding at least one type II Cas9 protein
comprising at least one nuclear localization signal."b. With regard to the invention, Cited Document 1 discloses the configuration
comprising introducing into a eukaryotic cell or embryo at least one guide RNA or
DNA encoding at least one guide RNA ([Claim 13], [0005]), among which the guideRNA comprises three regions; i.e., a first region at the 5' end that is complementary toa targeted site in a chromosomal sequence, a second internal region that forms a stem
loop structure, and a third 3' region that remains essentially single-stranded ([0067]),
wherein the guide RNA is operably linked to a promoter control sequence forexpression of the guide RNA in the cell or embryo of interest ([0074]), and the DNA
sequence encoding the guide RNA is part of a vector ([0075]).
Thus, Cited Document 1 discloses "a vector comprising a promoter control
sequence operably linked to DNA encoding at least one guide RNA comprising a firstregion at the 5' end that is complementary to a targeted site in a chromosomal
sequence in a eukaryotic cell, a second internal region that forms a stem loop structure,
and a third 3' region that remains essentially single-stranded."c. Cited Document 1 discloses that the invention comprises introducing at least
one donor polynucleotide into a eukaryotic cell or embryo ([Claim 13], [0005]), anddiscloses the configuration that in an embodiment in which the donor polynucleoti deis circular, it can be part of a vector ([0085]).
Thus, Cited Document 1 discloses "a vector comprising at least one donor
polynucleotide".
d. Cited Document 1 discloses that the invention is a "vector system."e. Cited Document 1 discloses that the invention has a function of culturing a20

eukaryotic cell or embryo such that each guide RNA directs a RNA-guided
endonuclease to a targeted site in a chromosomal sequence where the RNA-guided
endonuclease introduces a double-stranded break in the targeted site, and the doublestranded break is repaired by a DNA repair process such that the chromosomal
sequence is modified ([Claim 13], [0005]).
Thus, Cited Document 1 discloses that "the guide RNA directs a type II Cas9
protein to a targeted site in a chromosomal sequence in a eukaryotic cell where thetype II Cas9 protein introduces a chromosomal DNA double-stranded break in thetargeted site, and the double-stranded break is repaired by a DNA repair process suchthat the chromosomal sequence is modified."f. According to the foregoing, Cited Document 1 discloses the above
configurations a to e.

Therefore, it can be found that Cited Document 1 discloses theinvention as found in the decision of the present case (Cited Invention 1).
(4) Comparison between the Present Invention and Cited Invention 1
a. The Present Invention is as the above No. 2, 2 and can be divided into theconstituent features as follows:
A. An engineered, non-naturally occurring clustered regularly interspaced shortpalindromic repeats (CRISPR) - CRISPR-associated (Cas)
G. (CRISPR-Cas) vector system;
C. comprising one or more vectors comprising;B-a. a first regulatory element operably linked to a nucleotide sequence
encoding a CRISPR-Cas system polynucleotide sequence comprising a guidesequence, tracrRNA, and a tracr mate sequence, wherein the guide sequencehybridizes with one or more targeted sequences in polynucleotide loci in a eukaryotic
cell;B-b. a second regulatory element operably linked to a nucleotide sequence
encoding a type II Cas9 protein; andB-c. a recombination template;
D. wherein components a), b), and c) are located on the same or different
vectors of the system;
E. the system further comprises one or more nuclear localization signals (there
can be more than one) (NLS (there can be more than one)) to be expressed together
with the nucleotide sequence encoding the Cas9 protein;
F. whereby the guide sequence targets the one or more polynucleotide loci inthe eukaryotic cell and the Cas9 protein cleaves the one or more polynucleotide loci,whereby sequence of the one or more polynucleotide loci is modified.
21

b. Constituent Feature A(C)(a) The Present Invention is an engineered, non-naturally occurring clusteredregularly interspaced short palindromic repeats (CRISPR) - CRISPR-associated (Cas)(CRISPR-Cas) system.
On the other hand, Cited Invention 1 uses an engineered, non-naturally
occurring Cas9 protein which is derived from a naturally occurring type II
CRISPR/Cas system and is modified to include a nuclear localization signal ([0014],[0063]).

Thus, this portion of Cited Invention 1 corresponds to a portion where thevector system is "clustered regularly interspaced short palindromic repeats (CRISPR)- CRISPR-associated (Cas) (CRISPR-Cas)" among constituent feature A of the
Present Invention.
(b) Among constituent feature A of the Present Invention, the portion of"vector system" (the same applies to constituent feature C) corresponds to theconstituent feature "vector system" of Cited Invention 1.
c. Constituent Feature B-a"A first region at the 5' end that is complementary to a targeted site in achromosomal sequence in a eukaryotic cell" in Cited Invention 1 corresponds to "theguide sequence" which "hybridizes with one or more targeted sequences i npolynucleotide loci in a eukaryotic cell" in the Present Invention.
The second and third regions which combine "a second internal region that
forms a stem loop structure" and "a third 3' region that remains essentially single stranded" in Cited Invention 1 correspond to a sequence of "loop" in addition to"tracrRNA" and a "tracr mate sequence" in the Present Invention, in view of thestatement "the portion is provided in the lower portion of Figure 13B (Judgment Note:
this is a typographical error of ‘Figure 8B’), where the portion of the sequence 5' ofthe final "N" and upstream of the loop corresponds to the tracr mate sequence, and the
portion of the sequence 3' of the loop corresponds to the tracr sequence" in [0076] ofthe description of the present application.
Thus, "at least one guide RNA comprising a first region at the 5' end that iscomplementary to a targeted site in a chromosomal sequence in a eukaryotic cell, asecond internal region that forms a stem loop structure, and a third 3' region th atremains essentially single-stranded" in Cited Invention 1 corresponds to "a CRISPRCas system polynucleotide sequence comprising a guide sequence, tracrRNA, and a
tracr mate sequence", wherein "the guide sequence hybridizes with one or moretargeted sequences in polynucleotide loci in a eukaryotic cell" in constituent feature
B-a of the Present Invention.
22

Therefore, "(ii) a vector comprising a promoter control sequence operably
linked to DNA encoding at least one guide RNA comprising a first region at t he 5' endthat is complementary to a targeted site in a chromosomal sequence in a eukaryotic
cell, a second internal region that forms a stem loop structure, and a third 3' region
that remains essentially single-stranded" in Cited Document 1 corresponds to "one ...
vectors comprising" "a) a first regulatory element operably linked to a nucleotide
sequence encoding a CRISPR-Cas system polynucleotide sequence comprising a
guide sequence, tracrRNA, and a tracr mate sequence, wherein the guide sequencehybridizes with one or more targeted sequences in polynucleotide loci in a eukaryotic
cell" in the Present Invention.
d. Constituent Feature B-b
The second regulatory element in the Present Invention comprises one
encoding a CRISPR enzyme comprising a nuclear localization sequence (NLS)([0071]), and thus is a regulatory element operably linked to an enzyme-coding
sequence encoding a CRISPR enzyme comprising a nuclear localization sequence([0012]).
On the other hand, "(i) a vector comprising a promoter control sequence
operably linked to a nucleic acid encoding at least one type II Cas9 protein
comprising at least one nuclear localization signal" in Cited Invention 1 corresponds
to "one ... vectors comprising" "b) a second regulatory element operably linked to anucleotide sequence encoding a type II Cas9 protein, and further comprising "one ormore nuclear localization signals (there can be more than one) (NLS (there can be
more than one)) to be expressed together with the nucleotide sequence encoding theCas9 protein" in the Present Invention.
e. Constituent Feature B-c"(iii) at least one donor polynucleotide" in Cited Invention 1 corresponds to "asequence or a template which can be used for recombination into the locus to betargeted, including a targeted sequence" in the Present Invention (an explanation tosuch matters is given in [0065] of the description of the present application), and thus,
corresponds to "a recombination template."

Therefore, "a vector comprising at least

one donor polynucleotide" in Cited Document 1 corresponds to "one .. vectors
comprising" "c) a recombination template" in the Present Invention.
f. Constituent Feature D
Cited Document 1 discloses not only an embodiment in which the vectors (i) to(iii) are different from each other, but also an embodiment in which the vectors (i) to(iii) are the same ([0005], [0088]).

Thus, these embodiments correspond to23

constituent feature D of the Present Invention.
g. Constituent Feature E
As mentioned in the above d, Cited Document 1 comprises at least one nuclear
localization signal to be expressed together with the nucleotide sequence encoding a
Cas9 protein, and thus, this corresponds to constituent feature E of the Present
Invention.
h. Constituent Feature F
In Cited Invention 1, the above three vectors (i) to (iii) are the constituent
features, and a vector system comprising these three vectors is provided with afunction in which "the guide RNA directs a type II Cas9 protein to a targeted site in achromosomal sequence in a eukaryotic cell where the type II Cas9 protein introduces
a chromosomal DNA double-stranded break in the targeted site, and the doublestranded break is repaired by a DNA repair process such that the chromosomal
sequence is modified."Thus, "a targeted site in a chromosomal sequence in a

eukaryotic cell", "break", and "modify" of Cited Invention 1 correspond to "the one ormore polynucleotide loci in a eukaryotic cell", "cleave", and "modify" of the Present
Invention, respectively.
Thus, the above function of Cited Invention 1 is the same as the function of the
Present invention in which "a guide sequence targets the one or more polynucleotideloci in the eukaryotic cell and the Cas9 protein cleaves the one or more polynucleotide
loci, whereby sequence of the one or more polynucleotide loci is modified", andtherefore, corresponds to constituent feature F.
i. Constituent Feature G
It is clear that Cited Invention 1 is a CRISPR-Cas vector system, whichcorresponds to constituent feature G of the Present Invention as well.
j. According to the foregoing, it can be found that the Present Invention is
identical with Cited Invention 1.
(5) Assertions by the Plaintiffs
a. The Plaintiffs assert that Cited Document 1 is not supported by experimental
data that a targeted site sequence was modified and does not provide a rational basis
for assuming that the CRISPR-Cas9 system could be applied to eukaryotic
applications.

Accordingly, the Plaintiffs assert that [i] it cannot be deemed that the

Present Invention is substantially identical with Cited Invention 1, because Cited
Invention 1 does not comprise a function of the Present Invention; i.e. "a guide RNA
directs a type II Cas9 protein to a targeted site in a chromosomal sequence in a
eukaryotic cell where the type II Cas9 protein introduces a chromosomal DNA
24

double-stranded break in the targeted site, and the double-stranded break is repairedby a DNA repair process such that the chromosomal sequence is modified", and that[ii] it cannot be deemed that Cited Document 1 has an effect of excluding a later
application stipulated in Article 29-2 of the Patent Act, because a system disclosed in
Cited Document 1 cannot solve the problem of the Present Invention.
b. Assertion of the above a[i](a) Example 4 and Example 5
Cited Document 1 is a Patent Document in which the main purpose is toprovide new genome-editing techniques; concretely, to provide an RNA-guided
endonuclease directed by a guide RNA.

In this regard, Cited Document 1 inTreatment D of Example 4 discloses a method of introducing DNA encoding a protein
as part of a vector, as a method of introducing into a eukaryotic cell an RNA -guided
endonuclease (a Cas9 protein) comprising a nuclear localization signal, and discloses
using U6-chimeric RNA plasmid DNA as a method of introducing a guide RNA into aeukaryotic cell.
Fluorescence-activated cell sorting (FACS) was performed in Example 4, and a
PCR experiment was performed in Example 5.
(b) Fluorescence-Activated Cell Sorting (FACS)
Introducing a donor sequence into a targeted sequence resulted in a fusion
protein of the first 107 amino acids of PPP1R12C and a turbo GFP (Example 3), andwhen the fusion protein (expressed GPF protein) was irradiated with laser light, itemitted fluorescence at a specific wavelength (Exhibit Ko 103, page 5, lines 2 to 5).
By measuring the fluorescence of the GFP protein, it is possible to presume that the
GFP protein is expressed in the cell and that a donor sequence (such as a GFP gene) is
incorporated into the targeted sequence.
Cited Document 1 discloses in Example 4 that in Treatment D (Treatments E
and F are the control treatment groups), fluorescence-activated cell sorting (FACS)
was performed using the Cas9, the guide RNA, and the donor polynucleotides
prepared in Examples 1 to 3.
According to Figures 4-1 to 3 (as shown in Attachment 2), which show theresults of the experiments in Example 4, a numerical value of Treatment D was 7.47%.
This value is considerably higher than values of the control treatment groups; i.e.,
Treatment E (1.92%) by using only "AAVS1-GFP plasmid DNA" (donor DNA) and
Treatment F (0.159%) by using no reagents.

According to this, it can be found tha t adonor sequence (such as a GFP gene) was incorporated into a targeted sequence.
(c) Results of PCR experiment25

In PCR experiment, the presence or absence of a PCR product obtained in theexperiment can be detected by bands on a gel, whereby it is possible to presume that adonor sequence (GFP gene) is incorporated into or near a targeted sequence (Exhibit
Ko 103, page 18, line 10 to page 20, line 16).
Example 5 discloses that the PCR experiment was performed using the same
samples as in Example 4.

According to Figure 5 (as shown in Attachment 2), whichshows the results of the experiments in Example 5, a band of 1388 bp (base length),which was expected for Treatment D, was not detected.
However, in Treatment D in Example 5, it cannot be denied that a band on thepredetermined gel could not be detected, as a result of insufficient efficiency ofgenome modification due to differences in guide RNAs, targeted sequences, etc.,
similarly to the chimeric RNA with n of +48 (a tracr sequence is 26 nucleotides inlength) and the chimeric RNA with n of +54 (a tracr sequence is 32 nucleotides inlength) of the description of the present application.

Therefore, it is not denied thatthe vector system of the Cited Invention 1 has a function of incorporating a donor
sequence (such as a GFP gene) into a targeted sequence, notwithstanding the resultsof Treatment D in Example 5.
(d) According to the foregoing, it can be understood from the disclosure of
Cited Document 1 that an RNA-guided endonuclease directed by a guide RNA has afunction of modifying a chromosomal sequence at a targeted site in a eukaryotic cell.
(e) As vector (i), Cited Document 1 uses a vector which was obtained by
optimizing a Cas9 gene obtained from a Streptococcus pyogenes strain for enhancing
translation in mammalian cells and adding the nuclear localization signal PKKKRKV(SEQ ID NO: 1) to the C-terminus, followed by placing it under the control of apromoter (Example 1).
In addition, as vector (ii), Cited Document 1 uses a vector which was obtainedby placing a chimeric RNA comprising nucleotides 1 to 32 of the crRNA, a GAAA
loop, and nucleotides 19 to 45 of the tracrRNA under the control of human U6
promoter (Example 2).
Further, as vector (iii), Cited Document 1 uses "AAVS1-GFP plasmid DNA"by which a target gene is integrated into a targeted sequence by homologous
recombination (Example 3).
Furthermore, by showing experimental examples, Cited Document 1 discloses
in Example 4 that the vector system comprising the vectors disclosed in Exam ples 1
to 3 has a function of cleaving a targeted sequence in a eukaryotic cell and modifying
the targeted sequence.
26

Thus, each vector (i) to (iii) in Cited Invention 1 has a technical means
necessary for appropriate transcription, translation, and nuclear translocation, etc. in a
eukaryotic cell, and a technical means necessary for appropriate modification of atargeted sequence in a eukaryotic cell.

Therefore, it can be understood that it is also

disclosed that such vectors have functions of cleaving a targeted sequence in a
eukaryotic cell and modifying the targeted sequence.
(f) Summary
According to the foregoing, it should be deemed that Cited Document 1
discloses not only formally but also substantially that "a guide RNA directs a type II
Cas9 protein to a targeted site in a chromosomal sequence in a eukaryotic cell where
the type II Cas9 protein introduces a chromosomal DNA double-stranded break in thetargeted site, and the double-stranded break is repaired by a DNA repair process suchthat the chromosomal sequence is modified."

It can be understood that the vector

system of Cited Invention 1 is disclosed as a system which also includes the above
functions.
c. Assertion of the above a[ii](a) Article 29-2 of the Patent Act provides that if an invention claimed in apatent application is identical with an invention or a device stated in the description or
the drawings (hereinafter referred to as "the description, etc. of an earlier
application") originally attached to a request of another application for a patent or ofanother application for a utility model registration which was filed before the date of
the patent application and for which the Patent Gazette or the Utility Model Gazette
was published after filing the patent application, a patent shall not be granted for theinvention.
An object of this Article is as follows.

An invention stated in the description,etc. of the earlier application, even if the invention is not stated in the Scope ofClaims, is laid open to the public in a publication, etc. of the earlier application.
Thus, even if a later application is filed before the publication, etc. of the earlier
application, when the invention of the later application is identical with the inventionof the earlier application, no new technology is laid open to the public in the
publication, etc. of the later application.

Therefore, granting a patent to suchinvention is inappropriate from the viewpoint of the patent system which intends toprotect an invention as a reward for laying a new invention open to the public.
The "invention" stated in the description, etc. of the earlier application in this
Article is construed as an invention which is understood from a matter stated in thedescription, etc. of the earlier application and from a matter equivalent to that stated27

in the description, etc. of the earlier application.

The "matter equivalent to thatstated" means a matter which can be derived from the stated matter by taking into
consideration common general technical knowledge as of the time of filing.
Thus, even if there is no particular statement of the description, etc. of theearlier application, the invention of the earlier application can be found by taking
common general technical knowledge into consideration in order to understand theinvention of the earlier application.

On the other hand, in the case where an

invention lacks concreteness or technical content of an invention is insufficiently
disclosed even while taking into consideration common general technical knowledgeof a person ordinarily skilled in the art, such invention does not fall under the"invention" mentioned above, and does not have an effect of excluding the later
application stipulated in this Article.

In addition, it should be deemed that thedegree of disclosure of technical content required here should be sufficient if thetechnical content is disclosed to the extent that a person ordinarily skilled in the art
can understand that the invention of the earlier application is shown in the description,etc. of the earlier application and the person ordinarily skilled in the art is enabled towork the disclosed invention of the earlier application.
(b) In the present case, with regard to the above, Examples 1 to 3 in Cited
Invention 1 disclose in detail a method for producing each vector (i) to (iii) in Cited
Invention 1, and Example 4 clearly discloses a concrete test method for confirming
whether a donor sequence (GFP gene) is incorporated in or near a targeted sequence.
In addition, as mentioned above, from the experimental results of Exa mple 4, it
can be understood that a combination of the RNA-guided endonuclease comprising
the nuclear localization signal, the guide RNA, and the donor polynucleotide isincorporated into the eukaryotic cell and that the double-stranded break and repair
occur in the targeted site.

The experimental results of Example 5 are not to beinterpreted as a bar to the above understanding.
Further, a vector system comprising the above vectors (i) to (iii) has a technicalmeans necessary for appropriate transcription, translation, and nuclear translocation,etc. in a eukaryotic cell, and a technical means necessary for appropriate modificationof a targeted sequence in a eukaryotic cell.

Thus, it can be understood that even if

the vector system is used, such vector system has functions of cleavage of a targeted
sequence and modification of the targeted sequence in a eukaryotic cell, as mentioned
above.
From the above, it can be deemed that there is a disclosure in Cited Document
1 to the extent that a person ordinarily skilled in the art can understand that the28

invention of the earlier application is disclosed in the description, etc. of the earlier
application, and the person ordinarily skilled in the art is enabled to work the shown
invention of the earlier application.

Therefore, it is proper to find that in Cited

Document 1, a technology was laid open to the public to the extent that thetechnology was sufficient to exclude the later application, in which the technology
includes a portion of the function such that "a guide RNA directs a type II Cas9
protein to a targeted site in a chromosomal sequence in a eukaryotic cell where thetype II Cas9 protein introduces a chromosomal DNA double-stranded break in thetargeted site, and the double-stranded break is repaired by a DNA repair process suchthat the chromosomal sequence is modified."d. Other Assertions by the Plaintiffs(a) The Plaintiffs assert as follows.

According to the description on which thepriority claim is based with regard to Cited Document 1 (Exhibit Ko 105), there are
no statements "integration of the donor sequence and expression of the fusion protein
were confirmed" in Example 4.

In addition, it cannot be deemed that the FACS

alone performed in Example 4 shows that the fluorescence detected is not due to otherfactors than incorporation into the targeted site.

Further, it should be understood thatincorporation into the targeted site does not occur unless approximately 10 times ormore fluorescence is detected as compared to the control.

The Plaintiffs submit awritten opinion by a researcher along these lines (Exhibit Ko 103).

However, itcannot be deemed that the technical content of the experimental results stated in Cited
Document 1 is insufficiently disclosed.
The Plaintiffs assert that the results of the PCR experiment in Example 5 showthat a PCR product was not confirmed in Treatment D, indicating that the gene of thedonor sequence was not incorporated into the targeted site.

However, in Treatment

D in Example 5, as mentioned in the above b(c), there is a possibility that the band on
the predetermined gel was not detected, as a result of insufficient efficiency ofgenome modification due to differences in guide RNAs and targeted sequences.
Therefore, from the result of Treatment D in Example 5, it should be construed that it
is not denied that the vector system of the Cited Invention 1 has a function ofincorporating a donor sequence (such as a GFP gene) into a targeted sequence.
(b) The Plaintiffs assert that a person ordinarily skilled in the art would havereviewed the conflicting results obtained by the FACS experiment and the PCR
experiment in Cited Document 1 and concluded that a question of whether the
CRISPR-Cas9 system functions in a eukaryotic cell is unresolved.

Alternatively, thePlaintiffs assert that it was not also possible to deduce that the experiments in Cited29

Document1 were successful on the basis of the fact that other scientists could achieve
success in a eukaryotic cell using other techniques or constructs.
However, the results of the experiment in Example 4 are sufficient tounderstand that the RNA-guided endonuclease comprising the nuclear localization
signal, the guide RNA, and the donor polynucleotide were introduced into theeukaryotic cell and that the double-stranded break and repair occurred in the targetedsite.

In addition, the results of the experiment in Example 5 are not to be construed

as a bar to the above understanding.
Further, a vector system comprising vectors (i) to (iii) in Cited Invention 1 has
a technical means necessary for appropriate transcription, translation, and nuclear
translocation, etc. in a eukaryotic cell, and a technical means necessary for
appropriate modification of a targeted sequence in a eukaryotic cell, as mentioned in
the above b(e).

Thus, it can be understood that the vector system of Cited Invention

1 has a function of cleaving a targeted sequence in a eukaryotic cell and modifying
the targeted sequence.

That is, in Cited Document 1, there is sufficient disclosure tounderstand that Cited Invention 1 can be worked.
Therefore, it cannot be deemed that the issue of whether a CRISPR-Cas9
system functions in a eukaryotic cell is unresolved in Cited Invention 1.
(c) The Plaintiffs assert that the Present Invention discloses guidance foradapting a CRISPR-Cas9 system to eukaryotic genomic DNA in a eukaryotic cell
environment, and emphasize a difference in the function from the CRISPR-Cas9
system of Cited Document 1.

However, as determined from the statement of Claim 1,

the Present Invention does not make a selection such as adding multiple NLSs to Cas9,
making tracrRNAs longer, and inserting, e.g., poly-T as a terminator at the 3' end of achimeric RNA.

Rather, the Present Invention and Cited Invention 1 use a vector

system containing common vectors in the configuration of the invention.
The Plaintiffs' assertion emphasizing the difference between the two inventions
is unfounded.
(6) Summary
As mentioned above, the Present Invention is identical with the Cited Invention
1, and thus is not allowed to be granted a patent under the provision of Article 29 -2 ofthe Patent Act.
Therefore, Reason 1 for Rescission is unfounded.
3. Conclusion
According to the foregoing, the decision of the present case did not err inconcluding that the request for the appeal of the present case shall be dismissed.
30

The

Plaintiffs’ claim for seeking a rescission of the decision is unfounded and thus shallbe dismissed.

Therefore, the judgment is rendered as mentioned in the main text.

Intellectual Property High Court, First Division
Presiding Judge

TAKABE Makiko

Judge KOBAYASHI YasuhikoJudge SEKINE Sumiko

31

Attachment 1 (List of the Drawings of the Present Invention)[Figure 8]

human
EMX1
locus

protospacer (1)protospacer (3)protospacer (5)pre-crRNA + tracrRNA processing
spacer(30bp)
pre-crRNA

protospacer (4)protospacer (2)protospacer
targetchimeric RNA
guide sequence (20bp)
indel (%)

Figures 8A-C

32

[Figure 10]

chimeric RNA

chimeric RNA architecture
guide sequence (20bp)

loop sequence

Figure 10A

protospacer 1 (EMX1)

protospacer 2 (EMX1)chimeric RNA

indel (%)

Figure 10B

33

protospacer 3 (EMX1)

protospacer 4 (PVALB) protospacer 5 (PVALB)

chimeric RNA

Figure 10C

indel (%)

indel (%)

[Figure 11]

protospacer 1(EMX1)

protospacer 2(EMX1)

protospacer 3(EMX1)

Figures 11A-B

34

protospacer 4(PVALB)

protospacer 5(PVALB)Attachment 2 (List of the Drawings, etc. of Cited Invention 1)[Table 7]

Table 7 Transfection Treatments
Modified Cas9
Treatment

Guide RNA

Donor sequence

Cas9 mRNA transcribedwith an Anti-Reverse
Cap Analog (10 μg)

Pre-annealed crRNAtracrRNA duplex (0.3nmol)

AAVS1-GFP plasmid
DNA (10 μg)

Cas9 mRNA transcribedwith an Anti-Reverse
Cap Analog (10 μg)

Chimeric
nmol)

RNA

(0.3

AAVS1-GFP plasmid
DNA (10 μg)

Cas9 mRNA capped via
post-transcription
capping reaction (10 μg)

Chimeric
nmol)

RNA

(0.3

AAVS1-GFP plasmid
DNA (10 μg)

Cas9 plasmid DNA (10μg)

U6-chimericRNA
plasmid DNA (5 μg)

AAVS1-GFP plasmid
DNA (10 μg)

None

None

AAVS1-GFP plasmid
DNA (10 μg)

None

None

None

[Table 8]

Table 4. Guide RNAsSEQ ID NO:

5'-3' Sequence

Chimeric RNA

35

[Figure 4][Figure 4-1]

[Figure 4-2]

[Figure 4-3]

[Figure 5]36