Regents of the University of Minnesota

Patent Trials and Appeals Board

Jun 24, 2021

2020006123 (P.T.A.B. Jun. 24, 2021)

UNITED STATES PATENT AND TRADEMARK OFFICE
UNITED STATES DEPARTMENT OF COMMERCE
United States Patent and Trademark Office
Address: COMMISSIONER FOR PATENTS
P.O. Box 1450
Alexandria, Virginia 22313-1450
www.uspto.gov
APPLICATION NO. FILING DATE FIRST NAMED INVENTOR ATTORNEY DOCKET NO. CONFIRMATION NO.
14/629,859 02/24/2015 Daniel F. Voytas 09531-0335002 9831
26191 7590 06/24/2021
FISH & RICHARDSON P.C. (TC)
PO BOX 1022
MINNEAPOLIS, MN 55440-1022
EXAMINER
UYENO, STEPHEN G
ART UNIT PAPER NUMBER
1662
NOTIFICATION DATE DELIVERY MODE
06/24/2021 ELECTRONIC
Please find below and/or attached an Office communication concerning this application or proceeding.
The time period for reply, if any, is set in the attached communication.
Notice of the Office communication was sent electronically on above-indicated "Notification Date" to the
following e-mail address(es):
PATDOCTC@fr.com
PTOL-90A (Rev. 04/07)

UNITED STATES PATENT AND TRADEMARK OFFICE
BEFORE THE PATENT TRIAL AND APPEAL BOARD
Ex parte DANIEL F. VOYTAS,
PAUL ATKINS, and NICHOLAS J. BALTES
Appeal 2020-006123
Application 14/629,859
Technology Center 1600
Before DONALD E. ADAMS, FRANCISCO C. PRATS, and
TAWEN CHANG, Administrative Patent Judges.
CHANG, Administrative Patent Judge.
DECISION ON APPEAL
Pursuant to 35 U.S.C. § 134(a), Appellant1 appeals from the
Examiner’s decision to reject claims 1, 11, 15, and 22.2 We have
jurisdiction under 35 U.S.C. § 6(b).
We AFFIRM.

1 We use the word “Appellant” to refer to “applicant” as defined in 37
C.F.R. § 1.42. Appellant identifies the real party in interest as Regents of
the University of Minnesota. Appeal Br. 2.
2 An oral hearing was held on April 7, 2021. A transcript (“Tr.”) of this
hearing was entered into the record April 27, 2021.
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STATEMENT OF THE CASE
“Precise genome targeting technologies . . . enable systematic reverse
engineering of causal genetic variations by allowing selective perturbation of
individual genetic elements, as well as to advance synthetic biology,
biotechnological, and medical applications.” Zhang3 1:62–67; ’527
application4 ¶ 2. In particular, targeted double-stranded breaks (DSBs) can
“activate cellular DNA repair pathways, which can be harnessed to achieve
desired DNA sequence modifications near the break site.” Spec. 3:18–19.
The CRISPR/Cas5 system provides “a relatively simple, effective tool
for generating modifications in genomic DNA at selected sites” by creating
targeted DSBs in a cell through the use of targeting RNAs and an
endonuclease that induces a double strand break at or near the sequence to
which the targeting RNAs are directed. Spec. 2:7–13, 2:18–24. More
specifically, “[d]irecting DNA DSBs [using the CRISPR/Cas system]
requires two components: the Cas9 protein, which functions as an
endonuclease, and CRISPR RNA (crRNA) and tracer RNA (tracrRNA)
sequences that aid in directing the Cas9/RNA complex to target DNA
sequence.” Id. at 4:5–8.

3 Zhang, US 8,697,359 B1, issued Apr. 15, 2014.
4 U.S. Provisional Application No. 61/736,527, filed Dec. 12, 2012, attached
to the Appeal Brief as Appendix A of the Evidence Appendix.
5 CRISPR stands for “Clustered Regularly Interspersed Short Palindromic
Repeats,” and Cas stands for “CRISPR-associated.” Spec. 1:13–15. In the
context of the claims, Cas refers to the Cas9 endonuclease molecule of
Sreptococcus pyogenes. Appeal Br. 17 (Claims App.).
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The Specification states that the invention relates to gene targeting in
plants, in particular gene targeting using CRISPR/Cas systems. Spec 1:13–
15.
CLAIMED SUBJECT MATTER
The claims are directed to a method of modifying the genomic
material in a plant cell. Claim 1, the only independent claim, is illustrative:
1. A method for modifying the genomic material in a plant
cell, comprising:
(a) introducing into the plant cell a nucleic acid sequence
encoding a Clustered Regularly Interspersed Short Palindromic
Repeats RNA (crRNA) and a trans-activating RNA (tracrRNA),
or a sequence encoding a chimeric cr/tracrRNA hybrid, wherein
the crRNA and tracrRNA, or the chimeric cr/tracrRNA hybrid,
is targeted to a sequence that is endogenous to the plant cell;
(b) introducing into the plant cell a nucleic acid molecule
comprising a sequence encoding a Cas9 endonuclease molecule
of Streptococcus pyogenes, wherein the Cas9 endonuclease
molecule induces a double strand break at or near the sequence
to which the crRNA and tracrRNA sequence is targeted, or at or
near the sequence to which the chimeric cr/tracrRNA hybrid is
targeted, and wherein the plant cell is a dicotyledonous plant
cell.
Appeal Br. 17 (Claims App.).
REJECTION(S)
Claims 1, 11, 15, and 22 are rejected under pre-AIA 35 U.S.C. 103(a)
as being unpatentable over Zhang and Klimyuk.6 Ans. 4.

6 Klimyuk et al., US 2005/0091706 A1, published Apr. 28, 2005.
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OPINION
A. Issue
The Examiner finds that Zhang teaches all of the elements of claim 1,
except that Zhang does not specifically teach transformation of dicots. Ans.
5. However, the Examiner finds that “Klimyuk teaches transformation of
dicots, such as tomato,” as well as transgenic Arabidopsis plants. Id. at 6.
The Examiner concludes that it would have been obvious, and within the
scope of a skilled artisan at the time of invention, to modify Zhang’s
CRISPR/Cas9 system with Klimyuk’s geminivirus and tobravirus viral
vectors to express a transgenic CRISPR/Cas9 system in dicots. Id.
More particularly, the Examiner finds that a skilled artisan
would have been motivated [to] generate viral vectors for the
purposes of expressing a CRISPR/Cas9 system because Zhang
suggests generating transgenic plants comprising a
CRISPR/Cas9 system. As is illustrated by Zhang, precise
genome editing technologies, like that of CRISPR, enable a
wide range of biological disciplines including: synthetic
biology, biotechnology, and medical biology by allowing the
study of targeted genome perturbations. Zhang would have
motivated the ordinary artisan to apply CRISPR/Cas9 in plants
because Zhang teaches that the binding of a CRISPR complex
to a target sequence in a cell can lead to targeted inactivation of
the sequence. Furthermore, Zhang teaches that the inactivated
target sequence may include a deletion mutation, an insertion
mutation, or a “knockout” of the target sequence. Given the
flexibility demonstrated by the CRISPR/Cas9 system and the
explicit statement of Zhang to adapt their CRISPR/Cas9 system
in transgenic plants, one of ordinary skill in the art would have
been motivated to modify the vectors taught by Zhang and
utilize plant based vectors comprising a synthetic guide RNA
and a Cas9 endonuclease.
One of ordinary skill in the art would have had a
reasonable expectation of success modifying the transformation
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CRISPR/Cas9 vectors of Zhang and employing the plant
transformation vectors of Klimyuk because methods of
expressing RNA, such as the synthetic guide RNA of Zhang,
and . . . methods of expressing heterologous proteins, such as a
Cas9 endonuclase of Zhang, were methods that were well
known to one of ordinary skill in the art at the time of filing.
Furthermore, given that CRISPR/Cas9 system is originally a
prokaryotic system found within the bacterium Streptococcus
pyogenes as illustrated in Figure 1, one of ordinary skill in the
art would have had a reasonable expectation of success in plants
because Zhang demonstrates success in a different eukaryotic
cell. It was well known at the time the invention was made that
prokaryotic cells are structurally distinct from eukaryotic cells,
such as plants and mammals.
Ans. 6–7 (citations omitted).
Appellant contends that a skilled artisan would not have had a
reasonable expectation of success in carrying out the claimed methods based
on the combination of cited references. Appeal Br. 5.
Appellant does not separately argue the claims. We therefore focus
our analysis on claim 1 as representative. The issue with respect to this
rejection is whether a skilled artisan would have had a reasonable
expectation success in modifying the genomic material in a dicotyledonous
plant cell using the CRISPR/Cas9 system.
B. Analysis
Unless otherwise noted, we adopt the Examiner’s findings of fact and
reasoning regarding the rejection of claim 1 as obvious over Zhang and
Klimyuk (Final Act. 3–33; Ans. 3–33). We are not persuaded by
Appellant’s arguments, as explained below. Only those arguments timely
made by Appellant in the briefs have been considered; arguments not so
presented in the briefs are waived. See 37 C.F.R. § 41.37(c)(1)(iv)
(2015); see also Ex parte Borden, 93 USPQ2d 1473, 1474 (BPAI 2010)
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(informative) (“Any bases for asserting error, whether factual or legal, that
are not raised in the principal brief are waived.”).
Appellant contends that “the Examiner’s rationale [for the rejection] is
unsupported, deficient, and based on legal error.” Appeal Br. 12–15
(emphasis omitted); see also Reply Br. 4–6. Appellant first contends that
the Examiner erroneously suggests that “methods for modifying
CRISPR/Cas9 vectors for plants are known in the art.” Appeal Br. 12; see
also Reply Br. at 5 (arguing that “[n]either Zhang nor Klimyuk discloses
using any type of method to modify a CRISPR/Cas9 vector for use in plants,
and the Examiner has not cited any other prior art document disclosing the
modification of a CRISPR/Cas9 vector for use in plants”).
We are not persuaded. Zhang discloses a method of modifying a
target polynucleotide in a eukaryotic cell using a CRISPR/Cas system and
vectors for use in such a system. Zhang 3:44–62, 4:20–21, 7:34–43; ’527
application ¶¶ 4, 11. Zhang further teaches that its vectors may be used to
produce a transgenic plant and that “[m]ethods for producing transgenic
plants . . . are known in the art, and generally begin with a method of cell
transfection, such as described [in Zhang].” Zhang 26:3–5, 26:8–11; ’527
application ¶ 79; see also Zhang 14:6–10; ’527 application ¶ 44 (stating that
“[t]he practice of [Zhang’s] invention employs, unless otherwise indicated,
conventional techniques of immunology, biochemistry, chemistry, molecular
biology, microbiology, cell biology, genomics and recombinant DNA”).
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Klimyuk likewise teaches that methods of plant cell transfections are
known in the art.7 See, e.g., Klimyuk ¶¶ 15–26 (describing a “process of
controlling a biochemical process . . . in a plant” comprising steps of
“infecting the plant with one or more vectors”). Furthermore, although
Appellant disputes that a skilled artisan would reasonably expect
transformation of plant cells with CRISPR/Cas vectors to “induce[] a double
strand break at or near the sequence to which [a] crRNA and tracrRNA
sequence [or the chimeric cr/tracrRNA hybrid] is targeted,” Appellant does
not appear to dispute that, at the time of the claimed invention, a skilled
artisan would have been able to transform plant cells, which are eukaryotic
cells, with the CRISPR/Cas vectors suggested by the combination of Zhang
and Klimyuk. Tr. 3:25–4:19.
Thus, that the Examiner does not cite to specific examples where
CRISPR/Cas9 vectors were used in plants does not suggest that methods of
modifying CRISPR/Cas9 vectors for plants are not known the prior art. In
re Merck & Co., 800 F.2d 1091, 1097 (Fed. Cir. 1986). (“Non-obviousness
cannot be established by attacking references individually where the
rejection is based upon the teachings of a combination of references. . . .
[The reference] must be read, not in isolation, but for what it fairly teaches
in combination with the prior art as a whole.”).

7 We note that claim 1 is directed specifically to a method “wherein the plant
cell is a dicotyledonous plant cell.” Appeal Br. 17 (Claims App.). However,
Appellant has not argued that a skilled artisan would lack reasonable
expectation of success specifically with respect to dicotyledonous plant cells
as compared to other plant cell types. Moreover, as the Examiner points out,
Klimyuk teaches that its methods are applicable to dicotyledonous plants.
Ans. 6 (citing Klimyuk ¶ 285).
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Appellant contends that Zhang contains “extremely limited disclosure
. . . regarding the use of CRISPR/Cas systems in plants” and “nonexistent
disclosure . . . regarding the use of CRISPR/Cas systems in dicotyledonous
plants,” and that “Klimyuk simply discloses methods that involve the
interaction between a heterologous DNA sequence in a transgenic plant and
a heterologous DNA sequence in plant viral transfection vector,” without
ever mentioning CRISPR/Cas. Appeal Br. 6; see also Reply Br. 3–4. In
particular, Appellant contends that the only disclosures in Zhang that relate
to plants and that may be considered prior art to the claims on appeal only
“stat[e] that ‘one or more vectors described herein are used to produce a
non-human transgenic animal or transgenic plant’” and that “methods ‘for
producing transgenic plants and animals are known in the art, and generally
begin with a method of cell transfection, such as described herein.’” Appeal
Br. 6; see also Reply Br. 3 (arguing that there are no “specific details
describing the modification of genomic material in a dicotyledonous plant
cell as claimed”).8

8 Appellant contends that “Zhang issued after Applicant’s priority filing,
while claiming priority from two U.S. provisional applications . . . that were
filed before Applicant’s priority filing. Thus, it is only the material
disclosed in these two U.S. provisional applications that is properly
considered when looking to Zhang.” Appeal Br. 6. We note that it is the
issued patent, not the provisional applications, that is considered prior art.
See 35 U.S.C. § 102(a)(2). We further note, however, that
[f]or purposes of determining whether a patent . . . is prior art to
a claimed invention under [35 U.S.C. § 102(a)(2)], such patent .
. . shall be considered to have been effectively filed, with
respect to any subject matter described in the patent . . . —
. . .
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Appellant contends that,
[g]iven the extremely limited amount of information specific
for the use of CRISPR/Cas in plants provided by the
combination of Zhang and Klimyuk, a person having ordinary
skill in the art at the time of filing and reading the combination
of cited references would not have concluded that the presently
claimed methods could have been carried out with a reasonable
expectation of success.
Appeal Br. 6–7; see also Reply Br. 5.
We are not persuaded for reasons similar to those discussed above.
The presence of a reasonable expectation of success is measured from the
perspective of a person of ordinary skill in the art at the time the invention
was made. Life Techs., Inc. v. Clontech Labs, Inc., 224 F.3d 1320, 1326
(Fed. Cir. 2000). On this record, Zhang discloses that the CRISPR/Cas
system, and the vectors used therein, may be used to modify a target
polynucleotide in a eukaryotic cell. Zhang 3:44–62, 4:20–21, 7:34–43; ’527
application ¶¶ 4, 11. Zhang and Klimyuk further suggest that a skilled
artisan would reasonably expect such vectors may be transfected
successfully into plant cells, which are eukaryotic cells, to create transgenic
plant cells that express the necessary components of the CRISPR/Cas

(2) if the patent . . . is entitled to claim a right of priority under
section 119 . . . based upon 1 or more prior filed applications
for patent, as of the filing date of the earliest such application
that describes the subject matter.
35 U.S.C. § 102(d). Thus, we understand Appellant’s position to be that the
only subject matter in Zhang that is “described” by its two earlier-filed
provisional applications are the materials actually disclosed in those
provisional applications.
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system. Zhang 26:3–5, 26:8–11; ’527 application ¶ 79; Klimyuk Abstract
(describing “plant viral transfection vector”), ¶¶ 15–26.
Appellant contends that the issue is not whether a skilled artisan
would have had a reasonable expectation of success for transfecting plant
cells with vectors or for generating a transgenic plant, but rather
whether there would have been a reasonable expectation of success
for using a CRISPR/Cas system to modify the genomic material in a
dicot plant cell by using Cas9 to induce a double strand break at or
near a sequence to which crRNA and tracrRNA (or a chimeric
cr/tracrRNA hybrid) are targeted, as required by Appellant’s claims.
Reply Br. 3. Appellant contends that “[t]here is no tie between Zhang’s
mention of transgenic plants and Zhang’s disclosure of deletion, insertion,
and nonsense mutations at target sites, because Zhang does not teach or
suggest that a CRISPR/Cas system could be used to induce a double strand
break in a plant.” Id. Appellant contends that none of the possible
CRISPR/Cas targets listed in Zhang are identified plant sequences. Id.
We are not persuaded. As discussed above, Zhang teaches that the
vectors described in its disclosures (i.e., vectors for use in the CRISPR/Cas
system) maybe used to produce a transgenic plant and that methods for
producing such plants are known. Zhang 26:3–5, 26:8–11; ’527 application
¶ 79. Zhang further teaches that the CRISPR system is useful in creating
double-stranded breaks at a target site in a cell. See, e.g., Zhang 4:25–29
(teaching that, “[i]n some embodiments, the CRISPR enzyme directs
cleavage of one or two strands at the location of the target sequence”); ’527
application ¶ 92 (teaching that, “[t]ypically, the CRISPR complex . . . , when
introduced into a cell, creates a break (e.g., a single or double strand break)
in the genome sequence”). More specifically, Zhang teaches that its
invention provides for methods of “modifying a target polynucleotide in a
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eukaryotic cell” (which as discussed above encompasses plant cells),
wherein the method
comprises allowing a CRISPR complex to bind to the target
polynucleotide to effect cleavage of said target polynucleotide
thereby modifying the target polynucleotide, wherein the
CRISPR complex comprises a CRISPR enzyme complexed
with a guide sequence hybridized to a target sequence within
said target polynucleotide, wherein said guide sequence is
linked to a tracr mate sequence which in turn hybridizes to a
tracr sequence.
Zhang 7:34–43; ’527 application ¶ 80. Zhang further teaches that “[t]he
CRISPR complex of [its] invention has a wide variety of utility including
modifying (e.g., deleting, inserting, translocating, inactivating, activating) a
target polynucleotide in a multiplicity of cell types.” Zhang 28:6–9; ’527
application ¶ 91. In light of the above, we agree with the Examiner that
Zhang suggests that a CRISPR/Cas system could be used to induce a double
strand break at or near the target nucleic acid sequence in a plant cell, as
recited in claim 1. As to Appellant’s contention that none of the possible
CRISPR/Cas targets listed in Zhang are identified plant sequences, we note
that “a reference must be considered not only for what it expressly teaches,
but also for what it fairly suggests.” In re Baird, 16 F.3d 380, 383 (Fed. Cir.
1994) (quoting In re Burckel, 592 F.2d 1175, 1179 (CCPA 1979)).
Appellant takes issue with the Examiner’s statements that, “given that
CRISPR/Cas9 is a bacterial system, its ability to retain its endogenous
function in a mammalian cell as taught by Zhang would have led one of
ordinary skill in the art to conclude that CRISPR/Cas9 would similarly be
functional in plants” and that a skilled artisan “would have recognized that
the cellular differences between prokaryotes and eukaryotes are much larger
than the differences between plant cells and animal cells.” Appeal Br. 12,
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14; see also Reply Br. 5. Appellant also contends that “what Zhang expects
is irrelevant to the obviousness inquiry” because “obviousness is assessed
from the perspective of a person having ordinary skill in the art at the time of
filing, not the author of a prior art reference.” Appeal Br. 14; Reply Br. 6.
We are not persuaded. That the CRISPR/Cas9 system functions in
both prokaryote cells and certain eukaryote cells does not necessarily mean
that it will function similarly in plant cells. However, “the expectation of
success need only be reasonable, not absolute,” Pfizer, Inc. v. Apotex, Inc.,
480 F.3d 1348, 1364 (Fed. Cir. 2007), and we agree with the Examiner the
fact that the CRISPR/Cas9 system functions in very different cell types (i.e.,
prokaryote and certain eukaryotic cells) is evidence supportive of a prima
facie case that a skilled artisan would have had reasonable expectation of
success in using CRISPR/Cas9 in plant cells despite their differences with
prokaryotic and mammalian eukaryotic cells.
Likewise, although we agree with Appellant that the likelihood of
success is assessed from the perspective of a person having ordinary skill in
the art at the time of filing, we agree with the Examiner that such a person
would have had a reasonable expectation of success of using the
CRISPR/Cas system in plants upon reading the disclosures in Zhang (i.e.,
that the vectors of the CRISPR/Cas system may be used to modify a target
polynucleotide in a eukaryotic cell through creating double-stranded breaks,
Zhang 3:44–62, 4:20–21, 4:25–29, 7:34–43; ’527 application ¶¶ 4, 11, 80,
92; and that such vectors may be used to produce a transgenic plant
(consisting of eukaryotic cells) using methods known in the art, Zhang 26:3–
5, 26:8–11; ’527 application ¶ 79), notwithstanding that Zhang himself may
be more than an ordinarily skilled artisan.
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Appellant contends that, in fact, a skilled artisan would not have had a
reasonable expectation of success of using the CRISPR/Cas system in plants
at the time of the invention in light of the “meaningful differences between
mammalian and plant cells.” Appeal Br. 7; see also Reply Br. 4. Appellant
contends that these differences include
the presence of a cell wall, a large vacuole, plastids, chloroplast,
plasmodesmata, and glyoxysomes in plant cells, the absence of
centrioles in most plant cells, the significantly larger genome
that typically is present in plant cells, and differences in DNA
methylation patterns, amino acid production and usage,
developmental regulation, mechanisms to maintain genome
integrity, defense systems against foreign nucleic acids, and
optimal growth temperatures between plant and animal cells.
Appeal Br. 7. Appellant further contends that a skilled artisan “would have
been well aware of the attempted implementation of other RNA-based
systems in plants, contributing to a conclusion that Zhang and Klimyuk fail
to provide a reasonable expectation of success for carrying out the claimed
methods.” Id. at 9–10.
We are not persuaded. With the exceptions discussed further below,
Appellant does not provide any specific reasons why the cited differences
between mammalian and plant cells would have lead a skilled artisan to lack
a reasonable expectation of success in using the CRISPR/Cas system in plant
cells. Likewise, Appellant does not point to any particular similarity
between the CRISPR/Cas9 system and “other RNA-based systems” that
have allegedly failed to be implemented in plants, such that a skilled artisan
would have lacked a reasonable expectation of success in implementing
CRISPR/Cas9 system in plants.
Appellant contends more specifically that Zhang describes the use of
CRISPR/Cas9 in mammalian cell lines grown, transfected with CRISPR/Cas
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plasmid DNA, and incubated at 37ºC, whereas “plant cells are grown and
transformed at lower temperatures,” e.g., at room temperature, or 20–25ºC.
Appeal Br. 7–8; see also Voytas Decl.9 ¶ 12. Appellant contends that,
because Streptococcus pyogenes, the facultative anaerobe that is the source
of the Cas9 recited in the claims, is grown at 37ºC, the Cas9 enzyme recited
in the claims “most likely evolved to function optimally at ~37ºC.” Appeal
Br. 7 (citing Gera10 for the teaching that S. pyogenes is grown at 37ºC).
We are not persuaded. “The reasonable expectation of success
requirement refers to the likelihood of success in combining references to
meet the limitations of the claimed invention.” Intelligent Bio-Systems, Inc.
v. Illumina Cambridge Ltd., 821 F.3d 1359, 1367 (Fed. Cir. 2016). Claim 1
does not require the method for modifying the genomic material in a plant
cell to be performed at 20–25ºC. Similarly, although Appellant submits
evidence that mammalian cells are generally cultured at 37ºC whereas plant
cells are commonly grown and transformed at lower temperatures, see, e.g.,
Voytas Decl. ¶ 12, Appellant has not provided persuasive evidence that a
skilled artisan would not have been able to, or would not have reasonably
expected to be able to, grow or transform plant cells at temperatures above
20–25ºC.11

9 Declaration under 37 C.F.R. § 1.132 of Daniel F. Voytas (September 27,
2013).
10 Kanika Gera & Kevin S. McIver, Laboratory Growth and Maintenance of
Streptococcus pyogenes, (The Group A Streptococcus, GAS) (Oct. 2, 2014)
(author’s manuscript, published in final edited form as 30 CURRENT
PROTOCOLS MICROBIOLOGY 9D.2.1 (2013)), attached to the Appeal Brief as
Appendix D of the Evidence Appendix.
11 Appellant cites a number of references for the proposition that
transformation of various types of dicotyledonous cells are “commonly” or
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Moreover, as discussed further below, assuming for the sake of
argument that a skilled artisan would not reasonably expect that plant cells
may be grown and transformed at temperatures higher than 20–25ºC, we
remain unpersuaded that a skilled artisan would thereby lack reasonable
expectation of success with respect to the claimed method.
Appellant cites to, e.g., the Voytas Declaration in support of its
argument that “differences in cellular temperature could affect various
aspects of CRISPR function in plant cells compared to mammalian cells”
and “would have led to uncertainty as to whether CRISPR/Cas systems
would work in plants.” Id. at 7–8. In particular, Appellant argues that (1)
enzyme function is temperature dependent; (2) “temperature affects RNA
folding,” and “RNA – specifically the noncoding guide RNA (gRNA) – is an
important component of the CRISPR system”; and (3) “temperature affects

“generally” performed at between 20ºC and 25ºC. Appeal Br. 7–8 (citing
Steven J. Clough & Andrew F. Bent, Floral Dip: a Simplified Method for
Agrobacterium-mediated Transformation of Arabidopsis thaliana, 16 PLANT
J. 735 (1998), attached to the Appeal Brief as Appendix E of the Evidence
Appendix; Sang-Dong Yoo et al., Arabidopsis Mesophyll Protoplasts: a
Versatile Cell System for Transient Gene Expression Analysis, 2 NATURE
PROTOCOLS 1565 (2007), attached to the Appeal Brief as Appendix F of the
Evidence Appendix; Imogen A. Sparkes et al., Rapid, Transient Expression
of Fluorescent Fusion Proteins in Tobacco Plants and Generation of Stably
Transformed Plants, 1 NATURE PROTOCOLS 2006 (2019), attached to the
Appeal Brief as Appendix G of the Evidence Appendix; and Yong Zhang et
al., Transcription Activator-Like Effector Nucleases Enable Efficient Plant
Genome Engineering, 161 PLANT PHYSIOLOGY 20 (2013), attached to the
Appeal Brief as Appendix H of the Evidence Appendix). However,
Appellant does not argue that these references suggest that a skilled artisan
would not have had a reasonable expectation of success of culturing and/or
transforming dicotyledonous plant cells at higher temperatures.
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RNA:DNA pairing,” and lower temperatures increase “the potential for
CRISPR/Cas to recognize off-target sequences . . . resulting in undesired
double-strand breaks and ultimately increasing toxicity of the CRISPR/[Cas]
system,” which may be further exacerbated by the generally larger size of
the plant genome as compared to mammalian genomes. Appeal Br. 8–9.
We acknowledge that the function of an enzyme may be temperature
dependent. However, we are not persuaded that a skilled artisan would
thereby lack reasonable expectation that the Cas9 endonuclease molecule
would, when introduced into a plant cell at 20–25ºC, “induce[] a double
strand break at or near the [target] sequence,” as recited in claim 1.
Appellant submits evidence that, “as temperature decreases, enzyme
function often decreases due to reduced kinetic energy.”12 See, e.g., Voytas
Decl. ¶ 12; Appeal Br. 8. Appellant also contends that, “[f]or some proteins,
small decreases in temperature can result in significant loss of enzyme
activity” and that “[s]uch temperature sensitivity can be seen . . . with DNA-
modifying enzymes, including restriction endonucleases and DNA ligases.”
Appeal Br. 8 (citing Pohl).13 Claim 1, however, does not recite any

12 Appellant also asserts that an increase in temperature will initially lead to
increased enzyme function until the enzyme begins to denature, resulting in
loss of function. Appeal Br. 8. However, as Appellant argues that plant
cells are typically cultured and transformed at lower temperatures than
mammalian cells, we understand Appellant’s argument regarding lack of
reasonable expectation of success to be focused on the alleged decrease in
enzyme activity corresponding to decreased temperatures.
13 Fritz M. Pohl et al., Temperature Dependence of the Activity of DNA-
Modifying Enzymes: Endonucleases and DNA Ligase, 123 EUR. J.
BIOCHEMISTRY 141 (1982), attached to the Appeal Brief as Exhibit I of the
Evidence Appendix.
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particular level or degree of enzyme function, and Appellant’s evidence does
not suggest a skilled artisan would not reasonably expect Cas9 endonuclease
to function at least to some extent at the temperatures at which plant cells are
typically cultured and transformed.14
In this regard, we note that Appellant has disputed the Examiner’s
finding that “the successful use of zinc finger nucleases (ZFNs) and
transcription activator-like effector nucleases (TALENs) in plant cells after
their successful use in animal cells would have provided a reasonable
expectation for using CRISPR/Cas successfully in plant cells.” Appeal Br.
11. Citing to the Voytas Declaration, Appellant contends that “a person of
ordinary skill in the art at the time of filing would have appreciated the
differences between protein-based genome editing tools [such as ZFNs and
TALENs] and the protein:RNA CRISPR/Cas system.” Id. In particular, Dr.
Voytas states:
The active genome editing molecules for the ZFN and TALEN
technologies are proteins. Neither technology requires proper
RNA:RNA duplexing, proper RNA:protein complexing, or
proper RNA:target DNA binding to function. In contrast,
CRISPR/Cas systems require a Cas endonuclease protein and a
CRISPR RNA component, which guides the Cas endonuclease
to the target DNA sequence. For the system to function, the
CRISPR RNA must survive in the cell without being degraded
or silenced and must effectively complex with the Cas

14 Appellant specifically cites to data relating to PstI, shown in Pohl’s Figure
7D. However, while Figure 7D shows that the activity of PstI is dependent
on temperature, it also shows that PstI in fact cleaved closed circular DNA
(pBR322 DNA) at incubation temperatures of 20–25ºC, albeit not as
efficiently as at incubation temperature of 40ºC, and that the fraction of
closed circular DNA remaining decreased as incubation time increased.
Pohl 148, Fig. 7D.
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endonuclease in order to form an active protein:RNA molecule
that successfully guides the Cas endonuclease to the appropriate
target DNA sequence. Nothing provided in the combinations of
cited references nor from the successes with ZFNs and
TALENs in plants leads a person having ordinary skill in the art
to conclude that it is reasonable to expect CRISPR/Cas success
in plants simply because CRISPR/Cas worked in animal cells.
This is especially true given all the meaningful differences
pointed out above.
Voytas Decl. ¶ 16.
We acknowledge the differences between the ZFN and TALEN
technologies and the CRISPR/Cas system. However, Appellant has
suggested that a skilled artisan would not have had a reasonable expectation
of success of implementing the CRISPR/Cas system in plant cells in view of
Zhang and Klimyuk, because, among other things, the temperature at which
plant cells are cultured may affect enzyme function. That ZFN and TALEN
technologies can be implemented in plant cells is supportive of a finding that
various enzymes are able to function at such temperatures.
Neither are we persuaded by Appellant’s arguments regarding
temperature effects on RNA folding and RNA:DNA pairing. Dr. Voytas
states that “[t]he CRISPR RNA molecules contain several hairpin structures
and form RNA:RNA duplexes” and that “[i]t was not known how a > 10ºC
decrease in temperature as discussed above would impact the CRISPR RNA
structure and its interaction with the Cas endonuclease.” Voytas Decl. ¶ 13.
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Appellant also cites to Behrouzi15 and Li16 as teaching, respectively, that
“noncoding RNAs form unique 3D structures, which perform many
regulatory functions” and that “temperature can affect the structure and
folding of RNA complexes, and that many tertiary interactions are weak
enough to be disrupted using temperatures or solutions that are not too far
from physiological conditions.” Appeal Br. 9.
However, as discussed above, “the expectation of success need only
be reasonable, not absolute.” Pfizer, Inc. v. Apotex, Inc., 480 F.3d 1348,
1364 (Fed. Cir. 2007). Appellant’s proffered evidence, which does not
explain how—or even whether—the particular temperature at which plant
cells are generally cultured would be expected to affect the particular RNA
components of the CRISPR/Cas system and the interaction of such RNA
components with the Cas endonuclease, does not suggest that a skilled
artisan would have lacked a reasonable expectation of success regarding use
of the CRISPR/Cas system in plant cells merely because plant cells are
generally cultivated at room temperature rather than the 37ºC at which
Zhang cultured the mammalian cells used in its examples.17

15 Reza Behrouzi et al., Cooperative Tertiary Interaction Network Guides
RNA Folding, 149 CELL 348 (2012), attached to the Appeal Brief as
Appendix J of the Evidence Appendix.
16 Pan T.X. Li et al., How RNA Unfolds and Refolds, 77 ANN. REV.
BIOCHEMISTRY 77 (2008), attached to the Appeal Brief as Appendix K of the
Evidence Appendix.
17 For example, we note that Dr. Voytas focuses on the hairpin structures and
RNA:RNA duplexes of the CRISPR RNA molecules. Voytas Decl. ¶ 13.
While Li does teach that “[m]any [RNA] tertiary interactions are sufficiently
weak that they can be disrupted using temperatures or solutions not too far
from physiological conditions,” Li also teaches that “[e]xperimental
investigations of RNA secondary structure have been constrained by the
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We further acknowledge that Dr. Voytas and Appellant cite to Evans18
as demonstrating “[t]he importance of . . . temperature differences with
respect to the use of RNA-based systems [(e.g., ribozymes)] in plants” and
as suggesting that “temperature or other cellular differences may have
played a role in the inability of [ribozyme-mediated cleavage] to work in
plants.” Voytas Decl. ¶ 14. Appellant cites to the following portion of
Evans in support of its arguments:
In vitro, the [ribozyme-mediated cleavage] reaction takes place
most efficiently at 37°C and above whereas the optimum
temperature for propagation of protoplasts is 25°C. Whilst we
have shown that cleavage is seen in vitro at 25°C these
conditions are suboptimal. Also, groups who have observed in
vivo cleavage in animal cells have found that a high molar
excess of ribozyme over target mRNA is required. Our
experiments still showed no effect with up to 100 fold excess of
ribozyme over target GUS molecules.
Appeal Br. 10 (quoting Evans 344S).
We are not persuaded, however, that a skilled artisan would have
lacked a reasonable expectation of CRISPR/Cas system functioning in plant
cells based on the disclosures in Evans. As an initial matter, Evans indicates
that ribozyme-mediated cleavage reaction does in fact occur at 25ºC, at least
in vitro, even if Evans acknowledges that 25ºC is not the optimal
temperature for the reaction and that “there has been no evidence of
ribozyme mediated cleavage in vivo as determined by GUS assay and by

stability of the folded state” and that “as few as three base pairs [may be]
sufficient for duplex stability at room temperature.” Li 81, left column; 78,
right column.
18 Gary J. Evans et al., The Effects of Ribozymes on Gene Expression in
Plants, 20 BIOCHEMICAL SOC’Y TRANSACTIONS 344S (1992), attached to the
Appeal Brief as Appendix L of the Evidence Appendix.
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assaying for the presence of cleavage products.” Evans 344S, right column.
As the Examiner points out, Evans appears to suggest that it may be the
inefficient PEG inoculation of protoplasts, resulting in insufficient copy
numbers of ribozyme in vivo, that leads to the lack of in vivo ribozyme-
mediated cleavage reaction. Id.; Ans. 21. Furthermore, the RNA at issue in
Evans functions as an enzyme, and Appellant has not explained why a
skilled artisan would have extrapolated results relating to ribozyme to the
RNA component of the CRISPR/Cas system, which are not the enzymatic
component of the system. Tr. 8:17 (“The CAS enzyme is the enzyme part of
that component.”).19
We are similarly unpersuaded by Appellant’s argument that lower
“[t]emperatures . . . tend to promote annealing [of nucleic acids] at off-target
sites,” which “would be unfavorable for accurate functioning of
CRISPR/Cas systems in plants, especially given the fact that many plant
genomes are larger than mammalian genomes.” Voytas Decl. ¶ 13; see also

19 For similar reasons, we are not persuaded by Appellant’s citation to Rob
de Feyter & Judith Gaudron, Expressing Ribozyme in Plants, in METHODS IN
MOLECULAR BIOLOGY™: RIBOZYME PROTOCOLS 403 (Philip C. Turner ed.,
1997). That is, Appellant is correct that de Feyter states that “there are
relatively few reports of synthetic ribozyme activity in plant cells or whole
plants compared to the numerous publications of activity in animal and
human cells.” Id. at 403 (citation omitted). However, we are not persuaded
that a skilled artisan would have concluded, from this general statement
regarding the “relatively few” reports of synthetic ribozyme activity in
plants as compared to animal and human cells, that the CRISPR/Cas system
would not reasonably be expected to function in plant cells despite the
disclosures in Zhang and Klimyuk. We further note that de Feyter describes
the fewer reports of synthetic ribozyme activity in plants as surprising, given
that “[t]ransformation of many plant species, particularly dicotyledonous
plants, is routine in many laboratories.” Id.
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Appeal Br. 9. In particular, although the claim requires a double strand
break at or near the sequence to which the crRNA and tracrRNA sequence is
targeted, the claim does not require any particular level of performance for
the CRISPR/Cas 9 system with respect to plant cells. Appellant’s evidence
does not persuasively suggest that a skilled artisan would not reasonably
expect the CRISPR/Cas system to function accurately at least in some,
perhaps many, instances at 20–25ºC. In this regard, we repeat that “[t]he
reasonable expectation of success requirement refers to the likelihood of
success in combining references to meet the limitations of the claimed
invention,” Intelligent Bio-Systems, 821 F.3d at 1367 (emphasis added), and
furthermore does not require absolute predictability of success, In re
O’Farrell, 853 F.2d 894, 903–04 (Fed. Cir. 1988).
Finally, Appellant contends that a skilled artisan would not have had a
reasonable expectation of success of using the CRISPR/Cas system in
dicotyledonous plant cells, based on the combination of Zhang and Klimyuk,
because “plants possess unique RNA-silencing mechanisms” that “facilitate
silencing of foreign DNA and RNA (e.g., transgenes and viruses) or
aberrantly produced RNA” and that are not present in mammalian cells.
Appeal Br. 10–11. In particular, Dr. Voytas states in his declaration that
one mechanism of RNA silencing involves amplification of the
foreign/aberrant RNA by RNA-dependent RNA polymerases
(RdRPs), resulting in double-stranded RNA that is cleaved by
dicer-like proteins into short interfering RNA (siRNA)
duplexes. The siRNA duplexes are then incorporated into a
complex that facilitates silencing of additional foreign/aberrant
RNA molecules. It was known at the time [of] the priority filing
that as a result of this mechanism, a single aberrant RNA
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species within a plant cell can generate many dsRNAs, which
can then silence even more target molecules.
Voytas Decl. ¶ 15. Dr. Voytas asserts that, “given the knowledge that the
RNA required for CRISPR/Cas activity could be subjected to RNA-
silencing” in plant cells, “it is unreasonable to expect that a system that
requires a complex of both protein and RNA to cleave nucleic acid would be
successful within plant cells . . . simply because of success within
mammalian cells that completely lack those RNA-silencing defense
mechanisms.” Id.
As an initial matter, and as we understand Appellant to agree,
although the RNA-silencing mechanisms in animals and plants differ, RNA-
silencing mechanisms also exist in animal cells. Baulcombe 357 (stating
that “[a]n ancient origin of these three pathways of RNA silencing is likely
because there are examples of each type in animals, fungi and plants,”
although “all the examples of natural silencing found so far [in mammals]
involve miRNAs”); Tr:12:12–13:3. Thus, Zhang suggests that CRISPR/Cas
system can function in cells despite the existence of at least certain types of
RNA silencing mechanisms. Furthermore, the evidence Appellant cites does
not suggest that all foreign RNAs introduced into plant cells would be
silenced, and, as discussed above, claim 1 does not require any particular
degree of efficiency in the functioning of the CRISPR/Cas system. Finally,
as the Examiner points out, and Appellant has not persuasively disputed,
despite the existence of the RNA silencing mechanism discussed in the
Voytas Declaration and in Baulcombe,20 heterologous expression of RNA in

20 David Baulcombe, RNA Silencing in Plants, 431 NATURE 356 (2004),
attached to the Appeal Brief as Exhibit N of the Evidence Appendix.
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plants is well known in the art. Ans. 23–24. Thus, we are not persuaded
that a skilled artisan would lack reasonable expectation of successfully
implementing CRISPR/Cas system in plants merely because of the
knowledge of the existence of such RNA silencing mechanisms.
In sum, we find that the Examiner has established a prima facie case
that claim 1 is obvious over Zhang and Klimyuk. We acknowledge that
Appellant has provided evidence of various differences between plant and
animal cells. However, we are not persuaded that, in view of the breadth of
the claim and how a skilled artisan would have expected the CRISPR/Cas
system to function in view of Zhang’s disclosures, that these differences,
individually or in combination, would have led a skilled artisan to lack a
reasonable expectation that the CRISPR/Cas system may be implemented in
a dicotyledonous plant cell. Accordingly, for the reasons discussed above,
we affirm the Examiner’s rejection of claim 1 as obvious over Zhang and
Klimyuk. Claims 11, 15, and 22, which are not separately argued, fall with
claim 1.
CONCLUSION
In summary:
Claim(s)
Rejected
35 U.S.C. § Reference(s)/Basis Affirmed Reversed
1, 11, 15, 22 103(a) Zhang, Klimyuk 1, 11, 15,
22

TIME PERIOD FOR RESPONSE
No time period for taking any subsequent action in connection with
this appeal may be extended under 37 C.F.R. § 1.136(a). See 37 C.F.R.
§ 1.136(a)(1)(iv).
AFFIRMED