CRISPR Crops--a talk by Sophien Kamoun at Science Portal BD

CRISPR Crops—
Plant Genome Editing:
Principles and Applications
http://KamounLab.net
@KamounLab

Ü Genome sequencing of mutagenized plants
Ü TILLING — targeted sequencing of mutant plants
Ü T-DNA or transposon mutagenesis
Ü Gene knock-down using RNAi (VIGS, artificial
miRNA, hairpin constructs)
Ü Site-specific nucleases (e.g. ZFNs and TALENs) for
gene editing
Reverse genetics in plants circa ~2012

Sequence-specific nucleases (SSNs)
• Zinc-Finger Nucleases
• TAL-Effector Nucleases
• CRISPR/Cas9
PY56CH22_Kamoun ARI 23 June 2018 11:9
a ZFN
b TALEN
c CRISPR-Cas9
N
N
Spacer
(5–7 bp)
Left TALE
Spacer
(12–21 bp)
N
N
Right TALE
sgRNA
Cas
Left ZFP
Right ZFP
FokI (+)
FokI (+)
FokI (–)
FokI (–)
Figure 2
Schematic representation of three classes of programmable nucleases. (a) Illustration of zinc-ﬁnger nuclease
(ZFN) pairs bound to DNA. Each ZFN is composed of a zinc-ﬁnger protein (ZFP) DNA-binding domain at
the N terminus and the FokI nuclease domain at the C terminus. The linker between the DNA-binding and
catalytic domains is represented by a blue line. Target sequences of ZFN pairs are typically 18–36 bp in
length, excluding spacers. (b) Representation of a transcription-activator-like effector nuclease (TALEN)
thol.2018.56.Downloadedfromwww.annualreviews.org
tralianNationalUniversityon07/06/18.Forpersonaluseonly.
Langner et al. 2018

Cut
Cut
www.sciencemag.org SCIENCE VOL 341 23 AUGUST 2013 833
BACTERIA MAY NOT ELICIT MUCH SYMPA-
thy from us eukaryotes, but they, too, can get
sick. That’s potentially a big problem for the
dairy industry, which often depends on bac-
teria such as Streptococcus thermophilus to
make yogurts and cheeses. S. thermophilus
breaks down the milk sugar lactose into tangy
lactic acid. But certain viruses—bacterio-
phages, or simply phages—can debilitate the
bacterium, wreaking havoc on the quality or
quantity of the food it helps produce.
In 2007, scientists from Danisco, a
Copenhagen-based food ingredient com-
pany now owned by DuPont, found a way to
boost the phage defenses of this workhouse
microbe. They exposed the bacterium to
a phage and showed that this essentially
vaccinated it against that virus (Science,
23 March 2007, p. 1650). The trick has
enabled DuPont to create heartier bacterial
strains for food production. It also revealed
something fundamental: Bacteria have a
kind of adaptive immune system, which
enables them to fight off repeated attacks
by specific phages.
That immune system has suddenly
become important for more than food scien-
tists and microbiologists, because of a valu-
able feature: It takes aim at specific DNA
sequences. In January, four research teams
reported harnessing the system, called
CRISPR for peculiar features in the DNA of
bacteria that deploy it, to target the destruc-
tion of specific genes in human cells. And in
the following 8 months, various groups have
used it to delete, add, activate, or suppress tar-
geted genes in human cells, mice, rats, zebra-
fish, bacteria, fruit flies, yeast, nematodes,
and crops, demonstrating broad utility for the
TheCRISPRCrazeA bacterial immune system yields a potentially
revolutionary genome-editing technique
CREDIT:EYEOFSCIENCE/SCIENCESOURCE
PublishedbyAAAS
Downloadedfr
Cas9 and sgRNA are the two
components of the CRISPR system
35S Cas9 NosT

Report
Bacterial and archaeal CRISPR systems rely on crRNAs in complex
with Cas proteins to direct degradation of complementary sequences
present within invading viral and plasmid DNA (1–3). A recent in vitro
reconstitution of the S. pyogenes type II CRISPR system demonstrated
that crRNA fused to a normally trans-encoded tracrRNA is sufficient to
direct Cas9 protein to sequence-specifically cleave target DNA sequenc-
es matching the crRNA (4). The fully defined nature of this 2-
component system suggested that it might function in the cells of eukar-
yotic organisms such as yeast, plants, and even mammals. By cleaving
genomic sequences targeted by RNA sequences (4–6), such a system
could greatly enhance the ease of genome engineering.
Here we engineer the protein and RNA components of this bacterial
type II CRISPR system in human cells. We began by synthesizing a
human codon-optimized version of the Cas9 protein bearing a C termi-
nus SV40 nuclear localization signal and cloning it into a mammalian
fluorescent. Homologous recombina-
tion (HR) using an appropriate repair
donor can restore the normal GFP se-
quence, enabling us to quantify the
resulting GFP+
cells by flow activated
cell sorting (FACS).
To test the efficiency of our system
at stimulating HR, we constructed two
gRNAs, T1 and T2, that target the
intervening AAVS1 fragment (Fig. 1B)
and compared their activity to that of a
previously described TAL effector
nuclease heterodimer (TALEN) target-
ing the same region (11). We observed
successful HR events using all three
targeting reagents, with gene correc-
tion rates using the T1 and T2 gRNAs
approaching 3% and 8% respectively
(Fig. 1C). This RNA-mediated editing
process was notably rapid, with the
first detectable GFP+
cells appearing
~20 hours post transfection compared
to ~40 hours for the AAVS1 TALENs.
We observed HR only upon simultane-
ous introduction of the repair donor,
Cas9 protein, and gRNA, confirming
that all components are required for
genome editing (fig. S2). While we
noted no apparent toxicity associated
with Cas9/crRNA expression, work
with ZFNs and TALENs has shown
that nicking only one strand further reduces toxicity. Accordingly, we
also tested a Cas9D10A mutant that known to function as a nickase in
vitro, which yielded similar HR but lower non-homologous end joining
(NHEJ) rates (fig. S3) (4, 5). Consistent with (4) where a related Cas9
protein is shown to cut both strands 6 bp upstream of the PAM, our
end of the target sequence (fig. S3B). We also confirmed that mutating
the target genomic site prevents the gRNA from effecting HR at that
locus, demonstrating that CRISPR-mediated genome editing is sequence
specific (fig. S4). Finally, we showed that two gRNAs targeting sites in
the GFP gene, and also three additional gRNAs targeting fragments from
homologous regions of the DNA methyl transferase 3a (DNMT3a) and
DNMT3b genes could sequence specifically induce significant HR in the
engineered reporter cell lines (figs. S5 and S6). Together these results
RNA-Guided Human Genome
Engineering via Cas9
Prashant Mali,1,5
Luhan Yang,1,3,5
Kevin M. Esvelt,2
John Aach,1
Marc Guell,1
James E. DiCarlo,4
Julie E. Norville,1
George M. Church1,2
*
1
Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. 2
Wyss Institute for Biologically
Inspired Engineering, Harvard University, Cambridge, MA 02138, USA. 3
Biological and Biomedical
Sciences Program, Harvard Medical School, Boston, MA 02115, USA.. 4
Department of Biomedical
Engineering, Boston University, Boston, MA 02215, USA.
5
These authors contributed equally to this work.
*To whom correspondence should be addressed. E-mail: gchurch@genetics.med.harvard.edu
Bacteria and archaea have evolved adaptive immune defenses termed clustered
regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas)
systems that use short RNA to direct degradation of foreign nucleic acids. Here, we
engineer the type II bacterial CRISPR system to function with custom guide RNA
(gRNA) in human cells. For the endogenous AAVS1 locus, we obtained targeting
rates of 10 to 25% in 293T cells, 13 to 8% in K562 cells, and 2 to 4% in induced
pluripotent stem cells. We show this process relies on CRISPR components, is
sequence-specific, and upon simultaneous introduction of multiple gRNAs, can
effect multiplex editing of target loci. We also compute a genome-wide resource of
~190k unique gRNAs targeting ~40.5% of human exons. Our results establish an
RNA-guided editing tool for facile, robust, and multiplexable human genome
engineering.
onJanuary4,2013www.sciencemag.orgDownloadedfrom
Report
Bacterial and archaeal CRISPR systems rely on crRNAs in complex
with Cas proteins to direct degradation of complementary sequences
present within invading viral and plasmid DNA (1–3). A recent in vitro
reconstitution of the S. pyogenes type II CRISPR system demonstrated
that crRNA fused to a normally trans-encoded tracrRNA is sufficient to
direct Cas9 protein to sequence-specifically cleave target DNA sequenc-
es matching the crRNA (4). The fully defined nature of this 2-
component system suggested that it might function in the cells of eukar-
yotic organisms such as yeast, plants, and even mammals. By cleaving
genomic sequences targeted by RNA sequences (4–6), such a system
could greatly enhance the ease of genome engineering.
Here we engineer the protein and RNA components of this bacterial
type II CRISPR system in human cells. We began by synthesizing a
human codon-optimized version of the Cas9 protein bearing a C termi-
fluorescent. Homologous recombina-
tion (HR) using an appropriate repair
donor can restore the normal GFP se-
quence, enabling us to quantify the
resulting GFP+
cells by flow activated
cell sorting (FACS).
To test the efficiency of our system
at stimulating HR, we constructed two
gRNAs, T1 and T2, that target the
intervening AAVS1 fragment (Fig. 1B)
and compared their activity to that of a
previously described TAL effector
nuclease heterodimer (TALEN) target-
ing the same region (11). We observed
successful HR events using all three
targeting reagents, with gene correc-
tion rates using the T1 and T2 gRNAs
approaching 3% and 8% respectively
(Fig. 1C). This RNA-mediated editing
process was notably rapid, with the
first detectable GFP+
cells appearing
~20 hours post transfection compared
to ~40 hours for the AAVS1 TALENs.
We observed HR only upon simultane-
ous introduction of the repair donor,
Cas9 protein, and gRNA, confirming
that all components are required for
genome editing (fig. S2). While we
noted no apparent toxicity associated
with Cas9/crRNA expression, work
with ZFNs and TALENs has shown
that nicking only one strand further reduces toxicity. Accordingly, we
also tested a Cas9D10A mutant that known to function as a nickase in
vitro, which yielded similar HR but lower non-homologous end joining
(NHEJ) rates (fig. S3) (4, 5). Consistent with (4) where a related Cas9
protein is shown to cut both strands 6 bp upstream of the PAM, our
end of the target sequence (fig. S3B). We also confirmed that mutating
the target genomic site prevents the gRNA from effecting HR at that
locus, demonstrating that CRISPR-mediated genome editing is sequence
specific (fig. S4). Finally, we showed that two gRNAs targeting sites in
the GFP gene, and also three additional gRNAs targeting fragments from
homologous regions of the DNA methyl transferase 3a (DNMT3a) and
DNMT3b genes could sequence specifically induce significant HR in the
RNA-Guided Human Genome
Engineering via Cas9
Prashant Mali,1,5
Luhan Yang,1,3,5
Kevin M. Esvelt,2
John Aach,1
Marc Guell,1
James E. DiCarlo,4
Julie E. Norville,1
George M. Church1,2
*
1
Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. 2
Wyss Institute for Biologically
Inspired Engineering, Harvard University, Cambridge, MA 02138, USA. 3
Biological and Biomedical
Sciences Program, Harvard Medical School, Boston, MA 02115, USA.. 4
Department of Biomedical
Engineering, Boston University, Boston, MA 02215, USA.
5
These authors contributed equally to this work.
*To whom correspondence should be addressed. E-mail: gchurch@genetics.med.harvard.edu
Bacteria and archaea have evolved adaptive immune defenses termed clustered
regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas)
systems that use short RNA to direct degradation of foreign nucleic acids. Here, we
engineer the type II bacterial CRISPR system to function with custom guide RNA
(gRNA) in human cells. For the endogenous AAVS1 locus, we obtained targeting
rates of 10 to 25% in 293T cells, 13 to 8% in K562 cells, and 2 to 4% in induced
pluripotent stem cells. We show this process relies on CRISPR components, is
sequence-specific, and upon simultaneous introduction of multiple gRNAs, can
effect multiplex editing of target loci. We also compute a genome-wide resource of
~190k unique gRNAs targeting ~40.5% of human exons. Our results establish an
RNA-guided editing tool for facile, robust, and multiplexable human genome
engineering.
/ http://www.sciencemag.org/content/early/recent / 03 January 2013; / Page 1/ 10.1126/science
cell line bearing a genomically integrated GFP coding sequence disrupt-
ed by the insertion of a stop codon and a 68bp genomic fragment from
he AAVS1 locus that renders the expressed protein fragment non-
site positions, further validating the sequence specificity
process (figs. S7 to S9). Interestingly, simultaneous intr
T1 and T2 gRNAs resulted in high efficiency deletion o
onJanuary4,2013g.org
Research
non-cod
CRISPR
nucleotid
under th
promote
the U6
sion of a
a single
(Fig. 1B
spacer to
(protosp
locus tha
site prot
(Fig. 1C
To t
pression
(SpCas9
pre-crRN
cleavage
we trans
ent com
nents. Si
are part
forming
(NHEJ)
SURVE
endogen
and fig.
four re
resulted
protospa
which
Sanger s
ingly, S
for clea
1D), and
cessed in
Le Cong,1,2
* F. Ann Ran,1,4
* David Cox,1,3
Shuailiang Lin,1,5
Robert Barretto,6
Naomi Habib,1
Patrick D. Hsu,1,4
Xuebing Wu,7
Wenyan Jiang,8
Luciano
Marraffini,8
Feng Zhang1
†
1
Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02142, USA and McGovern
Institute for Brain Research, Department of Brain and Cognitive Sciences, Department of Biological
Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
2
Program in Biological
and Biomedical Sciences, Harvard Medical School, Boston, MA 02115, USA.
3
Harvard-MIT Health
Sciences and Technology, Harvard Medical School, Boston, MA 02115, USA.
4
Department of Molecular
and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
5
School of Life Sciences, Tsinghua
University, Beijing, 100084, China.
6
Department of Biochemistry and Molecular Biophysics, College of
Physicians and Surgeons, Columbia University, New York, NY 10032, USA.
7
Computational and Systems
Biology Graduate Program and Koch Institute for Integrative Cancer Research, Massachusetts Institute of
Technology, Cambridge, MA 02139, USA.
8
Laboratory of Bacteriology, The Rockefeller University, 1230
York Avenue, New York, NY 10065, USA.
*These authors contributed equally to this work.
†
To whom correspondence should be addressed: zhang@broadinstitute.org
Functional elucidation of causal genetic variants and elements requires precise
genome editing technologies. The type II prokaryotic CRISPR (clustered regularly
interspaced short palindromic repeats) adaptive immune system has been shown to
facilitate RNA-guided site-specific DNA cleavage. We engineered two different type
II CRISPR systems and demonstrate that Cas9 nucleases can be directed by short
RNAs to induce precise cleavage at endogenous genomic loci in human and mouse
cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-
directed repair with minimal mutagenic activity. Finally, multiple guide sequences
can be encoded into a single CRISPR array to enable simultaneous editing of
several sites within the mammalian genome, demonstrating easy programmability
and wide applicability of the CRISPR technology.
Fig. 1. The Type II CRISPR locus from Streptococcus pyogenes SF370 can be reconstituted in mammalian cells to
facilitate targeted DSBs of DNA. (A) Engineering of SpCas9 and SpRNase III with NLSs enables import into the
mammalian nucleus. (B) Mammalian expression of SpCas9 and SpRNase III are driven by the EF1a promoter,
whereas tracrRNA and pre-crRNA array (DR-Spacer-DR) are driven by the U6 promoter. A protospacer (blue
highlight) from the human EMX1 locus with PAM is used as template for the spacer in the pre-crRNA array. (C)
Schematic representation of base pairing between target locus and EMX1-targeting crRNA. Red arrow indicates
putative cleavage site. (D) SURVEYOR assay for SpCas9-mediated indels. (E) An example chromatogram showing a

E/SCIENCESOURCE
Fighting invasion. When
viruses (green) attack
bacteria, the bacteria
respond with DNA-targeting
defenses that biologists
have learned to exploit
for genetic engineering.
onAugust23,2013www.sciencemag.orgDownloadedfrom
www.sciencemag.org SCIENCE VOL 341 23 AUGUST 2013 833
lactic acid. But certain viruses—bacterio-
phages, or simply phages—can debilitate the
bacterium, wreaking havoc on the quality or
quantity of the food it helps produce.
In 2007, scientists from Danisco, a
Copenhagen-based food ingredient com-
strains for food production. It also revealed
something fundamental: Bacteria have a
kind of adaptive immune system, which
enables them to fight off repeated attacks
by specific phages.
That immune system has suddenly
tion of specific genes in human cells. And in
the following 8 months, various groups have
used it to delete, add, activate, or suppress tar-
geted genes in human cells, mice, rats, zebra-
fish, bacteria, fruit flies, yeast, nematodes,
and crops, demonstrating broad utility for the
CREDIT:EYEOFSCIENCE/SCIENCESOURCE
PublishedbyAAAS
infects plant scientists

NbPDS
1 2Target: Primer F
70 bp
Primer R1Sac I
3
Primer R
400 bp
sgRNA_PDS2
Cas9
+
+sgRNA_PDS1
+
-
+
+
1 2
sgRNA_PDS3 - -
sgRNA_GFP - +
-
-
+
3
-
-
-
+
+
-
+
+
4 5
+ -
- +
-
-
+
6
-
-
650 -
500 -
400 -
850 -
1000 -
1650 -
Sac IGenomic DNA:
RBGGGALB
Cas9sgRNA2 L3E
ACTA
sgRNA1
TTACGCAATGCC
Small deletions with adjacent sgRNAs…
Belhaj et al. Plant Methods 2013, 9:39

Targets
Solyc04g007030 Solyc04g007050 Solyc04g007060 Solyc04g007070 Solyc04g007080
NRC4a NRC4b NRC4cNRC4-like
Solyc04g007080
CRISPR targets
labeled with
A 53 kb deletion of a four gene cluster in tomato using CRISPR/Cas
NRC4F
NRC4R
53 Kb
Roger Castells-Graells
Vladimir Nekrasov

1. Bioinformatics — design sgRNAs and cloning
strategy
2. Molecular biology — cloning of transformation
vectors (Cas9 + sgRNAs)
3. Plant tissue culture and transformation
4. Genotyping — Genotype mutants in T0 and
subsequent generations
How to CRISPR genes in plants?

LETTERS
https://doi.org/10.1038/s41587-020-0703-0
The potential of genome editing to improve the agronomic
performance of crops is often limited by low plant regenera-
tion efficiencies and few transformable genotypes. Here, we
show that expression of a fusion protein combining wheat
GROWTH-REGULATING FACTOR 4 (GRF4) and its cofactor
GRF-INTERACTING FACTOR 1 (GIF1) substantially increases
the efficiency and speed of regeneration in wheat, triticale
and rice and increases the number of transformable wheat
genotypes. GRF4–GIF1 transgenic plants were fertile and with-
out obvious developmental defects. Moreover, GRF4–GIF1
induced efficient wheat regeneration in the absence of exog-
enous cytokinins, which facilitates selection of transgenic
plants without selectable markers. We also combined GRF4–
GIF1 with CRISPR–Cas9 genome editing and generated 30
edited wheat plants with disruptions in the gene Q (AP2L-A5).
Finally, we show that a dicot GRF–GIF chimera improves regen-
eration efficiency in citrus, suggesting that this strategy can
be applied to dicot crops.
Recent studies have reported improvements in the efficiency of
plant regeneration from tissue culture by overexpression of plant
developmental regulators, including LEAFY COTYLEDON1 (refs. 1,2
),
LEAFY COTYLEDON2 (ref. 3
), WUSCHEL (WUS)4
and BABY
BOOM (BBM)5
. These genes promote the generation of somatic
embryos or the regeneration of shoots. For example, overexpression
of the maize developmental regulators BBM and WUS2 produces
high transformation frequencies in previously non-transformable
maize inbred lines and other monocot species6–8
. Another strategy
uses different combinations of developmental regulators to induce
de novo meristems in dicotyledonous species without tissue cul-
ture9
. However, there remains a need for new methods that provide
efficient transformation, increased ease of use and suitability for a
broader range of recalcitrant species and genotypes.
or in plants overexpressing miR396 (refs. 11–13,18,19
), while overex-
pression of GIF genes promotes organ growth and can boost the
activity of GRFs12,13,15,20–22
. Furthermore, simultaneous increases in
the expression of Arabidopsis GRF3 and GIF1 promote the devel-
opment of larger leaf sizes than are observed when the expression
of these genes is increased individually15
. Based on the observation
that GRFs and GIFs interact to form a protein complex15
, we evalu-
ated the effect of a GRF–GIF chimeric protein. Here we show that
expression of a sequence encoding a chimera composed of a GRF
transcription factor and its GIF cofactor substantially increases
regeneration efficiency in both monocotyledonous and dicotyle-
donous species, increases the number of transformable cultivars
and results in fertile transgenic plants.
We began by identifying ten GRF genes in the wheat genome
(Supplementary Fig. 1a) and selected GRF4 based on its homology
to OsGRF4, a rice gene that promotes grain and plant growth in rice
and wheat23–27
. Among the three wheat GIF cofactors, we selected
the closest homolog of Arabidopsis and rice GIF1 (Supplementary
Fig. 1b), because members of this clade have been shown to con-
trol growth in Arabidopsis, rice and maize12,13,21,22
. We then com-
bined GIF1 and GRF4 to generate a GRF4–GIF1 chimera including
a short intergenic spacer (Fig. 1a) using the primers described
in Supplementary Table 1 (Methods). Transgenic plants overex-
pressing the GRF4–GIF1 chimera under the control of the maize
UBIQUITIN promoter (Ubi::GRF4–GIF1; Methods) were fertile
and showed normal phenotypes (Fig. 1b). However, they exhibited
a 23.9% reduction in the number of grains per spike and a 13.7%
increase in grain weight (Supplementary Table 2).
We performed 18 transformation experiments in the tetraploid
wheat Kronos (Methods) and estimated regeneration frequencies as
the number of calli showing at least one regenerating shoot per the
total number of inoculated embryos (Supplementary Table 3 sum-
A GRF–GIF chimeric protein improves the
regeneration efficiency of transgenic plants
Juan M. Debernardi1,2
, David M. Tricoli3
, Maria F. Ercoli 4,5
, Sadiye Hayta6
, Pamela Ronald 4,5
,
Javier F. Palatnik 7,8
and Jorge Dubcovsky 1,2 ✉
LETTERS NATUREBIOTECHNOLOGY
a
c
b
Q (AP2L-A5) = free threshing and floret development
GRF4 GIF1ZmUbi
GRF4–GIF1
CRISPR–Cas9
on same T-DNA
Removed
together after
editing
Control
GRF4–GIF1/
CRISPR–Cas9–gRNA–Q
d
e
gRNA
Gene
Target
Cas9
PAM LB Hyg. GRF4–GIF1 Cas9 gRNA RB
StyI
PAM
WT
a1
a1
a2
a1
a2
a1
a2
a1
a1
a2
a1
a2
a1
a1
a2
a1
a2
T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
1 cm

Ü Improved resistance to pathogens and pests
Ü Resistance to drought and abiotic stress
Ü Improved yield
Ü Increased nutritional value
Ü Removal of allergens and toxins
Ü de-novo domestication
Applications of CRISPR to agriculture

PP70CH28_Gao ARjats.cls February 26, 2019 14:44
Cross breeding (8–10 years) Mutation breeding (8–10 years)
Transgene breeding (8–12 years) Genome editing (4–6 years)
5–7 cycles of backcross
Calli
Chemicals/
radiation
Foreign gene integration Endogenous gene modification
Elite variety
(disease susceptible)
Selection and backcross
Mutants
Elite variety
with disease resistance
Regenerated plants
Elite variety
Elite variety
Elite variety
Plasmid
Genome CRISPR
3%
6%
50%
12%
25%
×
Donor variety
(disease resistant)
Calli
Figure 1
Comparison of breeding methods used in modern agriculture. Cross breeding: improving a trait (e.g., disease resistance) through
Annual Review of Plant Biology
CRISPR/Cas Genome Editing
and Precision Plant Breeding
in Agriculture
Kunling Chen,1,∗
Yanpeng Wang,1,∗
Rui Zhang,1
Huawei Zhang,1
and Caixia Gao1,2
1
State Key Laboratory of Plant Cell and Chromosome Engineering, Center for Genome
Editing, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences,
Beijing, China 100101; email: cxgao@genetics.ac.cn
2
University of Chinese Academy of Sciences, Beijing, China 100864
28.31
online at
ant-050718-
s.
o this article
Keywords
genome editing, CRISPR/Cas, precision plant breeding, trait improvement
Abstract
Enhanced agricultural production through innovative breeding technology
is urgently needed to increase access to nutritious foods worldwide. Recent
advances in CRISPR/Cas genome editing enable efficient targeted modifi-
cation in most crops, thus promising to accelerate crop improvement. Here,
we review advances in CRISPR/Cas9 and its variants and examine their
applications in plant genome editing and related manipulations. We high-
light base-editing tools that enable targeted nucleotide substitutions and
describe the various delivery systems, particularly DNA-free methods, that
have linked genome editing with crop breeding. We summarize the applica-
tions of genome editing for trait improvement, development of fine-tuning
gene regulation, strategies for breeding virus resistance, and the use of high-
throughput mutant libraries. We outline future perspectives for genome
editing in plant synthetic biology and domestication, advances in delivery
systems, editing specificity, homology-directed repair, and gene drives. Fi-
nally, we discuss the challenges and opportunities for precision plant breed-
ing and its bright future in agriculture.

Applications of CRISPR to plant health
Ü S genes – disease resistance through loss-of-
function
Ü Allele replacement – new disease resistance alleles
Ü Knock-ins – promoter fusions, domain engineering…
Ü Basic research – genetic analysis of previously
intractable crops and pathogens

Annual Review of Phytopathology
CRISPR Crops: Plant Genome
Editing Toward Disease
Resistance
Thorsten Langner, Sophien Kamoun,
and Khaoula Belhaj
The Sainsbury Laboratory, Norwich Research Park, Norwich, NR4 7UH, United Kingdom;
email: sophien.kamoun@tsl.ac.uk
2.1–22.34
is online at
yto-080417-
ws.
Keywords
genome editing, disease resistance, sequence-specific nucleases, TALENs,
CRISPR-Cas9, susceptibility genes
Abstract
Genome editing by sequence-specific nucleases (SSNs) has revolutionized
biology by enabling targeted modifications of genomes. Although routine
plant genome editing emerged only a few years ago, we are already witnessing
the first applications to improve disease resistance. In particular, CRISPR-
Cas9 has democratized the use of genome editing in plants thanks to the ease
and robustness of this method. Here, we review the recent developments in
plant genome editing and its application to enhancing disease resistance
against plant pathogens. In the future, bioedited disease resistant crops will
become a standard tool in plant breeding.
22.1
PY56CH22_Kamoun ARI 23 June 2018 11:9
PTI
Negative regulator
of immunity
Plant cell
Cytoplasmic
effectors
Pattern
recognition
receptors
Apoplastic
effectors/PAMPs
Pathogen
ImmunitySusceptibility
Effector
targets
Susceptibility S genes!

Pipeline to
target
conserved
S genes
a putative function based on evolutionary origin and published information. MLO members of
Clade I
Clade II
Clade III
Clade IV
Clade V
Clade VI
Clade VII
Powdery
mildew
interactions
Green algae
>490 Mya
Apoplast
Cytoplasm
Calmodulin
binding
COOH
NH2
MLO
Plasma
membrane
Plasma
membrane
a b
c d48-bp deletion
SlMLO1
...NNNNGGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGGNNNN...
PAM
sgRNA1
PAM
sgRNA2
e
WT
2 4 6
kb
8 10 12
slmlo1 8-2
slmlo1 8-4
slmlo 8-6
(T-DNA)
T-DNA
LB RB
g
ACATAGTAAAAGGTGTACCTGTGGTGGAGACTGGTGACCATCTTTTCTGGTTTAATCGCCCTGCCCTTGTCCTATTCTTGATTAACTTTGTACTCTTTCAGG
ACATAGTAAAAGGTGTACCTGTGGTGGAGACTGGTGACCATCTTTTCTGGTTTAATCGCCCTGCCCTTGTCCTATTCTTGATTAACTTTGTACTCTTTCAGG
ACATAGTAAAAGGTGTACCTGTGGTGGA------------------------------------------------CTTGATTAACTTTGTACTCTTTCAGG -48
ACATAGTAAAAGGTGTACCTGTGGTGGA-------------------------------------------------TTGATTAACTTTGTACTCTTTCAGG -49
WT
Plant 1
*Plant 2
*Plant 8
*Plant 10
PAM PAMTarget 1 Target 2
f
500
400
300
bp
1 2 8 10WT
SlMLO1 (WT)
slmlo1
Annu.Rev.Phytopathol.2018.56.Downloadedfromwww.annualreviews.org
AccessprovidedbyAustralianNationalUniversityon07/06/18.Forpersonaluseonly.
Langner et al. 2018

11-9 11-20 11-24
1-1 1-14 1-24
mlo1
MLO1
Tomelo – resistant to powdery mildew fungus

Blast resistant wheat varieties for
Bangladesh by genome editing
Ü ~12 S genes are known in rice to mediate
susceptibility to rice blast fungus
Ü Identify orthologs in wheat
Ü CRISPR genes in wheat
Ü Create mutant populations
Ü Wheat blast infection assays
with Emma Wallington, Toffazal Islam and Nick Talbot
Diana Gomez & Thorsten Langner

CCN CCN
gRNA1 gRNA2
ATG TAA
sHMA
PAM gRNA2
TACTACTACCCGCACAGCTACTACCACCATCAGTACCAGCCGCCGCACATG
TACTACTACCCGCACAAGCTACTACCACCATCAGTACCAGCCGCCGCACATG +1
TACTACTACCCGCAC-------ACCACCATCAGTACCAGCCGCCGCACATG -7
WT
T1-B
WT
T2-B.1
T2-B.6 T2-B.12
Infections in T2 plants
Parental genotype
Proof-Of-Concept—CRISPR KO of sHMA
gene (effector target) in barley

Strategy for CRISPR-ing sHMA1 and sHMA7
homeoalleles in wheat

~120 T0-plants for genotyping and propagation from 3 independent transformations
Genotyping by multiplex amplicon sequencing

PAMsgRNA PAM sgRNA
Coverage
Examples of edited copies of sHMAs in wheat

PAMsgRNA PAM sgRNA
Coverage
Lack of reads mapping to
this region might indicate
larger chromosomal
deletions
Examples of edited copies of sHMAs in wheat

WT WT
Preliminary phenotype screens of T1 plants
Infections of plants with edits - Bangladeshi isolates

Acknowledgements
Khaoula Belhaj Angela Chaparro Garcia Joe Win Vladimir Nekrasov
Vladimir Nekrasov
Diana Gomez Adeline Harant Thorsten Langner

CRISPR Crops--a talk by Sophien Kamoun at Science Portal BD

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