This document discusses the use of CRISPR-Cas9 genome editing in crop improvement. It begins with an introduction to CRISPR-Cas9 and its mechanism of action. It then discusses the discovery of CRISPR and key scientists involved. Several case studies on using CRISPR to edit rice genes for disease resistance and hybrid seed production are summarized. Achievements using CRISPR in rice, horticulture crops, and other field crops are briefly outlined. The document concludes that CRISPR provides a simple and efficient tool for genome editing in plants.

1. CRISPR
[Clustered Regularly Interspaced Short
Palindromic Repeats]
in Crop Improvement
PARTHASARATHI.G
2018-630-812
II M.Sc(Ag)
Dept of PBG

2. Contents
Introduction
Mechanism of CRISPR/Cas9 complex
Discovery of CRISPR
Case Studies
Achievements in CRISPR
Conclusion

3. Introduction
• CRISPR-Cas9 is used to edit parts of
the genome by removing, adding or altering
sections of the DNA sequence, based on
bacterial adaptive immune system .
• Key Elements
i) CRISPR sequence
[clustered regularly interspaced short
palindromic repeats]
ii) Cas9 (nucleases)

4. Mechanism of CRISPR/Cas9 complex
• Creating Nuclease induced DSBs (Double stranded
breaks) .
• Repairing DSBs in either one of the two pathways.
»NHEJ
[Non-homologous End Joining]
»HDR repair
[Homology Directed Repair]
• It is unique and flexible owing to its dependence
on RNA as the moiety that targets the nuclease to
a desired DNA sequence.

5. Components
• crRNAs - each harboring a variable sequence
transcribed from the invading DNA, known as the
“protospacer” sequence, and part of the CRISPR
repeat.
• Each crRNA hybridizes with a second RNA, known as
the transactivating CRISPR RNA (tracrRNA), and
these two RNAs complex with the Cas9 nuclease.
• The protospacer-encoded portion of the crRNA
directs Cas9 to cleave complementary target-DNA
sequences, if they are adjacent to short sequences
known as protospacer adjacent motifs (PAMs).

7. Cas9 variants

8. Targeting range and choice of
gRNAs
 RNA polymerase III–
dependent U6 promoter or
the T7 promoter require a G
or GG, respectively, at the 5′
end of the sequence of the
RNA that is to be
transcribed.
 Standard full-length or tru-
gRNAs expressed from these
promoters are limited to
targeting sites that match
the forms GN16-19NGG or
GGN15-18NGG; such sites are
expected to occur every 1 in
32 bp or 1 in 128 bp,
respectively, in random DNA
sequence.

9. Neccessity
Nuclease
platforms
ZFN TALEN CRISPR/Cas9
Source
Bacteria,
Eukaryotes
Eukaryotes
Bacteria
(Streptococcus sp.)
DNA binding
determinant
Zinc finger protein
Transcription-
activator-like
effector
crRNA/sgRNA
Binding specificity 3 Nucleotides 1 Nucleotide
1:1 Nucleotide
pairing
Mutation rate (%) 10 20 20
Target site length
(bp)
18–36 24–40 22
Endonuclease Fok I Fok I Cas9
Double-stranded
break pattern
Staggered cut (4–5
nt, 5′ overhang)
Staggered cut
(Heterogeneous
overhangs)
Sp Cas9 creates
blunt ends; Cpf1
creates staggered
cut (5′ overhang)
Off-target effects High Low Variable

10. Timeline of Discovery
2007
• Experimental demonstration of adaptive immunity
2008
• Spacer sequences are transcribed into guide RNAs
• CRISPR acts on DNA targets
2010
• Cas9 cleaves target DNA
2011
• Discovery of tracrRNA for Cas9 system
2012
• Biochemical characterization of Cas9-mediated cleavage
2013
• CRISPR-Cas9 harnessed for genome editing

11. Scientists on CRISPR
Jennifer Doudana (June,2012)
Emmanuelle Charpentier (June,2012)
Feng Zhang (January,2013)

12. Case Studies

14. Background of the Work
• Xanthomonas oryzae pv. oryzae (Xoo) pathogenicity depends on
transcription activator-like (TAL) effectors.
• Host disease-susceptibility genes - Os8N3 (also known as
OsSWEET11).
• EBEs - DNA polymorphisms in the so-called TAL effector binding
elements (EBEs), located at the promoter region of the
susceptibility gene, lead to no development of the disease.
• TAL effector PthXo1 from Xoo strain PXO99 directly activates
Os8N3 through recognition of TAL EBEs located at the promoter
region of Os8N3.
• Nipponbare & Kitaake having 100 % identical promoter region
including PthX01 EBE sites.

16. T-DNA recombinant
Promoter - OsU6a with vector OsU6a::pHAtC
Target site - xa13m in the first exon of Os8N3 (20-bp nucleotide sequence)
Binary plasmid - OsU6a::xa13m-sgRNA/ pHAtC, carrying xa13m-sgRNA targeting
the Os8N3 gene.

17. Kitaake Transgenic Plants (T0) - 1A, 2A, 3A, 4A
Genotypes - homozygote,
- biallele,
- heterozygote,
- chimera,
- Wild type.
Homozygous and Biallelic showed robust resistant.
PCR based selection using the Cas9-specific primers, Cas9_RT_F
and Cas9_RT-R

18. Segregation Pattern

21. Selection of Homozygous, transgene
clean mutants

22. Conclusion
• Os8N3 knockout mutants showed increased expressions
of several SWEET genes such as OsSWEET3a,
OsSWEET6b, OsSWEET13, and OsSWEET15.
• Mutant lines harboring the desired modification in
Os8N3 but without the T-DNA of the OsU6a::xa13m-
sgRNA/pHAtC were obtained.
• T-DNA-free homozygous mutant lines displayed
significantly enhanced resistance to Xoo and normal
pollen development.

24. Background of the Work
• Nongken58S –PGMS line having pms1, pms2 and pms3
encodes lncRNA called LDMAR
• Peiai64S is TGMS indica rice line from Nongken 58S, tms12-1
encodes a ncRNA of 21-nt smallRNA.
• carbon starved anthers is a rPGMS. CSA encodes R2R3 MYB
transcription factor.
• AnnongS-1 (AnS-1) indica TGMS line identified in1987. TGMS
gene tms5 encodes RNase ZS1 controls TGMS trait by
degrading temperature sensitive UbL40.
• Using CRISPR/Cas9 editing technology to knock out TMS5,
which is of great value in new commercial TGMS line
applications.
• Based on this system, they developed 11 commercial
“transgene clean” TGMS rice lines within only one year.

25. Mutation types and frequencies in T0 plants with 10 target sites

27. Development of commercial
TGMS lines in rice
• 11 Elite rice cultivars are target edited in the genome using the
TMS5ab construct.
• Except all other YNSMS showed no homozygous mutants, it
indicates that editing efficiency of CRISPR/Cas9 differs based on
genetic background.
• After LT treatment of the T0 sterile individuals, plants with
restored fertility were selected.
• Selection of Transgene clean plants - plants with restored fertility
planted at HT. And sterile plants without hygromycin phopshate
tranferase (hpt) and Cas9 obtained through PCR analysis and
Southern blotting.
• These “transgene clean” sterile plants were grown under
conditions, and T2 seeds were obtained.

30. Conclusion
• They tested CSITs of 7 TGMS lines generated using the TMS5ab
editing system and found that varieties with different genetic
backgrounds possessed different CSITs.
• These results suggested that the CSIT of tms5 TGMS lines was
determined according to their genetic backgrounds but not
tms5.
• Furthermore, TGMS lines with higher CSITs could be crossed
with lower CSIT lines to select new TGMS lines with lower
CSITs.
• Using the TMS5ab construct, the TMS5 gene was edited in 11
elite rice lines to develop the “transgene clean” TGMS lines
within only one year whereas in conventional breeding requires
several years or decades.

32. Background of the Work
• Coeliac disease is an autoimmune disorder triggered in
genetically predisposed individuals by the ingestion of gluten
proteins from wheat, barley and rye.
• The a-gliadin family is the main protein group associated with
the development of coeliac disease.
• In bread wheat, a-gliadins are encoded by approximately 100
genes and pseudogenes organized in tandem at the Gli-2 loci of
chromosomes 6A, 6B and 6D.
• The a-gliadin gene family of wheat contains four highly
stimulatory peptides, of which the 33-mer is the main
immunodominant peptide in patients with coeliac.

33. Continue…
• Two sgRNAs (sgAlpha-1 and sgAlpha-2) to target conserved
regions adjacent to the coding sequence for the
immunodominant epitope in wheat gluten in a-gliadin genes.
• sgAlpha-2 was more effective than sgAlpha-1. It was noted that
lower regeneration was observed in transgenic plants containing
sgAlpha1 (0.3% transformation frequency) than plants with
sgAlpha2.
• The CRISPR/Cas9 constructs were transformed into
i)BW028
ii)TAH53
iii)DP cultivars

34. sgRNAs design and plasmid construction

35. a) a- gliadin genes, b)deletions and c) indels in T0
mutants

36. Illumina sequencing of alpha-gliadins in 18 T1 bread and
durum wheat transgenic lines

37. Cont..

38. A-PAGE & MALDI-TOF analysis of Gliadins of
sgAlpha-1 and sgAlpha-2

39. Conclusion
• Gluten content was determined by competitive ELISA assays
using two monoclonal antibodies R5 and G12.
• Immunoreactivity of the CRISPR-edited wheat lines was reduced
by 85%, as revealed the R5 and G12 ELISA tests.
• Twenty-one mutant lines were generated, all showing strong
reduction in a-gliadins.
• Transgene-free lines were identified, and no off-target mutations
have been detected in any of the potential targets.

40. Mugui Wang1,3, Yanfei Mao1,3, Yuming Lu1,
Xiaoping Tao1 and Jian-kang Zhu1,2,*

41. What is CRISPR-Cpf1?
• Clustered Regularly Interspaced Short Palindromic Repeats from
Prevotella and Francisella 1 (CRISPR-Cpf1, also known as Cas12a)
is a class II type V endonuclease.
• The first identified Cpf1 is from Francisella novicida (FnCpf1)
• Most commonly used Cpf1s are LbCpf1 (Lachnospiraceae
bacterium ND2006 Cpf1) and AsCpf1 (Acidaminococcus sp. BV3L6
Cpf1)

43. Target genes and it sequences
• They had chosen 22–24 nt target sequences to induce
mutations at six sites of three endogenous genes:
 5-Enolpyruvylshikimate 3-Phosphate Synthase (OsEPSPS,
LOC_Os06g04280),
 Bentazon Sensitive Lethal (OsBEL, LOC_Os03g55240),
 Phytoene Desaturase (OsPDS, LOC_Os03g08570).
• Results showed that both FnCpf1 and LbCpf1 with their
own mature DRs can introduce targeted gene mutations in
transgenic plants.
• The LbCpf1 system exhibited a higher editing efficiency than
FnCpf1 in all of the six tested target sites, and both of them
showed big differences in the frequency of induced mutations
between two target sites within the same gene.

45. Cont..
• FnCpf1 to edit four members related to receptor-like
kinases (OsRLKs):
 OsRLK-798 (LOC_ Os02g04430),
 OsRLK-799 (LOC_Os02g07960),
 OsRLK-802 (LOC_Os01g39600),
 OsRLK-80 (LOC_Os06g04370),
• LbCpf1 to edit four OsBEL genes of the CYP81A family:
 OsBEL-230 (LOC_Os03g55230),
 OsBEL-240 (LOC_Os03g55240),
 OsBEL-250 (LOC_Os03g55250),
 OsBEL-260 (LOC_Os03g55260)

47. The phenotypes of rice wild type (WT), OsPDS chimera (Chi) and bi-
allele (Bi) T0 mutants derived from Cpf1 gene editing.

48. Off Target effects
• Different from the common 1–2 bp short indels generated by
Cas9 in rice, most of the mutations derived from multiplex gene
editing by both FnCpf1 and LbCpf1 in rice were 3–30 bp
deletions at 30 of the target sequence.
• No off-target mutations were found at potential off-target sites,
when LbCpf1 was used to edit several single genes in rice .
• However, slight off-target effects were found at highly
homologous sequences that have only one mismatch with their
on-target sites, when some single loci were edited using FnCpf1
in rice.

49. Conclusion
• Multiplex gene editing provides a powerful tool for targeting
members of multigene families.
• The Cas9 system requires large constructs to express multiple
sgRNA cassettes, which are more laborious to construct and
may cause instability and reduce transformation efficiency.
• Their study has demonstrated the feasibility of high-efficiency
multiplex gene editing in plants using engineered CRISPR-Cpf1
with a simple short DR guide array.

50. Achievements in
CRISPR

51. Rice Improvement

53. Revolutionizing in Horticulture
Crops

54. Cont..

55. CRISPR in Field Crops

56. Cont..

57. Conclusion
• The emergence of the CRISPR/Cas9 technology provides a simple,
cheap and efficient genome editing platform for researchers.
• New Cas9 variant proteins and homologous proteins, such as Cas9-
VQR, Cas9-VRER, Cpf1-RR, Cpf1-RVR and SaCas9, have been created
and applied in genome editing, which has greatly expanded its
editing range.
• In terms of transgenic safety, transgene-free technology also
achieves a great breakthrough.
• Mutants without transgenic ingredients can be obtained in their
progeny through an instantaneous editing or screening system.
• As a advantage CRISPR edited plants does not come under GMO
crops.

58. References
• Kim, Y.-A., Moon, H., & Park, C.-J. (2019). CRISPR/Cas9-targeted mutagenesis of
Os8N3 in rice to confer resistance to Xanthomonas oryzae pv. oryzae. Rice, 12(1),
1-13.
• Sander, J. D., & Joung, J. K. (2014). CRISPR-Cas systems for editing, regulating and
targeting genomes. Nature biotechnology, 32(4), 347.
• Wang, M., Mao, Y., Lu, Y., Tao, X., & Zhu, J.-k. (2017). Multiplex gene editing in
rice using the CRISPR-Cpf1 system. Molecular plant, 10(7), 1011-1013.
• Zhou, H., He, M., Li, J., Chen, L., Huang, Z., Zheng, S., . . . Zhao, B. (2016).
Development of commercial thermo-sensitive genic male sterile rice accelerates
hybrid rice breeding using the CRISPR/Cas9-mediated TMS5 editing system.
Scientific reports, 6, 37395.
• Fiaz, S., Ahmad, S., Noor, M. A., Wang, X., Younas, A., Riaz, A., . . . Ali, F. (2019).
Applications of the CRISPR/Cas9 system for rice grain quality improvement:
Perspectives and opportunities. International journal of molecular sciences,
20(4), 888.

59.  Jun, R., Xixun, H., Kejian, W., & Chun, W. (2019). Development and
Application of CRISPR/Cas System in Rice. Rice Science, 26(2), 69-76.
 Li, M., Li, X., Zhou, Z., Wu, P., Fang, M., Pan, X., . . .Li, H. (2016).
Reassessment of the four yield-related genes Gn1a, DEP1, GS3, and IPA1 in
rice using a CRISPR/Cas9 system. Frontiers in Plant Science, 7, 377
 Shimatani, Z., Kashojiya, S., Takayama, M., Terada, R., Arazoe, T., Ishii, H., . .
. Miura, K. (2017). Targeted base editing in rice and tomato using a CRISPR-
Cas9 cytidine deaminase fusion. Nature biotechnology, 35(5), 441.
 Wang, F., Wang, C., Liu, P., Lei, C., Hao, W., Gao, Y., . . . Zhao, K. (2016).
Enhanced rice blast resistance by CRISPR/Cas9-targeted mutagenesis of
the ERF transcription factor gene OsERF922. PLoS One, 11(4), e0154027.