This is PPT about genome editing by using Base editor

1. Base Editing in Crops: Current Advances, Limitations
and Future Implications
Speaker– HarikantYadav
Ph.D.(GeneticsandPlantBreeding)
DOCTORALSEMINAR
(AGP-789)
ID No-53969
fb: hkyadav@live.com

2. Content of the seminar…
Introduction
CRISPR/Cas9 and its limitations
Cytosine and Adenine Base Editors (C:G to
T:A; A:T to C:G)
Recent Improvement in Base Editors
Plant Base Editors (PBE)
Applications of Base Editors For Crop
Improvement
Limitations of Base Editors
Future perspectives of Base Editors
Conclusion

3. Crop
Improvement
Genetic
Engineering
Transgenic
Approach
CRISPR
Cas 9
Base Editing
Bt Brinjal,
DMH-11
Bio safety
issue
Indel
Single
base

4. Alexis Komor & Nicole
Gaudelli: Developers of
BEs (CBEs & ABEs);
Komor Asst. Prof
(Department Of Chemistry
and Biochemistry At The
University Of California,
San Diego, USA);
Gaudelli joined Beam
Therapeutics

5. CRISPR/Cas9 : Mushroom
(Cultivated in USA)
White button mushroom
cultivated in USA developed by
using CRISPR/Cas9.
Edited for PPO (polyphenyl
oxidase) gene.

6. Limitation of CRISPR/Cas9
PAM (20 bp)
Plant Biotechnology :Mishra et al. July,
2019

7. Emmanuella Charpentier (A), Jennifer Doudna (B),
Feng Zhang (C) & David Liu (D)
A B C D

8. David R Liu: The Gene Corrector:
“Nature’s 10 who mattered in 2017”
In 2016, David Liu & his team at
Harvard University developed C to
U to T Base editors using Cytosine
Deaminases, These were
successively improved thus giving
four generations of BEs (BE 1, BE 2,
BE 3 &BE 4)
Nature News : Dec,
2017

9. Base Editing vs Other techniques of
Genome Editing
Base Editing approach enables the
conversion of one target base into another
in programmable (C to A or A to G) manner
using Deaminases (removes amino
group), without DSBs or a donor template.

10. BEs are Hybrids: Vertebrate AID + Bacterial CRISPR/Cas9= Target AID
Nishida et al. 2016

11. Cytidine Deaminase for Base Editor (BE1 to BE4)
CG to UG to TA conversion
Komor et al. Nov. 2017
H2O
NH3

12. Conversion of Adenosine (A) in Inosine (I)
Nature : Liu et al. Dec 2018

13. Base Editors: Generation 1 (BE1)
BASE EDITING: Cas9 to dCas9 (Asp10Ala, His840Ala) + Cytidine
Deaminase (rAPOBEC1: C to U)
Activity window is 3 to 6 nt; 50%, 80%
Nature: Komor et al. May 2016

14. Major Challenge For The use of BE1
Nature :Liu and Rees et al. Dec. 2018

15. Base Editor: Generation 2 (BE2 with UGI)
Limitation with BE1: Base Excision (BER) Reverts U:G back to
C:G due to U Glycosylase
Solution in BE2: BE1 + Uracil Glycosylase Inhibitor (UGI)
Result: (i) 3-Fold increased efficiency
(ii) Indels formation rates <0.1% in BE1 and BE2
Nature : Kim et al; 2017

16. Base Editors – Generation 3
dCas to nCas + UGI
NICKASE will nick the non edited strand
MisMatch Repair (MMR)
C:G to U:G to U:A to T:A
Nature :Holly et al. Dec 2018

17. Large number of BE3
variants (including for
those non-canonical PAM)
Table : BE3 variants with different Cas9 variants
(including those for non-canonical PAM )
Sr.
No.
BE3 Plasmid name Cas9 (PAM)
1 pJL-SaBE3 SaCas9 (NNGRRT)
2 pJL-SaKKH-BE3 SaCas9 (NNNRRT)
3 pBK-VQR-BE3 VQR-Cas9 (NGA)
4 pBK-EQR-BE3 EQR-Cas9 (NGAG)
5 pBK-VRER-BE3 VRER-Cas9
(NGCG)
6 pBK-YE1-BE3 SpCas9 (NGG)
7 pBK-EE-BE3 SpCas9 (NGG)
8 pBK-YE2-BE3 SpCas9 (NGG)
9 pBK-YEE-BE3 SpCas9 (NGG)
10 pET42-HF-BE3 HF-Cas9
11 pCMV-HF-BF3 HF-Cas9
Nature :Holly et al. Dec 2018

18. Base Editors: Generation 4: 2 Copies of UGI
Two copies of UGI increased product purity;
Decreased indels formation
APOBEC1 16aa Cas9n(D10A) 4aa UGI
APOBEC1 16aa Cas9n(D10A) 9aa UGI UGI
9aa
BE3
BE4
Nature : Kim et al 2015

19. Enhanced Base Editors (eBEs):
(More than one copy of UGI)
Nature : Kim et al; 2017

20. HF-BE with dCas9-HF& Gam (Protein) from phage mu
(2 Mutations Red for dCas 9 & 5 for HF 2 (Black)
Gam 16aa 16aa
Gam 16aa 12aa
BE3
Gam
BE4
Gam
Protein and Cell: Liang et al April
2017
Schematic representation of the Tyr locus and gRNA target sites. The codon to be modified
with the nucleotide to be deaminated in red.
APOBEC1
APOBEC1
UGI
UGI

21. DNA Adenine Base Editors (ABE)
There is no known enzyme that converts Adenine to
Inosine in DNA.
But RNA Deaminases are available: How to convert it into
DNA Adenine Deaminase
Tad A is an enzyme, which converts A to I in E. Coli tRNA;
this was used for developing DNA adenine deaminase.

22. Steps in Developing Adenine Deaminase
E. coli strain developed, which die, when treated with
chloremphenicol, unless it can convert A to I.
Select enzyme for A to I conversion in RNA; E. Coli Tad A millions
of variants were created.
Tad A variants tested on this E. Coli strain, and selected one which
allowed E. Coli to survive in chloremphenicol.
Tad A variants tested on this E. Coli strain, and selected one which
allowed E. Coli to survive in chloremphenicol.

23. Nature: Komor et al , Nov. 2017.
Programmable base editing of A:T to G:C and G:C to A:T in
genomic DNA without DNA cleavage
Adenine Base Editor (ABE):
Adenine Deaminase synthesized in Lab

24. Left to Right John
Evans (CEO);
David Liu; Keith
Joung & Feng
Zhang (Co-
Founder)
Beam Therapeutics & RNA Base Editor

25. RNA Base Editing with CRISPR – Cas13
ADAR= A family of Adenine Deaminase Acting on RNA
Nature: Cox et al. Oct (2017)

26. Adenine base editing in DNA and RNA
(Antisense oligonucleotide directed A to I RNA editing )
A
D
C
B
Nature : Liu et al. Dec (2018)
Using ADAR
By gRNA

27. Plant Base Editors: dCas9/nCas9-PBE
Nature : Yuan Zong et al.(2018)

28. Application of Base Editing For Improvement Of Economic Traits in crop
Base editing techniques are showing positive results in improving the yield in
several important crops including rice and wheat.
In case of rice the gene OsSPl- 14 has been targeted by using the technique of
Adenine Base Editor (ABE) to increase yield (Hua et al., 2018).
In rice a gene (s) GL-2/OsGRF-4, OsGRF-3 has been targeted to improve both
grain size and yield by using ABE technique (Hao et al., 2019).
In case of wheat the gene (s) TaDEP-1 and TaGW-2 has been targeted by using
the technique of Adenine Base Editor (ABE) to improve spike length and grain
weight(Li et al., 2018).

29. Crop name Targeted genes Type of base
editor used
Functions References
Oryza sativa NRT1.1B and
SLR1
CBE Enhance nitrogen use efficiency Lu and Zhu, 2017
C287 CBE Herbicide resistant Shimatani et al., 2017
OsPDS, OsSBEIIb CBE Nutritional improvement Li et al., 2017
OsCDC48 CBE Regulate Senescence and death Zhong et al., 2017
OsSPL14 CBE Herbicide resistance Tian et al., 2018
OsMPK6 ABE Pathogen responsive gene Yan et al., 2018
Pi-d2 CBE Blast resistance Ren et al., 2018
Triticum
aestivum
TaLOX2 CBE Lipid metabolism Zong et al., 2017
TaDEP1, TaGW2 ABE Spike length and grain weight Li et al., 2018
Zea mays ZmCENH3 CBE Chromosomal segregation Zong et al., 2017
S.tuberosum StALS, StGBSS CBE Herbicide resistance, Starch synthesis Zhong et al., 2018
S
lycopersicum
and S.
tuberosum
SLALS1 CBE Herbicide resistance Veillet et al., 2019
List of Genes targeted by Cytidine and Adenine Base Editors in
different Crops
Plant Biotechnology :Mishra et al. July,
2019

30. Mutation Induction for crop improvement
This CRISPR/Cas9-xyr5APOBEC1 base editing system was then used to induce
point mutations in two rice genes NRT1.1B and SLR1 (Lu and Zhu, 2017).
NRT1.1B gene encodes a nitrogen transporter and SLR1 gene encodes a
DELLA protein.
Earlier studies showed that nitrogen use efficiency in rice was enhanced with a
C to T substitution (Thr327Met) in NRT1.1B (Hu et al., 2015) and reduced
plant height with an amino acid substitution in or near its TVHYNP motif
(Asano et al., 2009; Hu et al., 2015).
Point Mutation
Base Substitution
A point mutation in Acetolactate synthase (ALS) gene results in herbicide
resistance in plants (Yu and Powles, 2014). In rice, the C287T mutation of ALS
homolog gene results in an A96V amino acid substitution in the encoded protein
that confers resistance to the herbicide imazamox (IMZ).

31. Codon Optimization
 In this study, the rice Codon-optimized TadA XTEN-TadA* was cloned into
pHUN411 binary vector under the control of a maize ubiquitin promoter.
The rice amylose synthesis gene Wx was targeted by this vector. Wx-mq is a mutant
allele that results in low amylose content in rice endosperm (Sato et al, 2002) and this
allele contains a point mutation (T to C) at position 595, resulting in the replacement
of tyrosine by histidine at 191 positon.
Rice codon optimized ABE-nCas9 toll was synthesized to induce targeted A:T to
G:C point mutation in the Rice genome (Li et al., 2019).

32. Limitations Of Base Editing
Targeting limitations
Successful base editing requires the presence of a specific PAM sequence
(NGG PAM for SpCas9) and the target base must be present within a narrow
base-editing window (Gaudelli et al., 2017; Komor et al., 2016).

33. Cont…
To broaden the PAM compatibility and expand the scope of base editing, we have to
developed novel ABE and CBE base editors using Cas9 variants which recognize
PAMs other than the NGG motif. (Endo et al., 2019).
These optimized base editors can improve the base-editing efficiency and expand its
scope in targeting different sites in crop plants.

34. Size of catalytic window
Cytosine deaminase base editors can potentially edit any C that is present in the
wide activity window of approximately 4–5 nucleotides (or up to 9 nt).
Therefore, we have to generate high-precision base editors with narrow catalytic
windows that can precisely edit a single cytidine residue within the catalytic window
with high accuracy and efficiency. (Tan et al., 2019).

35. Cont….
 Base editing on this target may be possible by
using base editor that recognizes different PAM
(protospacer adjacent motif) sequence.
 Thus, these highly precise base editors with high
efficiency can be used as valuable tools for precision
crop breeding.

36. Off-target editing
In the base- editing systems, off-targets occur when additional cytosines
proximal to the target base gets edited.

37. These mutations were usually the C to T type of single nucleotide variants
(SNVs). The study also indicates that to minimize the off-target mutations, it is
necessary to optimize the cytidine deaminase domain and/or UGI
components.
Furthermore, use of improved variants of CBEs, YEE-BE3, could also be
employed to minimize the off-target editing in plants (Jin et al., 2019)
Cont…

38. Comparison between CRISPR Cas9 and Base
Editing
Particulars CRISPR Cas9 BASE EDITING
Discoverer Yoshizumi Ishino (1987),
Emmanuella Charpentier and Jennifer
Doudna(wolf prize, June 2020)
David Liu (2016)
Sequence
Information
Prior to editing Prior to editing
Component  gRNA
 Cas9
PAM
gRNA
 Cas9
PAM
Deaminase (AID)
Donor Template Require (HDR) Do not Require
gRNA RNA:DNA
(R Loop)
RNA:DNA
(R Loop)
Nick DSB Single strand
Precision DSB, Indel,
(low precision)
Single stranded (high precision)

39. Comparison between CRISPR and Base Editing
Particulars CRISPR Cas9 BASE EDITING
Repair NHEJ and HDR BER and MMR
Catalytic window Large, 21-25 bp Small, 4-5 bp
Maximum up to 9 bp
Off target editing More Less
Multiple target By using different nCAS9 gRNA ligated with different aptamers
Used Generation of mutation, Insertion of sequence,
correction of mutation etc.
Precise base change transition, Point
mutation, Base substitution

40. Future perspectives of Base Editing
The sgRNAs could be ligated with different aptamers (MS2, PP7, COM and box B
(Ma et al., 2016; Zalatan et al., 2015) to facilitate simultaneous base conversions (C-
T and A-G) and correct point mutations related to important agricultural traits (Li et
al., 2018).
Most recently, a CRISPR/Cas-based-directed evolution platform (CDE) was
developed for plants to evolve the rice (Oryza sativa) SF3B1 spliceosomal protein for
resistance to splicing inhibitors (Butt et al., 2019).

41. This directed evolution platform can be used to engineer crop traits for better
performance and develop resistance to biotic and abiotic stresses.
 It offers possibilities for breeding climate resilient crops that can enhance
global food security.
Thus, base-editing diversification strategies for direction evolution need to be
explored in the future that can increase genetic diversity in plants.
Cont…

42. Conclusion