Gene editing 1

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5/7/22 1 WELCOME

Transcript of Gene editing 1

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WELCOME

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Efforts to produce more food for survival have never going to stop

BY the end of the 18th century, more than 1000 species of plants have been domesticated around the world, of which approximately 100-200 now constitute the major components of the human diet, such as rice, wheat, maize, yam,cocnut,banana, etc.

Domestication &Selection

Hybridization breeding

Mutation breeding

Transgenic breeding

Xiong et al.,(2015)

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Hybridization breeding technology:Two compatible plants in the same or closely related species/genus.Undesirable traitsPerennial horticultural cropsMutation breeding

technology:

May not heritableChimeras and require ‘‘purification,’’Low frequency of chance mutations in nature.Transgenic technology in

horticultural crop breeding:Recalcitrant to genetic transformation and regeneration.Opposition in recent years

Xiong et al.,(2015)

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Gene editing

Kittur rani channamma college of horticulture, Arabhavi

University of Horticultural Sciences, Bagalkot

Presented by: VEERESHA S M

UHS15PGM552Dept. of Biotechnology and Crop Improvement

Seminar- 1 on

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What is gene editing?

Genome/gene editing is a type of genetic engineering in which precise genome modifications are brought about using artificially engineered nucleases in organisms.

Use of engineered nucleases to introduce DNA insertions, Deletions, or replacements at sequence specific sites in a genome

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These engineered nucleases contains

1. A non specific DNA cleavage module ( Nucleases) 2. Sequence specific DNA binding domains ( ZFN or TALE

protein or CRISPR- gRNA)

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Lotus Nelumbo nucifera 929 2013 (Ming et al., 2013)

Bladderwort plant Utricularia gibba 82 2013 (Ibarra-laclette et al.,2013)

Bladderwort plant Utricularia gibba 82 2013 (Ibarra-laclette et al.,2013)

Hot pepper Capsicum annuum 3480 2014 (Kim et al., 2014)

Pineapple Ananas comosus 526 2014 (Zhang et al., 2014)

Eucalyptus Eucalyptus gramdis 640 2014 (Muburg et al., 2014)

Wild tomato Soanum pennellii 1207 2014 (Bolger et al., 2004)

Tobacco Nicotiana tobacum 4500 2014 (Sierro et al., 2014)

Oilseed Brassica napus 1130 2014 (Chalhoub et al., 2014)

Sweet orange Citrus sinensis-2 319 2014 (wu et al., 2014)

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As we already knowing the

DNA sequence of some

organism’s we can edit the

gene by using certain

techniques & tools

ZFNs

TALENs

CRISPR/cas9

Zinc finger nucleases

Transcription activator like effector nucleases

Clustered regularly interspaced short palindromic repeats

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DSBs pose a uniquely high threat to the integrity of the genetic information of a cell. Not only can the unfaithful repair of a DSB result in unwanted modifications of genetic information, but a failure to repair such a break could also lead to the loss of larger chromosomal fragments (Agarwal et al. 2006). Thus, eukaryotic cells have evolved elaborate mechanisms to repair DSBs faithfully and efficiently. Two major mechanisms of DSB repair are known, NHEJ and HR. both of which can be exploited to achieve targeted genome engineering such as gene knockout and gene addition.

Principle

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Breaks induce DNA repair and increases mutagenesis frequencies by >1000 folds

DSB

Donar DNA

NHEJ=Non-homologous end joiningTargeted mutagenesis/deletion

HDR=homology directed repairTargeted insertion /replacement

GENOME EDITING PROCESS:- Double strand breaks

Double stranded DNA

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ZFNs: The pioneer of SSNsCys2-His2 zinc finger (ZF) domine is the most

abundant DNA-binding motif in eukaryotes

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Structure of ZF

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1. ZFNs

Hybrid nuclease Induces double strand break Function as site-specific endonucleases They specify 18bp of DNA per cleavage

ZFP DNA binding domain

Cleavage domain of Fok I Endonuclease

ZFN

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DSB

Donar DNA

NHEJ=Non-homologous end joiningTargeted mutagenesis/deletion

HDR=homology directed repairTargeted insertion /replacement

GENOME EDITING PROCESS:- Double strand breaks

Fok IZink-fingerZFNs

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NNNNNNNNNNNNNNNNNNNCATCC

NNNNNNNNNNNNNNNNNNNGTAGG5’3’ 5’

3’

NucleaseDNA-binding

Fok I

Flavobacterium okeanokoites N-terminal: DNA-binding domain C-terminal: nonspecific DNA-cleavage activity

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Fok I nucleases

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Construction of ZFN

Randomized Library and a Phage DisplayBipartite Library and Two-Finger ArchiveModular AssemblyBacterial-Based SelectionContext-Dependent Assembly (CoDA)

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1.Targeted gene (disruption) knockouts: Targeted induction of minor insertions and deletions

2. Targeted gene integration: To place exogenous genetic sequences precisely into targeted sites of the genome

3.Targeted gene correction: Transfer of single-nucleotide changes and short heterologous stretches from donor to the chromosome.

Mode of action

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Figure 7: (A) ZFPAP3 target sequence (boxed) and its position in the AP3 5 UTR. Numbers indicate the distance from the ATG translation initiation codon. The arrowed boxes indicate the exon of AP3. (B) DNA recognition helix sequences of the ZFPAP3 protein. The underlined amino acids are the components of the new zinc fingers that provide specificity for the selected nucleotide sequences indicated in A. The recognition helices of fingers 1–6 (F1–F6) are underlined. Guan et al., 2002, San Diego, CA

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Figure 8: Activation of the silenced endogenous AP3 gene in Arabidopsis leaf cells. RT-PCR was used to detect AP3 expression in Arabidopsis leaf protoplasts transformed with GFP (control), ZFPm4-VP64 (nonspecific activation control), and ZFPAP3-VP64 (AP3-specific activation). The last lane is no template control amplification.

Guan et al., 2002, San Diego, CA

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Figure 9: GUS staining flowers of background plant with AP3::GUS only and double transgenic plant with both AP3::GUS and activation construct AP1::ZFPAP3-VP64 nos. (A). Predicted GUS staining patterns of AP3::GUS and AP1::GUS

Guan et al., 2002, San Diego, CA

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May 3, 2023 26Guan et al., 2002, San Diego, CA

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Figure 11: Floral phenotypic changes in double transgenic plant expressing AP3::GUS and AP1::ZFPAP3-VP64nos simultaneously. A seven-petal flower is shown here. Two extra petals are fully converted (f), and the third one is partially converted (p).

Guan et al., 2002, San Diego, CA

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IN PLANTA REPRESSION OF ENDOGENOUS AP3

UBQ3 – Promoter was used to drive the expression of AP3 specific ZFP

UBQ3:: sid-ZFPAP3//nos

Guan et al., 2002, San Diego, CA

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Figure 12: Repression of endogenous AP3 expression by the constitutive repression construct UBQ3::Sid-ZFPAP3nos in transgenic plant ND0052–2e. (A) RT-PCR identification of transgene ZFPAP3 in transgenic event ND0052–2e and wild-type control plant. (B) RT-PCR evaluation of endogenous gene AP3 expression level in transgenic event ND0052–2e and wild-type plant. In plant ND0052–2e, the expression of AP3 is significantly repressed by the expression of repressor Sid-ZFPAP3 fusion protein. Quantitative PCR indicated 46-fold repression. Guan et al., 2002, San Diego, CA

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AP3::GUS only AP3::GUS and UBQ3::Sid-ZFPAP3nos

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2.TALENsTranscription activator-like effector nucleases

( Core genomic cruise missile for SSNs)

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Alternative to ZFNs for gene editing

Since the discovery of “ the Code ’’ connecting the repetitive regions of transcription activation- like effector protiens with the DNA bases to which they bind ( Boch et al. 2009; Moscou and Bogdanove 2009), TALENs have become the current reagent of choice for efficiently modifying eukaryotic genomes in a targeted fashion ( Baker 2012).

Chen,K., & Gao C., 2014

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Structure of TALENsN-terminal : Secretion and translocation signals

C- terminal : Nuclear localization signals (NLS) and an acidic transcription activation domine (AD), and @ central DBD

HD= CNG=TNI=ANN=G or A

NK & NH = Guanine

Chen,K., & Gao C., 2014

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CONSTRUCTION METHODS

Standard cloning assembly methodsGolden gate assembly methodsSolid- phage assemble method

Chen,K., & Gao C., 2014

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May 3, 2023 35Xiong et al, 2015

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May 3, 2023 36Lor et al., 2014

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May 3, 2023 37Lor et al., 2014

Strategy for regenerating pTAL425/6 TALEN- induced mutants

NO β-estradiol

Induced with β -estradiol

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Characterization of TALEN-induced mutations

Lor et al., 2014

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Figure S4. The coding region of PRO showing the sequences of the pro and one of the pro_7 alleles. The 88bp that are deleted in one of the pro_7 alleles (Figure 1B) are highlighted. The insertion sequence of pro allele is shown. TALEN_1 TALEN_1

Lor et al., 2014

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May 3, 2023 40Lor et al., 2014

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3.CRISPRClustered regularly

interspaced short palindromic repeats

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CRISPRs: New SSN troops

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Structure of CRISPR/Cas9

Guided RNA

Three types:Type I and type III involve multiple proteins forming large cas complex

Type II from Streptococcus pyogenes, which relies on a single endonucleage,Cas9

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List of Primers used in the experimentPrimer Name Gene Name Primer sequence(5’-3’) Application

GNbPDS-FNbPDS guide sequence

TCGGGCCGTTAATTTGAGAGTCCAGuide sequenceGNbPDS-R AAACTGGACTCTCAAATTAACGGC

GStIAA2-FStIAA2 guide sequence

TCGGGATGTTTAGCTCCTTTACTAGuide sequenceGStIAA2-R AAACTAGTAAAGGAGCTAAACATC

NbPDS-FNbPDS

GCTTTGCTTGAGAAAAGCTCTCPCR assay and sequencingNbPDS-R ACATAACAAATTCCTTTGCAAGC

Cas9ID-FCas9

ATGGCCCCTAAGAAGAAGAGRT-PCR assayCas9ID-R GAGATTCCCGAACAAGCCG

StIAA2-FPGSC0003DMB000000125

GTGTCAGAGGTCCATACGCTAGAACPCR assay and sequencingStIAA2-R CCTTGGGACCCATAATTACCTGC

StoffT-FPGSC0003DMB000000384

GGTAGGGCACAAGTTGTGGGTPCR assay and sequencingStoffT-R GGTGTCAACTGACTCTCCTTCG

StUbi3-FPGSC0003DMG400005199

AAAGAAACCCTAAAAGCCGCRT-PCR assayStUbi3-R TGTCGGAAGACTCAACCTCT

25296596FNiben.v0.3.Scf25296596

GTATCACCCCAAGACCAAGCPCR assay25296596R TGTTCAACTATGCTTGTTTATTCC

24888932FNiben.v0.3.Scf24888932

GCAACATGAAGTTAGTTTGAGCAPCR assay24888932R GGGGCAGAAGGTAATTAGCAG

23457768FNiben.v0.3.Ctg23457768

TCCTGTAATTTTCTTTGTGATTCGPCR assay23457768R TTCCCCTTTCTTGAGTTCCA

Wang et al., 201546

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CRISPR/Cas9-mediated gene editing in stable transgenic potato plants.

a ) Schematic illustrating the engineered CRISPR/Cas9 vector. b ) Schematic illustrating the guide RNA (sgRNA, red arrows) targeting the

StIAA2 coding sequence. Black arrows indicate the PCR primers used to evaluate the mutation types.

c) RT-PCR for the transcripts of Cas9 in wild-type and transgenic potatoes.

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sgRNA – Targeting stIAA2

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Target gene mutagenesis in N.benthamiana using CRISPR/Cas9 system

a. Schematic and sequence of the PHYTOENE DESATURASE(PDS) gene of N.benthamiana with one target(red) in it and primers(green) for PCR assay.

b. DNA gel detection amplificons using primers flanking the target site within the PDS gene. In lanes 1-6 the genomic DNA was digested with MlyI. Lane 7 shows the control genomic DNA. Wang et al., 2015

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Alignment of reads in PDS obtained from lane 1 • The sequence of wild type is shown at the top. • The guide sequence is highlighted in red and the mutants in blue. • The changes in length and sequence are shown to the right. "G">"T" means that the

nucleotide G is converted to T, T(x4) indicates that four clones have this insertion.

Wang et al., 2015

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Native U6 promoter of potato is efficient for targeted mutagenesis in N. benthamiana

stIAA2, CRISPR/cas9 construct was transferred To check induced knockouts in potato

stIAA2::CRISPR/cas9 POTATO

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PCR genotyping of six representative StIAA2 mutation plants showing the types of DNA lesions generated

Wang et al., 2015

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Complete sequence of potato U6 promoter and gRNA scaffold. The potato U6 promoter is highlighted in grey and the gRNA scaffold is highlighted in green. Two AarI sites are in red. The guide sequence can be inserted between the two AarI sites using annealed oligonucleotides.

Wang et al., 2015

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May 3, 2023 54Brooks et al., 2015

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CRISPR/Cas9-mediated gene editing in stable transgenic tomato plants. A Schematic illustrating two sgRNAs (arrows) targeting the SIAGO7 coding sequence. Cas9/sgRNA2 Were expressed from the same plasmid (Belhaj et al., 2013). Black arrows indicate PCR primers used to evaluate mutation type and efficiency.

B, A wild-type (WT) tomato plant at 9 weeks of age, and fourth produced leaf from the primary shoot (insect).

C, The strong wiry2-1 allele of slago7. First formed leaves have leaflets petioles (blue arrow), and later formed leaves are radialized (white arrows).

Brooks et al., 2015

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D and E, a Representative crispr/Cas9-slago7 (CR-slago7) plant (D) and its first five leaves showing the distinctive loss-of-function recessive wiry syndrome (E). Bars=1cm.

Brooks et al., 2015

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H .Germline transmission and heritability of the small and large deletions from plant 3 ( left) and 2 small deletions from plant 5 ( Right ) in the absence of inductive cas9 / sg RNA 1 /sgRNA 2 trasgene. All 4 progeny from plant 3 lacked the CRISPR/cas9 transgene, and all four were heterozygous for deletions, three of which were heterozygous for the large deletion and one heterozygous for the smaller deletion. of the 10 progeny from plant 5, 2 plants lacked the CRISPR/cas9 transgene, and sequencing showed that both were heterozygous for the wild type allele and one of the two deletions( bottom ). Brooks et al., 2015

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Advantages

Speed

Simplicity

Acc

urac

y

or s

pecificit

y

Abse

nce

of tra

ns

ge

nes

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ZFN TALEN CRISPR

Binding principle Protein-DNA Protein-DNA RNA-DNA

Core component ZFN-Fok1 fusion protein TALE-Fok1 fusion protein

sgRNA and Cas9

Work mode (pair) Pair Pair No

Construction Difficult Easy Very easy

Time construction (days)

5-7 5-7 1-3

Cost High Moderate Low

Efficiency Variable High High

Length of target ~18 to 24bp including 4-7 spacers

~30 to 60bp including 13-33 spacers

~20bp

Comparison between ZFN,TALEs and CRISPR/cas9 system of gene editing

Chen, K. & Gao,C.,2014

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CONCLUSION

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THANK YOU