EDITING COMPLEMENTING OR MERGING IN ACTION 2 ND iPLANTA CONFERENCE - - PowerPoint PPT Presentation

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EDITING COMPLEMENTING OR MERGING IN ACTION 2 ND iPLANTA CONFERENCE - - PowerPoint PPT Presentation

Dec 25, 2023 284 likes •541 views

CA15223 IPLANTA GORITSA RAKLEOVA 1 , LIDIA PETROVA 2 , ATANAS ATANASSOV 1 , IVELIN PANTCHEV 2 * 1 Joint Genomic Center, 8 Dragan Tzankov, 1164, Sofia, Bulgaria 2 Department of Biochemistry, Sofia University, 8 Dragan Tzankov, 1164 Sofia, Bulgaria *

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RNA INTERFERENCE AND GENE EDITING – COMPLEMENTING OR MERGING IN ACTION

GORITSA RAKLEOVA 1, LIDIA PETROVA 2, ATANAS ATANASSOV 1, IVELIN PANTCHEV 2 *

1 Joint Genomic Center, 8 Dragan Tzankov, 1164, Sofia, Bulgaria 2 Department of Biochemistry, Sofia University, 8 Dragan Tzankov, 1164 Sofia, Bulgaria * email: ipanchev@abv.bg

2ND iPLANTA CONFERENCE RNAi: THE FUTURE OF CROSS TALK Focus on RNAi Technology (WG1) and Applications (WG2) Poznan (Poland) February 14-16, 2018

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Controlled changes of genetic background and/or gene expression are among the key targets in modern biology. This goal might be achieved via several ways – through introducing entire new genes, modulating expression levels of existing genes or modifying existing gene sequence(s).

Controlling gene expression – an overview

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CA15223 IPLANTA AAAAAAAA Epigenetic modifications Transcription factors Cell signaling RNAi Protein modifications

Controlling gene expression – an overview

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New gene introduction…

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Transgenic technologies

First, in early 80-ies, the introduction of novel genes was developed, which led to the rise of the transgenic techniques. The target genome was supplemented with novel gene(s), often from very distant species. The transgenic organism gained traits that are impossible to obtain through classic mating. While promising from scientific point of view, the new technology soon faced strong public unacceptance. As a result, a race to develop new approaches for gene modification took place.

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Allele replacement…

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Transgenic technologies

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From early genetic research it was realized that recombination can be stimulated by induction of double-stranded breaks in DNA. DSB can be repaired via two pathways – HR and non-homologous end-joining. While HR is characteristic mainly for the cells in germinate lines, NHEJ pathway is active in almost all cell types. Meanwhile, first mechanisms relying on allele replacement via DSB-induced recombination were described and first examples of endonucleases with unique sites (homing restrictases) appeared. This scientific finding acted as a ‘proof-of-concept’ for the viability of endonuclease-directed gene editing (EDGE) through DSB repair. Allele replacement, recombination, etc…

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Allele modification…

AAAAAAAA AAAAAAAA

+

DSB HR NHEJ*

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Research on the phenomenon of “gene silencing” leaded to the discovery of a natural mechanism for controlling gene expression through modulation of mRNA stability. Soon, details of the molecular mechanism were revealed – miRNA, siRNA, DROSHA, DICER, RISK . . . RNA interference had gained its place as an important tool of functional genomics. Allele expression control …

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Allele expression control …

AAAAAAAA No protein synthesis RISK miRNA siRNA

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To evaluate the future of the technology, one should compare its pros and cons with existing “rivals”. First, who are the “rivals”? CRISPR/Cas is the “word of choice”, but it is just a member of a bigger family of approaches, exploring DSB repair mechanisms in the cell. pro`s, con`s and rivals . . .

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What could be achieved through DSB repair:

  • Allele replacement
  • Allele modification
  • Allele disruption

How could this be achieved:

  • Random introduction of DSB in presence of DNA source
  • Oligonucleotide-directed mutagenesis (with or w/o DSB)
  • Site-specific endonucleases (meganucleases, ZnF

, TAL, CRISPR/Cas) with or w/o presence of DNA source Allele modification through DSB repair

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CA15223 IPLANTA AAAAAAAA AAAAAAAA

+

DSB introduction

(ODN, TALEN, ZnF, CRISPR/Cas)

HR NHEJ*

OR

Allele modification through DSB repair

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What could be achieved through RNAi:

  • RNA inactivation
  • Possibly, chromatin rearrangement

How could this be achieved:

  • Introduction of RNA substrate
  • Introduction of gene for guide RNA

pro`s, con`s and rivals . . .

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CA15223 IPLANTA AAAAAAAA No protein synthesis RISK miRNA siRNA

RNA interference and gene expression control …

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Next, one should compare the origins of both approaches. Well, where they come from? RNAi and CRISPR/Cas appeared to be members of a bigger functional superfamily of RNA-guided enzymes . . . . . . and probably, remnants from the times when RNA ruled this world… pro`s, con`s and rivals . . .

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ancestral systems? sn(o)RNP telomerase RNaseP gRNA-dependent editing RdRP ADAR CRISPR/Cas pseudouridine synthase RNAi

pro`s, con`s and rivals . . .

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Burroughs AM, Ando Y and L Aravind (2014) New perspectives on the diversification of the RNA interference system: insights from comparative genomics and small RNA sequencing. WIREs RNA, 5: 141-181. doi: 10.1002/wrna.1210

RNA-guided endoDNase RNA-guided endoRNase ? ancestor ? prokaryotes eukaryotes protection against DNA viruses protection against RNA viruses chromatin remodeling Regulation of cellular processes through: mRNA stability mRNA translation target degradation (DNA) target degradation (RNA)

pro`s, con`s and rivals . . .

modular organization

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RNAi:

  • Native machinery exploited
  • No additional genes except

RNAi

  • Functional gene inactivation
  • Possible chromatin

rearrangement

  • Uniform action through cells
  • No nucleotide changes
  • Guide RNA kept in genome

DNA editing systems:

  • Sequence changes
  • Target specificity
  • Stably inherited
  • Heterologous genes required
  • Cell type dependency

Advantages and disadvantages…

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The future of RNA interference as a tool

RNAi cannot replace gene editing tools . . .

but

can be used for chromatin remodeling

. . . this might also be achieved by CRISPR/Cas ???

Unniyampurath U, Pilankatta R and MN Krishnan (2016) RNA Interference in the Age of CRISPR: Will CRISPR Interfere with RNAi? Int J Mol Sci 17(3): 291

  • r

still can be used for RNA targeting

. . . this cannot be readily achieved by CRISPR/Cas

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GENOME ORGANIZATION GENE SEQUENCE

RNA

PROTEIN

CRISPR/Cas etc. RNAi

Ψ Synthase RNaseP ADAR RdRP (RNA editing)

The future of RNA interference as a tool

RNA editing DNA editing

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How RNAi can complement gene editing tools . . .

. . . introducing novel regulatory stage(s) in gene expression

. . . targeting non-coding RNAs

The future of RNA interference as a tool

. . . regulated specific changes in mRNA codons . . . as a foundation of novel hybrid effectors for RNA editing

Retargeting pseudouridine synthase or ADAR toward directed modifications of bases in mRNA

RNAi can be transformed into RNA editing platform . . .

. . . designing novel complex regulatory networks . . . establishing persistent changes in chromatin organization

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CA15223 IPLANTA CRISPR/Cas Target gene (non-edited) RNAi system Cas Target gene (edited) Signal

  • r

Stimuli

The future of RNA interference as a tool

Merging CRISPR/Cas and RNAi appears to be possible even in this classic way…

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