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 * email: ipanchev@abv.bg RNA INTERFERENCE AND GENE EDITING – COMPLEMENTING OR MERGING IN ACTION 2 ND iPLANTA CONFERENCE RNAi: THE FUTURE OF CROSS TALK Focus on RNAi Technology (WG1) and Applications (WG2) Poznan (Poland) February 14-16, 2018
Controlling gene expression – an overview CA15223 IPLANTA 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 CA15223 IPLANTA Transcription factors Epigenetic modifications Cell signaling AAAAAAAA RNAi Protein modifications
New gene introduction… CA15223 IPLANTA 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 AAAAAAAA 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.
Allele replacement… CA15223 IPLANTA Transgenic technologies AAAAAAAA AAAAAAAA
CA15223 IPLANTA Allele replacement, recombination, etc… 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.
CA15223 IPLANTA Allele modification… + DSB HR NHEJ* AAAAAAAA AAAAAAAA
Allele expression control … CA15223 IPLANTA 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 … CA15223 IPLANTA miRNA AAAAAAAA siRNA RISK No protein synthesis
pro`s, con`s and rivals . . . CA15223 IPLANTA 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.
Allele modification through DSB repair CA15223 IPLANTA 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
CA15223 IPLANTA Allele modification through DSB repair + DSB introduction (ODN, TALEN, ZnF, HR CRISPR/Cas) NHEJ* AAAAAAAA AAAAAAAA OR
pro`s, con`s and rivals . . . CA15223 IPLANTA 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 •
RNA interference and CA15223 IPLANTA gene expression control … miRNA AAAAAAAA siRNA RISK No protein synthesis
pro`s, con`s and rivals . . . CA15223 IPLANTA 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 . . . CA15223 IPLANTA ADAR gRNA-dependent editing telomerase RdRP sn(o)RNP ancestral CRISPR/Cas systems? pseudouridine synthase RNAi RNaseP
pro`s, con`s and rivals . . . CA15223 IPLANTA modular organization prokaryotes eukaryotes ? ancestor ? RNA-guided endoDNase RNA-guided endoRNase target degradation target degradation (DNA) (RNA) Regulation of cellular protection against RNA viruses processes through: mRNA stability chromatin remodeling mRNA translation protection against DNA viruses 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
Advantages and disadvantages… CA15223 IPLANTA RNAi: DNA editing systems: Native machinery exploited Sequence changes • • No additional genes except Target specificity • • RNAi Stably inherited • Functional gene inactivation • Possible chromatin • rearrangement Uniform action through cells • No nucleotide changes Heterologous genes required • • Guide RNA kept in genome Cell type dependency • •
The future of RNA interference as a tool CA15223 IPLANTA RNAi cannot replace gene editing tools . . . but can be used for chromatin remodeling . . . this might also be achieved by CRISPR/Cas ??? or still can be used for RNA targeting . . . this cannot be readily 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
The future of RNA interference as a tool CA15223 IPLANTA GENOME ORGANIZATION DNA editing GENE SEQUENCE CRISPR/Cas etc. Ψ Synthase RNAi RNA RNaseP ADAR RNA editing RdRP (RNA editing) PROTEIN
The future of RNA interference as a tool CA15223 IPLANTA . . . introducing novel regulatory stage(s) in gene expression . . . designing novel complex regulatory networks How RNAi can complement gene editing tools . . . . . . targeting non-coding RNAs . . . establishing persistent changes in chromatin organization . . . as a foundation of novel hybrid effectors for RNA editing . . . regulated specific changes in mRNA codons Retargeting pseudouridine synthase or ADAR toward directed modifications of bases in mRNA RNAi can be transformed into RNA editing platform . . .
The future of RNA interference as a tool CA15223 IPLANTA CRISPR/Cas Signal RNAi or system Stimuli Cas Target gene Target gene (non-edited) (edited) Merging CRISPR/Cas and RNAi appears to be possible even in this classic way…
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