PEPS ExoMod 27/02/17. NeoGene: Caractérisation de nouveaux gènes par expansion du code génétique chez les levures du genre Lachancea

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1 PEPS ExoMod 27/02/17 NeoGene: Caractérisation de nouveaux gènes par expansion du code génétique chez les levures du genre Lachancea Gilles Fischer - Biology of Genomes Shixin Ye-Lehmann - Genetic code reprograming and synthetic biology Laboratory of Computational and Quantitative Biology

2 Introduction How are new gene created? To create is to recombine New functions...are merely variations on previous themes Evolution does not produce novelties from scratch

3 Introduction How are new gene created? Duplication/Divergence Fusion/Fission TE Exaptation Exon shuffling Long et al, 2003

4 Introduction How are new gene created? 2006 de novo emergence of a gene from a non-coding sequence

5 Goals of the NeoGene project How are new gene created? 1) Quantify de novo gene emergence at a large scale - Choice of the species - Bio-informatic detection at the genome scale 2) Characterize the function of de novo genes - Detection of native de novo proteins - Identification of interacting proteins

6 A broad evolutionary range in yeast 7 Saccharomyces cerevisiae Saccharomyces paradoxus Saccharomyces mikatae * Saccharomyces kudriavzevii Saccharomyces bayanus 85 6 Saccharomyces exiguus Saccharomyces servazzii Homo sapiens Saccharomyces castellii Candida glabrata Zygosaccharomyces rouxii Mus musculus 5 Lachancea thermotolerans Lachancea waltii 2 4 Lachancea kluyveri Kluyveromyces lactis Kluyveromyces marxianus Ashbya gossypii Pichia angusta Debaryomyces hansenii Pichia sorbitophila Takifugu rubripes Tetraodon negroviridis 1 3 Candida guilliermondii Candida lusitaniae Candida tropicalis Candida parapsilosis Candida albicans Candida dubliniensis Yarrowia lipolytica Ciona intestinalis Euascomycetes %Similarity between orthologous proteins Dujon et al., and Jaillon et al., Nature, 2004

7 The genus Lachancea Sequencing, assembly and annotation of 10 Lachancea genomes Vakirlis, Sarilar, Drillon et al. Genome Res (2016)

8 Reconstruction of gene repertoire evolution Nikos Vakirlis Ingrid Lafontaine 850 Taxonomically Restricted Genes Sequencing, assembly and annotation of 10 Lachancea genomes

9 The bioinformatics pipeline for the detection of de novo genes 850 Taxonomically Restricted Genes: HMM Profiles protein simulation missed homology de novo spurious ORF Synteny

10 The bioinformatics detection pipeline 850 Taxonomically Restricted Genes: missed homology spurious ORF de novo

11 The bioinformatics detection pipeline 850 Taxonomically Restricted Genes: missed homology spurious ORF de novo

12 Experimental detection 239 de novo gene candidates missed homology spurious ORF de novo Mass Spectrometry proteomics de novo 8.7 % Collaboration with Chris Hittinger and Joshua Coon University of Wisconsin-Madison 21 de novo proteins with unknown function

13 Intrinsic properties of neogenes and encoded proteins

14 Goals of the NeoGene project How are new gene created? 1) Quantify de novo gene emergence at a large scale - Choice of the species - Bio-informatic detection at the genome scale 2) Characterize the function of de novo genes - Detection of native de novo proteins - Identification of interacting proteins

15 Genetic code expansion requires 4 ingredients: 1. Introduction of a targeted amber stop codon 2. Suppressor trna recognizing amber stop codon 3. Engineered trna synthetase (RS) 4. Unnatural amino acids

16 1. Introduction of a targeted amber STOP codon with CRISPR/Cas9 technology Guide RNA Structural grna Genomic target 23 base pairs PAM sequence DiCarlo et al., 2013

17 1. Introduction of a targeted amber STOP codon with CRISPR/Cas9 technology Targeted cut Aubin Fleiss Alma Chapet-Battle grna pgz110-kan Leu2 transformation break: 99% Cas9 select S. cerevisiae cells with Leu2 phenotype 1~2 colonies CRISPR/cas9 cuts the genome with very high efficiency (lethal)

18 1. Introduction of a targeted amber STOP codon with CRISPR/Cas9 technology Targeted cut Aubin Fleiss Alma Chapet-Battle grna pgz110-kan Leu2 transformation break: 99% Cas9 select S. cerevisiae cells with Leu2 phenotype 1~2 colonies CRISPR/cas9 cuts the genome with very high efficiency (lethal) Targeted repair grna CAN1 ADE2 YFP + Cas9 pgz110-kan STOP Leu2 transformation select S. cerevisiae cells with Leu2 phenotype ~100 colonies break: 99% repair : 94% high efficiency cut and repair : cells can be rescued with custom DNA sequence Targeted integration of a STOP codon without any associated marker

19 2. Suppressor trna and 3. Engineered trna Synthetase aars amino acids Yuting Chen Shixin Ye-Lehmann Meilin Tian trnas peptide ribosome mrna L. Wang, Science, 2001 J.W. Chin, Science, 2003 Adapted from Lehmann J., Springer-Verlag. 2006

20 Confirm the AzF-RS sequence Obtained from P. G. Schultz laboratory of Scripps Institute

21 4. Unatural amino acids 20 natural amino acids Unnatural amino acids (UAAs) translation mrna UAG amber stop codon protein containing an UAA Wang L. and Schultz PG, Angew. Chemie Liu CC and Schultz PG, Annu.Rev. Biochem. 2010

22 Incorporation of p-azido-l-phenylalanine (AzF) Engineered Tyr-tRNA synthetase Modifying the Tyr binding pocket Chin J et al., Science. 2003

23 Experimental plan for 2017 Proof of principle: encoding Uaa in S. cerevisiae - Construct amber stop mutants in reporter genes using CRISPR/Cas9 - Incorporate AzF in reporter genes

24 Experimental plan for 2017 Proof of principle: encoding Uaa in S. cerevisiae - Construct amber stop mutants in reporter genes using CRISPR/Cas9 - Incorporate AzF in reporter genes Encoding Uaa in L. kluyveri - Construct a genetically tractable strain (ura3 -, leu2 - ) - Construct amber stop mutants in reporter and neogenes using CRISPR/Cas9 - Incorporate AzF in reporter and neogenes

25 Experimental plan for 2017 Proof of principle: encoding Uaa in S. cerevisiae - Construct amber stop mutants in reporter genes using CRISPR/Cas9 - Incorporate AzF in reporter genes Encoding Uaa in L. kluyveri - Construct a genetically tractable strain (ura3 -, leu2 - ) - Construct amber stop mutants in reporter and neogenes using CRISPR/Cas9 - Incorporate AzF in reporter and neogenes Site-specific fluorescent detection of proteins by bioorthogonal labeling - AzF mediated copper-free azide-alkyne 1,3-dipolar cycloaddition with Alexa647-DIBO (protocol established) - Visualization of proteins using confocal microscope and SDS-PAGE gel

26 Experimental plan for 2017 Proof of principle: encoding Uaa in S. cerevisiae - Construct amber stop mutants in reporter genes using CRISPR/Cas9 - Incorporate AzF in reporter genes Encoding Uaa in L. kluyveri - Construct a genetically tractable strain (ura3 -, leu2 - ) - Construct amber stop mutants in reporter and neogenes using CRISPR/Cas9 - Incorporate AzF in reporter and neogenes Site-specific fluorescent detection of proteins by bioorthogonal labeling - AzF mediated copper-free azide-alkyne 1,3-dipolar cycloaddition with Alexa647-DIBO (protocol established) - Visualization of proteins using confocal microscope and SDS-PAGE gel Identify interactive proteins with AzF mediated photo-cross-linking - Establish UV-crosslinking protocol using model protein GST-amb based on existing reports - Apply to L. kluyveri neoproteins and identify interactive partners by western blotting protein ligand Crosslinked complex Tian M. and Ye S. Scientific Reports. 2016

27 People involved in the work Gilles Fischer - Biology of Genomes Alma Chapet-Battle (Master student) Aubin Fleiss (PhD student) Nikos Vakrlis (PhD student) Ingrid Lafontaine (assistant professor) Shixin Ye-Lehmann - Genetic code reprograming and synthetic biology Yuting Chen (PhD student) Meilin Tian (PhD student) Collaboration with Chris Hittinger and Joshua Coon University of Wisconsin-Madison