školní rok 2016/2017, kurz Bi6405 Metody molekulární biologie - cvičení Petr Beneš Ustav experimentální biologie Přírodovědecká fakulta MU il cvičeni_ PotlaČení/umlČení exprese vybraného genu a) mutageneze v genomu pomocí metody CRISPR/Cas9 a) post-transkripČím umlčením exprese pomoci shRNA —> funkce genu C\\er\á mutageneze přímo v genomech Targeted Tar mutagenesis rs Targeted 1 Targeted gene mutagenesis replacement Cílena mutageneze přímo v genomech Nuclease-induced double-strand break mY \ Deletions template Insertions Variable length in dels T 1 ■ ■ Precise insertion or modification od 80. let - Četnost homologické rekombinace je zvýšena indukcí dsDNA zlomů - snaha o vývoj systému pro sekvenčně specifickou tvorbu dsDNA zlomů CRISPR/Cas9 CRISPR-associated protein 9 - nukleáza zzStreptococcuspyogenes x adaptivní imunita bakterií proti virům (obecně cizorodé DNA) x RGN - RNA-guided nuclease x sekvenční specif ita je dána interakcí ĎNA-RNA Bakterie - inkorporace cizorodé DNA do CRISPR repetic v genomu - tyto následně přepsány do RNA (crRNA) crRNA - photospacer - fragment cizorodé DNA - CRISPR repetici CkISPk/Cas9 crRNA poté hybridizuje s transaktivující CRISPR RNA (tracrRNA) tento komplex RNA interaguje s Cas9 nukleázou photospacer RNA navede celý komplex k cizorodé DNA (komplementarita sekvencí) výsledný ribonukleoproteinový komplex štěpí cizorodou komplementární DNA Protospacer tracrRMA I CRISPR repeats crRNA tracrRNA crRNA;1racrRNA hybrids ivJP ni Cas9:crRNA tracrRNA complex Target DNA site cleavage by Cas9:crRWA-tracrRNA complex CkISPk/Cas9 celý systém modifikován pro cílenou mutagenezi Vektor: - guideRNA (gRNA) = crRNA + tracr RNA - součástí gRNA i 20nt komplementární úsek k cílovému místu v genomové DNA + koexprese Cas9 nukleázy crRNA tracrRNA Fusion of crRNA + tracrRNA V\J gRNA Cas9:gRNA complex Target DNA site cleavage by Cas9:gRNA complex CkISPk/Cas9 PAM - photospacer adjaccnt motif - sekvence v těsném sousedství s gĎNA - nutná pro účinné štěpení Cas9 nukleázou - původní systém „NGG" (ale vývoj systémů s jinými sekvencemi) - dle systému cílová sekvence musí být ve formátu N20GG Genomic Target Site CasS-RuvC f* H N N H N H N H N H N H N r; N H N f; V , Hl C3í9 HNH ....... N N n H N H M H H N N Aue lllllllllll 3ŕ S' NMNNNNNNHNNN Your gRNA target sequence (most cntital residues for specificity in red) r 3' tracrRNA CkISPk/Cas9 - zvýšení specif ity Pouze 20 nt naváděcí sekvence off-target efekty Cas9 - 2 nukleázové domény (RuvC, HNH) Cas9 ni kázy - mutanti v jedné doméně - indukce cílených SSB -design - 2x SSB blízko sebe - a na opačných řetězcích DSB vysí specif i ta gRNA L gRNA 2 i ^^^^^ C omplex fermat ion y CasÉligRNA coniplex 1 C as 9 g R NA ccmplex ľ J,. Targal Binding í' r....... 1 JJIIIIIIf Target 1 + PAM 1 □ual nic k i.DSBí I Target 2+ PAM ? .........ty iiiiiiii minu uiiiiiimimuimii JIIIIIIIIIIJI Ml HíflM .................. NHEJ II r......Illlllltlllll mu.....itm n.....iiiiiimirmmimiimm Indels lead to insertíons. deletions oř fra meshift CRISPR/Cas9 hŤtp://qenome-enqineerinq.orq/qecko/ http://www.addqene.orq/crispr/ ">^| ■ GgCKO Ikl 1 JITI! ' - dostupné vektory i R^d^^j knihovny gDNA pro různé organismy - validovanégRNA ■ 0 |l|fl MIT ZhangLab - různé firmy - různé platformy (modifikace původního systému) pro design lze využít dostupný software http://crispr.mit.edu/ http://www.e-crisp.orq/E-CRISP/ hŤŤps://omicŤools.com/crispr-cas9-cateqory + všechny větší i menší Lifetech firmy http://www.addgene.org/52961/ lentiCRISPRv2 cPPT psiv- RRE U6 ŕ kb Wer EFS $pCas9.. Fl AG PPA Rjru WPRf lentiCRISPRv2 design CRISPR/Cas9 Sekvence genu včetně znalosti kde začínají a končí exony https://www.ncbi.nlm.nih.gov/ Soubor Úpravy Zobrazení Historie Záložky Nástroje Nápověda @ Ienticrisprv2 - Hledat Googjem X Optimized CRISPR Design T % National Center for Biotechno... X 1 NCBI BlastiNucleotide Sequen... 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Clear all Show additional filters I Nucleotide ; b-myb human mrna | Create alert Advanced Help Summary 20 per page - Sort by Default orders Send:- Filters: Manage Filters See MYBL2 ÍBMYB1 MYE <„pQto on&ar, ]ene like 2 in the Gene database bmyb reference sequence Transcript (21 protein (21 Items: 1 to 20 of 45 Page [i | of 3 Next s Last ss Ü Found 67 nucleotide sequences. Nucleotide (45) EST (22) □ Mus musculus strain C57BU6J chromosome 2. GRCm38.p4 C57BL/6J i: 182,113,224 bp linear DNA Accession: NC_000068.7 Gl: 372033108 GenBank FASTA Graphics □ Xenopus laevis strain J chromosome 9_1 OS. Xenopus_laevis_v2. whole genome shotgun sequence z 104,563,341 bp linear DNA Accession: NC_030741.1 Gl: 1051343558 GenBank FASTA Graphics □ Homo sapiens chromosome 20. GRCh38.p7 Primary Assembly' 3- 64,444,167 bp linear DNA Accession: NC_000020.11 Gl: 568815578 GenBank FASTA Graphics □ Homo sapiens chromosome 20. alternate assembly CHM1 1.1. whole genome shotgun sequence A- 62,914,916 bp linear DNA Accession: NC_01 8931.2 Gl: 528470541 GenBank FASTA Graphics □ Danio rerio strain Tuebingen chromosome 11. GRCzIO 5- 45,107,271 bp linear DNA Accession: NC_0071 22.6 Gl: 685508231 GenBank FASTA Graphics □ Aspergillus fumigatus Af293 chromosome 1. whole genome shotgun sequence' G- 4,918,979 bp linear DNA Accession: CM0001 69.1 Gl: 67471107 GenBank FASTA Graphics 1 Asnprnillus fuminstus Af293 rhrnmnsnmp 1 whnlp npnnmp shntnun spnupnrp Results by taxon Top Organisms ITreel Homo sapiens (25) Oryza saliva Japonica Group (5) Mus musculus (3) Aspergillus fumiqatus Af293 (3) Gallus gallus (2) All othertaxa (I) More... Find related data e Database: Select Search details b-myb[All Fields] AND ("Homo sapiens" [Organism] OR hunian[All Fields]) AND rarna[All Fields] Search I Recent activity Q, b-myb human mrna (45) Q, c-myb human mrna (979) Q, c-Myb (S323) Q, c-Myb mrna (2877) Turn Off Clear ŕ Výkazy práce ... ^1 Navod_cvika_... I b-myb human ... RS Lentiviral CRIS... ^ SchránkaOB -1... Ějíj trop2_myb_cri.. Soubor Úpravy Zobrazení Historie Záložky Nástroje Nápověda Ienticrisprv2 - Hledat Googlem X Optirnized CRISPR Design ■ J> RefSeq RNA Links for Gene (S... X W NCBIBIast:NucleoüdeSequen... X +. ^ ^ A https://www.ncbi.nim.nih.gov/nuccorey?LinkName=gene_nuccore_refseqrna&frürn_uid=4605 NCI3! Resources 0 How To 0 Nucleotide Species Animals.(2) Customize'.. Molecule lypes mRNA (2) Customize ... soiii ce il.it .il Nis-e-s RefSeq (2) Customize ... Sequence lei nil 11 Custom range... Release il.lt* Custom range... Revision date Custom range... Clear all Show additional filters- [Nucleotide y| Advanced Summary t Sort by Default orders Items: 2 □ Homo sapiens MYB proto-oncogene like 2 (MYBL2). transcript variant 2. mRNA 1- 2,713 Pp linear mRNA Accession: NM_001 27861 0.1 Si: 519666782 GenBank FAST "□ Homo sapiens MYB proto-oncogene like 2 fMYBL.21, transcript variant 1. mRNA 2- 2,785 bp linear mRNA Accession: NM_002466.3 Gl: 519666781 GenBank FASTA Graphics Google ♦ Ä Z = Sign in to NCBI Help Send: Filteis: Manage Filters Analyze these sequences Run BLAST Find related data Database: Select Recent activity SB Turn Off Clear 6^ RefSeq RNA Links for Gene (Select 4605) (2) Nucleotide B Homo sapiens MYB proto-oncogene like 2 (MYBL2), transcript variant 1, mRN/ Nucleotide mybl2 mrna (586) Nucleotide 3 MYBL2 MYB proto-oncogene like 2 [Homo Sapiens] Gene Q, b-myb AND (alive[prop]) (71) See more.. 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X + Resources @ Hew To @ Nucleotide I Nucleotide y| Advanced Search Nucleotide I Help GenBank- Send: - Homo sapiens MYB proto-oncogene like 2 (MYBL2),(tianscript variant 1, mRNA NCBI Reference Sequence: NM_002466.3 FASTA Graphics Go to: R LOCUS DEFINITION ACCESSION VERSION KEYWORDS SOURCE ORGANISM REFERENCE AUTHORS JOURNAL PUBHED REMARK REFERENCE AUTHORS TITLE JOURNAL PUBHED REHARK REFERENCE AUTHORS TITLE NM_002466 2785 bp mRNA linear PRI 07-OCT-2016 Homo sapiens MYB proto-oncogene like 2 (MYBL2), transcript variant 1, mRNA. NM_00246£ NM_00246 6.3 RefSeq. Homo sapiens (human) Homo sapiens Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; Haplorrhini; Catarrhini; Hominidae; Homo. 1 (bases 1 to 278S) Krakstad C, Tangen IL, Hoivik EA, Halle HK, Berg A, Werner HM, Raeder MB, Kusonmano K, Zou JX, Oyan AM, Stefansson I, Trovik J, Kailand KH, Chen HW and Salvesen HB. ATAD2 overexpression links to enrichment of B-MYB-translational signatures and development of aggressive endometrial carcinoma Oncotarget 6 (29), 28440-23452 (2015) 2 6308378 G-eneRIF: Data indicate that gene expression alterations in endometrial carcinoma samples with high ATAD2 expression showed upregulation of several cancer-related genes including B-MYB gene. 2 (bases 1 to 2785) Tao D, Pan Y, Jiang G, Lu H, Zheng S, Lin H and Cao F. B-Myb regulates snail expression to promote epithelial-to-mesenchymal transition and invasion of breast cancer cell Med. Oncol. 32 (1), 412 (2015) 25502082 GeneRIF: Ue found that B-Myb upregulated expression of the key epithelial-to-mesenchymal transition regulator snail and that it mediated epithelial-to-mesenchymal transition activation and cell invasion by B-Myb. 3 (bases 1 to 2785) Tao D, Pan Y, Lu H, Zheng S, Lin H, Fang H and Cao F. B-myb is a gene implicated in cell cycle and proliferation of Change region shown Customize view Analyze this sequence Run BLAST Pick Primers Highlight Sequence Features Find in this Sequence Articles about the MYBL2 gene '—1 A human interactome in three quantitative dimensions organized by stoichiomet [Cell. 2015] ATAD2 overexpression links to enrichment of B-MYB-translational signature [Oncotarget. 2015] The BioPlex Network: A Systematic Exploration □f the Human Interactome. [Cell. 2015] See all... Pathways for the MYBL2 gene 8 TFAP2A acts as a transcriptional repressor during retinoic acid induced cell differentiation Transcriptional regulation by the AP-2 (TFAP2) family of transcription factors Polo-like kinase mediated events See all Reference sequence information RefSeq alternative splicing See the other reference mRNA sequence splice variant for the MYBL2 gene (NM_001278610.1) I Doručená pošt... Výkazy práce ... S| Navod_cvika_... 1 Homo sapiens ... ŘS Lentiviral CRIS... 5chľánka04 -1... Ějj tfop2_myb_cri... Soubor Úpravy zobrazení Historie Záložky Nástroje Nápověda ínticrisprv2 - Hledat Googlem Ptirnized CR.I5PR Design Homo sapiens MYB proto-onc. £3 NCBI Blast:Nuc!eotideSequen... X ^ A rittps;//www.ncbi,nlm,nih.gov/nuccore/NH_Ú0246&,3 I * Google / it * ft 2 = 286..379 /gene="MYBL2" /ge ne_s yno nym="B-HYB; / inf erence= "alignment 380..451 /gene="MYBL2" /ge ne_s yno nym="B-HYB; /inference="alignment; 452..544 /gene="HYBL2" /ge ne_s yno nym="B-HYB; /inference="alignment 545..765 /gene="HYBL2" / ge ne_s y no nym=rr B - H YB; /infecence="alignment i 766..928 /gene="HYBL2" /gene_synonym="B-HYB; /inference="alignment: 929. . 1216 /gene="MYBL2" / ge ne_s y no nym=rr B - H YB; /inference="alignment: 1217..1630 /gene-"HYBL2" /ge ne_s yno nym="B-HYB; /infecence="alignment: 1631..1770 /gene="HYBL2" / ge ne_s y no nym=,r B - M YB; /inference="alignment: 1771..1870 /gene="HYBL2" /ge ne_s yno nym="B-HYB; /inference="alignment: 1871..1984 /gene="MYBL2" / ge ne_s y no nym=rr B - H YB; /inference="alignment: 1985..2089 /gene="HYBL2" / ge ne_s y no nym=rr B - H YB; /inference="alignment: 2090..2239 /gene="HYBL2" /ge ne_s yno nym="B-HYB; /inference="alignment: 2240..2777 /gene="MYBL2" / ge ne_s y no nym=rr B - H YB; /inference="alignment: 2379..2574 B HYB " Splign:1. B HYB " Splign:1. B HYB " Splign:1. B HYB " Splign:1. B HYB " Splign:1. B HYB " Splign:1. B HYB " Splign:1. B HYB " Splign:1. B HYB " Splign:1. B HYB " Splign:1. B HYB " Splign:1. B HYB " Splign:1. B HYB " Splign:1. StBft E Doručená pošt... E Výkazy práce ... & CRISPR £| Navod_cvika_... I Horno sapiens ... g* Lentiviral CRIS... f J Schránka05 -1... E| trop2_myb_cri... CS ^'HQř 10:16 y nuiiyin- Li-niiJ, uii i u upstream in-frame stop codon CDS 23 68 y^>GI: 519666782 GenBank Fftgffi—&m Homo sapiens MYB proto-oncogene like 2 (MYBL2). transcript variant 1. mRNA 2,785 bp lingar mPNA Accessioi<^NMJ02466T)3l: 51 96667S1 GenBank FA"5TC TjTaphics ^ Bhttps:i/blast.ncbi.nlm.nih,oov/Blast.cai?PAGE_TYPE=BlastSearch&ELAST_5PEC=blast2seqSiINK_LOC=alion2seq IT Google P Ů Ě + U.S. National Library of Medicine NCBI National Center for Biotechnology Information BLAST' » blastn suite j blaatn | blnatp | blast« | tblastn | tbl.istx | Align Sequences Nucleotide BLAST Home Recent Results Saved Strategies Help Enter Query Sequence Fnlrr ii",i"iiTTJrt|'L-iiiiiiili "i \r~\ fli(s|. or FASTA se<|iience(s) ^ ^ in^001g7861oTl^^ BLAU(ii piograms seal=200 1 550 1100 1650 2200 2750 266_ atgtctcggcggacgcgctgcgaggatctggatgagctgcactaccaggacacagattcagatgtgccggagcag agggatagcaagtgcaaggtcaaatggacccatgaggaggacgagcagctgagggccctggtgaggcagtttgga cagcaggactggaagttcctggccagccacttccctaaccgcactgaccagcaatgccagtacaggtggctgaga gttttgaatccagaccttgtcaaggggccatggaccaaagaggaagaccaaaaa| - u transkriptu 2 chybi 3. exon (zeleny) ^ crispr.rnit.edu CRIS PR Design I T Google P\ it £ * # Help Forum Submit Submit a single sequence for CRISPR design and analysis search name * [A B-myb email address ; (Jb enestasci.muni.cz 2 sequence type 0 other region (23-500 nt) O unique genomic region (23-500 nt) \ demp| target genome ^^^uman (h^^^ O'mouse (mrn9) O zebrafish (danRer7) sequence CTcggcggacgcgctgcgaggatctggatgagctgcai aggacacagattcagatgtgccggagcagagggatagcaagtgc aaggtcaaatggacccatgaggaggacgagcagctgagggccct ggtgaggcagtttggacagcaggactggaagttcctggccagccac ttccctaaccgcactgaccagcaatgccagtacaggtggctgaga Batch Mode Single Sequence By submitting your query, you agree that the results obtained from this web tool will only be used as a reference for nnn-nlinical research purposes. 0 Ägre e and Submit 3-:-jt-:-f Upr b.: Z-l-br aJtrn Hitf-jr it Z - N a*.-1:i ■:■ It f J a£_td a Ienticrisprv2 - Hledat Googlem X^^H":^-/-f;:'::'^tr^;:J:-T"f="v--'^' ^omo saP'ens MVB proto-on... X NCBI Blast:Nucleotide Sequ... > I Vyhledäväni osob-Kontakty-... X E T^l] Tereza Dohnalova Danhofer... X + = ) ♦ 1 ^ crispr.mit.edu/job/716734579046491 J I'll" Google P| * & * * Z CRIS PR Design 1ob"'B-myb' Help Forum CRISPR Job Submission "B-myb" Status: Job is complete. Results View online results for B-myb Downloads Job Info Results Guides & offtargets / Nickase analysis The "zippcr-likc" annealing mechanics may explain why mismatches; between the target sequence in tlie 3' seed sequence completely abolish target cleavage, whereas mismatches toward the 5' end are permissive for target cleavage. Zhang Lab, MIT 2015 O Optimized CRISPR De... 3 Lentmral CRISPR tool... C'orucenä posta - Mic... ?-:-ut-:-r i.'c-t ä - Z-:-b-»-3~r-rn Hi-.-.f.-.ne Z-blor! fJb-.-r.r f J bpo^edb @ Ienticrisprv2 - Hledat Googlem X ^^H:'"i^^T^t^^ ^omo saP'ens MVB proto-on... X ^§ NCBI Blast:Nucleotide Sequ... > Q Vyhledäväni osob-Kontakty-... X [1 HE "ereza Dohnalova Danhofer... X ES + £ & + A Z = ;(jt crispr.mit.edu/nick/716734579046491 J ftf T Google P CRISPRDesign Job"B~myb" Nickase Help Forum job is not mapped to the genome Double Nickase Design All Pairs Sorted by score. Export all pairs to GENBANK #9 range 47 .. 109 g3 .112 g3 .133 lge iea lf'7 169 ■ 103 .159 score 44 Top Pairs View top pairs by overhang subregion. © No filter (finds all guide pairs in the query sequence) O Guide pairs overlapping a subregion from base(s) 112 to 112 (drag below specify a region) Explain this View 47 .. 109 (clickto select47..103) Export to GENBANK _ _=^__ _>_ Overhang region Guide A Guide B Pair score, A S B VTGTCTCGGCGGACGCGCTGCGAGGA DCTGG. ŕAGC TGC AC TAG CAGGAC AC A ATGTG< quality high cuts after position 69 in query sequence_ AC TTGC TATC C C TC TGC TC C GG oTftargots--■ 237 tt genie offtargets 33 Export off-targets to .csv GAGCAGAGGGATAGCAAGTG JA= GG TCAAATC GACCCATGAGG quality high cuts after position 112 in query sequence_ GGTCAAATGGAC C CATGAGC r&fftw y u u--■ 151 = genie offtargets 16 Export off-targets to .csv GCCCTC quality high ° offtarget pairs of A & B o = genie OT pairs of A & B o obě sekvence lze vklonovat do jediného vektoru s mutantní formou Cas9 (nikázou) 3-:-jt-:-f Upr b.: Z-l-br aJtrn Hi*.-1: vi it Z - N 3*.-f.r ■:■ it r J äf_td 3 Ienticrisprv2 - Hledat Googlern X^^H":^-/-f;:^t^;^-^-;^ ^omo saP'ens MVB proto-on... X NCBI Blast:Nucleotide Sequ... > I Vyhledäväni osob-Kontakty-... X E Tereza Dohnalova Danhofer... X + = ; ♦ 1 crispr.mit.edu/job/716734579046491 J I'll" Google P| * & * ft- Z CRIS PR Design 1ob"'B-myb' Help Forum CRISPR Job Submission "B-myb" Status: Job is complete. Zhang Lab, MIT 2015 O Optimized CRISPR De... 3 Lentiviral CRISPR tool... Dorucenä posta - Mic... "B-myb" Interactive results: mouse over a guide or explore below for details all guides scored by inverse likelihood of offtarget binding mouse over for details ... show legend A score Guide #1 86 3uide #2 85 Suide #3 $2 (;uide#4 $0 C iuide #5 SO C uide#6 75 G uide#7 73 Guide #8 71 Guide #9 70 C uide#10 70 C uide#11 70 (iuide#12 70 5uide #13 $7 Buide #14 65 Guide #15 $2 Guide #16 61 sequence ^^attgc tggtcagtgc ggtt TCTGGATGAGCTGCACTACC ATTCAGATGTGC C GGAGCAG CTGAC CAGCAATGCCAGTAC CAAGGTCAAATGGAC CCATG GCCACCTGTAC TGGCATTGC GGTCAAATGGAC C CATGAGG ACCAGCAATGCCAGTACAGG GCGGACGCGCTGCGAGGATC GGTCAGTGC GGTTAGGGAAG TTCAGATGTGC C GGAGC AGA GCTGTCCAAACTGCCTCACC ATTGC TGGTCAGTGC GGTTA GGTGAGGCAGTTTGGACAGC GGACACAGATTCAGATGTGC agg agg agg agg agg agg tgg agg tgg tgg tgg ggg agg ggg agg cgg guide #1 quality scor $6 guide sequence: cattgctggtcagtgcggtt agg on-target locus: unknown number of offtarget sites: 100(11 are in genes) top 20 genome-wide off-target sites (j^^ATTTC TGTAC AGTGC GGTTCA^ TvTc '1'bL. 1AG1 c bG 1 bl_ trti'lTcAS TAGTGC TGGGCAGTGC TGTTAGG CATGGTTTCTCAGTGCGGTTGAG CTTTGC TC GTCAGGGCGGTTGGG CATTGC TGGC CAGTGC TGTAAGG GAGTGC TGGTGTGTGC GGTTCAG c GGTGC TGC TCAGTGCAGTTCAG C C TTGC TGGTGAGTGC GC TTAGG CACTACTGATCAGTGC TGTTAAG C TC TGC TTGTCAGTGC GATTGGG c TC TGC TTGTCAGTGC GATTGGG cATTTGTGGACAATGC GGTTAAG GATGGC TGGC CAGTGCGTTTGGG score 0.8 0.7 0.6 0.5 0.4 0.4 0.4 0.4 0.4 0.4 0.3 0.3 0.3 0.3 rnismatcJies 4MMs [ 4"ífe [■ 4MMs [ 4MMs 3MMs 3MMs [ 4MMs [ 4MMs [ 3MMs 4MMs [ 4MMs [ 4MMs [ 4MMs [ 4MMs [ □ show all exonic UCSC gene 1:5:9:10] 1:3 !8TÍ2] 1:3 : 10: 17] [4:6:8:9] [2:8:14] 10:17:20] 1:3:11: 12] 2:3:9:17] [2:11:18] 3:5:9:17] 2:3:8:18] 2:3:8:18] 5:6:10:13] 1:4:10: 18] NR_027018 NM 201551 HM 013402 locus chr3:+128839272 chr3:+193871562 chrX:+64899769 chr9:+126101702 chrll:-3829338 chrll:+20949992 chrl2:+110541575 chrll:-61579938 chrll:+68869605 chrl:+76254707 chrl2:+123202933 chrl2 :+123189486 chrY:+17926685 chr20:-62085902 266_ at_ agga aggacacagattcagatgtgccggagcag ^^^^^^^^J^^^^^^^^^^^^^^^^^^^^^^Jacgagcagctgagggccctggtgaggcagtttgga cagcaggactggaagttcctggccagccacttccct§ ccagtacaggtggctgaga gttttgaatccagaccttgtcaaggggccatggaccaaagaggaagaccaaaaagtcatcgagctggttaagaag CATTGCTGGTCAGTGC GGTT GTC TCGGC GGACGCGC TGCG TCTGGATGAGC TGCAC TACC ok CACCG TCTGGATGAGCTGCACTACC AAÄC GGTAGTGCAGCTCATCCAGA C Target Guide Sequence Cloning Protocol In order to clone the target sequence into the lentiCRISPRv2 or lentiGuide-Puro backbone, synthesize two oligos of the following form. All plasmids have the same overhangs after BsmBI digestion and the same oligos can be used for cloning into lentiCRISPRv2T lentiGuide-Puro or lentiCRISPRvl. Target Sequence; 1 2 3 4 S 6 7 8 9 1Q (1 12 13 14 15 (6 17 1919 20 NGG RAM Oligo 1 —5' - CACCGNNNNNNNNNNNNNNNNNNNN - 3' 3' - CNNNNNNNNNNNNNNNNNNNNCAAA -5'«- Oligo 2 Example oligo design: Note that the nc-c- PAM is not included in the designed oligos. "■' NGG PAM Genomic 5' - . . , GACCACAGTCTGATCAGTTTTCCTTGGGCTGCAA... - 3' Sequence 3' - . . . CTGGTGTCAGACTAGTCAAAAGGAACCCGACGTT . . . - 5' Oligo 1 —5' - CACCGCAGTCTGATCAGTTTTCCTT - 3' 3' - CGTCAGACTAGTCAAAAGGAACAAA - 5' «- Oligo 2 cPPT lentiCRISPRv2 P kb fUter ffS S^CasS I~l AG PPA Purrj WPFír caccgtctggatgagctgcactacc aaac ggtagtgcagctcatccagac RE BsmBI (ESp3I) ♦ cacaccaCGTCTCa nnnn gtgtggtGCAGAGt nnnn CACCGTCTGGATGAGCTGCACTACC CAGACCTACTCGACGTGATGGCAAA ligace, transformace E. coli ligacní směsí, expanze klonů, izolace plazmidové DNA, sekvenace další postup ... přenos plazmidu do eukaryotických buněk (transfekce) selekce a expanze puromycin-rezistentních buněk příprava jednotlivých klonů metodou limitního ředění analýza exprese cílového genu na úrovni proteinu (westernův přenos) izolace genomové DNA klonu(ů) s nulovou expresí cílového proteinu amplif ikace cíleného úseku genomové DNA (200-400 nt) f Genornová DNA: caataaaaataagtgaagcagaagtgcctatgtcttaaaaagaagaaaaagtaagagcctctctcccccagacct ttccctagggtgggctggccgctgcctacacgttctccattatgtaaaacatatggacacaccatccttgaccct tggcctgctttcci i il m I^^^^KS aggacacagattcagatgtgccggagcaga ^^^^^^^^^^^^^^^^^^^^^JI^^^^^^Jl 'i i111 111 1 11 ■ | ■ | ■ | ■ | i i ■ | i ■ | i ■ l ■ l ■ 11 I ■ l 11 ■ l ■ l ■ ľ ľ I 11 ggggctgtccagaggaactgagcctggagtgtatttgatgtctcatcagatgggatgaagaggaggagccggtga aggaagggatgggtcctctgattgtcctcatgcctcttctcagagcattctgggggattctcaattttgaggact cagtgggtgggaaggggatgtgtgtcctagacctcagggcccattgtgtctcctgagcagctgccatgttctatg tcatggacattgattgtgctttttgccttttgactgctaggaggtttggccaaatctgtaactccccccaccctt Xhol gag c c tc gag aagt gaagc agaagtgc c tat g Igagc aagctt acctcctagcagtcaaaaggc HindlII další postup ... - klonování zacíleného úseku genomové DNA do plazmidu vhodného pro sekvenaci (napr. p£L3) - sekvenace určitého počtu klonů (2 alely genu - min. 4 klony) - analýza sekvenaČních výstupů, identifikace mutací a jejích významu (ORF) Jaké mutace lze očekávat? -jakékoli (nejčastěji malé inzerce/delece) - Cas9 štěpí DNA 3-4 nukleotidy upstream od PAM sekvence ■ ideální jsou malé posunové mutace, které mění Čtecí rámec —> předčasný stop kodon https://www.ncbi.nlm.nih.gov/orffinder Soubor Úpravy Zobrazení Historie Záložky Nástro]e Nápověda Optimized CRI5PR Design Tajemnice Fakulty-Vedení fak... X ■ % Home - ORFfinder - NCBI ^ , ^ A https://www.ncbi.nim.nih.gov/orFFinder ORF finder searches for open reading frames (ORFs) in the DNA sequence you enter. The program returns the range of each ORF, along with its protein translation. Use ORF finder to search newly sequenced DNA for potential protein encoding segments, verify predicted protein using newly developed SMART BLAST or regular BLASTP. This web version of the ORF finder is limited to the subrange of the query sequence up to 50 kb long. Stand-alone version, which doesn't have query sequence length limitation, is available for Linus x64. Examples (click to set values, then click Submit button) : • NC_011604 Salmonella enterica plasmid pWES-1; genetic code: 11; 'ATG'and alternative initiation codons; minimal ORF length: 300 nt • NM_000059; genetic code: 1; start codon: ATG only'; minimal ORF length: 150 nt PI -Cr Ě * it 2 I = Enter Query Sequence '® From Choose Search Parameters '** Minimal ORF length |nt): | 75 v| Genetic code: 1. Standard ORF start codon to use: ® "ATG" only O "ATG" and alternative initiation codons O Any sense codon '■Q Ignore nested ORFs: EH Start Search / Clear ( [•Submitj*j)rlear ] A I Home - ORFFinder - N... [ Doručená pošta - Mic... £^ 5chránkal3 - IrfanView lř Metody_2017 j Microsoft PowerPoint ... ^ trop2_rnyb_crispr_no... CS *;B0ŕ H:í B-Myb Open Reading Frame Viewer Sequence ORFs found: 10 Genetic code: 1 St.nt cm Inn: 'ATG' only ö 1:1-2-IK (2.lKbp) - 1 Find: l<3 g>iq.[t> ÍÍL. nTc JjJ Tools - # 1 £ Tracks £ ^ - ORF1 S 1199 |280 |300 |400 |E08 |BB0 |700 |800 909 11 K 11,100 |1,S00 |1,30B j 1,400 11,500 |1,6B0 |1,700 |1,B00 |1,900 |2 K ]RFfindeE_l.18.145630180 □a) LTD Ä — ~ ORFS - 0RF7 ~ 0RF3 iJS^H 1199 |200 |300 |400 |EB9 |B90 |700 |800 0RF18 HI ■ 0RF4 90B |1 K 11,100 |1,300 |1,300 j 1,400 11,500 |1,600 |1,700 11,800 |1,900 |2 K Add six-frame translation track ORF1 (700 aa) Display ORF as.. Mark Mul k subset... Marked: 0 Download marked set as Protein FASTA s-lcl I 0RF1 HSEETRCEDLDELHYQDTDSDVPEQEDSKCKVEtTTHEEDE Q L RAL VRQ F GQQDWKF LASHF PHRTDQQC QYRTBL RVLNPD LVKGPWTKE EDQKVIE LUKKYCTKQMTLIAKHLKGRL GKQ C RE RFHIIHLNPEUKKS CTflTEEED RIICEAHKVL GNRWAEI AEHL P GRTIJUAUKHHTOIS T IEREOTi TGGF LSF SED CEP P V YLLLELEDKDGLQ.SAQPTEGQGSLLTNWPSVPPTIKEEEN SEHE LAAAT T SKE Q E PIGTD LDAVRTPE P L E E F PKRED Q E GSPPETSLPYKWWEAAIILLIPAVGSSLSEALDLIESDPD AWCTJ L SEFD L PEE P SAEIi SI HITS LVQLQASHQQQVLPPRQ PSALVPSVTEYRLDGHTISDLSRSSRGELIPISPSTEVGG S GlGT P P SVLKRQ EKRRVAL S P VT EHST S L S F LI> S CNS L T PKSTPVKTLPFSPSQFLNFWHKQDTLELESPSLTSTPVCS QKWVT T P LHEDKT P LHQKHAAF VT PDQKYSHDNT PHT P T SmartBLAST ORF1 ] BLAST ORF1 l I BLAST marked set Label I Strand Frame Start Stop Length (nt | aa) ORF1 + 1 c <1 2103 2103 1 700 — --■ ORF5 - 2 2093 1521 573 1 190 ORF8 - 2 437 42 396 I 131 ORF4 - 1 1500 1135 366 I 121 ORF7 - 2 770 522 249 I 82 ORF6 - 2 1208 1053 156 |51 ORF2 + 2 32 127 96 |31 ORF3 + 3 204 296 93 I 30 ORF9 - 3 1423 1337 87 I 28 '— nncin inno n-iT a-i i no BLAST Database: i UniProtKB/Swiss-Prot (swissprot) v| B-Myb - mutovaný Open Reading Frame Viewer Sequence ORFs found: 10 Genetic code: 1 Start codon: 'ATG' only 1: 1..2.1K (2.1Kbpl - Find: V \

|etQ>- = fit XTools - # 10Jracks 5? ^ - i |iea |saa |3ea 4BB ÜRF3 S |EBB |700 |S88 988 |1 K |1,1BB |1,288 11,300 |1,488 |1,S00 11,688 11,788 |1,S00 11,988 |2 K ORFfinder_l.18.15037995 0RF3 1 H 1 1 H 1 * l |18B |288 |38B 4BB ^T^^^ 0RF7 |588 |SBB |7BB |888 ^™ 0RF18 0RF4 988 |1 K |1,1BB |1,288 |1,3BB |1,488 |1,EBB 11,688 11,788 |1,SBB 11,988 |2 K ORF3 (538 aa) Display ORF as... Mark í-lcl I 0RF3 MLP GRTDNAVKHHTOWS TIKRKVD TGGFLSE SKD CKP P VYL LLELEDKDGLQSArjPTEGQGSLLTNWPSVPPTIKEEENSE EELAAATTSKEQEPIGTDLDAVRTPEPLEEFPKREDQEGS P P E T S L P YKSJWEAAHL LIPAVGS S L S BALD LIE SD PDAW CD L SKFD L P E E P SAED SIIIHS L VQ L QASHQ Q Q VL P P RQ P S ALVPSVTEYRLDGHTISDLSRSSRGELIPISPSTEVGGSG IGTPPSVLKRQ RKRRVAL SPVTENST S L S F LD S CNS L T PK STPVKTLPFSPSrjFLHFWIIKQDTLELESPSLTSTPVCSQK VWTTPLHPDKTPLHQKHAAFVTPDQKYSMDNTPHTPTPF KNALEKYGPLKPLPQTPHLEEDLKEVLRSEAGIELIIEDD IRP EKQKRKP GLRRSPIKKVRKS LALDIVD ED VKLHHS T L PKSLSLPTTAPSHSSSLTLSGIKEDHSLLHQGFLQAKPEK AAVAQKP RSHFTTPAPHSS AWKTVAC GGTRDQLFHQ EKAR SrnartBLAST ORF3 BLAST ORF3 BLAST marked set I Add six-frame translation track Mark subset.. Marked: 0 Downlead marked set ] as Protein FASTA v Label Strand Frame Start Stop Length (nt | aa) ORF3 + 3 486 >2102 1617 I 538 A ORF5 - 2 2092 1520 573 I 190 ORFS - 2 436 41 396 I 131 ORF4 - 1 1499 1134 366 I 121 ORF7 - 2 769 521 249 I 82 ORFB - 2 1207 1052 156 I 51 ORF1 + 1 c <1 126 126 I 41 ORF2 + 2 203 295 93 I 30 ORF9 - 3 1422 1336 87 I 28 nnirin ■=> mm nic ov i no V BLAST Database: I UniProtKB/Swiss-Prot (swissprot) Organizace cvičení (CRISPR) skupiny po 12 lidech (2x 6 hod praktických cvičení) Každá skupina ... před praktickou částí cvičení... - přidělen jeden cílový gen - návrh strategie cílené mutageneze tohoto genu metodou CRISPR (miniprojekt) - zaslání nejpozději v poslední neděli před začátkem cvik na pbenes@sci.muni.cz ... po praktické části cvičení... - výsledky získané na cvičeních budou zpracovány do tohoto miniprojektu včetně analýzy sekvenaČních výstupů - včetně vyhodnocení významu vzniklých mutací pro translaci cílového genu Miníprojekt (.doc) - stručný úvod + cíl - detailní popis návrhu gRNA sekvencí pro vybraný gen stručný popis výsledků dílčích kroků cílené mutageneze z praktických cvičení - analýza výstupů ze sekvenace genomové DNA získaných klonů - závěr RNA interference_ http://www.naturexom/nrg/multimedia/rnai/animation mechanismus post-transkripčního umlčování genů využívá dvouřetězcové RNA pro interferenci s genovou expresí (dsRNA efektivnější než ssRNA) podstatou je enzymová degradace nebo zastavení translace specifické mRNA miRNA vs siRNA siRNA malé nekódující dsRNA o velikosti 21-25 nukleotidů negativní regulátory genové exprese, které obvykle zprostředkovávají degradaci cílové mRNA (úplná homologie) exogénni původ (viry, syntetické) v cytoplazmě procesovány proteinem Dicer siRNA se váže k RISC, jiedno vlákno se degraduje a druhé zprostředkovává degradaci nebo inhibici translace příslušné mRNA Double overhang dsRNA Dicer b ÍR NA Y RISC protein components RISC O. SiRNA unwinding 4 Activated RISC Ass« at on with target m RNA Sense Antjsense Target rnRNA cleavage ■ Target rnRNA /PO&X&OOWX&CS gDNA 11 Pre-miRNA Pri-miRNA NUCLEUS CYTOPLASM miR/miR* or siRNA duplex (21-25 nt long small RNA) asymmetric RISC assembly P-BODiBS capped target m R N A P-BODIES partial homology » TRANSLATIONAL REPRESSION most animal mi R N As target rnRNA perfect homology » rnRNA CLEAVAGE most plant miRNAs, and siRNAs miRNA versus siRNA obě RNA negativně regulují translaci miRNA je endogenní, siRNA je exogénni miRNA může, ale obvykle není úplněJ