školní rok 2021/2022 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 b) post-transkripčím umlčením exprese pomocí shRNA —► funkce genu ... Cílená mutageneze přímo v genomech _1-1- i i 1 L y i f v -i \ -1 >- Targeted mutagenesis Targeted gene replacement Cílená mutageneze přímo v genomech Nuclease-induced double-strand break Deletions template Insertions - /- Variable length in de Is \ Precise insertion or modification od 80. let - Četnost homologické rekombinace je zvýšena indukcí dsDNA zlomů - snaha o vývoj systému pro sekvenčně specifickou tvorbu dsĎNA zlomů CkISPk/Cas9 CRISPR-associated protein 9 - nukleáza ze Streptococcuspyogenes x adaptivní imunita bakterií proti virům (obecně cizorodé DNA) 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 (pre-crRNA —► crRNA) crRNA - protospacer - fragment cizorodé DNA - CRISPR repetice CRISPR/Cas9 crRNA poté hybridizuje s transaktivující CRISPR RNA (tracrRNA) tento komplex RNA interaguje s Cas9 nukleázou protospacer RNA navede celý komplex k cizorodé DNA (komplementarita sekvencí) výsledný ribonukleoproteinový komplex štěpí cizorodou komplementární DNA Protospacer tracrRNA I CRISPR repeats crRNA tracrRNA crRNA:tracrRNA hybrids wT i vu Cas9:crRNA-tracrRNA complex Target DNA site cleavage by Cas9:crRNA-tracrRNA complex CRISPR/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 ■ Cas9 Protospacer px RuvCr) LAM li M ^ ni i 3' 5 iiiiiimuiii >< 3--V HNhO.T-5 jnEDkMi crRNA Linker j"^!yi'nimiiiMiiimii > loop sgRNA tracrRNA crRNA tracrRNA Fusion of crRNA + tracrRNA Cas9:gRNA complex Target DNA site cleavage by Cas9:gRNA complex CRISPR/Cas9 PAM - protospaccr adjacent 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 N2qGG Genomic Target Site 51- 3'< Ca&9-RuvC NHNNHNHNMNNfJN N N N H N N \ PAM C«9-HNH "X NNNNNNNNNNNNNNNNNNN i f i í i f i i i i t i m r i i <• N t < IIIIII III Your gRNA target sequence (most critical residues for specificity in red) r 3' 3 tracrRNA 3' CkIS?k/Cas9 - zvýšení specif ity - ni kázy Pouze 20 nt naváděcí sekvence off-target efekty Cas9 - 2 nukleázové domény (RuvC, HNH) Cas9 nikázy - mutanti v jedné doméně - indukce cílených SSB -design - 2x SSB blízko sebe - a na opačných řetězcích DSB vyšší specif i ta (?) gRNA 1 gRNA 2 »* 5 9 Complex-formation Cas9 gRNA complex 1 Cas9 gRNA complex 2 \, Target Binding Target 1 +• PAM \ Du.il nick (DSB) Target 2 * PAM i G«n« of irtttvsl III..... MitiiniinTm *■......r...........^ imilllllllllf.111,111 ■""«»""■'" i iiiiMinin riTTTTTTTTX i NHEJ llllllll lllllllllllllll fTTTTTTTTI......INU......... Indels lead to insertions, deletions or frameshift nebo využití upravených (více specifických forem Cas9) - SpCas9 - HF, eSpCas9 To KO gene - all you need is ... U6 promoter sgRNA Mammalian promoter 1 crR^A I tracrRMA hCas? 3CX Poly{A) J NNNNNNNNNNNNNNN 3' 3' CNNNNNNNNNNNNNNNNNNN 5" Plasmid - gRNA, Cas9 promotor (konstitutivní nebo inducibilní) Virový vektor Transfekce RNA po in vitro -transcňpá Transfekce komplexu Cas9 + gRNA 10 http://www.addgene.org/52961/ entiCRISPRv2 cPPF psu nnr lentiCRISPRv2 U6 ? kb filler ITS SpCasS l~l AG PJ'A Pwo WPRf a.77?£j' BemOi 112.flu I: hill 71 [tZ.DSJ] Hiirfll I'D.Bfrii Pnitl (SHZ) PflFI - 7M1 11J lllllll |t4,Jil| Pvul flbMCI : LEl:■ 31 Alc-E (1SH) "■el (zba) tit p. Li I £1*1 jh»1i Ntirl NUii! ■mtl' ^BKl Aval (5Mfl> Ittl] CRISPR/Cas9 http://www.addqene.org/crispr/ dostupné vektory Různé LifeTech firmy - různé platformy (modifikace původního systému) knihovny gDNA pro různé organismy validované gRNA Pro design lze využít dostupný software (více než 30 online softwarů) https://en.wikipedia.org/wiki/CRISPR/Cas_Tools http://crispor.tefor.net/ https://crispr.cos.uni-heidelberq.de/ + všechny větší i menší Lifetech firmy design CkISPk/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 Googlem X Optimized CRISPR Design T % National Center for Biotechno... X | NCEI Blast:Nucleotide Sequen... X + ^ J ^ A https://www.ncbi.nlnri.nih.gov □ e I T Google % NCBI Resources© How To© %NCBI National Center for Biotechnology Information Nucleotide human mRNA .A NCBI Home R eso li ice List (A-Z) All Resources Chemicals & Bioassays Data & Software DNA & RNA Domains & Structures Genes & Expression Genetics & Medicine Genomes & Maps Homology Literature PrntpinQ Welcome to NCBI The National Center for Biotechnology Information advances science and health by providing access to biomedical and genomic information. About the NCBI I Mission I Organization I NCBI News I Bloa Submit Deposit data or manuscripts into NCBI databases Download Transfer NCBI data to your computer Learn Find help documents, attend a class or watch a tutorial Popular Resources PubMed Bookshelf PubMed Central PubMed Health BLAST Nucleotide Genome SNP Gene Protein PubChem % NCBI Resources 0 How To 0 Sign in to NCBI ^NCBI National Center tor Biotechnology Intormation NCBI Home Resource List (A-Z) All Resources Chemicals & Bioassays Data & Software □ NA & RNA Domains & Structures Genes & Expression Genetics & Medicine Genomes & Maps Homology Literature Proteins Sequence Analysis Taxonomy Training & Tutorials Variation B-Myb human| Welcome to NCBI The National Center for Biotechnology Information advances science and health by providing access to biomedical and genomic information. About the NCBI | Mission | Organization | NCBI News & Blog Submit Deposit data or manuscripts into NCBI databases Develop Use NCBI APIs and code libraries to build applications Download Transfer NCBI data to your computer Analyze Identify an NCBI tool for your data analysis task Learn Find help documents, attend a class or watch a tutorial Research Explore NCBI research and collaborative projects Á Popular Resources PubMed Bookshelf PubMed Central BLAST Nucleotide Genome SNP Gene Protein PubChem NCBI News & Blog Save and Share in PubMed Labs! 18 Jan 2019 We've recently added save and share options to PubMed Labs. From your PnhMprl I ahc. Qpsrrh rpciiltQ list vnn -T- Improved ClinVar search quickly connects you to information about variants 17 Jan 2019 lfunii\*p hppn cpprrhinn in Plin\/pr uriii February 6 Webinar: New Variation Services for Normalizing, Remapping, and Annotating Variants 16 Jan 2019 More 14 CD A https://YiHA™,ncbi,nlrin,nih,gov/^ ^ Seznam - najdu tarn,, c. Přírodovědecká fakult. % NCBI Resources © How To 0 Nucleotide Nucleotide - B-myb human mRNA Create alert Advanced ■ The Nucleotide database will include EST and GSS sequences in early 2019. Read more. S p 6 CIS S Animals (63) Plants (7) Fungi (3) Bacteria (1) Customize... Molecule types genomic DNAIRNA (16) mRNA (53) Customize... Source databases INSDC(GenBank) (34) RefSeg (40} Customize... Sequence Type Nucleotide (52) EST (22) Sequence length Custom range-Release date Custom range... Revision date Custom range... Clear all Summary^ 20perpage-» Sort by Default order ■ Send to" Filters: Manage Filters GEI iE Was this helpful? i* MYBL2 - MYB proto-oncoqene like 2 Homo sapiens (human) Also known as: B-MYB. BMYB GenelD: 4605 <^eföeqtranscripts(2T) RefSeq proteins (2) PubMed (108) Genome Browser j BLAST Download RefSeq transcripts + RefSeq proteins + Items: 1 to 20 of 74 Results by taxon Top Organisms ITreel Homo sapiens 142! Mus musculus (15) Oryza sativa Japonica Group (Ö) Aspergillus fumigatus Af293 (3} Xenopus laevis (2) All other taxa (7) More... Find related data Database: Select Search details 3-myb[All Fields] AND ("H sapiens"[Organism] OR hum Fields]) AND mRNA]All Fie Search Page 1 of 4 Next > Last» $Í NCBI Resources 0 How To 0 Sian in to NCBI Nucleotide Nucleotide 1 Search 1 Advanced Help 1 (Q) The Nucleotide database will include EST and GSS sequences in earlv2019_ Read more. Species Summary-r Sort by Default order ^ Send to: - Filters: Manaae Filters Customize ... Molecule types mRNA [2) Customize ... Source databases RefSeq (2) Customize ... Sequence Type Nucleotide (2) Sequence length Custom range... Release date Custom range... Revision date Custom range... Clear all Show additional filters Items: 2 □ Homo sapiens MYB proto-oncoqene lite 2 (MYBL2), transcript variant 1. mRNA 1- 2.668 bp linear mRNA Accession: NM_002+66.4 Gl: 1519243668 Protein PubMed Taxonomy GenBank FASTA Analyze these sequences Run BLAST Find in these sequences Find related data Database: Select Recent activity H Turn Oft Clear Q, B-myb human mRNA (74) Nucleotide B myb human mRNA (12980) Nud-eotide Q, B myb human [13312) 3 Homo sapiens mucin 1, cell surface associated (MUC1), transcript variai Nucleotide 3 Homo sapiens mucin 1, cell surface associated (MUC1), transcript variai Nucleotide See more. 5oubor Úpravy Zobrazení Historie Záložky Nástroje Nápověda ["■""] fď~][~X~] @ Ienticrisprv2 - Hledat Googlem X Gptimized CRI5PR Design X ^ft NCBI BlastiNucleotide Sequen... X + ^ y fl https://www.ncbi,nlm.nih.gov/nuccore/NMJ0246rj,3 lve I T Google P £ z = % NCBI Resources 0 How To 0 Nucleotide Nucleotide Advanced I Search Nucleotide] GenBank^ Send: ~ Homo sapiens MYB proto-oncogene like 2 (MYBL2J, transcript variant 1, mRNA NCBI Reference Sequence: NM_002466.3 FASTA Graphics Go to: Homo sapiens ... r 2 Lentiviral CRI5... £J 5chránka05 -1... *§_ trop2_myb_cri... C5 '< B0^ 10:16 / yene—s yiiuiiyin-—u-ri i jj ,—uri i jj— /note="upstream in-frame stop codon" 2 6 6^2368 y^erfie="HYBL2" /gene_synonym="B-MYB; BMYB" /note="isoform 1 is encoded by transcript variant 1; MYB-related protein B; myb-like protein 2; v-rnyb myeloblastosis viral oncogene homo log-like 2; v-rnyb avian myeloblastosis viral oncogene homo log-like 2" /codon_start=l /product="myb-related protein B isoform 1" /protein id="NP 002457.1" /dta xref="CCD3:CCD313322■1" /db_xref="GeneID:4605" /db_xref="HGNC:HGHC:754S" /db_xref="HPRD:03247" /dh xref="MIM:601415" /1 r ans1at i o n="MS RRTRC E D L D E L HYQD TD S DVP E QRD S KC KVKWTHE E D E QL RA LVRQF GCjCjD WKF L AS HF PNRTD CjCjC CjYRWL RVLNP D LVKGP WTKE E D QKVIE L VKKYG TKQWTLIAKHL KGRL GKQC RE RWHNHLNP EVKKS C UTE E E D R11C E AHKVL GNRWAEI AKML P GRTDNAVKNHWNS TIKRKVD TGGF L S E 3 KD C KP P V YL L L E L E D KD GL QS AQP T EGQGSLLTWWPSVPPTIKEEENSEEELAAATTSKEQEPIGTDLDAVRTPEPLEEFPKR EDQEGSPPETSLPYKWVEAANLLIPAVGSSLSEALDLIESDPDAWCDLSKFDLPEEP SAEDSINNSLVCjLCjASHCjCjCjVLPPRCjPSALVPSVTEYRLDGHTISDLSRSSRGELIPI S P S TEVGGSGIGTP P SVL KRQRKRRVAL S PVTEN3T3L3FLD3CNS L TP KS TPVKTLP FSPSCjFLNFWNKCjDTLELESPSLTSTPVCS CjKWVTTP LHRDKTPLHQKHAAFVTPDQ KYSHDNTPHTPTPFKNALEKYGPLKPLPCjTPHLEEDLKEVLRSEAGIEL IIEDDIRPE KCjKRKPGLRRSPIKKVRKSLALDIVDEDVKLHHSTLPKSLSLPTTAPSNSSSLTLSGI KE DNS L LNQGF L Cj AKP E k A AV AQKP RS HF TTP AP MS S AUKTV AC GGTRD QL F MQE kAR CjLLGRLKPSHTSRTL ILS " Soubor Úpravy Zobrazení Historie Záložky Nástroje Nápověda [_ ][o1 ][X] © lenticrisprvS - Hledat Googlem X Optimized CRI5PR Design X ^fc § NCBI Blast:Nucleotide 5equen... X + / ycnc—3 yiiuTiyiLI™ Umi!l LU, actcaggccc ggagatagaa cgcgggacct gcagcccggg gcgcggcgcg gctgcactac caaatggacc ggactggaag gtggctgaga aaaagtcatc cctgaagggc ggtgaagaag gctgggcaac gaagaatcac gtccaaagac ccagagtgcc tcctaccata ggaacaggag attcccgaag ggtggtggag ggacttgatc ggaaccatct gcagcaagtc cctggatggc ctcccccagc gcagaggaag cctggattcc gccctcccag gctgacatcc caagacaccc ggacaacact gaagcccctg ggctggcatc tgggctgcgg tgaggatgtg cccttcaaac ccagggcttc cttcacgaca ggaccagctt cacatctcgg tttacagggg ctgcagggag atgtgctgcc cacttccagg aagcccacgt ggtgctcctg ctttgtgctg ctcccccggc aagtgcttca gctgacacgc tcctgacccc gtccgggccg caggacacag catgaggagg ttcctggcca gttttgaatc gagctggtta cggctgggga tcttgctgga cgctgggccg tggaactcta tgcaagcccc cagcccacgg aaggaggagg cccatcggta cgtgaggacc gcagctaacc gagtcggacc gcagaggaca ctgccacccc cacaccatct actgaagtcg aggcgtgtgg tgtaacagcc tttctgaact accccagtgt ctgcaccaga ccccacacgc ccacagaccc gaactcatca cggagcccca aagctgatga tcttccagcc ttgcaggcca cctgccccta ttcatgcagg accctcatct ttgtgggggc ccttctgcca ctgttgccga tctgcctggt caggcctggc tgctcaccct gtctggaaaa gcccgcgcgc acccgcgccg tgacgccttc ggcccggctc gggggatgtc attcagatgt acgagcagct gccacttccc cagaccttgt agaagtatgg agcagtgcccj ccgaggagga agatcgccaa ccatcaaaag cagt.gt.actt. aaggccaggg aaaacagtga cagatctgga aggaaggctc tcctcatccc ctgatgcttg gtatcaacaa gccagccttc cagacctgag ggggctctcjcj ctctgtcccc tcacgcccaa tctcjcjaacaa gcagccagaa aacatgctgc caaccccgtt cgcacctgga tcgaggacga tcaagaaagt tgtccacact tcaccctgtc agcccgagaa tgtccagtgc agaaagcccg tgtcctgagg agagggggtc ccagcccctc gcccagctcjt tccctcccca ctcatctcag ctcttggtgc aaaaa caggactggg gcggcgactg gagcgcggcc ccgctccggg tcggcggacg gccggagcag gagggccctg taaccgcact caaggggcca cacaaagcag tgaacgctgg ggaccgcatc gatgttgcca gaaggtggac gctgctggag aagtcttctg ggaggaactt cgcagtgcga cccaccagaa tgctgtgggt gtgtgacctg cagcctagtg cgccctggtg ccggagcagc cattggcaca tgtcactgag gagcacacct acaggacaca ggtggtggtc gtttgtaacc caagaacgcc ggaggacttg catcaggccc ccggaagtct gcccaagtct aggtatcaaa ggcagcagtg ctggaagacg gcagctcctg tgttgagggt tgtgaatctg cccagactct cjggcggctcc aggccacagg accctgctta atttttttgg cggcgccgac cagttcctgc cggggcccgg ctctgccggc cgctgcgagg agggatagca gtgaggcagt gaccagcaat tggaccaaag tggacactga cacaaccacc atctgcgagg gggaggacag acaggaggct ctcgaggaca accaactggc gcagcagcca acaccagagc acgagcctgc tctagcctct agtaaatttg cagctgcaag cccagtgtga cggggcgagc aatagcacca gttaagaccc ttggagctgg accacaccac ccagatcaga ctggagaagt aaggaggtgc gagaagcaga ctggctcttg ctatccttgc gaagacaaca gcccagaagc gtggcctgcg ggccgcctga gtcacgagcc agagtcattc caggtggagg tggtgctaac gagctccgtc ggatggggga aagaataaaa gcgcttggcg gagcgaggag agcggccgga gggcgggcga atctggatga agtgcaaggt ttggacagca gccagtacag aggaagacca ttgccaagca tcaaccctga cccacaaggt acaatgctgt tcttgagcga aggacggcct cctccgtccc ccacatcgaa ccttggagga cttacaagtg ctgaagccct acctccctga cgtcacatca ccgagtaccg tgatccccat tgctcaagcg gtctgtcctt tgcccttctc agagcccctc tgcaccggga agtactccat acggacccct tgcgttctga agaggaagcc acattgtgga cgacaactgc gcttgctcaa cccgaagcca gggggaccag agcccagcca cattctcatg aggtgacctc caacagggcc aacaaagttc agcttctccc tgtggccagg ttgcctctct SÍ3FÍ E3 Doručená pošt.,, r V Výkazy práce ... CRISPR r S| Navod_cvika_... O Horno sapiens ... _ Lentiviral CRIS... fíS 5chránka05 -1... Ejj trop2_rnyb_cri... CS ^B0t 10:17 Homo sapiens MYB proto-pncpqene like 2 (MYBL2), transcript variant 1, mRNA 2,66 8 toJiaeaf-mMUi Acces(jon: NM_Q02466.4^ 15192+3668 Protein P^hMprl_feiffffTomv GenBank FASTA Graphics Homo sapiens MYB proto-pncogene like 2 (MYBL2), transcript variant 2. mRNA 2,713 bp Nj>wr mUNA----- Accessior^lM_u01278610.1 ^>: 519666782 Protein Pu?rvte3—^nurffifw GenBank FASTA Graphics https://blast.ncbi.nlm. nih.gov/Blast.cgi?PRO 6RAM=blastn<£PAGE_ TYPE=BlastSearch<&LI NK LOC=blasthome tu delší sekvenci ft https://blastncbi.nlm.nih.QQv/Blastcgi?PAGE_TVPE=Blast5eaiľ^ I' Google •C? ě * * z UUJ]^ U.S. National Library of Medicíne ^ > ncbi National Center for Biotechnology Information Sign in to NCBI BLAST »blastn suite Home Recent Results Saved Strategies Help [ blastn j l>lasti> | htasti | thlastn | thlastx |_ H Enter Query Sequence h Fnjrr íi'i^rtininjiTimlľľff) gi(s), or FASTA sequence(s) jgt1 C HH_001Z78610.1^ Align Sequences Nucleotide BLAST,^? BLASTN programs search nucleotide subjects usinti a nucleotide <|uei y. more... Clear Query subrange ^ Reset page Bookmark From [ T.f Oi. upload file Jol> Title Procházet... j Soubor nevybrán. NM_001 278610:Homo sapiens MYB proto-oncogene... ■Eülaj^rJescMptive title foryour BLAST search >Qi 0 Align two or more sequences j^^^ Enter Subject Sequence Ent^p accegjP" numbers), gi(s), or FASTA sequences) j$ Subject stil».inge J From j Or, upload file Procházet... J Soubor nevybrán. Program Selection Optimize foi I (•) Highly similar sequences (megablast) O More dissimilar sequences (discontiguous megablast) O Somewhat similar sequences (blastn) Choose a BLAST algorithm # WilFAii 3> Search nucleotide sequence using Megablast (Optimize for highly similar sequences] □ Show results in a new window J Distribution of the top 2 Blast Hits on 1 subject sequences # Mouse over to see the title, click to show alignments ■ <40 Color key for alignment scores ■ 40-50 50-so ■ 80-200 B>=200 1 550 1100 1650 2200 2750 Homo sapiens MYB proto-oncogene like 2 (MYBL2), transcript variant 2, mRNA Sequence ID: NM 00127SE10.1 Length: 2713 Number of Matches: 2 Range 1: 330 to 2713 GenEank Graphics V Next Match Score 4311 bit3(2334] Expect 0.0 Identities 2334/2334(100%) Gaps 0/2334(0%) Strand Plus/Plus Query/ 452\ aaccGsoT:-A::i:-:iii:-::Ä:-ii:i:-:-i:-:-:i:-i:-i:-iiii:-jiTrrAEAc:rjiisic 511 I I 111111111111111111111111111111111111111111111111111111111111 sbjcta 330 j aaccgcacigaccagcaaigccagiaraggiggcigagagiiiigaaiccagacciigic 439 5l2 aaggggccai ggaccaaagaggaagaccaaaaagi caicgagc1gg11aa&aa&iai ggc 571 i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i 440 aa;-;-;-;-;;ai :-:-a: :aaa;-a;-;-aa;-a;;aaaaa;-t;ai;;-a;-;i;-;-ttaagaasiaigg; 499 Query sbjct Query 572 AGAaA;-"A;-iG;-A;A;i;-Aii;-;;ia;-"A;;i;-Ai;-;-;-;;;-;-;i;-;-;-;-AA;-;A;-T;-;;;-i E3i 111111111111111111111111111111111111111111111111111111111111 Sbjct 500 ACAAAGCAGIGGACACIGAIIGCCAAGCACCIGAAGGGCCGGC1GGGGAAGCAGIGCCGI 559 Query 632 GAACGCTGGCACAACCACCICAACCCTGAGGIGAAGAAGICI1GCTGGACCGAGGAGGAG 691 i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i Sbjct 560 GAACGC1GGCACAACCACCICAACCC1GAGGIGAAGAAGICI1GC1GGACCGAGGAGGAG 619 Query 692 GACCGCATCAICIGCGAGGCCCACAAGGIGCIGGGCAACCGCIGGGCCGAGAICGCCAAG 751 i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i Sbjct 620 GACCGCA1CAICI&CGAGGCCCÄCÄAGGIGCI&GGCAACCGCIGGGCCGA&AICGCCAAG 679 Query 752 AIG11GCCAGG&A&GACAGACAA1GCIGIGAA&AAICACIGGAACICIACCAICAAAAGG 211 i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i Sbjct 630 AIGTTGCCAGGGAGGACAGACSiATGCIGIGAAGAAICACIGGMCICIACCAICAAMGG 739 Query 312 AAGG1GGACACAGGAGGCIICIIGAGCGAGICCAAAGACIGCAAGCCCCCAGIGIACIIG S71 i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i Sbjct 740 AAGG1GGACACAGGAGGCIICIIGAGCGAGICCAAAGACIGCAAGCCCCCAGIGIACIIG 799 Query 372 CIGC1GGAGCICGAGGACAAGGACGGCCICCAGAGIGCCCAGCCCACGGAAGGCCAGGGA 931 i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i Sbjct 300 CIGC1GGAGCIC&AGGACAAGGACGGCCICCA&AGIGCCCAGCCCACGGAA&&CCAGGGA 359 Query 932 AGICIICIGACCAACIGGCCCICCGICCCICCIACCAIAAAGGAGGAGGAAAACAGIGAG 991 iii i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i Sbjct 360 AGICIICIGACCAACIGGCCCICCGICCCICCIACCAIAAAGGAGGAGGAAAACAGIGAG 919 Query 992 GAGGMCIIGCÄGrjAGCffiCCaCATCGMGGAACAGGAGCCCATCGGIACAGAICIGGAC 1051 i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i Sbjct 920 GAGGAACTTGCAGCAGCCACCACA1CGAAGGAACAGGAGCCCA1CGGTACAGAICIGGAC 979 Query 2672 Sbjct 2600 Query 2732 Sbjct 2fi60 CCC1GCIIAGGA1GGGGGAIG1GGCCAGGGGIGCICCIG1GCICACCC1CICIIGGIGCA I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I CCCIGCIIAGGAIGGGGGAIGIGGCCAGGGGIGCICCIGIGCICACCCICICIIGGIGCA tttttttGGAA&AAIAAAAII&CCICICICIIIGIGCIG&ICIGGaaaaaaaaa 27SS I I I I I I I I I IIIIIIIGGAAGfiAIfiÄÄAIIGCCICICICIIIGIGCIGGICIGGAAAAAAAAA 2713 Range 2: 1 to 373 BenBank Graphics a Previous Match 4 First Match Score 701 bits(379) Expect 0.0 Identities 379/379(100%) Gaps 0/379(0%) S t h a n d Plus/Plus Quer Sbjc 0 acicaggccccicccccggcgcccgcgcgccaggacigggcggcgccgacgcgciiggcg i I I I I i i i i i I I I I i i i i i I I I I i i i i i I I I I i i i i i I I I I i i i i i I I I I i i i i i i I I I i ACICAGGCCCCICCCCCGGCGC22C-; 2-2 2-2: a:-:-a2 i 2-2-2-2 2-2-2 2-22 2-a.2 2-2 2-2112-2-2 2- Query fil GGAGAIAGAAAAGIGCIICAACCCGCGCCGGCGGCGACIGCAGIICCIG!::-.-.:22: I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I Sbjct il GGAGAIAGAAAAGIGCIICAACCCGCGCCGGCGGCGACIGCAGIICCIGCGAGCGAGGAG 120 Query Sbjct Query Sbjct Query Sbjct Query Sbjct Query Sbjct 121 122 2 iL 2 42 3:2 3:2 3i2 2i2 CGCGGGACCIGCIGACACGCIGACGCCIICGAGCGCGGCCCGGGGCCCGGAGCGGCCGGA 130 i I I I I i i i i i I I I I i i i i i I I I I i i i i i I I I I i i i i i I I I I i i i i i I I I I i i i i i i I I I I CGCGGGACCIGCIGACACGCIGACGCCIICGAGCGCGGCCCGGGGCCCGGAGCGGCCGGA 130 GCAGCCCGGGICCIGAccccggcccggctcccgctccgggctctgccggcgggcgggcga 2 4 0 1111111111111111111111111111111111111111111111111111111 11111 GCAGCCCGGGICCIGACCCCGGCCCGGCICCCGCICCGGGCICIGCCGGCGGGCGGGCGA 240 gcgcggcgcggtccgggccggggggatgtctcggcggacgcgct gcgAGGAICIGGAIGA 300 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I GCGCGGCGCGGICCGGGCCGGGGGGAIGICICGGCGGACGCGCIGCGAGGAICIGGAIGA 300 GCIGCACIACCAGGACACAGA11CAGAIGIGCCGGAGCAGAGGGAIAGCAAGIGCAAGGI 360 i I I I I i i i i i I I I I i i i i i I I I I i i i i i I I I I i i i i i I I I I i i i i i I I I I i i i i i i I I I I GCIGCACIACCAGGACACAGAI1CAGAIGIGCCGGAGCAGAGGGAIAGCAAGIGCAAGGI 360 CAAAIGGÄCCCAIGAGGAG R7s\ I I I I I I I I I I I I I I I I I I I I J CAAAI C-2-A2T2AT GAGGAG \37 9/ 22 Distribution of the top 2 Blast Hits on 1 subject sequences # Mouse over to see the title, click to show alignments ■ <40 Color key for alignment scores ■ 40-50 50-80 ■80-200 B>=200 1 550 1100 1650 2200 2750 266_ atgtctcggcggacgcgctgcgaggatctggatgagctgcactaccaggacacagattcagatgtgccggagcag agggatagcaagtgcaaggtcaaatggacccatgaggagqacgagcagctgagggccctggtgaggcagtttgga cagcaggactggaagttcctggccagccacttccctaaccgcactgaccagcaatgccagtacaggtggctgaga gttttgaatccagaccttgtcaaggggccatggaccaaagaggaagaccaaaaagtcatcgagctggttaagaag - u transkriptu 2 chybi 3. exon (zeleny) bttps://www.^cbi.nlm.nih.gov/nuccore/HM 0024 6 6.4 Chci-li zacílit obě varianty mRNA - cílím na kratší variantu Homo sapiens MYB proto-ojacjogejge like 2 (MYBL2), kanscÓEt variant 1, mRNA ■ (Ctrl}- 171 181 241 301 ;ei -.2. 481 541 601 661 ":2 i 781 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 1981 2041 2i;i 2161 2221 ccacctcaac a^cjcjcttct^tg ajcgajtcca aa^actrjcaa gcccccčyjtg ta^ttg^ctgc aa^ctccaga ^t^cccagcc c^acggjyigcjc ca^g^g^aagtc ggagg^yiaac; a^ag^tcaaat gcja^cccaj^ga gga^jacgag cagctga^ggg ca^cag^jact ^gaa^ttcct ^^cca^ccac ttccctaacc t^acagtjtg^tjc t^dg^dgtt-EJi 3ááJÍ-E£áaá-E ££-E3£-Eáá33 gaccdaaaag t^dtcgagct g^ttdd^ddg^ tdto13cacaa aagcacctga a^gtj^cctjcjct ^^g^aa^cagi tgccgtgaac cct^agg^tga a^gaa^tcttg ct^gacctjacj cctOjCcttac aactccctcc tccacececc tascctcctc at_ccct_cjctcj t^g^ttctdg_ cct^tctgdd g^ccctggd^ct t^dtcgd^tc gg^accctgdt tjcty^ggj^gtcj tčKjtgca^ct gcaa^cg^tca catcajcarj^c aagtcctjcc accccgccaj ccttccgccc tggtgccca^ t^tgaccgag ta_cccjcct£tj a^tgg^ccacac catctcagac ctg^agcccjtja 3£aaccg333 c.gagct.g a t_c cccatctccc cacctjttaa g^accctg^ccc tt^tcgccct ccca^rj^tttct g^a^acttctg^ aca^cattgga g^ct^g^ajagc ccct^g^ctga catccacccc agt^tjcagc cag^aacjgtjjcj tgtjtca^ccac a^ccactgcjac cggg^caaga ca^cccctgca ccap(aaacat gct^cjtttg taaccccaja tca^gjjajjt^ac t^cat^jaca acactcccca cacgccaacc ccg^tcaaga acgccct^ga g^aa^tacgga cccctjaagc ccctg^ccaca g^accccgcac ctg^aggajjcj ag^tctctggc tcttcjacatt cjtggatgagc atgtgaagqt gat^at^tcc acactgccca ag^t^t^tatc ctt^ccg^aca a^ctg^cccctt. caaactcttc cagcctcacc E^tg^tcag^tjta gg^ccaagccc g^i_jaag^cacj g^aagccccg^a tcctg^jgccg^ cctgaajjccc apjCcacacat ^tcjjaccct ca^tcttgtcc tga Online target prediction tools _ r r . . protospacer PAM (-NGG) Of f-targets ,_i_, ^ 5'- GTGTTCATCTTTGGTTTTGTGGG 3' non-seed region seed region (12 nt) Efficiency - „pravděpodobnost Štěpení v cílovém místě" Out-of-frame score - „pravděpodobnost vzniku posunové mutace" - různé softwary různé metody/algoritmy scoringu 25 Chci-li mutovat gen —> předčasný stop kodon, obvykle volím začátek genu ... cca do 500 bp od A TG ... ale pozor na alternativní iniciační kodóny (tedy spíše ne první exon) vs nonsense mediated decay Pravidlo 50 nt - neumísťuji cílové místo štěpení blízko místa sestřihu - aby mutace nezpůsobila vznik alternativního místa sestřihu CDS ■ ■ 171..2201 /gene="MYBL2" /gene_synonym="B-MYB; BMYB" exon 191. .284 /gene="MYBL2" /gene_synonym="B-MYB; BMYB" /inference="alignment:Splign:2.1. 0" exon 285..377 /gene="MYBL2" /gene_synonym="B-MYB; BMYB" /inference="alignment:Splign:2.1. 0" exon 378.. 598 /gene="MYBL2" /gene_synonym="B-MYBj BMYB" /inference="alignment:Splign:2.1. 0" exon 599..761 /gene="MYBL2" /gene_synonym=,,B-MYB; BMYB" /inference="alignment:Splign:2.1.0" exon 762..1049 /gene="MYBL2" /gene_synonym="B-MYB; BMYB" /inference="alignment:Splign:2.1.0" exon 1050..1463 /gene="MYBL2" /gene_synonym="B-MYB; BMYB" /inference="alignment:Splign:2.1.0" 27 http://crispor.tefor.net/ Step 1 Planning a lentiviral gene knockout screen? Use CRISPOR Batch Sequence name (optional): Enter a single genomic sequence, <2000 bp, typically an exon U Clear Box- Reset to default Etc atcgagctgg ttaagaagta tjggcacaaajg cagtegacac 421 J£3ttgccaa gcacctgaag ggccggctgg ggaagcagtjg ccgtgaacgc tggcac^jacc: 481 acctcaaccc tgaggtgaag aagtcttgct ggaccgag&a Ega££acc£c ^41 agi&ccacga £gt£ctgggc aaccgctggg ccgagatcgc caggatgttjg S£SSSMSS. Text case is preserved, e.g. you can mark ATGs with lowercase. Instead of a sequence, you can paste a chromosome range, e.g. chr 1:1 1,130,540-11,130,751 sleet a genome me: pre-calculated exonic guides for this species are on the LI CSC Genome Browser. We hafre«&32_genomes, but. not yours? Search NCBI assembly and send a GCF^JSes ID to CRISPOR support. Step 3 a Select a Protospacer Adjacent Motif [PAM] | 20bp-NGG - Sp Cas9, SpCas9-HF1, eSpCas9 1.1 SUBMIT 28 io sapiens (hct19), chr20:42315492-42315712, forward genomic strand Your input sequence is221 bp long. It contains 36 possible guide sequences. Shown below are their PAM sites and the expected cleavage position located -3bp 5' of the PAM site. Click on a match for the PAM NGG below to show its20 bp-long guide sequence. [Need help? Look at the CRISPOR manual) Colors green, yellow and red indicate high, medium and low specificity of the PAM's guide sequence in the genome. Gene Models: Do not show Variant database: 11 □□□ Genomes 05-2013 v| Min. frequency: 0.0 Missing a variant database? We can add it. 0 10 20 30 40 50 60 70 80 90 100 Hi Position Variants .........................................................T...................................................... Sequence gtcatcgagctggttaagaagtatggcacaaagcagtggacactgattgccaagcacctgaagggccggctggggaagcagtgccgtgaacgctggcacaaccacctcaacc —TGG —TGG CCA--- —AGG —TGG CCG--- CCA--- CC CCT--- CCG--- ---TGG CCT--- —-GGG —GGG C ---CGG ---GGG Download for: SerialCloner (free) - ApE (free) - GenomeCompiler- Benchling- SnapGene - Geneious - Vector NTI - LaserGene - Genbanfc- FASIA 29 Position/ Guide Sequence + PAM MIT CFD Predicted Efficiency Outcome Off-targets for Genome Browser links to matches sorted by Strand n + Restriction Enzymes Specificity Spec. H 0-1-2-3^ score 0 + Variants £ Score ff score Show all scores in jz u c OJ O o £ mismatches D exons on ly D ch r2G on ly □ □nlyG- □□nlyGG- D Only A- U 11 Mor.-Mate Out-of-Fra Lindel + next to PAM U 84/ rev GGTGGTTGTGCCAGCGTTCA CGG 90 95 41 69 49 76 0-0-0-11- 3:mtergenic:RN7SKP277-PNPT1P2 4:im.ergenic:PARD3-AS1-SS18L2P1 4:intergenic:CTD-2521M 24.5-EST2 show all... A Inefficient. Enzymes: BceAl HpyWSll Mwol Cloning / PCR primers 90 0-0-0-0-0 101 off-targets 94/fw GGAAGCAGTGCCGTGAACGC TGG 90 93 56 36 59 64 0-0-0-7-93 4:intron:CDK11B 4:intergenic:CDK1 WRP1-283E3.8-RP1-283E3.8/CDK11 Enzymes: Hpy 16611 Mwol BstCSl Cloning / PCR primers 0-0-0-0-0 100 off-targets 4:exon:AC104695.4 show all... 143 / f w GAAGTCTTGCTGGACCGAGG AGG .....G. . .1.....1....... Enzymes: BssRl Taqlt BssDl Cloning / PCR primers 90 91 66 64 40 79 0-0-0-5-95 0-0-0-0-1 100 off-targets 4:imron:RAPGEF4 4:intergenic:IGHVII-26-2-IGHV7-27 4:mtergenic:AC024590.1-RP11-481 E4.1 show all... 164/fw GGAGGACCGCATCATCTGCG AGG S9 93 65 43 48 83 0-0-0-3-47 4:exon:DTX1 3:intron:DCP1A Enzymes: BshFl BstAPl Mwol PspPl Cloning / PCR primers 0-0-0-0-0 50 off-targets 4:exon:DTX2 show all... 186/rev TCTTGGCGATCTCGGCCCAG CGG Enzymes: BssYl M^AilLpnPl PspPl 87 94 70 74 67 83 0-0-1-7-55 0-0-0-1-1 4:im.ergenic:RP1 1-380l10.4-RPL5P22 4:intergenic:RNU6-596P-RP1 1-756K15.2 4:im.ron:RP11-544L8_B.4 show all... Pozor na možné mutace/polymorf ismy 30 https://cc1^p.cos.uni-heidelberg.de:8043/ be notified by email PAM type ©: Target selection I—|-S ' l±m±tat±on 51 miNNNNNNNNNNNNNNNHHHHGG 3 ' liitiitation-'-' NGG (Streptococcus pyogenes) Off-target prediction PAM MM- PAM UNNNNNNiniiiiiiiiiiiinnnnnmGG max 2 MM core target site length © : target site 5' limitation ©: target site 3' limitation 20 v NN NN max. total mismatches (J): 4v Q core length ©: 12 v| In vitro transcription © T7 *U6 O Custom. max. core mismatches 2): 2v fwd overhang: CACCG rev overhang: AAAC species 'I': J Animals J Prim homo rimate Human (Homo sapiens GRCh37/ng19) Human~{Horfio sapiens t*KChJ57hg38) Human (Homo sapiens) Ensembi V 99 * Bacteria Reset Submit _IL T33 T14 T16 T31 T36 T17 T18 T27 T25 T23 T34 T32 T20 T15 T19 T13 42315500 42315544 42315588 42315632 chr20 _ P T29 T10=_□ T9'_ T12_ T21 T11 T6 42315676 Legend for off-target site positon: MBflgHHI: I = intronic; - = intergenic Legend for the CRISPRater score: LOW efficacy (score<0 56); MEDIUM efficacy (0.56<=score<=0.74); HIGH efficacy (score>0.74) Tl out of 36 ^Previous Next> S e quenc e: GGAGGAC CGCAT CATCTGC GAGG Efficacy score by CRISPRater: 0.S1 HIGH OHgo pair fwd: CACCGGAGGACCGCATCAT CTGCG rev: AAACCGCAGATGAT GCGGTCCTCC Top 20 offiarget sites out of 27 (including on target; for full list see ids file) Coordinates strand MM targetseq PAM distanc e gene name gene id chr20:42315635-42315657 + 0 GGAGGACC[GCATCATCTGCG] AGG 0 E MYRL2 ENSG00000101057 chrl:l 56457648-156457670 - 4 CAAGGGCC[GGATCATCTGCG] AGG 2634 I MEF2D ENSG00000116604 32 84/ rev 94/fw GGTGGTTGTGCCAGCGTTCA CSG A Inefficient Enzymes: ScsAt Hpy166ll Mwol Cloning / PGR primers GGAAGCAGTGCCGTGAACGC TGG EnzyFritdi: Hjjy looll, iVlwuTBstCS! Cloning / PGR primers GAAGTCTTGCTGGACCGAGG A6G .....G. . . T.....I....... Enzymes: BseRl Taqll BseD! Cloning / PGR primers GGAGGACCGCATCATCTGCG AGG Enzymtii: bsllH Bs-lAFi, Mwol, PspPi Cloning / PGR primers 90 90 95 93 41 56 69 36 49 59 76 64 0-0-0-11 90 o-o-o-o-o 101 off-targets 0-0-0-7-93 0-0-0-0-0 100 off-targets 143 / f W 164/fw 90 91 66 64 89 93 65 43 40 79 0-0-0-5-95 0-0-0-0-1 100 off-targets 48 S3 0-0-0-3-47 0-0-0-0-0 50 off-targets Tl out of 36 Sequence: I T2 out of 36 j C^ggaggaccgcat cat ct gcgagg^> Sequence: Efficacy score by CPJSPRater: 0.81 HIGH I T3 out of 36 I ggaagcagt gccgt gaacgct gg_ I Efficacy score by CPJSPRater: 0.74 HIGH T4 out of 36 ^Previous Next> Sequence: ggt ggttgt gc cagc gt t c ac gg S e quenc e: gaagt c t t gc t ggac c gaggagg Efficacy score by CPJSPRater: 0.71 MEDIUM lEfficacy score by CPJSPRater: 0.83 HIGH 33 CPP1 psit nnr lentiCRISPRv2 U6 ? kb finer rrs SpCtiss n ag p?a Puro wpnr J_L1_L BsmB1 fl>-.7?S/ 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 lentiCRISPRv2. lentiGuide-Puro or lentiCRISPRvl Oligo 1 -5' - CACCGNNNNNNNNNNNNNNNNNNNN - 3' 3' - CNNNNNNNNNNNNNNNNNNNNCAAA -5'* Oligo 2 Example oligo design: Note that the i: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 84/ rev GGTGGTTGTGCCAGCGTTCA CGG j_ Inefficient Enzymes: BceAl HpylSStl Mwol Cloning / PGR primers 90 95 41 69 49 76 0-0-0-11-90 0-0-0-0-0 101 off-targets 94/fw GGAAGCAGTGCCGTGAACGC TGG Enzymes: ttpyl&Sll Mwol, Bs{C8l Cloning / PCR primers 90 93 56 36 59 64 0-0-0-7-93 0-0-0-0-0 100 off-targets 143/fw GAAGTCTTGCTGGACCGAGG AGG .....G. . .1.....1....... Enzymes: BssRI, TaqSl BseDl Cloning / PCR primers 90 91 66 64 40 79 0-0-0-5-95 0-0-0-0-1 100 off-targets 164/fw GGAGGACCGCATCATCTGCG AGG ^nzyme^Nfo/iH BstAPl Mwol PspPl Cloning /PCR primers 89 93 65 43 48 83 0-0-0-3-47 0-0-0-0-0 50 off-targets return to the list of all guides Guide sequence: GGAAGCAGTGCCGTGAACGC TGG Contents: • Cloning or expression of guide RNA o T7 in vitro expression from a plasmid q T7 in vivo expression from overlapping oligonucleotides ^~/gene="MY"BL2" /gene_synonym="B-MYB j BMYB" /inference="alignment:Splign:2.1.0" 671..833 /gene="MYBL2" /gene_synonym="B-MYB; BMYB" /inference="alignment:Splign:2.1.0" Homo sapiens MYB proto-oncogene like 2 (MYBL2), transcript variant 2, mRNA Sequence ID: NM 001278610.2 Length: 2596 Number of Matches: 1 Range l: 451 to 473 GenBank Graphics Score Expect Identities Gaps Strand 46.1 bits(23)_6e-04 23/23(100%)_0/23(0%)_Plus/Plus_ Query 1 GGAAGCAGTGCCGTGAACGCTGG 23 Sbjct 451 GGAAGCAGTGCCGTGAACGCTGG 473 , ,. .....—. - . - r■ ■ - ■ - ■ ~ exon 285..377 /gene="MYBL2" /gene_synonym="B-MYB; BMYB" /i n fer en ce="a 1ignment:Splign:2.1.0" exon (^"^378. .598 " 7g^rl^="MYBL~2" /gene_synonym="B-MYBj BMYB" /inference="alignment:Splign:2.1.0" exon 599..761 /gene="MYBL2" /gene_synonym="B-MYB; BMYB" /inference="alignment:Splign:2.1.0" cPPf psi* Rnr lentiCRISPRv2 U6 PkbfMcrrrS SpCas9 n AG Pí}A Puro WPRr QtrvD' DstiBI cac c ggaagcagtgc cgtgaacgc AAAC gcg tt cacggc a c tgc t tc c RE BsmBI (ESp3I) a nnnn cacacca gtgtggtGCAGAGt nnnn 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 (ev. FACS) - 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 + sekvenace 84/ rev ggtggttgtgccagcgttca CGG _l Inefficient Enzymes: BcsAt Hpy166ll Mwol Cloning / PGR primers 90 95 41 69 49 76 0-0-0-11-90 0-0-0-0-0 101 off-targets 94/fw ggaagcagtgccgtgaacgc TGG Enzymes: Hpy 16611 Mwol BslCSl Cloning / PCR primers 90 93 56 36 59 64 0-0-0-7-93 0-0-0-0-0 100 off-targets 143 / fw gaagtcttgctggaccgagg AGG .....g. . . t.....t....... Enzymes: BseRl Tsqll, BsaDl Cloning / PCR primers 90 91 66 64 40 79 0-0-0-5-95 0-0-0-0-1 100 off-targets 164/fw ggaggaccgcatcatctgcg AGG Enzymes: BgfiPl BstftHMwol PspPl Cloning /PCR primers 89 93 65 43 48 83 0-0-0-3-47 0-0-0-0-0 50 off-targets return to the list of all guides Guide sequence: GGAAGCAGTGCCGTGAACGC TGG Contents: • Cloning or expression of guide RNA o T7 in vivo expression from a plasmid o T7 in vivo expression from overlapping oligonucleotides o U6 expression from an Addgene plasmid o Direct PCR for C. intestinalis o Lentiviral vectors: Cloning with Gibson assembly o Summary of main cloning/expression primers C^»~PCR to amplify the on-target site ~~^^> • Restriction sites tor hlk validation ^ • PCR to amplify off-target sites ♦—— • Saturating mutagenesis using all guides PCR to amplify the on-target site Use these primers to amplify a genomic fragment around the on-target site: OntargetGuideRna 164fwLeft ACTGATTGCCAAGCACCTGA Tm 59.839 OntargetGuideRna164fwRight GACAGAAGACAGGGCCTTCC Tm 60.036 Genome fragment with validation primers (underlined) and guide sequence (yellow) Maximum amplicon I en j Genomic sequence chr20:42315634-42315657 including primers, genomic forward strand: ACTGATTGCCAAGCACCTGAAGGGCCGGCTGGGGAAGCAGTGCCGTGAACGCTGGCACAACCACCTCAACCCTGAGGTGA AGAAGTCTTGCTGGACCGAGGAGGAGGACCGCATCATCTGCGAGGCCCACAAGGTGCTGGGCAACCGCTGGGCCGAGATC GCCAAGATGTTGCCAGGGAGGTAAGCTGTCTTCTTGGGGGTTGGGACAGGTTCCCGGGAGGCCAGGCCCGTGTTTCTGAT GGAGGAGGGTTCCTGGGTGGGTATTGTGGTCCTGTGCTCTTGTTCTGTAACACCCAGCCTCGGCTCCAGGGCTCCCAGAG GCATGCATAGTCAGGAGGAAGGGTGGTTCCCCAGGGAAGGCCCTGTCTTCTGTC ACTGATTGCCAAGCACCTGAAGGGCCGGCTGGGGAAGCAGTGCCGTGAACGCTGGCACAACCACCTCAACCCTGAGGTGA AGAAGTCTTGCTGGACCGAGGAGGAGGACCGCATCATCTGCGAGGCCCACAAGGTGCTGGGCAACCGCTGGGCCGAGATC GCCAAGATGTTGCCAGGGAGGTAAGCTGTCTTCTTGGGGGTTGGGACAGGTTCCCGGGAGGCCAGGCCCGTGTTTCTGAT GGAGGAGGGTTCCTGGGTGGGTATTGTGGTCCTGTGCTCTTGTTCTGTAACACCCAGCCTCGGCTCCAGGGCTCCCAGAG GCATGCATAGTCAGGAGGAAGGGTGGTTCCCCAGGGAAGGCCCTGTCTTCTGTC Sequence length: 373 Cílové místo Štěpení alespoň 100 bp od primerů kvůli případným delším delecím 44 Sequencing results File Edit Options Help 3 y # -[-n #4 I—r- Open Save Export Print Next Find Enhance Sample: 90063046 igIyr'M r W/t Mutated homozygot X Second allele not detected 45 ^c.C^S S:2:^;f: 6:2:ülü: - Chroiräs i 46JC45_8:2:2i7-:_6:2:2374 - Chromas File Edit Options Help V" Open Save Export Heterozygot 2 alleles Heterozygot 3 alleles X Not a single cell clone 46 http://crispid.gbiomed.kuleuven.be/ 22: □ □ □ Ü Ľ c i c a i 2í: □ D □ □ □ g a t c i 1 100 1 ------------------ATGGCGAGGGGCTTGGATCTAGCACCGCTGCTACTGCTACTGCTGGCGATGGCGACCCGCTTTTGCACGGCTCAGAGCAACT 82 1 ATTCTACTCCACCCCACCATGGCGAGGGGCTTGGATCTAGCACCGCTGCTACTGCTACTGCTGGCGATGGCGACCCGCTTTTGCACGGCTCAGAGCAACT 100 1 ATTCTACTCCACCCCACCATGGCGAGGGGCTTGGATCTAGCACCGCTGCTACTGCTACTGCTGGCGATGGCGACCCGCTTTTGCACGGCTCAGAGCAACT 100 101 200 83 GTACATGCCCCACCAACAAGATGACGGTCTGCGACACAAATGGCCCAGGCGGGGTCTGCCAATGTCGGGCAATGGGCTCACAGGTATTGGTCGACTGCTC 182 101 GTACATGCCCCACCAACAAGATGACGGTCTGCGACACAAATG G CCCAG G CGG G GTCTG CCAATG----------GGCT CACAG GTATT GGTCGACTG CTC 190 101 GTACATGCCCCACCAACAAGATGACGGTCTGCGACACAAATGGCCCAGGCGGGGTCTGCCAAT--CGGGCAATGGGCTCACAGGTATTGGTCGACTGCTC 193 201 300 183 CACGCTAACTTCCAAGTGCCTGCTGCTCAAGGCGCGCATGAGCGCCCGGAAGAGCGGCCGCAGCCTGGTGATGCCGAGCGAGCACGCGATACTGGACAAC 282 191 CAC GCTAACTTCCAAGTGCCTGCTGCTCAAGGCGCGCATGAGC GCCCG GAAG AG CGG C CGCAGCCTGGTGATGCCGAG CGAG CACGCGATACTGGACAAC 290 19 9 CACGCTAACTTCCAAGTG CCTG CTGCTCAAGGC GCGCATGAG C GCCCG GAAG AG CGG C CGCAGCCTGGTGATGCCGAG C GAG CA CGCGATACTGGACAAC 298 301 400 283 GATGGCCTCTACGACCCGGAGTGTGACGACAAGGGCCGCTTCAAGGCGCGCCAGTGCAACCAGACCTCGGTGTGCTGGTGCGTAAACTCGGTGGGCGTGC 382 2 91 GATGGCCTCTACGACCCG GAGTGTG ACGACGAG GGCCG CTTCAAGGCG CGCCAGTGCAACC--------------------------------------- 351 299 GAT GGCCT CT ACGACCCG GAGTGTG ACGACCAG GGCCG CTTCAAGGCG CGCCA----------------------------------------------- 351 Velmi dobrý je i - https://ice.synthego.eom/#/ 48 další postup ... - eventuálně klonování zacíleného úseku genomové DNA do plazmidu vhodného - pro sekvenaci - 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 ORFfinder 5oubor Úpravy Zobrazení Historie Záložky Nástroje Nápověda Optimized CRI5PR Design Tajemnice fakulty-Vedenífak... X f ri Home - ORFfinder - NCBI ^ ^ ft 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 doesnt have query sequence length limitation, is available for Linux 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 Enter Query Sequence Enter a££e^ion number, gi, or nucleotide sequence in FASTA format ® From Choose Search Parameters Minimal ORF length (nt): |75 Genetic code: 1. Standard @ ORF start codon to use: © "ATG" only O "ATG" and alternative initiation codons O Any sense codon '® Ignore nested ORFs: □ Start Search/ Clear ^ I Submit|*j)tle3r | O Home - ORFfinder - N... El Doručená pošta - Mio... Microsoft PowerPoint... öjj trop2_myb_crispr_no... CS <,BG^' ľľ Vložíte-1i sekvenci kódující sekvence genu (od A TG) Open Reading Frame Viewer Sequence ORFs found: 10 Genetic code: 1 Start codon: 'ATG' only f 1: 1..2.1K (2.1Kbp) - 1 Find: 1 <=, c> 1 <=t CD— -> st s X Tools T (Jj Tracks 1 |100 pee 1398 |4ee pee |eee pee pee pee Ii k luee |i,2ee |i,3ee |i,4ee |i,see |i,eee |i,?ee i,see psee p k DEFfinder_l.18.145630180 G±] ITTI » — = 0RF8 ES 0RF7 ^K^H ORF10 ■ 0RF4 0... ^^^H 0RF3 ^H^H ^ |iee pee pee H 0RF6 ORFS |i,4ee |4ee pee pee pee pee pee |i k |uee |i,2ee |i,3ee |i,see psee |i,7ee i,see |i,see B-Myb Add six-frame translation track ORF I (700 aa) Display ORF as.., Mark J í-lcl I 0EF1 HSKKTRCEDLDE LHYQDTD SDVP E Q ED SKCKVKWTHE ED E QLEALVEQFGOQDWKFLASHFPNETDQQCQYEWLEVLNFD LUKGPWTKEEDQKVIELUKKYGTKQWTLIÄKHLKGELGKQ CEE EUHHHLIIF EVKKSCWT E E ED EU C E AHKVLGHEWAEI AKHL P GETDNAVKHHWNS TIKEKVD T GGF L S ESKD CKP PV YL L L E L EDKD GL Q SAQPT E GQ GS L L T HUP £VPPTIKE E EN SEEELAAATTSKEQEPIGTDLDAVETFEPLEEFPKEEDQE GS P P E T S L P YKWVE AAHL LIPAVGS S L S E ALDLIE SD PD AWCD L SKFD L P E E E S AED SIIIHS LVQ LQA3HQQQVLPPEQ PSALVP SVT EYELD GHTISD L S ES S EGE LIPISPST EVGG SGI GT P P SVLKP.Q EKEEVAL S PVT EHS T S L S FLD S CHS L T PKSTPVKTLPFSPSQFLNFTJTIKQDTLELESPSLTSTPVCS QKWVT T P LHEIiKT P LHQKHAAFVT PD QKYSHDHT PHTPT SmartBLAST ORF1 BLAST ORFÍ~] | BLAST marked set | Mark subset... Marked: 0 Download marked set as | Protein FASTA v | Label Strand Frame Start Stop Length (nt | aa) ORF1 + 1 C <1 2103 2103 I 700 ORF5 - 2 2093 1521 573 I 190 ORF8 - 2 437 42 398 I 131 ORF4 - 1 1500 1135 366 I 121 ORF7 - 2 770 522 249 I 82 ORF6 - 2 1208 1053 156 I 51 ORF2 + 2 32 127 96 I 31 ORF3 + 3 204 296 93 I 30 ORF9 - 3 1423 1337 87 I 28 '— rinc-in ■3 OT i no BLAST Database: I UniProtKB/Swiss-Prot (swissprot) B-Myb - mutovaný Open Reading Frame Viewer Vložíte-li sekvenci kódující sekvence genu (od A TG) Sequence ORFs found: 10 Genetic code: 1 Start codon: "ATG- only *J 1:1..2.1K (2.1Kbp) - I Find: v | £l C$ | [T)— = Q ffl ^ Tools - # H> Tracks $ f f 1QE1 |200 300 |400 1 ÜRF3 |6MM |700 |800 |903 |1 K |1,100 |1,200 |1,300 |1,400 |1,E00 |1,S03 11,700 |1,S00 11,900 |2 K rjRFEíndéE_l 18.15037995 0RF2 ^K^H 0RF3 diJ UU 11 ~ 1 0RF7 - ORF10 - K^^^H 0RF4 *~~ 0RF9 EM |1,400 |1,E00 L jiee 288 300 |499 |E00 1699 |790 |800 |909 |1 K 1.133 |1,200 |1,300 |1,G00 11,799 |1,S90 11,999 |2 K ORF3 (538 aa) Display ORF as.. Mark >lclI0EF3 HL P GETDIIAVKHHWIS TIKEKVD TGGFLSE SKD CKP FVYL LL E L EDKD GL Q SAQ F T E GQ GS L L TNUP SVP P TIKE E ENS E EELAAATTSKEQEPIGTDLDAVETFEPLEEFPKEEDQEGS PP E T S L PYKWVE AANL LIP AVGS S L S E ALD LIE SD PD AH CD L SKFD LPEEPSAEDSINIJS LVQ L QASHQ Q QVL P P EQ P S ALVFSVTEYELDGHTISDLSESSEGELIFISFSTEVGGSG IGT F F SVLKEQ EKEEVAL S F VT ENS T S L S F LD S CNS L T PE ST P VET LPFSPSQF LNFTffNKQD T L E L E S P S L T S T PVC S QE VWT T P LHEDKT P LHQKHAAFVT PD QEYSHDHT PHT P T P F HNALEKYGP LKP L P Q T PHLE ED LKEVL ES EAGIE L11EDD IEP EEQKPKP GL EES PIKKVEKS LALDIVD EDVKLMHS T L PKS L S L P T TAP SIIS S S L T L S GIKEDNS L Llirj GF L Q AKP EK AAVAQKP ESHF T T PAPHS SAWKTVAC GGTRDQL FIIQ EKAE SmarlBLAST ORF3 BLAST ORF3 BLAST marked set Add six-frame translation track Mark subset.. Marked: 0 Download marked set | as Protein FASTA v Label Strand Frame Start Stop Length (nt | aa) ORF3 + 3 486 >2102 1617 | S38 ORF5 - 2 2092 1520 573|190 ORF8 - 2 436 41 396 | 131 ORF4 - 1 1499 1134 366 | 121 ORF7 - 2 769 521 249 | 32 ORF6 - 2 1207 1052 156 | 51 ORF1 + 1 < <1 126 126 | 41 ORF2 + 2 203 295 93 | 30 ORF9 - 3 1422 1336 87 | 28 - nnc-in -■> O-T I -lO BLAST Database: 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 týden před začátkem cvičení na email vyučujícího dané skupinky ... 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, oligonukleotidů a sekvencí primerů pro vybraný gen stručný popis výsledků dílčích kroků cílené mutageneze z praktických cvičení - analýza výstupů ze sekvenace genomové DNA získaných klonů - závěr RNA Interference mechanismus post-transkripčního umlčování genu 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 Dirjer siR NA RISC orotoin components RISC Qr SiRNA unwinding 4 4 Activated RISC Association wrth target mRNA 4 Target rnRMA cleavage /POOOOGOOWWCS gDNA Í Pre-miRNA Pri-miRNA NUCLEUS miFVmiR* or siRNA duplex (21-25 nt long small RNA) asymmetric RISC assembly P-BODIES capped target mRNA partial homology » TRANSLATIONAL REPRESSION most animal miRNAs P-BODIES target mRNA Ago (™) perfect homology » mRNA CLEAVAGE most plant miRNAs, and siRNAs Sense Antsense Target rriRNA miRNA versus siRNA obě RNA negativně regulují translaci miRNA je endogenní, siRNA je exogénni miRNA může, ale obvykle není úplněJ9 nt) x 30-60% GC x ne cílené proti intronům, UTR a blízko ATG a terminačního kodónu x homologie pouze k cílové mRNA (BLAST search) https://rnQÍdesiqner.lifetechnoloqies.com/rnQiexpress/ hŤŤps://eujdtdnQ.com/siŤe/order/desiqnŤool/index/ĎSIRNA CUSTO https://www.invivoqen.com/sirnQwizQrd/ http: //si d i rect 2.rnai.) p/ siRNA shRNA shRNA produkovány z cxogcnního vektoru promotor polymerázy III syntéza a zpracování v buňce obdobné jako i mikroRNA Starting molecule Processing Targets Gene suppression 'i 11 n i Mir 111 i n m i i dsRNAoligo AAAA mRNA ■ V DNA plasmid (RNA pal III promoter) 1IMI|I|IM'^ ri FU i i i i i^J ShRNA I 1.....r 1111 unii íl dsRNA d eamHI Hind 111 sense 9 n t loop sntisense TERM sľiRNA/PIkl 5'- GUGCUUCGAGAUCGGACU' 'dicing' siRNA/Plk1 i, _i_^5'- GUGCUUCGAGAUCGGACUU 3- UUCACGAAGCUCUAGCCUG^_UUCACGAAGCUCUAGCCUG -5' Pol III piomoler spacer TTTTT RUA folding shRNA cleavage 51 short hairpin RHb atgtctcggcggacgcgctgcgaggatctggatgagctgcactaccaggacacagattcagatgtgccggagcag agggatagcaagtgcaaggtcaaatggacccatgaggaggacgagcagctgagggccctggtgaggcagtttgga cagcaggactggaagttcctggccagccacttccctaaccgcactgaccagcaatgccagtacaggtggctgaga gttttgaatccagaccttgtcaaggggccatggaccaaagaggaagaccaaaaagtcatcgagctggttaagaag tatggcacaaagcagtggacactgattgccaagcacctgaagggccggctggggaagcagtgccgtgaacgctgg cacaaccacctcaaccctgaggtgaagaagtcttgctggaccgaggaggaggaccgcatcatctgcgaggcccac aaggtgctgggcaaccgctgggccgagatcgccaagatgttgccagggaggacagacaatgctgtgaagaatcac tggaactctaccatcaaaaggaaggtggacacaggaggcttcttgagcgagtccaaagactgcaagcccccagtg tacttgctgctggagctcgaggacaaggacggcctccagagtgcccagcccacggaaggccagggaagtcttctg accaactggccctccgtccctcctaccataaaggaggaggaaaacagtgaggaggaacttgcagcagccaccaca tcgaaggaacaggagcccatcggtacagatctggacgcagtgcgaacaccagagcccttggaggaattcccgaag cgtgaggaccaggaaggctccccaccagaaacgagcctgccttacaagtgggtggtggaggcagctaacctcctc atccctgctgtgggttctagcctctctgaagccctggacttgatcgagtcggaccctgatgcttggtgtgacctg agtaaatttgacctccctgaggaaccatctgcagaggacagtatcaacaacagcctagtgcagctgcaagcgtca catcagcagcaagtcctgccaccccgccagccttccgccctggtgcccagtgtgaccgagtaccgcctggatggc cacaccatctcagacctgagccggagcagccggggcgagctgatccccatctcccccagcactgaagtcgggggc tctggcattggcacaccgccctctgtgctcaagcggcagaggaagaggcgtgtggctctgtcccctgtcactgag aatagcaccagtctgtccttcctggattcctgtaacagcctcacgcccaagagcacacctgttaagaccctgccc ttctcgccctcccagtttctgaacttctggaacaaacaggacacattggagctggagagcccctcgctgacatcc accccagtgtgcagccagaaggtggtggtcaccacaccactgcaccgggacaagacacccctgcaccagaaacat gctgcgtttgtaaccccagatcagaagtactccatggacaacactccccacacgccaaccccgttcaagaacgcc ctggagaagtacggacccctgaagcccctgccacagaccccgcacctggaggaggacttgaaggaggtgctgcgt tctgaggctggcatcgaactcatcatcgaggacgacatcaggcccgagaagcagaagaggaagcctgggctgcgg cggagccccatcaagaaagtccggaagtctctggctcttgacattgtggatgaggatgtgaagctgatgatgtcc acactgcccaagtctetatccttgccgacaactgccccttcaaactcttccagcctcaccctgtcaggtatcaaa gaagacaacagcttgctcaaccagggcttcttgcaggccaagcccgagaaggcagcagtggcccagaagccccga agecacttcacgacacctgcccctatgtccagtgcctggaagacggtggcctgcggggggaccagggaccagctt ttcatgcaggagaaagcccggcagctcctgggccgcctgaagcccagccacacatctcggaccctcatcttgtcc tga https://rnaidesigner.thermofisher.com/rnaiexpress/ Thermo Fisher SCIENTIFIC All Categories Q Recently Viewed Items Contact Us t- | Sign In ' Quick Order Applications & Techniques Shop All Products Services & Support About Us &% Cloud BLOCK-iT™ RNAi Designer The easiest wary to design effective RNAi molecules for yreat results See also: BLOCK-iT™ RNAi Express: Simplified online ordering of pre-designed and validated Stealth Select RNAi™ siRNA. Synthetics foi in vivo RNAi Plate-Select™: Order made-on-demand RNAi in customizable plate format at 1 nmole scale! e |i,:u,..... ......I Target Design Cgt Options: ealth RNAi™ siRNA rX^sjRNA^) miR RNAi ^shRN^ SiRNA to Stealth RNAi'" SiRNA siRNA to sliRNA Find out more about Stealth RHAi™ siRNA. the next generation RNAi molecule or read about the benefits of BLOCK-iT™ siRNA. How to Order Step 1: Enter an accession number or provide a nucleotide sequence Accession number: tide sequence: Enter only A, C, G, T, and U. See the online Help for additional information atgtrtcggcggacgcgrtgcgaggatrtggatgagrtgcartaccag tggcacaaagcagtggacactgattgccaagcacctgaagggccggctggggaa^^ aaggtgctgggcaaccgctgggccgagatcgccaagatgttgccagggaggacagacaatgctgtgaagaatM tacttgctgctggagctcgaggacaaggacggcctccagagtgc^^ atcgaaggaacaggagcccatcg^acagatrtggacgcagtgcgaacac^ rtcatccrtgrtgtgggttrtagcrtrtrtgaagccrt^ lagtcrtgccaccccgccagccticcgccrtggtgcc^ ggcattggcaüv^cgccrtrtgtgrtcaagcggcagaggaagaggcgtgtgg^ I^TI^fH^t SearchA" [^Search Q 0 Contact Us Sign In - Quick Order \§ Q help BLOCK-iT™ RNAi Designer Ttie easiest way to design effective RNAi molecules for great results See also: BLOCK-iT™ RNAi Express: Simplified online ordering of pre-designed and validated Stealth Select RNAi"™ siRNA. Synthetics for in vivo RNAi Plate Select™: Order made-on-demand RNAi in customizable plate format at Inmole scale! Target Design Options: Stealth RNAi™ siRNA s i RNA mi R RNAi (^shRNA^ siRNA to Stealth RNAi™ siRNA siRNA to shRNA 1,,, ....... muni Find out more about Stealth RNAi™ siRNA. the next generation RNAi molecule. How to Order Step 1: Enter an accession number or provide a nucleotide sequence flccgs?i"" number: ^r;_0024S6.3M OR Nucleotide sequence: Enter only A, C, G, T, and U. See the online Help for additional information Step 2: If you entered an accession number in Step 1, select regions for target design g Open reading frame (ORF) □ 5'UTR □ 3' UTR h?5ss database for Blast Human - Homo sapiens vJ^NOTE: BLAST is used to compare input sequence with sequences in the database to find unique regions against which to design RNAi targets. The databases contain representative gene sequences for that species. Blast databases were updated on May 01, 200S and the design output reflects the most up-to-date designs. |Step 4: Choose minimum and maximum G/C percentage Minimum G/C percentage: 135%.v | Maximum G/C percentage: 155% v | Istep 5: Select siRNA design options and click "RNAi Design" to design siRNA. © Default motif pattern P: Proprietary (Recommended) 0 Tuschl's motif pattern: S □ A: AA(N19)TT □ B: NA(N19)NN C: NA(RN17Y)NN □ D: NA(N18Y)NN RNAi Design ^ Reset Form Guarantee: The BLOCK-iT™ RNAi Designer is such an effective tool for the design of siRNAs that if you order the three best siRNA sequences designed by the BLOCK-iT™ RNAi Designer, we guarantee that two of them will give greater than 70% knockdown of rnRNA, given that the transfection efficiency in your experiment is at least 80%. If two or more fail to knock down your target RNA by at least 70% under these conditions, Invitrogen will design and ship a fourth siRNA to your target for free* *Please contact Invitrogen Technical Services to take advantage of this offer (800-955-6288 ext 2). Please be prepared to fax or email your order reference number, oligo sequences and data showing your transfection efficiency and knockdown. This offer is good for one free duplex per target. How to Order: You must have an account with invitrogen to order custom oligos. After your oligos have been designed and added to the order form, you must enter a valid customer number that is keyed to your account, if you do not already have an account, contact your local representative to set up an account. If you already know youi RNAi oligo sequence: Order oligos online at Order Custom Primers or by e-mail/Fax siRNA - default No. Start 5equence(DNA] Region GC% 1 517 G CCATGG ACCAAAG AGG AA ORF 52 64 2 656 CCTGAGGTGAAGAAGTCTT □ RF 47 37 3 765 GGACAGACAATGCTGTGAA ORF 47 37 954 CCGTCCCTCCTACCATAAA □ RF 52 64 5 1177 GGGTTCTAG CCTCTCTGAA 52 64 6 1557 CCTTCCTGGATTCCTGTAA ORF 47 37 7 15B7 CCAAGAG CACACCTGTTAA ORF 47 37 S 1623 CCTCCCAGTTTCTGAACTT ORF 47 37 9 1644 GGAACAAACAGGACACATT ORF 42.11 10 1756 CCAGAAACATG CTG CGTTT □ RF 47 37 siRNA - Tuschl Select No. Start Target seq jerce(DNA] Region QC% Rank1 □ 1 517 G CCATGG ACCAAAG AGG AAGA ORF 52.39 ***** □ 2 570 GCACAAAG CAGTGG ACACTGA ORF 52.39 ***** GGACAGACAATGCTGTGAAGA 47 62 ***** □ 4 1230 GGTGTGACCTGAGTAAATTTG ORF 42S6 ***** GCAGAGGACAGTATCAACAAC 47 62 n_ 6 15S5 GCCCAAG AG CACACCTGTTAA ORF 52.39 ***** GGAACAAACAGGACACATTGG 47 62 ***** r]_ S 1922 GCTGGCATCGAACTCATCATC ORF 52.39 ***** GG CTCTTG ACATTGTGGATGA 47 62 ***** iFil 10 2024 GCTCTTGACATTGTGGATGAG ORF 47 62 ***** shRNA Iselect No. Start Target sequencefDNAJ Region GC% Rank1 □ 1 517 G CCATGG ACCAAAG AGG AAGA ORF 52.39 ***** 2 57D GCACAAAG CAGTGGACACTGA ORF 52.39 ***** □ 3 765 GGACAGACAATGCTGTGAAGA 47 62 ***** 4 1230 GGTGTGACCTGAGTAAATTTG ORF 42.86 ***** □ 5 1271 GCAGAGGACAGTATCAACAAC 47.62 6 15S5 GCCCAAGAG CACACCTGTTAA ORF 52.39 ***** 7 1644 GGAACAAACAGGACACATTGG 47 62 ***** 8 1922 GCTGGCATCGAACTCATCATC ORF 52.39 ***** 9 2023 GG CTCTTGACATTGTGGATG A 47.62 ***** □ 10 2024 GCTCTTGACATTGTGGATGAG ORF 47.62 ***** http://sidirect2.rnai.jp/ ... pro design siRNA Si DirGCt VerSiOn 2.0 highly rffrrt\vr. target .ipRrifir BiRMA nnling Hasign s\ta. (Help) ber and retrieve sequence: retrieve sequence e sequence: >aaxiple sequence ggctgccaag aacctgcagg aggcagaaga atggtacaaa tccaagtttg ctgacctctc tgaggctgcc aaccggaaca atgacgccct gcgccaggca aagcaggagt cc-actgagta ccggagacag gtgcagtccc tcacctgtga agtggatgcc cttaaaggaa ccaatgagtc cctggaacgc cagatgcgtg aaatggaaga gaactttgcc gttgaagctg ctaactacca agacaetatt ggccgcctgc aggatgagat tcagaatatg aaggaggaaa tggctcgtca ccttcgtgaa taccaagacc tgctcaatgt taagatggcc cttgacattg agattgccac ctacaggaag c-tgctggaag gcgaggagag craggatttc-t ctgcctcttc caaacttttc ctccctgaac ctgagggaaa ctaatctgga ttcactccct ctggttgata cccactcaaa aaggacactt ctgattaaga cggttgaaac tagagatgga caggttatca ac-gaaacttc tcagcatcac gatgaccttg aataaaaatt gcacacactc agtgcagcaa tatattacca 68 Soubor Úp ra vy Zo b razit Hi zt o ri e Zá 1 ožky N á stroj e N á p o věd a 1 1 ■=. i a ^ Open source design algorithn" X W siDirect X Homo sapiens MYB proto-one X + ■ -> c ft | 0 & sidirect2.rnaijp ■■■ © ir Q, Vyhledal lll\ El 8 if = O1 Nejnavštěvovanejší Q Jak začít tcagcatcac gatgaccttg aataaaaatt gcacacactc agtgcagcaa tatattacca design siRNA O ptinnC|Tclicrcherg^ ^ nctional siRNA selection algorithm by H\ Ui-Tei etaf., Nucleic Acids Res 32, 936-948 (2004)\rik [hide options] □ Reynolds et sL, Nat Biotechnoi 11t 326-330 (2004) Link □ Amarzguioui eta/,, BBRC 316, 1050-1053 (2.004) Link use combined rule: Ui-Tei -I- Reynolds + Amarzguiou Ui-Tei -I- Reynolds * Amarzguiou Ui-Tei x Reynolds * Amarzguiou Minimization of seed-dependent off-target effects Seed-duplex stability: Max Tm 21.5 aC new! (for reducing ■ ■ ■ I ■ I■ |■ ■ 11■ I■ n| ptH Imi i|i I I IT i I'l Ui-Tei eta/,, Nudeicteif£0fies36t 7100-7109 (200&) Link Specificity check: Homo sapiens [human) non-redundant database ▼ © □ Hide le^-spe^rfa^sjRNAs [hide options] hi Show number of off-target hits within three mismatche Other options Target range: from start to end Avoid contiguous G's or C's 4 ▼ nt or more (for chemically synthesized siRNA) ^jtlf^^oid contiguous A's or T's 4 T nt or more (for shRNA vectors with pol III promoter) □ GC content: from 0 % to 100 % Custom pattern: NNGNNNNNNNNNNNNNNNNNNNN © □ Exclude pattern: \y show siRNAs that match all checked criteria •siDirect v.2.0 | Last modified on Dec 3, 2009. Reynolds Hi9h| S k»—■ Off-target reduced siRNAs l™ am™* 3 cs & v o ^ r < • % *r 4» 10:47 4.2.2020 Si Dir6Ct VerSiOn 2.0 highly effective, target specific siRNA online design site. (JJ^P) Enter an accession number and retrieve sequence: NM 002466 retrieve sequence or Paste in a nucleotide sequence: >HH_0024 F-c. 4 Home aapien.3 Hl£3 p::ctc-c:iccge:ie like 2 IMYBL2), transcript variant I, m3NA GACTGCAGI I CCTGCGAGCGAGGAGCGCGGGA CCTGCTGA CA CGCI GACGCCTI CGAGCGCGGCCCGGGG CCCGGAC-CGGCGGGAC-CAGCCCGGGrCCrGACCCCGGCCCGGClCCCGCTCCGGGCrCIGCCGGCC-GGCG GGCGAGCGCGGCGCGGTCCGGGCCGGGGGGAIGTCTCGGCGGACGCGCTGCGAGGArcrGGATGAGCrGC ACTACCAGGACACAGAIICAGATGIGCCGGAGLAGAGGGATAGCAJiGr&CAA> CAAATGGACC CAT GA &GAG&ACGAGCAGCIGAGGGCCCTGGIGA&GCAGXTIGGACAGCAGGACXGGAA&IICCT GGCCAGCCAC IrCCCTAACCGCACTGACCAGCAAIGCCAGXACAGGIGGCIGAGAGT TIT GAAT CCAGACCITGI CAAGG GGCCATGGACCAAAGAGGAAGACLAAAAAGXCA.rCGAGCrGGTTAAGAAGrArGGGACAA^ ACI GAirGCCAA&CACGI GAAGGGCCGGCT G&C-GAA&CAC-rKCCTGAACGCrC-C-raC^J^ CCTGA&GI GAAGAAGTCIrGCTGGACCGAG&AGGAG&ACCGCATCArCTGCGAGGCCCACAA>GCT &G &CAACCGCIGGGCCGA&AT CGCCAAGAT GTIGCCAGGGAGGACAGACAAT GCT GT GAAGAAICACIGGAA CICTACL^.TCAAAAGGAAGGrGGALACAGGAGGCrXCXXG^ TACTXGCIGCTGGAGCICGAGGACAAGGACGGCCTCCAGAGIGCCCAGCCCACGGAAGGCCAGGGAAGrC XTCTGACCAACT&GCCCTCCGXCCCTCCXAC^ Jeg£!ACC^-CA^a^AA&&AACAGGA&CCCAI CGGTACAGATCIGGACGCAGIGCGAACACCAGAGCCCT TG ft i-1-1-- Ti T, t- t" i-" i" design siRNA -_J -•- ■-■ -_J i_- t t "v _-t. --«-t ----- 70 si Direct Version 2.0 re&ult page. (lill) 2020-02-04 18:55:53. siDirect v.2.0 Query (Ju cry na me: N M_0 024S6.4 Homo sapiens MV B p ro Co - o n c o g en e I i ke 2 [ MY BL2), tra nscriptvariant 1, m RN A Query sequence: 2668 bp Functional siRNA selectfon: Ui-Tei + Reynolds + Amarzguioui Seed-djplex stability - MaxTm; 21.5CC Specif icity check: Homo sapiens non-redundant database Avoid contiguous A'5 nrT's: 4 nt Effective siRNA candidates target position target sequence 21nt target + 2nt overhang F.N A oligo SEquEncES 21nt guide (5r—3') 2 Lnt passenger (53') 1562-1684 CAGAAACATGCTGCGTTTGTAAC UACAAACG CAG CAU G U U U CU G GAAACAU GCUGCGUUUG UAAC functional siRNA selection: tiji-Tei Reynolds Am a rzg u i o u i URA □ seed-duplex stabilty (Tm); specificity check: minimum number of mismatches against a ny o ff- ta rg ets; guide passengEr guide passengEr 19.8 "C 12.1^ 2.[detailJ 3 [detail] contiguous A's arT's constraint ok ale shRNA ??? www.invivogen.com/sirnawizard/ ^InvivoGen siRNA Wizard Software Fl NDSIRNA SEQUENCES ADVANCED SEARCH DESIGN HAIRPIN INSERT SCRAMBLE SI RNA SELECTI ON CRITERIA SIRNA DESIGN GUIDELINES Selection of si RNA/shRN A tar gets InvivoGen's siRNA Wizard1" is a software designed to help you select si RNA/shRN A sequences targeting your gene (s) of interest. This program selects siRNA/shRNA sequences that match criteria suggested by studies of RNA interference and which will have the best expression rate in psiRNA vectors, si RNA Wizard™ is composed of 3 parts: Find siRNA/shRNAsequence: Two types of sea rches can be performed to find s iRhJA/s hRN A sequences: Standard search utilizes a default set of criteria to analyze your gene of interest and provide the best sequences to silence gene expression. Advanced search lets you manually set the criteria for selecting the sequences against your target gene. 72 Motif size: Search 6 siRNA candidate target sequences of 21 nt found : There isnon putative siRNA without homology to other genes. Check the list of homolog genes to each putative siRNA to choose siRNA corresponding to your needs. Sequence Start GC36 # of Blast homologs Design Scrambled GGGAT AGCAAGTGCAAGGT CA 77 5Z38 3 □ □ GGATAGCAAGT GCAAGGTCAA 78 47.62 4 □ □ GCCCAAGAGCACACCTGTIAA 1320 5Z38 H □ □ GGCTGGCAICGAACT CATCAT 1656 5Z38 2 □ □ GGAAGT CT CT GGCT CTTGÄCA 1748 52.38 4 \ D / GAAGTCTCTGGCTCTTGACAT 1749 47.62 5 \ B / □ For generating the hairpin insert: Loop sequence: TCAAGAG psiRNA Vector for cloning: psiRNA-h75K Gl (cloning sites: Bbsl/Bbsl) For gene rati ng scrambled si rna, select database: human Design hairpin Generate scrambled siRNA 73 portals.broadinstitute.org/gpp/public/seq/search PrÄf? ™" GPP Web Portal Home | Search by Gene | Search by Clone Design Hairpins to Target a Transcript Sequence Scoring of candidate shRNAsequences available in 2 ways: 1. If the desired transcript is listed in NCBI RefSeq, you can find hairpin designs by: * Searchina for an NCBI gene ortranscript here or selecting "Search by Gene" from the Navigation bar above * Clicking on the "Transcript ID" link on the Search Results page to navigate to the "Transcript Details" page * Underthe "Hairpin Candidate Sequences" heading, clicking the linktitled "Show high scoring hairpin designs for this transcript' This will yield a table of the top shRNAdesigns for this transcript, pre-scored for predicted knockdown efficacy and for specificity vs. other genes of the same taxon. 2. If the desired target transcript is not found in NCBI RefSeq, or is from a taxon otherthan mouse or human (e.g. Rat) you can paste the sequence you wish to target in the box below (in DNAor RNA form), and receive a list of matching shRNA sequence designs, scored according to predicted knockdown efficacy. Note that specificity versus othertargets not is not considered in this score. For a description of the candidate-picking process and rules, please see the following link: shRNAdesiqn process Target transcript sequence (in DNAor RNA form): e.g. 'AC GTATTG ATG CCACAGAC GTATTG ATG CCACAGAC GTATTG ATG C C AC AG', etc. 74 web.stanford.edu/group/markkaylab/cgi-bin/ shRNA CUg 5'- GCU GCAAACAL>CCGACUGAAC: a 3 -UUCGACGUUUGUAGGGUGAC Dicer processing I Guide strand uucgacgu tail 3're°lon«ntrai 3'- ÜucgacguuüguAggcugac 5' Kay Lab siRNA/shRNA/Oligo Optimal Design shRNA (short hairpin RNAs) are artificial constructs that can be expressed endogenously, These expressed hairpins fold to form dsRNA, and Drosha and Dicer then act on these hairpins to create mature sequence,, used by the RISC complex to target genes, siRNA/shRNA/oligo Optimal Design considering off-target: Besides intended gene down-regulation, shRNAs can introduce off-target gene silencing. Our program design shRNA with higher on-target potency, and less off-target effect. Unlike many traditional design programs, which focus on seed complementarity, we systematically study the influence of different nucleotides in each shRNA guide strand position on unintended gene silencing, and finally assign a score matrix for determining the degree of off-target effect. When scanning a target sequence, This program calculates the score for every possible shRNA, and ranks them from highest to lowest score, Ranked top shRNAs are then selected for on-target potency, and less off-target effect, Please cite: Gu S, Zhang Y, Jin L, Huang Y, Zhang F, Bassik MC, Karnprnann M, Kay MA. Weakbase pairing in both seed and 3' regions reduces RNAi off-targets and enhances si/shRNA designs. Nucleic Acids Res. 2014 Sep 30. pii: gku854. Stepl: Providing sequence information (please provide either gene accession numbers or seguence in fasta format) 1.1 Accession Number species Human v accession number (maximum of 20, comma separated) 75 pRNA-U6.1/Neo (5078 bp) B-Myb shRNA BamH I f 5' GATCCGGCCATGGACCAAAGAGGAATTCAAGAGATTCCTCTTTGGTCCATGGCrTTTTTT GG 3' I Sense | Loop | Antisense \l Termination Signal 5'AGCTCCAAAAAAGCCATGGACCAAAGAGGAA7CrCrrG>4>4TTCCTCTTTGGTCCATGGCCG 3' Hind III BamH I GATCCGGCCATGGACCAAAGAGGAATTCAAGAGATTCCTCTTTGGTCCATGGCTTTTTT GG GCCGGTACCTGGTTTCTCCTTAAGTTCTCTAAGGAGAAACCAGGTACCGAAAAAACCTCGA Hind III ligace, transformace E. coli ligaČní směsí, expanze klonů, izolace plazmidové DNA, sekvenace další postup ... přenos plazmidu do eukaryotických buněk (přechodná transfekce) analýza exprese cílového genu na úrovni proteinu (westernův přenos) Organizace cvičení (shRNA) - skupiny po 12 lidech (2x 6 hod praktických cvičení) Každá skupina ... před praktickou částí cvičení... - přidělen jeden cílový gen (stejný jako pro CRISPR) návrh siRNA/shRNA sekvence (miniprojekt) - zaslání nejpozději týden před začátkem cvičení na email vyučujícího dané skupinky ... po praktické části cvičení... - výsledky získané na cvičeních budou zpracovány do tohoto miniprojektu Miniprojekt (.doc) stručný úvod + cíl detailní popis návrhu gRNA a siRNA/shRNA sekvencí pro vybraný gen stručný popis výsledků z praktických cvičení: a) dílčích kroků mutageneze cílového genu b) dílčích kroků posttranskripČního umlčování cílového genu analýza výstupů ze sekvenace genomové DNA získaných klonů závěr Cílové geny Mus musculus!!!!!!!!!!!! Tacstd2 (Gene ID: 56753) - 10. 3. 2022 - Knopfová (knopfova@sci.muni.cz) - Myb (Gene ID: 17863) - 17. 3. 2022 - Knopfová (knopfova@sci.muni.cz) - Mybl2 (Gene ID: 17865) - 24. 3. 2022 - Beneš (pbenes@sci.muni.cz) - Cxcll (Gene ID: 14825) - 31. 3. 2022 - Beneš (pbenes@sci.muni.cz) - Icaml (Gene ID: 15894) - 7. 4. 2022 - Kohoutek (jiri.kohoutek@sci.muni.cz) - Tnfsf9 (Gene ID: 21950) - 21. 4. 2022 - Kohoutek (jiri.kohoutek@sci.muni.cz) - CDK13 (Gene ID: 69562) - 28. 4. 2022 - Navrátilová (22031@mail.muni.cz) - ILla (Gene ID: 16175) - 5. 5. 2022 - Navrátilová (22031@mail.muni.cz) Přihlašování v ISu dnes od 17.00