Cas9 angl CRISPR associated protein 9 CRISPR asocijovanij bilok ce kerovana za dopomogoyu RNK gidiv endonukleaza pov yazana z adaptivnoyu imunnoyu sistemoyu CRISPR angl Clustered Regularly Interspaced Palindromic Repeats u ryadu bakterij zokrema Streptococcus pyogenes S pyogenes vikoristovuye Cas9 dlya zapam yatovuvannya 2 podalshogo rozpiznavannya ta rozrizannya chuzhoridnoyi DNK 3 napriklad DNK bakteriofagiv abo plazmid Kristalichna struktura S pyogenes Cas9 v kompleksi z sgRNA i jogo cilovoyi DNK pri rozdilnij zdatnosti 2 5 A hypothetical protein SPy 1046IdentifikatoriSimvoliSPy 1046hypothetical proteinSPy1046Zovnishni ID GeneCards 1 OrtologiVidi Lyudina MishaEntrez901176 n dEnsembl n d n dUniProt Q99ZW2 n dRefSeq mRNK n dn dRefSeq bilok NP 269215n dLokus UCSC n dn dPubMed search 1 n dVikidaniDiv Red dlya lyudejCas9 rozpiznaye chuzhoridnu DNK rozkruchuyuchi yiyi i viznachaye yiyi komplementarnist do dvadcyati sparenih osnov spejsera keruyuchoyi RNK Yaksho substrat komplementarnij keruyuchij RNK Cas9 rozsheplyuye chuzhu DNK U comu sensi mehanizm CRISPR Cas9 maye ryad paralelej z mehanizmom RNK interferenciyi RNAi u eukariot Bezpeka praktichnogo zastosuvannya danogo metodu viznachayetsya v tomu chisli i tim faktorom chi ye shukana poslidovnist dvadcyati sparenih osnov unikalnoyu v DNK sho modifikuyetsya Zmist 1 Vikoristannya Cas9 v genniyi inzheneriyi 1 1 MAGESTIC 2 Zastosuvannya dCas9 v epigenetici 3 Novi sposobi dostavki Cas9 v klitinu 4 Div takozh 5 PrimitkiVikoristannya Cas9 v genniyi inzheneriyi RedaguvatiKrim svoyeyi pochatkovoyi roli v bakterialnomu imuniteti bilok Cas9 aktivno vikoristovuyut dlya stvorennya tochkovih rozriviv podvijnoyi spirali DNK taki rozrivi mozhut prizvoditi do inaktivaciyi geniv abo stvorennya geterologichnih geniv za dopomogoyu z yednannya negomologichnih kinciv i vidpovidnoyi gomologichnoyi rekombinaciyi Razom z bilkami ZFN i TALEN Cas9 staye vazhlivim instrumentom v sferi redaguvannya genomu Bilsh togo stvorena tehnologiya sho dozvolyaye vnositi tochkovu mutaciyu ne rozrizayuchi lancyug DNK a shlyahom peretvorennya odniyeyi nukleotidnoyi osnovi na inshu 4 nbsp Mehanizm viborchogo ingibuvannya transkripciyi za dopomogoyu dCas9 shlyahom sterichnoyi pereshkodiDo 2012 roku Cas9 otrimav shiroku populyarnist tomu sho vin dozvolyaye rozrizati praktichno bud yaku nukleotidnu poslidovnist komplementarnu keruyuchij RNK 3 Oskilki vibirkovist Cas9 bazuyetsya na komplementarnosti keruyuchoyi RNK i DNK a ne modifikaciyi samogo bilka na vidminu vid vipadkiv TALEN i ZFN dlya novih DNK mishenej mozhlive viroblennya specifichnih Cas9 5 Variaciyi Cas9 yaki zv yazuyut DNK ale ne rozrizayut yiyi dCas9 mozhut buti vikoristani dlya dostavki transkripcijnih aktivatoriv abo represoriv do specifichnim poslidovnostej DNK z metoyu regulyuvannya transkripcijnoyi aktivaciyi i represiyi 6 7 Hocha prirodnij Cas9 vimagaye skladannya keruyuchoyi RNK z dvoh dokorinno riznih RNK CRISPR RNK crRNA i trans aktivacijnu RNK tracrRNA 3 nacilyuvannya Cas9 bulo sprosheno za dopomogoyu viroblennya yedinoyi himernoyi keruyuchoyi RNK Peredbachayetsya sho Cas9 mozhna bude vikoristovuvati dlya zmini genomu cilih populyacij organizmiv 8 U 2015 roci za dopomogoyu Cas9 vpershe buv modifikovanij genom lyudskogo embriona 9 Rozrobleno tehnologiyu immunogenomnoj inzheneriyi gibridom sho dozvolyaye shvidko pereprogramuvati specifichnist yih antitil za dopomogoyu Cas9 10 MAGESTIC Redaguvati Stvorena tehnologiya yaka dozvolyaye redaguvati okremi literi DNK i RNK sho dozvolit likuvati vrodzheni zahvoryuvannya viklikani tochkovimi mutaciyami 11 Stvorena tehnologiya MAGESTIC multiplexed accurate genome editing with short trackable integrated cellular barcodes yaka ne tilki rozsheplyuye DNK ale she i dostavlyaye do miscya rozrivu shmatok DNK neobhidnij dlya tochnoyi zamini za dopomogoyu gibridnogo zv yazannogo DNK bilka LexA Fkh1p sho pidvishuye tochnist i efektivnist redaguvannya 12 13 Zastosuvannya dCas9 v epigenetici Redaguvati nbsp Varianti Cas9 yaki zv yazuyutsya z DNK ale ne rozrizayut yiyi mozhut buti vikoristani dlya stvorennya shtuchnih faktoriv transkripciyi dlya viborchogoirkovogo regulyuvannya transkripcijnoyi aktivaciyi ta represiyiPoyednavshi inaktivovanu molekulu dCas9 yaka zv yazuye DNK ale ne rozrizaye yiyi z nukleazami FokI vdayetsya otrimati nukleazi i restriktazi dlya visokospecefichnogo rozrizannya DNK 14 15 16 Rozrobleno takozh sposib viborchogo epigenetichnogo pereprogramuvannya aktivnosti geniv za dopomogoyu inaktivovanoyi molekuli dCas9 z yednanoyi z fermentom sho zdijsnyuye demetilyuvannya DNK 17 Prichomu take pereprogramuvannya epigenomu mozhna provoditi navit in vivo 18 Piznishe z yasuvalosya sho yaksho vkorotiti keruyuchu RNK do 14 15 nukleotidiv to molekula Cas9 vtrachaye zdatnist rozrizati DNK 19 20 Vikoristovuyuchi cyu vlastivist vdalosya stvoriti sistemu dlya viborchoyi aktivaciyi pevnih geniv in vivo i pereviriti yiyi efektivnist shlyahom likuvannya mishej zi zmodelovanimi zahvoryuvannyami U cogo metodu ye tilki odna problema zazvichaj sistema CRISPR zavantazhuyetsya v neshkidlivij virus yakij nazivayut adenoasociovanim virusom AAV yakij perenosit sistemu v klitinu Ale ves bilok sho skladayetsya z dCas9 i keruyuchoyi RNK zanadto velikij shob pomistitisya v odin AAV Shob virishiti cyu problemu doslidniki zavantazhili dCas9 v odin virus a keruyuchu RNK v inshij Novi sposobi dostavki Cas9 v klitinu RedaguvatiOsnovnimi vimogami do sistemi dostavki Cas9 krim visokoyi efektivnosti dostavki ye konstrukciya sho sintezuye Cas9 ne povinna vbudovuvatisya v genom klitini i ne povinna buti v klitini postijno shob ne zavazhati yiyi roboti i ne sprovokuvati imunnih reakcij sposib dostavki povinen buti zdatnij vmistiti dosit velikij za rozmirami ferment Cas9 abo jogo koduyuchu mRNK a takozh odnu abo kilka keruyuchih RNK voni povinni buti zruchni dlya vikoristannya u viglyadi in yekcij takij sposib razom z Cas9 i keruyuchimi RNK maye buti dosit legko vidtvoryuvanim dlya velikomasshtabnogo virobnictva likarskih preparativ dlya borotbi z poshirenimi hvorobami Takim kriteriyam na vidminu vid virusnih sistem dostavki vidpovidayut lipidni nanochastinki Tak napriklad bula stvorena biodegraduyucha sistema dostavki Cas9 lipidnoyu nanochastkoyu yaka dozvolila pislya odnorazovogo vvedennya dosyagti rivnya ponad 97 ingibuvannya odnogo z bilkiv sirovatki krovi in vivo Take odnorazove vvedennya nezvazhayuchi na timchasovij harakter sistemi dostavki i komponentiv sistemi redaguvannya prizvodilo do trivalogo prignichennya sho prodovzhuvalosya protyagom 12 misyaciv 21 Div takozh RedaguvatiCRISPR Endonukleazi restrikciyiPrimitki Redaguvati Human PubMed Reference Heler Robert Samai Poulami Modell Joshua W Weiner Catherine Goldberg Gregory W Bikard David Marraffini Luciano A 12 bereznya 2015 Cas9 specifies functional viral targets during CRISPR Cas adaptation Nature 519 7542 s 199 202 ISSN 1476 4687 PMC PMC4385744 PMID 25707807 doi 10 1038 nature14245 Arhiv originalu za 10 sichnya 2019 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