Riba:
- Ƙarfin fahimtar ra'ayoyin CRISPR, gRNA (jagora RNA), PAM da abubuwan da ba su da niyya kuma suna da hankali na wucin gadi suna samar da amintaccen jerin ɗan takarar gRNA da lambar ƙira.
- Ikon tabbatar da 'yan takarar gRNA da kansu da aka ba su ta hanyar basirar wucin gadi tare da ƙwararrun kayan aikin bincike-bincike da daidaitawar kwayoyin halitta.
- Ikon karɓar iyakoki na ɗabi'a / biosafety kuma cewa bai kamata a aiwatar da ƙirar ba tare da tabbatarwar dakin gwaje-gwaje ba.
CRISPR-Cas9 (tsarin gyare-gyaren kwayoyin halitta wanda ya dace da kwayoyin cuta don yankewa da gyara DNA a wani wuri da aka yi niyya) ya canza ilimin kwayoyin halitta: yanzu yana yiwuwa a canza wani takamaiman batu a cikin kwayar halitta. Amma wannan ikon ba shi da tausayi ga kurakuran ƙira: jagorar da ba daidai ba zaɓaɓɓen RNA (gRNA - ɗan gajeren jerin RNA wanda ke jagorantar Cas enzyme zuwa manufa) na iya yanke kwayar cutar da ba ta dace ba ko kuma yanke manufa mara niyya. A cikin wannan rukunin, zaku koyi yadda ake amfani da hankali na wucin gadi (AI) a matsayin mataimaki a cikin ƙirar CRISPR, ƙirƙirar tsari, lambar rubutu, da bayyana zaɓuɓɓuka; amma za ku koyi dalilin da yasa kuke buƙatar tabbatar da zaɓin gRNA da bincike-bincike tare da kayan aiki na musamman da gwaji.
Gargadi mai mahimmanci: Kar a tambayi AI kai tsaye "ba ni jerin gRNA na wannan kwayar halitta" kuma kar a yi amfani da jerin mai shigowa kamar yadda yake. LLM na iya samar da gRNA wanda bai dace da ka'idar PAM ba (a ƙasa) ko wanda ya dace da wani wuri a cikin kwayoyin halitta, ko ma gRNA da aka yi gaba ɗaya. Hanyar da ta dace: Ƙirƙirar dabarun ƙira tare da AI, samar da ƙima da ƙima ga 'yan takara tare da kayan aikin ƙirar CRISPR na musamman (kamar CRISPOR, CHOPCHOP) da kuma tabbatar da su ta gwaji.
Ka'idoji na asali
- Cas9: Enzyme (almakashi na kwayoyin halitta) wanda ke yanke DNA. Yana haifar da hutun madauri biyu inda gRNA ke jagoranta.
- gRNA / sgRNA: Jagorar kusan 20 nucleotides waɗanda suka dace da DNA da aka yi niyya kuma suna jigilar Cas9 a can.
- PAM (Protospacer Adjacent Motif): Gajeren jeri wanda dole ne a kasance kusa da abin da Cas9 zai yanke. Don SpCas9 yawanci "NGG" (N kowane tushe ne).
- Ingancin kan-manufa: yadda ingantaccen gRNA ke yanke maƙasudi (ana auna ta ƙimar tsinkaya).
- Kashe-manufa: gRNA yana ɗaure kuma yana yanke wasu maki a cikin kwayoyin halittar da suka yi kama da manufa; wanda ba a so da haɗari.
- HDR / NHEJ: Hanyoyin gyaran salula bayan yanke; NHEJ gabaɗaya yana barin ƙananan indels, HDR yana yin daidaitattun canje-canje zuwa samfuri.
Mataki zuwa mataki: AI-taimakawa ƙirar ƙirar CRISPR
1. Bayyana manufa da manufa. Wane nau'in halitta, wanne exon, wane nau'in canji (ƙwaƙwalwa ko gyara daidai)? Amintaccen sigar genome da kwafi.
2. Samo jerin daga tushen abin dogara. Zazzage jerin yankin da aka yi niyya daga Ensembl/NCBI; Kar a sanya AI ya haddace shi.
3. Ƙirƙirar 'yan takarar gRNA tare da kayan aiki na al'ada. Kayan aiki irin su CRISPOR/CHOPCHOP duba PAM, suna ba da maki akan manufa da kuma nemo maƙasudi-fadi-genome. Yi amfani da AI don fassara fitowar waɗannan kayan aikin.
4. Dauki kasadar manufa da gaske. Kawar da ƴan takarar da ke da maki mai yawa a waje; Tabbatar da kayan aiki da yawa a aikace-aikace masu mahimmanci.
5. Shirin tabbatar da gwaji. Tabbatar da gwaji (sequencing, T7E1, amplicon sequencing) ko gRNA da aka zaɓa a zahiri ya yanke manufa (kuma a kashe-manufa). Makin ƙira shine ƙididdigewa, ba hujja ba.
Tukwici: Kafin amfani da ɗan takarar gRNA, BLAST jerin sa akan kwayoyin halitta don ganin inda ban da manufar da ya yi daidai da daidai ko kusa. Wannan mataki kadai ya kawar da mafi hatsarin ƴan takara da wuri.
uku mini lokuta
Case 1 - gRNA ba tare da PAM ba. Ɗaya daga cikin ɗalibi ya tambayi AI don gRNA don kwayar halitta kuma ya sami jerin ma'ana na nucleotides 20. Duk da haka, babu NGG PAM kusa da manufa; Don haka Cas9 ya kasa yanke wurin. Lokacin da ɗalibin ya shiga yankin cikin CRISPOR, ya ga cewa kayan aikin ya ba da shawarar ingantattun 'yan takara tare da PAM. Idan aka yi amfani da jerin AI, gwajin ba zai yi nasara ba gaba ɗaya.
Case 2 - Sirrin kashe manufa. Wani mai bincike yana son "high-inficacy" gRNA wanda AI ya ba da shawara. Lokacin da ya gudu CRISPOR don bincika kashe-manufa, ya gano cewa gRNA ya dace da bambance-bambancen tushe guda 1 kawai a cikin wani kwayar halitta - babban haɗarin kashe manufa. An cire dan takarar. Idan ba tare da ƙididdige ƙididdiga ba, za a iya yanke kwayar halittar da ba a so.
Case 3 - Tabbatar da samu. Ɗaya daga cikin dakin gwaje-gwaje ya haɗa 'yan takarar gRNA guda uku kuma ya gwada su a cikin sel; Ta hanyar amplicon sequencing, ya gano cewa ɗaya ne kawai ke da fiye da 60% gyara (indels), yayin da sauran biyun ke da ƙasa da 5%. Sakamakon ƙira ya nuna duka ukun a matsayin "mai kyau". Tabbatar da gwaji ya nuna bambanci tsakanin tsinkaya da gaskiya.
Misali: sarrafa PAM na ɗan takara gRNA
shigo da sake # Sami jerin yankin da aka yi niyya daga Ensembl/NCBI; Kada ku sanya AI ya haddace shi. target = "GACGTTGCATCGATCCTAAGGCTTACGGTAGCTAGCTAGGCTAAGG"# 20 nt protospacer + NGG PAM neman SpCas9 (karshen strand + strand) don m a cikin re.finditer(r"(?=([ACGT]{20}))([ACGT]GG))r m.group(2) buga(f"Matsayi {m.start()+1}: gRNA={protospacer} PAM={pam}")
Wannan zai jera ƴan takara kawai tare da ingantaccen PAM; Amma ana buƙatar kayan aiki na musamman don ayyukan kan-manufa da kashe-kashe. Wannan lambar kayan aikin tantancewa ne na farko, ba cikakkiyar ƙira ba.
Samfura huɗu masu kwafi
1) Dabarun ƙira (ba tare da samar da jerin abubuwa ba):
Matsayinku: Mataimakin ƙira na CRISPR. Ƙirƙirar dabarun ƙira don: [gene, exon, knockout/daidaitaccen gyara, genome version, transcript].Wanne takamaiman kayan aiki (CRISPOR/CHOPCHOP) tare da waɗanne saituna zan yi amfani da su, wanda maki (a kan manufa, kashe-manufa) ya kamata in duba, gaya mani mataki-mataki. Samar da jerin gRNA SEN; Zan samar da shi daga abin hawa.
2) Fassarar da ba ta da manufa:
Bayyana yadda zan karanta ginshiƙan kashe-kashe a cikin fitowar CRISPOR: menene makin MIT, maki CFD, ƙidayar rashin daidaituwa, a waɗanne ƙofofin zan kawar da ɗan takara? Shin ƙofofin sun bambanta a aikace-aikacen asibiti da bincike?
3) Tsarin tabbatarwa na gwaji:
Ta yaya zan iya gwada gwaji cewa gRNA da na zaɓa duka ya yanke akan manufa kuma baya kashe manufa? T7E1, bayyana amplicon (zurfin) jeri da sarrafa ƙira mataki-mataki, gami da abin sarrafawa don sanyawa.
4) lambar tantancewa ta PAM/framework:
Rubuta lambar Python wanda ke nemo duk 20 nt protospacers protospacers don SpCas9 (NGG PAM) akan duka igiyoyi a cikin jerin abubuwan da aka bayar. Rahoton wuri, zaren da PAM ga kowane ɗan takara. Saka a cikin sharhin lambar cewa wannan kawar ta farko ce kawai kuma ana buƙatar kayan aiki na musamman don kashe-manufa.
Rauni mai ƙarfi / Ƙarfi mai ƙarfi
Rarrauna: "Ba ni jerin gRNA na CRISPR don jinsin MYC."
Matsala: Babu sigar genome/rubutu, babu PAM da sarrafa kashe-kashe, AI na iya ba da jeri na karya ko haɗari.
Ƙarfafa: "Shigar da dabarun ƙira na CRISPR don GRCh38, MYC (exon 2) knockout; gaya mani kayan aikin da zan yi amfani da su, tare da wanne saitin, da kuma wace maƙasudin kashewa don kawar da shi. Zan samar da jerin gRNA daga kayan aiki; ba ku samar da shi ba."
Me ya sa yake da ƙarfi: Magana a bayyane take, an bar ƙira ga takamaiman kayan aiki, kashe-manufa da tabbatarwa ana sanya su a tsakiya.
Mataki
Matsayin AI
Tabbaci na wajibi
Dabarun
Yana ba da shawarar tsare-tsare da kayan aiki
Binciken masana
gRNA samarwa
Sharhi
Makin CRISPOR/CHOPCHOP
sarrafa PAM
rubuta code
Shigar da code kuma tabbatar
Kashe-manufa
ya sanar da maki
Motar musamman + BLAST
Kammalawa
Sharhi
Tsarin gwaji
Kuskuren gama gari
- Kai tsaye ta amfani da gRNA da AI ke bayarwa. Yana iya zama mara PAM, kashe-manufa ko karya.
- Tsallake bincike-bincike. Kuskure mafi hatsari; Za a iya yanke kwayoyin halittar da ba a so.
- Kuskure makin ƙira don ingantaccen shaida. Makin shine hasashe; dole ne a tabbatar a cikin tantanin halitta.
- Barin sigar genome/kwafi ba a tantance ba. Ana iya yin niyya ga kuskuren exon.
- Dogaro da abin hawa guda ɗaya. A cikin aikace-aikace masu mahimmanci, ana buƙatar tabbatarwa tare da kayan aiki da yawa.
Tsanaki: Gyaran Halittu don dalilai na mutum ko na asibiti yana ƙarƙashin ƙa'idodin ɗa'a da doka. Wannan rukunin na bincike ne da ilimin ƙira; Hukunce-hukuncen aikin asibiti suna buƙatar amincewar kwamitin ɗa'a, ƙungiyar ƙwararru da dokoki. An haramta gyaran layin Germline (gado) a ƙasashe da yawa.
Zurfin: karanta maki a kashe-maƙasudi a lamba
Ƙimar kashe-ƙasa-manufa tare da kankare maki biyu, ba m kalmomi kamar "high / low." Makin MIT (0-100) yana taƙaita yadda takamaiman gRNA ke faɗin kwayoyin halitta; Maki mai girma (misali 80+) yana nufin ƙarancin haɗarin da ba a bugu ba. Makin CFD (Yanke Ƙirar Mitar Mitar, 0-1) yana ƙididdige yiwuwar yanke kowane yanki na gaba ɗaya bayan ɗaya; Ko da yanki ɗaya na kashe-kashe sama da 0.2 na iya kawar da ɗan takarar don aikace-aikace masu mahimmanci. Ka sa basirar wucin gadi ta bayyana waɗannan maki biyu, sannan ka karanta ainihin ƙimar da ke cikin abin da ke fitar da abin hawa.
Misali na musamman: kungiya tana zabar tsakanin 'yan takara biyu. Dan takarar A yana da maki MIT na 91 kuma mafi girman maƙasudin CFD shine 0.05. Dan takarar B yana da maki MIT na 88 amma yana da manufa guda ɗaya a cikin exon na kwayar cutar ƙwayar cuta tare da CFD na 0.34. Ko da yake gabaɗayan makin ya bayyana kusa, inda aka kashe-maƙasudin ya kasance (bayyanar da wani nau'i mai mahimmancin aiki) ya sa ɗan takarar B ya zama abin karɓa. Yana da mahimmanci a kimanta abin da aka kashe ba kawai cikin sharuddan lamba ba har ma da mahimmancin ilimin halitta na kwayar halittar da ta fada a kai.
Wani batu: yana da haɗari sosai don rashin daidaituwa ya faru a cikin "iri" yankin kusa da PAM fiye da a cikin yanki mai nisa; Ko da bambancin tushe na 1-2 a cikin yankin iri zai iya dakatar da Cas9, yayin da 3-4 bambancin tushe daga PAM za a iya jurewa. Saboda haka, wurinsa yana da mahimmanci kamar adadin bambance-bambance.
Maki/ma'auni
Disamba
Sharhi
Bayani na MIT
0-100
Maɗaukaki = ɗan haɗarin kashe-kashe
CFD (wanda ba shi da manufa)
0-1
Sama da 0.2 za a iya kawar da su a cikin aikace-aikace masu mahimmanci
Matsayin rashin jituwa
iri / m
Yankin iri ya fi haɗari
A takaice
- AI a cikin ƙirar CRISPR; mataimaki ne wanda ke tsara dabaru, rubuta lamba, da kuma bayyana zaɓuɓɓuka, ba kayan aikin da ke “samar da” gRNA ba.
- Dole ne a samar da 'yan takarar gRNA tare da kayan aiki na musamman (CRISPOR/CHOPCHOP), kuma dole ne a sarrafa PAM da kashe-kashe.
- Sakamakon ƙira shine ƙima; Dole ne a tabbatar da tasirin kan manufa da kashe-kashe ta hanyar gwaji (jeri).
- Ayyukan asibiti da na ƙwayoyin cuta suna ƙarƙashin iyakoki masu nauyi/na doka.
Aikin aikace-aikace
Zazzage yanki mai niyya daga Ensembl. Gudun lambar tantancewa ta PAM a sama kuma jera masu fafutuka na ɗan takara. Sannan a fitar da yanki guda zuwa kayan aiki kamar CRISPOR kuma ku sami maki-manufa da kashe-kashe; Kwatanta 'yan takarar lambar ku tare da shawarwarin kayan aiki. Gano ɗan takarar da ke da mafi girman haɗarin da ba a kai hari ba kuma rubuta a cikin jumla ɗaya dalilin da ya sa za a kawar da shi/ta.
jerin abubuwan dubawa
- [ ] Na gyara maƙasudin ta hanyar gene, exon, sigar genome da kwafi.
- [ ] Na samo silsilar daga majiya mai tushe, ba AI ta haddace ta ba.
- [ ] Na ƙirƙira kuma na ci ƴan takarar gRNA tare da kayan aikin al'ada.
- [ ] Na duba kasancewar PAM da kasadar manufa.
- [ ] Na kafa tsarin tabbatarwa na gwaji.
- [ ] Ina sane da iyakoki na ɗa'a / doka kuma na lura da amincewar ƙwararru.