I-Clustered regularly interspaced short palindromic repeats (CRISPR) yindlela yokuhlela i-genome yesizukulwana sesithathu eguqule ihlabathi ngeziphumo zayo eziphezulu. Isetyenziselwe ukunyanga izifo ezahlukeneyo zebhayoloji kunye nosulelo. Iibhaktheriya ezahlukeneyo kunye nezinye iiprokaryotes (ezifana ne-archaea) nazo zineenkqubo ze-CRISPR/Cas9 zokuzikhusela kwi-phages. Kuye kwaxelwa ukuba amaqhinga asekelwe kwi-CRISPR/Cas9 anokuthintela ukukhula kunye nokuqhubela phambili komhlaza webele ongemuhle kathathu (TNBC) ngokujolisa kwiijini zokumelana ezinokuthi zitshintshe, ukubhalwa kwe-transcription, kunye nolawulo lwe-epigenetic. Olu ngenelelo lonyango lunokunceda ukujongana neengxaki ezinzima ezifana nokungamelani namayeza okubonwa nakwi-TNBC. Okwangoku, iindlela ezahlukeneyo zisetyenziswa ukuhambisa i-CRISPR/Cas9 kwiiseli ezijoliswe kuzo, ezifana neendlela zomzimba (i-microinjection, i-electroporation kunye ne-hydrodynamic), i-viral (i-adeno-associated virus kunye ne-lentivirus) kunye ne-non-viral (liposomes kunye ne-lipid nanoparticles). Nangona iimodeli ezahlukeneyo ziye zaphuhliswa ukuze kufundwe izizathu zeemolekyuli ze-TNBC, ukungabikho kweendlela ezinobuthathaka nezijolise ekunikezeni izixhobo zokuhlela i-genome kwi-vivo kuthintela ukusetyenziswa kwazo kwezonyango. Ngamafutshane, olu phononongo luhlola ngokupheleleyo inkqubela phambili, imingeni, imida, kunye namathuba onyango lwe-CRISPR/Cas9 lwe-TNBC ngokusekelwe kubungqina obukhoyo. Sikwagxininisa indlela indibaniselwano yobukrelekrele bokwenziwa kunye nokufunda koomatshini enokuphucula ngayo amaqhinga e-CRISPR/Cas9 kunyango lwe-TNBC.
Umhlaza ubonakaliswa kukwahlukana kweeseli okungalawulekiyo, ukuphazamiseka kweendawo zokujonga umjikelo weseli, kunye nokuguquka kwezakhi zofuzo ezithintela ithumba (ii-TSG) (uMatthews et al., 2022). Phakathi kweentlobo ezahlukeneyo zomhlaza, umhlaza webele lolona hlobo luqhelekileyo lomhlaza kwabasetyhini kwaye unezinga eliphezulu lokufa kwihlabathi liphela (uWaks noWiner, 2019). Umhlaza webele sisifo esahlukileyo esineempawu ezahlukeneyo, ezifana neempawu ze-histological nezebhayoloji, ukubonakaliswa kweklinikhi kunye nokuziphatha, kunye nempendulo kunyango (uWeigelt et al., 2010). Ukuhlelwa komhlaza webele kubonelela ngengqiqo echanekileyo yokuchongwa komhlaza webele kunye nokubikezela ithumba. Ukusetyenziswa kwee-biomarkers eziqhelekileyo kunye neempawu zeklinikhi kuye kwaba yeyona nto iphambili yokuhlelwa komhlaza webele (uTsang noTse, 2019). Ukubikezela kunye nempendulo kunyango lomhlaza webele kuphenjelelwa zizinto ezininzi, kubandakanya ubukho be-estrogen receptor (ER), i-progesterone receptor (PR), i-human epidermal growth factor receptor 2 (HER2/neu), kunye nenqanaba le-histological, uhlobo lwethumba, kunye nenqanaba. Ubungakanani kunye nokusasazeka kwe-lymph node (Al-Tubaiti, 2020). Kuchongwe iintlobo ezintlanu ze-intrinsic molecular subtypes zomhlaza webele, kuquka i-luminal A, i-luminal B, i-HER2-enriched, i-basal-like, kunye ne-claudin-low (Prat et al., 2015). I-TNBC luhlobo lwe-molecular of cancer olungabonisi zonke ii-ER, i-PR, kunye ne-HER2 (Yin et al., 2020). Ezi mpawu ze-pathological zidibene ne-TNBC, ziqinisekisa ukuqhubela phambili kwayo ngokukhawuleza kunye nohlobo lwayo olunoburhalarhume ngaphezu kwalo naluphi na olunye uhlobo lomhlaza webele (Feng et al., 2018). Ukongeza, uPeru et al (2000) basebenzise itekhnoloji ye-microarray ukwahlula umhlaza webele kunye nokuchonga iintlobo ezintlanu ze-intrinsic zomhlaza webele (Cadenas, 2012). Umhlaza webele ofana ne-basal luhlobo lwe-substitute yomhlaza webele olune-triple-negative phenotype kwaye ludibene nokuqhubela phambili ngokukhawuleza. Kufuneka kuqatshelwe ukuba zonke iintlobo zomhlaza webele ezifana ne-basal zihlala zingachongwa njenge-TNBC, kodwa yi-77% kuphela eyi-TNBC; Ngokwahlukileyo koko, i-71–91% ye-TNBC ifana ne-basal, nto leyo ebonisa ukuba ezi ntlobo zimbini zomhlaza webele ziyadibana kwaye zimele udidi olwahlukileyo (Wang D.-Y. et al., 2019). Oku kudala isidingo sokuchonga ukungafani kwe-TNBC ukuze kucaciswe i-prognosis kunye nokuchonga iimpendulo ezinokubakho kunyango lwangoku nolwexesha elizayo. Ukongeza, i-TNBC ibandakanya i-15–20% yazo zonke iimeko zomhlaza webele kwaye ixhaphake kakhulu kubasetyhini abangaphantsi kweminyaka engama-50 ubudala. Utshintsho lwe-BRCA1 okanye i-BRCA2 luye lwaxelwa malunga ne-20% yeemeko ze-TNBC (Xie et al., 2017; Tzikas et al., 2017). , 2020). Izifundo zikwabonise ukuba i-TNBC ine-immune microenvironment eyahlukileyo equka amanqanaba aphezulu e-vascular endothelial growth factors, ii-tumor-associated macrophages (TAMs), ii-tumor-infiltrating lymphocytes (TILs), kunye nezinye iimolekyuli ezibandakanyeka ekukhuleni nasekufudukeni kwe-tumor. Ke ngoko, ukuqonda i-microenvironment ye-TNBC kubalulekile kwi-prognosis yayo kunye nonyango (Fan and He, 2022).
Ukuvavanya ukubikezela kwangaphambili kwe-TNBC nokuqinisekisa unyango olusebenzayo, ukuxilongwa ngokuchanekileyo, okusekelwe kakhulu kwi-immunohistochemistry (IHC) ukufumanisa i-ER, i-PR kunye ne-HER2, kunye ne-mammography ukufumanisa amathumba ebele, kubalulekile. Nangona kunjalo, i-mammography ayinakubonisa ngokwaneleyo iimpawu zangaphakathi kwesisu ezifana ne-necrosis kunye ne-fibrosis (Deepak Singh et al., 2021). Ekubeni ukubikezela kwangaphambili okungalunganga kunye nokuxilongwa kuthintela oogqirha ekunikezeni amayeza afanelekileyo (Chaudhary, 2020), kusetyenziswa amaqhinga ahlukeneyo ukuphucula ukhathalelo lwezigulane ezine-TNBC. Okwangoku, amayeza amabini, i-doxorubicin kunye ne-cyclophosphamide, asetyenziswa kwizigulane ezine-TNBC kwaye abonise iziphumo ezilungileyo. Ukongeza, kusetyenziswa amanye amayeza eplatinum, afana ne-carboplatin kunye ne-cisplatin (Sikov et al., 2015). Ukongeza, ii-PARP inhibitors ezifana ne-Olaparib, i-Velaparib, kunye ne-PF-01367338 zisetyenzisiwe njengamayeza e-chemotherapy anokubakho kunyango lwe-TNBC (Ishino et al., 2018). Ekubeni iindlela zokubonisa iimpawu zeWnt/b-Catenin, NOTCH, kunye neHedgehog zixelwe ukuba zibandakanyeka ekwenzekeni nasekuqhubekeni kweTNBC, ukujolisa amayeza kwezi ndlela kunokuba licebo elibalulekileyo (Aysola et al., 2013). Nangona utyando, unyango lwemitha kunye ne-chemotherapy ziseyeyona nto iphambili kunyango lweTNBC namhlanje, inkqubela phambili enkulu yenziwe ekuphuhliseni unyango olutsha oluquka unyango olujoliswe kulo, i-immunotherapy, izixhobo ezahlukeneyo zokuhlela i-gene ezinxulumene neCRISPR ezifana neCas9n, dCas9., CRISPR/Cas12, i-prime editing, kunye ne-CRISPR/Cas9-target gene therapy. Apha siza kugxila kwizixhobo ezahlukeneyo zokuhlela i-gene ezinxulumene neCRISPR ezisetyenziswa kunyango lwe-TNBC. Phakathi kwazo, iCRISPR/Cas9 ifumene ingqwalasela ebanzi.
I-Cas9n, eyaziwa ngokuba yi-Cas9 nickase, lutshintsho olwenziwe ngokwezakhi zofuzo lweprotheyini ye-Cas9 ephuma kwinkqubo yokuhlela i-genome ye-CRISPR/Cas9 (Gupta et al., 2019). Kwimo yayo yokuqala, iprotheyini ye-Cas9 ineendawo ezimbini ze-nuclease: i-RuvC kunye ne-HNH, umsebenzi wayo ophambili kukuqhekeka kwemigca ye-DNA yomibini. Nangona kunjalo, kwi-Cas9n, enye yeendawo ze-nuclease, i-HNH, iguqulwa ngokwezakhi zofuzo kwaye ayisebenzi. Ke ngoko, indawo ye-HNH ye-Cas9n ihlala ingasebenzi. Yindawo ye-RuvC kuphela ehlala isebenza, ivumela i-Cas9n ukuba isike okanye idale ukuqhekeka kwimigca ye-DNA enye (Trevino kunye noZhang, 2014). Xa kuthelekiswa ne-Cas9, i-Cas9n inokunciphisa ngempumelelo iziphumo ezingajoliswanga kwaye iphucule ngokuchanekileyo iindlela zokulungisa iiseli. I-Cas9n ingasetyenziselwa ukusombulula iingxaki ezahlukeneyo, njengokudala ukuqhekeka kwiindawo ezithile kwi-DNA enemigca emibini. Ngenxa yesi sizathu, iimolekyuli ezimbini zeCas9n zadityaniswa zaza zasetyenziswa kunye (Yee, 2016). I-Mixed lineage kinase 3 (MLK3) yi-protein kinase esebenza kwi-mitogen esebenza njengomlawuli oyintloko ngexesha le-TNBC metastasis (Cronan et al., 2012).
I-MLK3 ingavula iindlela ezininzi zokubonisa ezikhokelela kwi-metastasis ye-TNBC. Umzekelo, indlela ye-c-Jun N-terminal kinase (JNK) ilawula ukuhamba kweseli kunye nokudilizwa kwe-extracellular matrix, kwaye i-MLK3 ivula indlela ye-JNK, ngaloo ndlela iphucula ukuhamba kweseli kwaye inike iipropati ezihlaselayo (uRattanasinchai noGallo, 2016). I-MLK3 ikwalawula i-EMT. Ukwenza oku, ivula izinto ezibangela ukubhalwa kwe-epithelial ezifana ne-Snail, i-Slug kunye ne-Twist. Ezi zinto zithintela ukubonakaliswa kweempawu ze-epithelial kwaye zikhuthaza ukubonakaliswa kweempawu ze-mesenchymal, okukhokelela ekufumaneni i-metastatic phenotype (uCasalino et al., 2023). I-MLK3 ibonwe idlala indima ekuguqulweni kwe-ECM kunye nokusebenza kwe-proteases ezifana ne-matrix metalloproteinases (MMPs) ezonakalisa i-ECM. Oku kwenza kube lula ukuhlasela kweseli ye-tumor kwaye kusasazeke kwiindawo ezikude (uKatari et al., 2019). Ke ngoko, izifundo zangaphambili zibonise ukuba i-MLK3 idlala indima ebalulekileyo kwi-TNBC. URattanasinchai noGallo basebenzise iimodeli zeTNBC ukufunda indima yeMLK3 baza bafumanisa ukuba ikhuthaza uphuhliso lomhlaza ngeendlela ezithile zokubonisa iimpawu. Basebenzise iCRISPR/Cas9n ukuhlela iMLK3 baza babona ukuncipha okukhulu kwi-metastasis yeTNBC (Rattanasinchai noGallo, 2016).
I-dCas9, edla ngokubizwa ngokuba yi-Cas9 engenamsebenzi, luhlobo oluguquliweyo lweproteni yeCas9. Ngokungafaniyo neCas9 esebenzayo, i-dCas9 ayinawo umsebenzi we-endonuclease, nto leyo eyenza ingakwazi ukubangela ukuqhekeka kwe-DNA double-strand. Ke ngoko, i-dCas9 ingasetyenziselwa ukujolisa ngqo kwiindawo ezithile ze-genome ngaphandle kotshintsho okanye utshintsho kulandelelwano lwe-DNA (Wang et al., 2016). Kwi-dCas9, iiproteni ezimbini ze-endonuclease, i-RuvC kunye ne-HNH, azisebenzi ngokucinezela ii-amino acid residues ezibalulekileyo. (Richter et al., 2016). Nangona ingenawo umsebenzi wokuqhekeka kwe-DNA, i-dCas9 idlala indima ebalulekileyo kuphando lwe-genetic kunye ne-biotechnology. Umzekelo, ivumela ukubonakala kweendawo ezithile ze-genome, iququzelela ukulawulwa kokubhalwa kwemibhalo, kwaye ikhuthaza utshintsho lwe-epigenetic (Brocken et al., 2018).
I-transcription factor ZEB1 (i-zinc finger E-box binding homeobox 1) idlala indima ethile nebalulekileyo ekulawuleni utshintsho lwe-epithelial-mesenchymal (EMT), inkqubo yeselula eyenzeka ngexesha lophuhliso lwe-embryonic, ukulungiswa kwezicubu, kunye nokuqhubela phambili komhlaza. Ibandakanya ukuguqulwa kweeseli ze-epithelial zibe ziiseli ze-mesenchymal, okubangela utshintsho kwimo yeseli, ukuhamba, ukungenelela, njl. (Wu et al., 2020). I-ZEB1 ithintela iimpawu ezininzi ze-epithelial, ezifana ne-E-cadherin kunye ne-Occludin, ezinoxanduva lokugcina ukunamathela kweseli kunye ne-polarity yeeseli ze-epithelial kwi-TNBC (Moreno-Bueno et al., 2008). Ukongeza, i-ZEB1 isebenzisa iimpawu ezithile ezifana ne-N-cadherin, i-vimentin kunye ne-fibronectin (Konradi et al., 2014) kwaye ikwalawula ii-genes ezibandakanyekayo ekuguqulweni kwe-cytoskeletal ezifana ne-Rho GTPases kunye ne-matrix metalloproteinases (MMPs) (Huang). et al., 2014). 2022), ukongeza, ikwachaphazela iindlela ezahlukeneyo zokubonisa iimpawu ezifana nokuguqula i-growth factor-β (TGF-β), i-Wnt signaling, njl.njl., ngaloo ndlela ikhuthaza ukuhlasela kwe-tumor kunye ne-metastasis kwi-TNBC (Chen et al., 2016). ). Izifundo ezininzi kunye nobungqina besayensi zibonisa ukuba i-ZEB1 inamandla amakhulu njengearhente exabisekileyo yokufumanisa kunye nokungenelela konyango lwe-TNBC. Kwisifundo sakutshanje, uWaryah et al. basebenzise imodeli ye-TNBC kwaye bafezekisa ucinezelo olupheleleyo lwe-ZEB1 yi-dCas9. Ngoko ke, babone ukucaciswa okuphezulu kakhulu kunye nokuthintelwa okuphantse kuphele kwe-ZEB1 kwiimeko ze-in vivo (Waryah et al., 2023).
I-CRISPR/Cas12 sisixhobo sokuhlela izakhi zofuzo esibizwa ngokuba yi-CRISPR/Cpf1. Ivela kwinkqubo ye-CRISPR/Cas, apho igama elithi Cas12 libhekisa kwiproteni enxulumene ne-CRISPR 12 (Bharathkumar et al., 2022), efana ngokupheleleyo ne-Cas9. Umahluko kuphela kukuba iqulethe iproteni ye-Cas12. Xa ithelekiswa ne-Cas9, i-Cas12 inemisebenzi eyahlukileyo efana nokuvelisa iziphelo ezinamathelayo ngexesha lenkqubo yokuhlela izakhi zofuzo, ngelixa i-Cas9 ivelisa iziphelo ezibuthuntu (Wang et al., 2021). Le propati ye-Cas12 inegalelo kubuchwepheshe bayo bokulawula i-DNA ethile. Njengeproteni ekwaziyo ukuqaphela ngokuchanekileyo nokusika i-DNA ekujoliswe kuyo, inokuguquguquka okumangalisayo, okwenza ibe sisixhobo esisebenzayo kwiindlela ezahlukeneyo zokusebenza, kubandakanya nokuhlela izakhi zofuzo (Pickar-Oliver kunye noGersbach, 2019).
Ngokufanayo nezinye iinguqulelo zeCRISPR, iCRISPR/Cas12 ikwakwazi ukukhupha okanye ukusebenzisa iijini kwi-TNBC, ijolise kwiijini ezidlala indima ebalulekileyo kwi-pathogenesis yayo okanye impendulo kunyango (uYang noZhang, 2023). Ukuze kufezekiswe le nkqubo, kwaveliswa i-RNA yesikhokelo (gRNA) eqondisa iCas12 kwijini ekujoliswe kuyo. Nje ukuba iCas12 ibopheleleke kwithagethi yayo, ingenisa ukuqhekeka kwemigca emibini kwiDNA kwaye isebenze iindlela zokulungisa iDNA. Ngexesha lenkqubo yokulungisa, iinucleotides ezingalunganga zinokufakwa, nto leyo ekhokelela ekuguqukeni kwejini kunye nokulahlekelwa ngumsebenzi (uZhang et al., 2021a). Ukongeza, inguqulelo ephuculweyo ye-enzyme yeCas12 (ebizwa ngokuba yi-dCas12) nayo yasetyenziswa ukwenza iijini zisebenze. Ngokuyidibanisa ne-transcription activator, kunokwenzeka ukuba kukhuthazwe iijini ezithile, ngaloo ndlela zisebenze. Le teknoloji inamandla amakhulu okukhuthaza ukusebenza kweejini ezithintela ithumba (uSultan et al., 2022).
I-Prime editing yiteknoloji yokuhlela i-genome entsha negqwesileyo kakhulu. Iyakwazi ukuguqula i-DNA yento ephilayo ngokuchanekileyo (uChen noLiu, 2023). I-Prime editing ifezekiswa ngokudityaniswa kwezinto ezimbini eziphambili: i-enzyme ye-CRISPR/Cas9 eguquliweyo kunye ne-reverse transcriptase. Indima ye-enzyme ye-CRISPR/Cas9 kukukhetha iindawo ezichanekileyo kwi-genome, ngelixa i-reverse transcriptase inceda ukuguqula ngokupheleleyo i-DNA kwindawo ekujoliswe kuyo (uHassan et al., 2021). Kwindlela yokuhlela i-primer, inyathelo lokuqala libandakanya ukudalwa kwe-primer editing guide RNA (pegRNA) (uStandage-Beier et al., 2021). Iqulethe ulandelelwano lwethagethi kunye ne-template ye-RNA ehambelana nendawo yokuhlela efunekayo kwi-DNA ekujoliswe kuyo. I-pegRNA emva koko ingeniswa kwiiseli ekujoliswe kuzo kunye ne-primer editing nuclease (PE2). I-PE2 yiprotheni ye-fusion equlathe i-enzyme ye-Cas9, i-reverse transcriptase, kunye ne-primer editing adapter (uMartín-Alonso et al., 2021). Ngaphakathi kwiseli, ii-pegRNA kunye nee-PE2 complexes zikhangela i-DNA ethile ezifuna ukuyiguqula. I-enzyme ye-Cas9 inqumla i-DNA kwaye yenze itemplate equlathe imicu enye (Choi et al., 2022). I-Reverse transcriptase isebenzisa le template ukulungiselela i-DNA ukuze ihlelwe. Kunye nokukopa i-DNA, imiyalelo yokuhlela itemplate ye-RNA nayo ifakiwe. Okokugqibela, i-DNA strand entsha edaliweyo isetyenziswa njengetemplate yokulungisa ikhefu kwi-DNA, nto leyo ekhokelela kulandelelwano lwe-DNA olutshintshileyo oluqulethe utshintsho olufunekayo (Ochoa-Sanchez et al., 2021). Iindlela zokuhlela ze-primer zinokukhokelela kwiinguqu ezibanzi kwiijini. Inokujolisa kwiinguqu zenqaku, ukufakwa, ukususwa, kwanokutshintshwa kwejini (Anzalone et al., 2019; Chen kunye noLiu, 2023). Ukuhlela kwe-Prime kunika iingenelo ezininzi kuneetekhnoloji zokuhlela i-genome zangaphambili. Oku kuquka ukuchaneka okwandisiweyo, iziphumo ezincitshisiweyo ezingajoliswanga, kunye nokukwazi ukuhlela i-DNA ngaphandle kokuxhomekeka kwiimpazamo ze-DNA double-strand (Anzalone et al., 2020).
Itekhnoloji yeCRISPR/Cas9 yaqalwa yaphuhliswa ukukhusela iintsholongwane kwi-plasmid transfer kunye nosulelo lwe-phage kwaye kamva yaphinda yasetyenziswa njengesixhobo esisebenzayo sokujolisa i-DNA esisekelwe kwi-RNA sokuhlela i-genome (uJiang noDoudna, 2017). Ukongeza, inkqubo yeCRISPR/Cas9 ibikwe ukuba ikhona kwi-50% kunye ne-87% ye-bacterial kunye ne-archaeal genomes, ngokulandelelana (uIshino et al., 2018). I-CRISPR/Cas9 sisixhobo esinokubakho sokususa, ukufaka kunye nokulungisa naluphi na ulandelelwano olungaqhelekanga lwe-genetic usebenzisa iindlela ze-in vivo kunye ne-in vitro (uSabit et al., 2021). Ngaphezu koko, i-CRISPR/Cas9 ibonakalisiwe ukuba yinxalenye yenkqubo yokuzikhusela eguquguqukayo ngenxa yobuchule bayo kwiijini ezijoliswe kuzo (uChen noZhang, 2018).
I-CRISPR/Cas9 ine-Cas9 kunye ne-RNA enye ekhokelayo (sgRNA). I-Cas9 yi-endonuclease eyenziwe ziinxalenye ezininzi zeprotheyini. Ukongeza, i-Cas9 ineempawu ezizodwa zesakhiwo kunye nokwakheka (Pacesa et al., 2022) njengoko inee-lobes ezimbini: ukuqatshelwa (REC) kunye ne-nuclease (NUC). I-REC lobe yahlulwe ngakumbi kwiindawo ezintathu: i-REC1, i-REC2, kunye ne-bridge helices (Cromwell et al., 2018). I-NUC inee-lobes ezintathu, ezizezi i-RuvC (RuvC I, RuvC II, RuvC III), i-HNH kunye ne-protospacer adjacent motif (PAM) interaction domain (Umzobo 1) (uSong et al., 2016). I-sgRNA ineenxalenye ezimbini, kubandakanya i-CRISPR RNA (crRNA) kunye ne-transcoding small RNA (tracer RNA) (Umfanekiso 1). I-sgRNA idibanisa iproteni yeCas9 kwi-connexins ukuze yenze i-complex esebenzayo, eyaziwa ngokuba yi-effector complex (Richter et al., 2012). I-crRNA yi-18–20 nucleotide base pair edlala indima ebalulekileyo ekuqondeni ulandelelwano lwe-DNA ekujoliswe kuyo. Ukongeza, i-crRNA idibana ne-DNA ekujoliswe kuyo, kwaye i-tracrRNA isebenza njenge-scaffold ye-Cas9 nuclease ukubopha i-DNA ekujoliswe kuyo (Manghwar et al., 2019). Kwelinye icala, ulandelelwano lwe-PAM lunee-nucleotides ezi-3, eziqinisekisa, zicacisa kwaye zilawula ukubopha kwe-effector complex kwi-DNA. Ii-subunits ze-Cas9 protein complex iRuvC kunye ne-HNH zinomsebenzi we-catalytic (Richter et al., 2012; Asmamaw kunye noZawdie, 2021). Indawo ye-HNH nuclease ye-subunit yeCas9 inqumla umtya we-DNA onxulumene ne-crRNA. I-RuvC nuclease domain iqhekeza eminye imicu ye-DNA kwaye ivelise ukuqhekeka kwemicu emibini (ii-DSB), emva koko iindlela ezimbini ezahlukeneyo zokulungisa ukuqhekeka kwe-DNA ziyavulwa (uJiang noDoudna, 2017) (Umfanekiso 1).
Umfanekiso 1. Isishwankathelo seCRISPR/Cas9 (A). Izinto eziyinxalenye yenkqubo yeCRISPR/Cas9: (i). I-Cas9 endonuclease inoxanduva lokusika ulandelelwano lweDNA ekujoliswe kuyo, (ii) i-RNA enye yesikhokelo (sg) ephuma ekudibaneni kwe-crRNA kunye ne-tra-crRNA chimeras. (ezimbini). I-Cas9 ibandakanya izinto ezininzi ezifana ne-Rec I, Rec II, NUC lobe (HNH kunye ne-Ruv C ziinxalenye ezincinci) kunye ne-PAM interaction domain (C) enemisebenzi ehambelanayo. I-CRISPR/Cas9 protein complex iqhekeza ulandelelwano lweDNA kwiifom ezingezizo ezongezayo nezihambelanayo (D). I-CRISPR/Cas9 ihlela i-genome ngamanqanaba amathathu: ukuqaphela, ukusika kunye nokulungisa. I-sg-RNA eyilwe ikhokela i-Cas9 kwaye iqaphela ulandelelwano olufunekayo nge-crRNA yecandelo elihambelanayo. I-Cas9 iqaphela ulandelelwano lwe-PAM kwi-5′-NGG-3′ kwaye inyibilikisa i-DNA, yenza i-DNA-RNA hybrid kwaye isebenze i-cleavage. I-HNH domain yeCas9 inqumla umtya oncedisayo, kwaye i-RuvC domain inqumla umtya ongenanto. I-CRISPR/Cas9 ilungisa i-dsDNA ngokuyiqhekeza ngeendlela ezimbini: i-non-homologous end joining (NHEJ) kunye ne-homology-directed repair (HDR). I-NHEJ ilungisa i-DNA enemicu emibini xa kungekho i-DNA yangaphandle enemicu ngokusebenzisa inkqubo ye-enzyme, indlela enokuphoswa ziimpazamo enokufaka okanye isuse ulandelelwano lwe-DNA olungaqhelekanga. I-HDR icacile kakhulu kwaye ifuna iitemplate ze-DNA ezinemicu efanayo.
Iindlela zokulungisa ezilawulwa yiCRISPR/Cas9 ziquka iindlela zokudibanisa isiphelo ezingahambelaniyo (NHEJ) kunye neendlela zokulungisa ezikhokelwa yihomology (HDR). I-NHEJ yindlela eyenzeka ngempazamo kuba ibandakanya ukufakwa okanye ukususwa kwaye ayifuni naluphi na uhlobo ngexesha lokulungisa. Isebenzisa iinucleotides ezingacwangciswanga ukuvelisa iiproteni ezisemgangathweni (Abbasi et al., 2021). Le nkqubo yokulungisa inee-complex ezine ezifana ne-KU complex, i-cross-complementing protein complex type 4 (XRCC-4), i-DNA end processing enzyme, kunye ne-protein kinase DNA-PKcs (Abbasi et al., 2021). Iprotheyini ye-KU complex inee-subunits ezimbini, i-Ku 70 kunye ne-Ku 80, kwaye idlala indima ebalulekileyo kwindlela ye-NHEJ, kuba indlela yokulungisa iqalwa ngokubophelela ii-subunits ezimbini (Ku 70 kunye ne-Ku 80) kwiziphelo ezibuthuntu okanye eziphantse zibe buthuntu ze-DNA ekujoliswe kuyo (Abbasi et al., 2021), esebenza njenge-scaffold yokufumana ezinye izinto ezinxulumene ne-NHEJ kwindawo yokwenzakala (Yang et al., 2020). I-XRCC-4 kunye ne-DNA ligase zenziwe ngama-amino acids angama-334 kunye nama-911, ngokulandelelana, kwaye i-XRCC4-DNA ligase IV complex ivuselela ukuxhuzula kweziphelo ze-DNA (Chatterjee et al., 2015). I-enzyme yokucubungula i-DNA end, eyaziwa ngokuba yi-polynucleotide kinase 3′-phosphate, yi-enzyme yokucubungula i-DNA end. Ingasusa iqela le-3′P kwi-DNA kwaye i-phosphorylate iqela le-5′OH ngexesha lokulungiswa kwe-DSB. Ikwabandakanya ukulungiswa kwee-single-strand breaks (SSBs) kusetyenziswa indlela yokulungisa i-SSB (Chatterjee et al., 2015). I-protein kinase I-DNA-PKcs yi-protein kinase exhomekeke kwi-DNA equlathe i-subunit ye-catalytic ye-PIKKs (phosphatidylinositol 3-kinase-related kinases) kunye nosapho lwe-ataxia telangiectasia mutated (ATM), kunye ne-ATM kunye ne-Rad3-related ATRs. ii-strand breaks (DSBs) kunye nee-single-strand breaks (Yue et al., 2020; Peng et al., 2016).
I-HDR yindlela yokulungisa echanekileyo nefanelekileyo kuba ulwazi lukopishwa kusetyenziswa uhlobo olupheleleyo lwe-homologous DNA duplex, nangona ifuna ukubakho kwee-chromatids ezingoodade. Oku kwenzeka ngexesha lesigaba se-S/G2 somjikelo weseli yezilwanyana ezincancisayo. I-HDR iyenzeka ikakhulu kwiintlobo zemvubelo, kodwa i-NHEJ ibalulekile kwizilwanyana ezincancisayo (Burma et al., 2006; Abbasi et al., 2021). Indlela yokulungisa epheleleyo iboniswe kuMfanekiso 1.
Ekubeni i-TNBC ibangelwa kukungasebenzi kakuhle kwezakhi zofuzo kunye ne-epigenetic, ukulungiswa kwezinto ezingalunganga ze-genomic/epigenomic kusetyenziswa i-CRISPR/Cas9 kunokuba yindlela efanelekileyo yonyango (Chen et al., 2019). Ukongeza, ezinye izinto ezichaphazela ukubhalwa kwezakhi zofuzo ezibandakanyekayo kulawulo lwe-transcriptional oluthile lweseli zinokubonisa iimpawu ezikhethekileyo zeeseli zomhlaza, nto leyo ebonisa ukuba ukulawulwa kwe-transcriptional kunokuba yindlela efanelekileyo yokunyanga umhlaza (Drost et al., 2017). Ukusebenzisa ezi mpawu ze-molecular ze-tumor, ezifana neziphene ze-genetic, i-epigenetic kunye ne-transcriptional, ukuphuhliswa kwamayeza kunokuphucula iziphumo zeklinikhi kwaye kunciphise iindleko zokuhlola. I-CRISPR sisixhobo esinokusetyenziswa ekuhleleni izakhi zofuzo esingenakukwazi kuphela ukubona nokuchonga iinjongo ze-genomic ezibangela umhlaza, kodwa sinokusetyenziselwa ukuhlela, ukucinezela, kunye nokuguqula i-oncogenes kwiiseli zabantu (Itheyibhile 1) (Ahmed et al., 2021). Kuye kwenziwa izikrini ezininzi ze-CRISPR ukukhangela izakhi zofuzo ezinxulumene ne-tumor suppressors, i-oncogenes, kunye nokumelana namayeza. Ukusetyenziswa kweCRISPR/Cas9 ukuhlela ii-oncogenes ezahlukeneyo ze-TNBC kuboniswe kuMfanekiso 2.
Umfanekiso 2. Ukuhlelwa kwezakhi zofuzo ze-TNBC oncogenes ezahlukeneyo kusetyenziswa i-CRISPR/Cas9 okubangela ukukhula okunciphileyo kwethumba kunye nokusasazeka kwe-metastasis. Ezi ntsholongwane zofuzo ziquka i-CDK7, i-NAT1, i-UBR5, i-YTHDF2, i-ITGA9, i-CXCR4 kunye ne-CXCR7, i-Crypto1, i-ROR1 kunye ne-ST8SIA, ezibandakanyekayo ekwenzekeni nasekusasazeka kwe-TNBC.
Ukufuduka kweeseli zomhlaza, ukuhlasela, kunye notshintsho lwe-epithelial-mesenchymal (EMT) kunxulunyaniswa ne-ITGA9. Izifundo zangaphambili zibonise ukuba i-ITGA9 ngumdlali ophambili kwindlela ye-Notch kwaye idlala indima enomdla kwi-rhabdomyosarcoma metastasis (Molist et al., 2020). Ukongeza, i-ITGA9 ifunyenwe inxulumene ngokusondeleyo nokubikezela kwesigulane kwiintlobo ezahlukeneyo ze-tumor, kubandakanya umhlaza webele (Wang Z. et al., 2019). Ukongeza, uhlalutyo lwe-bioinformatics lwe-ITGA9 lubonise ukuba ukubonakaliswa kwayo kwi-TNBC kwakuphezulu kakhulu kunakwezinye iintlobo ze-tumor cancer. Amanqanaba aphezulu e-ITGA9 anxulunyaniswa ne-tumor metastasis kunye nokubuyela umva kwizigulane ze-TNBC. Ukususwa kwe-ITGA9 yi-CRISPR/Cas9 kubangele iipropati ezifana ne-cancer stem cell (CSC), i-tumor angiogenesis, ukukhula kwe-tumor, kunye nokunciphisa i-metastasis ngokukhuthaza ukuchithwa kwe-β-catenin kwi-TNBC (Wang Z. et al., 2019).
I-Crypto-1 lilungu losapho lwe-TGF-β kwaye ibalulekile ekukhuleni kwe-embryogenesis kwangethuba, ukugcinwa kweeseli ze-stem, kunye nokusasazeka komhlaza (Ishii et al., 2021). Ikwaziwa ngokuba yi-Tdgf-1, yiprotheyini yokubonisa i-GPI e-oncogenic ebandakanyeka ekulawuleni ukwakheka kwe-primitive streak, i-mesoderm kunye ne-endoderm, kunye nokusekwa kwe-asymmetry yasekhohlo/ekunene ekuphuhlisweni kwamalungu omzimba ngexesha le-embryogenesis (Zhang et al., 2021b). Ukongeza, i-Cripto-1 ibonakalisiwe ukuba ibandakanyeka kwi-epithelial-mesenchymal transition (EMT) njenge-stem cell marker (Zhang et al., 2021b). I-EMT ayibalulekanga kuphela kwiinkqubo ezahlukeneyo ezifana nophuhliso lwe-embryonic, i-fibrosis kunye nokuphiliswa kwamanxeba, kodwa nakwi-cancer invasion kunye ne-metastasis. Ukongeza, i-Cripto-1 ibonakalisiwe ukuba isebenzisane nee-receptors ezine ze-Notch kwaye iphucula ukukhula kwazo emva kokuguqulwa (Brandstadter kunye ne-Maillard, 2019). Indlela yokubonisa iNotch iyaziwa ukuba ibandakanyeka ekugcinweni kweeseli zomhlaza wamabele zomntu. Izifundo zibonise ukuba i-CRISPR/Cas9-mediated knockout ye-Crypto-1 ithintela ukukhula komhlaza kunye nokusasazeka kwawo. Ke ngoko, i-Crypto-1 inokuba yinto ebalulekileyo ekujoliswe kuyo kwi-TNBC (Castro et al., 2015).
Iprotheyini ye-XCL12 kunye ne-chemokine receptor yayo i-CXC (CXCR4 kunye ne-CXCR7) zidlala indima ezahlukeneyo ekwandeni kweeseli zomhlaza, ukukhula, ukufuduka kunye nokuhlasela (Wu et al., 2015). Ezi chemoreceptors zidibene nophuhliso lwe-TNBC ngeendlela ezininzi zokubonisa iimpawu kuzo zombini iimodeli ze-in vivo kunye ne-in vitro (Wu et al., 2015). Ukongeza, ukusebenza kwe-CXCR4 kunye ne-CXCR7 kunxulunyaniswa nokuchaphazeleka okukhulu kokusasazeka kwesifo kunye nokubikezela okubi kwi-TNBC (Karn et al., 2022). Ke ngoko, ukuphuma kwe-CXCR4 kunye ne-CXCR7 kunokuba yi-genes ejolise kwiyeza ezisebenzayo kunyango lomhlaza webele, kubandakanya i-TNBC. Uphononongo lukaYang et al. Yang et al (2019) lusebenzise i-CRISPR/Cas9 ukukhupha ii-genes ze-CXCR4 kunye ne-CXCR7 kwaye lufumanise ukuba ukwanda kwe-TNBC, ukukhula, ukufuduka kunye nokuhlasela kwakuthintelwe kakhulu (Yang et al., 2019).
Amanqanaba aphezulu e-miR-3662, i-TNBC oncogene, abonwe kwiithishu zomhlaza webele (Yi et al., 2022). Ukuthotywa kwe-miR-3662 kuboniswe ukuba kuthintela ukukhula kwethumba lomhlaza webele kunye nokusasazeka kwesifo somhlaza webele kokubini emzimbeni nakwi-vitro (Agarwal kunye noGupta, 2021). I-HBP-1 sisithinteli esinamandla se-Wnt/-catenin signaling kwaye kusenokwenzeka ukuba sinoxanduva lokukhula kwe-miR-3662-mediated kweeseli ze-TNBC. Kutshanje, uYi et al. ufumanise ukuba i-miR-3662-HBP1 axis ilawula indlela ye-Wnt/-catenin signaling kwiiseli ze-TNBC (Yi et al., 2022). Ngenxa yokubonakaliswa kwayo ngokukodwa kwethumba, i-miR-3662 inokuba yindawo ekujoliswe kuyo yonyango lwe-TNBC. Ke ngoko, ukuthotywa kwe-miR-3662-mediated CRISPR/Cas9-mediated kunokuba yindlela entle yokuphuhlisa amayeza amatsha okunyanga i-TNBC.
I-UBR5 yi-nucleophosphoprotein engama-300 kDa echongiweyo njengomlawuli oyintloko we-tumorigenesis, i-metastasis, kunye nempendulo yomzimba kwiintlobo ngeentlobo zomhlaza (Shearer et al., 2015; Fu et al., 2023). I-UBR5 ibikwe ukuba inyuswe kakhulu kwiisampulu ze-TNBC kwaye ivuselela umsebenzi we-ERα ngokubangela ukwanda komsebenzi wayo we-ubiquitin ligase (Bolt et al., 2015). Izifundo zokulandelelana kwe-exome epheleleyo kwiisampulu ze-TNBC eziphambili nazo zibonise ukwanda kokubonakaliswa kwe-UBR5, nto leyo ebonisa indima yayo kuphuhliso lwe-TNBC. Ukongeza, ukususwa kwe-UBR5 okuqhutywa yi-CRISPR/Cas9 kubonise ukuthintela okukhulu kwe-metastasis ye-TNBC kunye nokukhula kwiimodeli zegundane zovavanyo. Ngaphezu koko, ukufakwa kwe-UBR5 kwimodeli yegundane yohlobo lwasendle kubuyisele ukusebenza kwayo okupheleleyo, ngelixa esi siphumo singakhange sibonwe kwiintlobo ze-mutant ezingasebenziyo (Liao et al., 2017). Ukungabikho kwe-UBR5 kunxulunyaniswa nokwanda kwe-apoptosis, i-necrosis, kunye nokuthintela ukukhula kwe-tumor kwi-TNBC ngenxa ye-angiogenesis embi. Ngenxa yokulahleka kwe-UBR5, ukusasazeka kwe-tumor kwizitho ezikude kuyancitshiswa, kwaye i-UBR5 ibangela i-EMT engaqhelekanga ikakhulu ngokunciphisa ukubonakaliswa kwe-E-cadherin (Zhang kunye noWeinberg, 2018). Kutshanje, i-UBR5 ibonakaliswe njengeyona nto ibalulekileyo kwi-IFN-γ-induced PDL1 transcription kwi-TNBC ngenxa yokungabikho komsebenzi wayo we-E3 ubiquitination. Uhlalutyo lwe-RNA transcriptome lutyhile ukuba i-UBR5 inokuba nemiphumo yenkqubo kwi-genes enxulumene nendlela ye-IFN-γ kwaye ikhuthaze i-PDL1 transactivation ngokunyusa amanqanaba okusebenza kwe-protein kinase RNA (PKR) kunye ne-signal transducers kunye ne-activators. i-transcription 1 (STAT1) kunye ne-interferon regulatory factor 1 (IRF1). Nangona kunjalo, ukususwa okudibeneyo kwe-UBR5 kunye ne-PD-L1 okubangelwa yi-CRISPR/Cas9 kunefuthe lokunyanga elihambelanayo kune-blockade yodwa, enefuthe elikhulu kwi-microenvironment ye-tumor (Wu et al., 2022). Ke ngoko, i-CRISPR/Cas9 inokuba sisixhobo esibalulekileyo sokuthintela umsebenzi we-UBR5, ngaloo ndlela icinezela i-metastasis ye-TNBC kunye nokukhula kwe-tumor.
I-ROR1 yiprotheyini ye-transmembrane yohlobo lwe-1 ebonakaliswa ngexesha lomhlaza kunye nophuhliso lwe-embryonic kwaye ichongiwe njengeprotheyini ye-oncofetal (Nicholas Borcherding, 2014). Ukuziphatha okungafunekiyo komhlaza wabantu abahlukeneyo kunxulunyaniswa nokunyuka kwe-ROR1. Iziphumo ezithembisayo zifunyenwe kwizifundo ze-in vivo kunye ne-in vitro ezibandakanya iikhompawundi zonyango ezijolise kwi-ROR1 (Chien et al., 2016). Amanqanaba aphezulu e-ROR1 mRNA kwii-biopsies zezicubu zebele nawo anxulunyaniswa neethumba zesifuba ezifana ne-basal (BL) ezihlaselayo kunye nokufudukela kwazo kwezinye iindawo zomzimba. Ngaphezu koko, ukuvezwa kakhulu kwe-ROR1 kuchongiwe njengophawu lokuxela kwangaphambili uphuhliso lwe-TNBC (Chien et al., 2016). Nangona kunjalo, ukuthulisa i-ROR1 kusetyenziswa i-CRISPR/Cas9 ukuthintela ukukhula kwe-TNBC kunye nokusasazeka kwe-metastasis kuya kuba licebo elisebenzayo.
Inkqubo ye-sialylation ibandakanya ukongezwa kwe-sialic acid kwi-glycoconjugates, ekhuthazwa yi-sialyltransferases (STs). I-ST8SIA1 ikwintsapho ye-ST kwaye idlala indima ebalulekileyo kwi-pathogenesis yezifo ezahlukeneyo ezifana ne-lymphocytic leukemia kunye nomhlaza we-colorectal (Chang et al., 2018). Uhlalutyo lolandelelwano lwe-RNA lubonisa ukuba i-ST8SIA1 ibonakaliswa kakhulu kwizicubu zebele zezigulane ze-TNBC kwaye inxulumene kakuhle notshintsho kwi-tumor suppressor gene p53, enokuba negalelo kwi-pathogenesis ye-TNBC (Battula et al., 2017). Ukongeza, i-ST8SIA1 ibandakanyeka kwi-metastasis kunye nokubuyela kwakhona kwe-TNBC, nto leyo ebonisa ukuba idlala indima ebalulekileyo ekwenzekeni nasekuphuhlisweni kwe-TNBC. Kwimodeli ye-in vitro ye-TNBC, ukunqotshwa kwe-ST8SIA1 yi-CRIPSR/Cas9 kuboniswe ukuba kuthintela ukukhula kunye ne-metastasis (Battula et al., 2017). Oku kuthetha ukuba i-CRISPR/Cas9 inokuba sisixhobo esibalulekileyo sokuthintela umsebenzi we-ST8SIA1 oncogene kunyango lwe-TNBC.
I-NAT1 yi-enzyme ye-metabolic ekhuthaza ukwakheka kwee-phase II xenobiotic compounds kwaye ibonakaliswa phantse kuzo zonke izicubu zomntu. I-NAT1 ingasebenzisa i-cofactor folate ukujolisa kwi-acetyl-CoA (acetyl-CoA) nokuba akukho substrate ye-aromatic amine (Stepp et al., 2015; Laurieri et al., 2014). Izifundo zibonise ukuba i-NAT1 ilawula umsebenzi we-matrix metalloproteinase 9 (MMP9) kwiimodeli zeeseli zomhlaza webele kwaye ikhusela kwiintlobo ze-oxygen ezisabelayo (ROS) ngexesha lokungabikho kweglucose (Wang et al., 2018). Ukususwa kwe-NAT1 kuboniswe ukuba kuthintela i-pyruvate dehydrogenase complex, okukhokelela ekungasebenzi kakuhle kwe-mitochondrial (Wang L. et al., 2019). Ukongeza, ezinye iingxelo ezahlukeneyo zibonise ukuba ukuthintelwa kwe-NAT1 ngokusebenzisa iimolekyuli ezincinci kunye nokuthulisa i-siRNA kunokunciphisa ukungenelela kunye nokwanda kweeseli zomhlaza webele (Stepp et al., 2018). Kutshanje, i-CRISPR/Cas9 isetyenzisiwe ukucinezela i-NAT1 kumgca weseli yomhlaza webele i-MDA-MB-231, ichaphazela imetabolism yeseli ngokuxhomekeke kwinqanaba lokubonakaliswa kwayo. Olu phononongo lukwabonise ukuba i-NAT-1 ibalulekile ekuqhubekeni nasekukhuleni kwe-TNBC (Carlisle et al., 2020).
Ukubhalwa kwe-oncogenes okudibeneyo kulawulwa zi-super enhancers, ii-transcription factors kunye nee-cofactors (Hnisz et al., 2015). Ukongeza, iqela le-cyclin-dependent kinases (ii-CDK), ezifana ne-CDK7, CDK8, CDK9, CDK12, kunye ne-CDK13, ziyafuneka ukuze kulawulwe ukubhalwa kwe-transcription. Phakathi kwazo, i-CDK7 ibandakanyeka kwi-phosphorylation ye-RNA polymerase II, ebaluleke kakhulu ekuqalisweni nasekunabiseni ukubhalwa kwe-oncogene kwi-pathogenesis ye-TNBC. Kwisifundo esiphambili, ukususwa kwe-CDK7 yi-CRISPR/Cas9 kuthintele i-TNBC, okubonisa ukuba i-pathogenesis ye-TNBC ixhomekeke kwi-CDK7 (Wang Y. et al., 2015).
Izifundo zibonise ukuba utshintsho kwi-MYC kunye ne-RBP (iprotheni yokubopha i-RNA) lunokukhokelela kwi-apoptosis, ngelixa utshintsho lwe-single gene kwi-MYC okanye kwi-RBP lungachaphazeli ukukhula kweeseli zomhlaza (Einstein et al., 2021). Ngaphezulu kwe-1000 RBPs kwi-genome yomntu ihlolwe kusetyenziswa ithala leencwadi elisekelwe kwi-CRISPR/Cas9. Phakathi kwazo, ii-RBP ezingama-57 zifunyenwe zibalulekile ekukhuleni kweeseli zomhlaza ezinamazinga aphezulu e-MYC (Wheeler et al., 2020). Ukongeza, i-YTHDF2 ibalulekile ekugcineni ukukhula kweeseli ze-TNBC kwaye inciphisa inani lee-translated ze-methylated ngexesha lokubhalwa nokuguqulwa kwezinga eliphezulu kwiiseli zomhlaza ezine-MYC expression ephezulu. Ukongeza, i-YTHDF2 ayibalulekanga kwiiseli zomhlaza, ezingaxhomekekanga kakhulu kumanqanaba aphezulu e-MYC ukuze kwandiswe ukusinda kwezigulane ze-TNBC (Einstein et al., 2021), nto leyo ebonisa ukuba inokuba yindawo ekujoliswe kuyo yonyango lwamayeza anokoyisa i-TNBC.
Iiproteni zeminwe yeZinc (ii-ZNF) zenza malunga ne-1% ye-genome iyonke yomntu. Izifundo zibonise ukuba i-ZNF inokulawula ukwanda kweeseli kwiintlobo ngeentlobo zomhlaza, ezifana nomhlaza wesibindi, umhlaza webele kunye nomhlaza we-colorectal (uZhang W. et al., 2021; uLi et al., 2017). Iilayibrari eziveliswe yi-CRISPR knockout zisetyenziselwe ukuhlola ii-genes ezahlukeneyo ze-tumor suppressor (ii-TSG) kwiiseli zomhlaza webele (uShalem et al., 2014). Ukususela ngoko, uphando lwe-transcriptomic lwee-CRISPR/Cas9 ZNF319-deleted breast cancer cells lubonise ukuba i-ZNF319 yi-gene ye-tumor suppressor enciphisa ukwanda komhlaza webele kwaye ngenxa yoko ibandakanyeka kwiindlela ezahlukeneyo zokubonisa iimpawu kunye neminye imisebenzi yebhayoloji (uWang L. et al., 2022).
I-Ferroptosis luhlobo lokufa kweeseli okucwangcisiweyo okuxhomekeke ekubeni kukho isinyithi. Kuyaziwa ukuba uphuhliso lwe-ferroptosis luphenjelelwa bubukho be-lipid peroxides. Ukongeza, i-PKCβII ibikwe ukuba ibonisa i-lipid peroxidation kwangoko, kwaye ukwanda kwe-lipid peroxidation kunxulunyaniswa ne-ferroptosis. Ukuhlolwa kwethala leencwadi le-CRISPR/Cas9-mediated kinase inhibitors kutyhile ukuba i-PKCβII ibandakanyeka kwinkqubo ye-lipid peroxidation, ebaluleke kakhulu kwi-ferroptosis kwiiseli ze-MDA-MB-231 (Zhang et al., 2022). Ke ngoko, oku kuthetha ukuba ukunqunyulwa kwe-PKCβII yi-CRISPR/Cas9 kunokuba yinto ekujoliswe kuyo kwi-tumor suppressor gene kunyango lwezifo ezinxulumene ne-ferroptosis (Zhang et al., 2022).
Kukholelwa ukuba ukunganyangeki ngamayeza kubangele malunga ne-90% yokufa kwabantu abanesifo somhlaza kwaye yenye yeengxaki ezinkulu kunyango lomhlaza (Bukowski et al., 2020). Iziphumo zibonisa ukuba inani elaneleyo leejini ezinxulumene nokuphuma kwamayeza, ukulungiswa kwe-DNA, i-apoptosis kunye neendlela ezahlukeneyo zokubonisa iiseli zidibene nokunganyangeki ngamayeza (Haider et al., 2020). Phakathi kwazo, iijini ezininzi zijoliswe kwizixhobo ze-CRISPR/Cas9 kwaye zibonise iziphumo ezithembisayo ekunciphiseni ukunganyangeki ngamayeza kunye nokwandisa ukusebenza kakuhle konyango lokulwa nomhlaza (Vaghari-Tabari et al., 2022). Ukongeza, iilayibrari zokuhlola i-CRISPR/Cas9 gene knockout eziphezulu ziye zabandakanyeka ekuguquleni umsebenzi weethagethi zejini zokunganyangeki ngamayeza (Shalem et al., 2015). Ukuchonga iijini zokuxhathisa i-paclitaxel, ukulandelelana kwe-RNA kunye nokuhlolwa kwelayibrari ye-sgRNA ye-genome wide kusetyenzisiwe ukuchonga iijini ezisibhozo ezikhethiweyo, kuquka i-histone deacetylase 9 (HDAC9), ezinxulunyaniswa nokuxhathisa amayeza kwizigulana ezine-TNBC ephindaphindayo (B et al., 2020). ). Kolunye uphando, ukwanda kokubonakaliswa kwe-diserine/threonine kunye ne-tyrosine protein kinase (DSTYK) kwabonwa ngexesha lokusinda kwezigulana ze-TNBC ezinyangwa ngamayeza okulwa nomhlaza. Ngaphezu koko, ukunqongophala kwe-DSTYK okubangelwa yi-CRISPR/Cas9 kwaphucula kakhulu i-apoptosis yeeseli zomhlaza ezinganyangekiyo ngamayeza kwi-vitro (iiseli ze-SUM102PT kunye neeseli ze-MDA-MB-468) kunye nemodeli ye-TNBC kwi-vivo (Ogbu et al., 2021).
Nangona i-TNBC ingasebenzi ngendlela engaqhelekanga kwindlela ye-MAPK, isiphumo seklinikhi sezonyango ezijoliswe kwi-MEK asisihle. Isikrini selayibrari ye-genomic ye-CRISPR/Cas9 sityhile ukuba ukuthintelwa kwe-PSMG2 (i-proteome assembly chaperone 2) kwenza iiseli ze-TNBC BT549 kunye ne-MB468 zibe nomdla kwi-MEK inhibitor AZD6244. Ukuwa kwe-PSMG2 yi-CRISPR/Cas9 kutshintshe umsebenzi oqhelekileyo we-proteasome, nto leyo ekhokelela ekuqhekekeni kwe-PDPK1 okwenziwa yi-autophagy, ngaloo ndlela kuphucuka ngakumbi iiseli ze-tumor kwiimodeli ze-TNBC mouse ezibangelwa yi-AZD6244 (MEK inhibitor) kunye ne-MG132 (proteasome inhibitor). Ke ngoko, ii-proteasome kunye ne-MAP kinase (MEK) inhibitors zinokusetyenziswa ngokubambisana ukunciphisa ukwanda kweeseli ze-tumor (Wang X. et al., 2022).
I-CRISPR/Cas9 ikwasetyenzisiwe ukuhlola ukulahleka komsebenzi wejini okukhokelela ekuxhathiseni kwe-TNBC (Shu et al., 2020). UGe et al bachaze indlela yokuxhathisa amayeza kwiiseli zomhlaza ezinyangwa nge-JQ1, i-BET bromodomain inhibitor (BBDI), kwi-TNBC. Besebenzisa i-CRISPR/Cas9, bafumanise ukuba ukususa i-rb1 gene kubangele ukuba i-TNBC ingaxhathisi kwiyeza lokulwa nomhlaza i-JQ1. Ke ngoko, umsebenzi we-rb1 ubalulekile ekuphenduleni amayeza e-JQ1 kwi-TNBC. Bakwaxele ukuba i-paclitaxel yi-CDK4/6 kinase/microtubule inhibitor, kwaye ukudibana kwayo nee-BBDI ezifana ne-JQ1 kunokubonelela ngeempendulo zonyango ezithembisayo kwi-TNBC engaxhathisi amayeza (Ge et al., 2020).
Imeko yokubhalwa kwe-RNA ende engabhalwanga (lncRNA) ibikwe njengebangela ukuxhathisa unyango lwe-neoadjuvant kwi-TNBC. Olu phononongo lubonisa ukuba ii-transcripts ezintlanu ezahlukeneyo ze-MALAT1 lncRNA zibonakaliswa kakhulu kwi-TNBC. Ukongeza, ukususwa kwe-MALAT1 okubangelwa yi-CRISPR/Cas9 kwandisa uvakalelo lweeseli ze-TNBC BT-549 kwi-paclitaxel kunye ne-doxorubicin, nto leyo ebonisa indima enokubakho yokuxhathisa kwe-MALAT1 kwi-TNBC (Shaath et al., 2021).
Utshintsho kwi-BRCA1 (BRCA1m) alufani kwaye ngenxa yoko kunzima ukulibona. Ukujolisa kwi-PARP1 (poly(ADP-ribose) polymerase), iqabane elibulalayo elenziweyo le-BRCA1, kunokunyusa uvakalelo lwe-chemosensitivity yamayeza e-TNBC. Ukusebenzisa i-CRISPR/Cas9, ukususwa kwe-PARP1 kwandisa uvakalelo lwamayeza okulwa nomhlaza afana ne-doxorubicin, i-gemcitabine kunye ne-docetaxel kwiiseli ze-TNBC eziguqulweyo ze-mBRCA1, nto leyo ebonisa ukuba i-PARP1 ikwabandakanyeka ekuchaseni amayeza kwi-TNBC (Vaghari-Tabari et al., 2022). Utshintsho kwi-genes ye-tumor suppressor BRCA1 okanye i-BRCA2 lwaziwa ngokunyusa amathuba okuba nomhlaza wamabele ebantwini. Unyango lwaba baguli lufuna ukusetyenziswa kwezithinteli ze-PARP. Ukongeza, kuye kwaboniswa ukuba ukususwa kwe-nucleotide salvage factor DNPH1 kusetyenziswa i-CRISPR/Cas9 kunokususa i-nucleotide 5-hydroxymethyldeoxyuridine (hmdU) monophosphate enetyhefu, ngaloo ndlela kuphucula impendulo yeeseli ezingenayo i-BRCA kwi-sensitivity kwi-PARP inhibitors (Fugger). et al., 2021). Ke ngoko, i-CRISPR/Cas9 kunye ne-PARP1 inhibitors zinokuba yindlela ebalulekileyo yonyango lwe-TNBC.
I-gene ye-penetrant glycoprotein (P-gp) yi-gene yokumelana namayeza amaninzi edla ngokunyuswa malunga ne-41% yazo zonke ii-TNBC (Sun et al., 2020). Ukuphuma kwamayeza abangelwa yi-P-gp kuye kwachongwa njengomlawuli ophambili wokuxhathisa amayeza kumhlaza webele. Izithinteli ze-P-gp zibonakalise ukwanda kovakalelo kumayeza okulwa nomhlaza kumhlaza webele (Famta et al., 2021). Ke ngoko, ukuncitshiswa okanye ukuncitshiswa kwe-P-gp kunye ne-P-gp ezibangelwa yi-CRISPR/Cas9 kunokuba ziindlela ezibaluleke kakhulu zokoyisa ukuxhathisa amayeza kwi-TNBC.
I-ATP-binding cassette transporter G2 (ABCG2) yaziwa ngokuba ibangela ukunganyangeki ngamayeza kwi-TNBC (Palasuberniam et al., 2015), nangona okwangoku kungekho ngxelo malunga nobudlelwane phakathi kwe-CRISPR/Cas9 kunye ne-ABCG2 silencing kunye nokungasebenzi kwe-tumor suppressor gene. I-PTEN isenokunyusa ukusebenza kwe-ABCG2 (Palasuberniam et al., 2015), (Deepak Singh et al., 2021). Ke ngoko, ukusebenzisa i-CRISP/Ca9 ukususa i-ABCG2 nokuyidibanisa ne-ABCG2 inhibitor kunokuba lunyango olufanelekileyo lokoyisa ukunganyangeki ngamayeza kwi-TNBC. Itheyibhile 2 ikhankanya i-CRISPR/Cas9 ejolise kuzo zonke ii-genes zokunganyangeki.
Itheyibhile 2. I-CRISPR/Cas9 ijolise kwiijini ezahlukeneyo zokuxhathisa i-TNBC ukuze zenze iiseli zikwazi ukufumana amayeza okulwa nomhlaza.
Iilayibrari zeCRISPR/Cas9 zizixhobo ezinokubakho zokuhlola utshintsho lwezakhi zofuzo olunxulumene ne-pathogenesis yomhlaza (Chan et al., 2022). Le ndlela yokuhlola ibandakanya amanyathelo amane anje (a) ukwakhiwa kwelayibrari, (b) ukudluliselwa kwe-lentiviral, (c) ukuhlolwa kwe-phenotypic, kunye (d) nohlalutyo lwezakhi zofuzo ekujoliswe kuzo. Nangona i-TNBC inomsebenzi ongaqhelekanga wendlela ye-MAPK, i-prognosis yeklinikhi yonyango olujoliswe kwi-MEK kwizigulane ze-TNBC ezithwala utshintsho kwiizakhi zofuzo ezithintela ithumba ezifana ne-PTEN, RB1, kunye ne-TP53 ayilunganga kakhulu.
Ukongeza, isikrini sokukhupha iijini sisebenzisa i-CRISPR/Cas9 sivavanye eyona molekyuli inamandla nekhethayo, i-dehydrocatrol, efumaneka kakhulu kwiincasa zentyatyambo namagqabi zaseGuatemala, ukuze kuqondwe isiphumo se-dehydrocatrol kwi-MDA-MB-231. Iindlela zokubulala iiseli ezikhethiweyo. Iimpawu ze-Mesenchymal stemness ze-TNBC subtypes. Esi sikrini sisekelwe kwi-CRISPR/Cas9 sikwatyhile ukuba i-HSD17B11, ijini efaka i-17β-hydroxysteroid dehydrogenase type 11, ibonakaliswa kakhulu kwiiseli ze-MDA-MB-231 kwaye iphumela kwi-dehydrofalcarinol ekhethekileyo kwiiseli ze-MDA-MB-231 (Grant et. al., 2020). Ke ngoko, oku kuthetha ukuba ukuhlolwa kwe-CRISPR/Cas9 genomic kunamandla amakhulu ekuchongeni iindlela eziphantsi kweempawu zokulwa nomhlaza zeekhompawundi zendalo ezinokubakho.
Ukongeza, ukuba sesichengeni kwe-TNBC kumhlaza kufundwe kusetyenziswa isikrini se-CRISPR/Cas9 esingakhethi cala, esibonisa unxibelelwano phakathi kweendlela ze-oncogenic kunye ne-tumor suppressor. Izinto eziphambili zeendlela ze-mTOR kunye ne-Hippo ziye zaxelwa ukuba zidlala indima ebalulekileyo ekulawuleni i-tumor kwi-TNBC. Ukongeza, izifundo zibonise ukuba ukuthintelwa kwe-mTORC1/2 kunye ne-YAP oncoprotein kwi-pharmacological kuthintela ngempumelelo i-TNBC kusetyenziswa imodeli ye-in vitro drug-matrix synergy kunye ne-xenografts ezivela kwi-vivo kwisigulane. Ngaphezu koko, ukuthintelwa kwe-mTORC1/2 okubangelwa yi-Torin-1 kwandisa i-macropinocytosis, ngelixa ukuthintelwa kwe-YAP okubangelwa yi-vertebrofin kukhokelela ekufeni kweeseli ze-TNBC. Xa zizonke, ezi ziphumo zibonisa amandla kunye nokuqina kokuhlolwa kwe-CRISPR kwi-vivo ukuze kuchongwe unyango olutsha nolusebenzayo lwe-TNBC (Dai et al., 2021).
Ukungasebenzi kakuhle kwenkqubo yomzimba yokuzikhusela komzimba yinto ebalulekileyo ekuveleni kwesifo sephepha. Iiseli zomhlaza ziphepha ukususwa kwesixhobo somzimba ngokuthintela iindlela zokuzikhusela, kuquka ukuphazamisana nomsebenzi weeseli zomzimba kwindawo encinci yesifo sephepha kunye nokubeka emngciphekweni inkqubo yomzimba yokuzikhusela. Ke ngoko, ukuphuhlisa inkqubo yomzimba yokuzikhusela ephuculweyo kunokuba yindlela ephambili yokulwa neentsholongwane. Ukusetyenziswa kokuguqulwa kwezakhi zofuzo okusekelwe kwi-CRISPR/Cas9 kujongana nemiba eliqela enxulumene nokungasebenzi kakuhle kwenkqubo yomzimba yokuzikhusela kwiimbono ezahlukeneyo. I-CRISPR/Cas9 isetyenzisiwe ukuphucula amandla omzimba okuzikhusela kwisifo sephepha nxamnye nomhlaza webele ngeendlela ezilandelayo (Umfanekiso 3).
Umfanekiso 3. I-CRISPR/Cas9 immunotherapy ijolise kwiiseli ze-TNBC. Ukulahlekelwa yi-CDK5 kunye nokudilizwa kwe-PDL1 kunye ne-CD155 kuphucula inkqubo yomzimba yokuzikhusela. Ngokufanayo, ukulahleka kwe-A2AR kunye nokudilizwa kwee-TAA ezifana ne-HER2, i-mucin 1 kunye ne-TEM8 kwandisa ukusebenza kakuhle kweeseli ze-CAR-T ekubulaleni iiseli zomhlaza. Ukuhlolwa kweeseli ze-T eziqhutywa yi-CRISPR/Cas9 kubonise ukuba ukuphazamiseka kwe-p38 kinase kuphucula umsebenzi wokulwa nethumba le-T. Iiseli ze-T eziguqulwe yi-CRISPR/Cas9 zonyusa ukubonakaliswa kwe-TCR (i-T cell receptor), nto leyo ebangela ukuba iiseli ze-T zibe nomsebenzi wokulwa nethumba.
Injongo yonyango lwe-immunotherapy kukukhuthaza inkqubo yomzimba yokuzikhusela ukuba ihlasele iiseli zomhlaza. Ukwanda kakhulu kweeproteni zokujonga amajoni omzimba kudla ngokuthintela ukusabela kwe-autoimmune kodwa kunokunceda umhlaza ukuba ungangeni kuzo (Topalian et al., 2015). Ezi proteni zithintela ukusabela kwe-immune ngokubophelela kwii-receptors kumphezulu weeseli zomzimba. Iiproteni ezininzi zokujonga amajoni omzimba (ezifana ne-CD155 kunye ne-PD-L1) zijolise kwi-PD-1 receptor kwiiseli zomzimba kwaye zibonakaliswa kumhlaza webele, ngakumbi i-TNBC (Li Y.-C. et al., 2020). Ukuthotywa kwe-PD-L1 okanye i-receptor yayo esebenzisa i-CRISPR/Cas9 kunokukhuthaza inkqubo yomzimba yokuzikhusela ukuba ihlasele iithumba ze-TNBC (Yahata et al., 2019). Ukongeza, ukunciphisa ukubonakaliswa kwe-PD-L1 ngokususwa kwe-CDK5 yi-CRISPR/Cas9 kuye kwabonakala kuthintela ukukhula kwethumba kwi-vitro nakwi-vivo (Deng et al., 2020). Ukuthintela ukukhula kwiimodeli zomhlaza webele kwi-in vitro nakwi-vivo kubonisa ukuba ukunciphisa i-CD155 okubangelwa yi-shRNA kunokuba nefuthe lokunyanga umhlaza webele (Gao et al., 2018).
Iiseli ze-CAR T zibonisa ii-CAR eziqaphela ii-antigens ezinxulumene ne-tumor (ii-TAA) kwaye zinokusebenzisa ukuthotywa kweeproteni zokujonga ukuphucula umsebenzi wazo (uLi C. et al., 2020). Kukho iindlela ezahlukeneyo ezinokuthi zijoliswe kuzo unyango lweeseli ze-CAR T kumhlaza webele, kubandakanya ii-TAA ezininzi ezifana ne-HER2, i-mucin1, kunye ne-TEM8 (uBajgain et al., 2018). Kukho ubungqina bokuba iiseli ze-CAR T ezijolise kwi-mesothelin (ezivezwa kakhulu kwiiseli ze-TNBC BT-459) zisebenza ngakumbi kumhlaza xa i-PD-1 ikhutshwa kusetyenziswa i-CRISPR/Cas9 (uHu et al., 2019). Nangona kunjalo, ukwahlula iiseli ze-T kwizigulana uze uzihlele ngaphandle komzimba yinkqubo enzima nethatha ixesha. Nangona iiseli ze-T ezipheleleyo zinokunciphisa iimfuno zokwahlulwa, iiseli ze-T ezinikelayo eziveza i-human leukocyte antigen (HLA) class I kunye ne-T cell receptor (TCR) kufuneka zisuswe ukuthintela ukungasebenzi kakuhle kwe-graft-versus-host (uRen et al., 2017). , 2017a). Ukusebenzisa i-CRISPR/Cas9, i-HDR inokususa i-TCR kunye ne-HLA ngaxeshanye kwaye isuse i-CAR edibanisa i-gene. Izifundo zibonise ukuba i-CRISPR/Cas9 ingasetyenziselwa ukungenisa ii-anti-CD19 CARs kwindawo ye-TCR, ngaloo ndlela ivelise ii-CARs ngokufanelekileyo ngaphandle kokunciphisa ii-T cells (Dimitri et al., 2022). Ubuchwepheshe be-Multiplex buphuhlisiwe ukuvelisa ii-allogeneic CAR T cells ngokuphelisa ngaxeshanye i-TCR, i-beta-2-microglobulin (B2M), i-HLA-I subunit kunye nezinye iiproteni ezifana ne-PD-1 kunye ne-CTLA-4, ezinokuba neempawu ezinkulu zokulwa nomhlaza. ngokuchasene ne-TNBC (Eyquem et al., 2017; Liu et al., 2017; Ren et al., 2017b; Dimitri et al., 2022).
Ukusebenzisa i-CRISPR/Cas9 kunokuphucula ukusebenza kakuhle kweeseli ze-CAR-T. I-Adenosine yaziwa ngokuba neempawu zokuthintela umhlaza kwaye inokunciphisa ukhuselo lomhlaza ngokuthintela ukusebenza kweeseli ze-T kunye nokwenza ii-receptors ze-adenosine A2A (A2AR) zisebenze (Vigano et al., 2019). Ukuthulisa i-A2AR usebenzisa i-CRISPR/Cas9 kuphucule kakhulu ukusebenza kakuhle kweeseli ze-CAR-T emzimbeni (Giuffrida et al., 2021). Iiseli ze-T zaziwa ngokubonisa iimpawu ezahlukeneyo ze-phenotypic ezifana nokwandiswa kweeseli, ukwahlukana, uxinzelelo lwe-oxidative kunye noxinzelelo lwe-genomic. Ukuhlolwa kweeseli ze-T ezisekelwe kwi-CRISPR/Cas9 kutyhile ukuphazamiseka kwee-kinase ze-receptor zeseli ze-T ezingama-25 ezahlukeneyo. Phakathi kwazo, ukususwa kwe-p38 kinase kuboniswe ukuba kuphucula umsebenzi we-antitumor weeseli ze-T, okubonisa ukuba i-p38 kinase ngumlawuli ophambili wolawulo lweeseli ze-CAR-T (Gurasamy et al., 2020).
Iiseli ze-T zinokuguqulwa ngokwemfuza kusetyenziswa i-CRISPR/Cas9 ukuvelisa ii-TCR eziveliswe kakhulu. Ukudlulisela iiseli ze-T eziguqulwe ngokwemfuza kwizigulana kubonakalise umsebenzi onamandla wokulwa nomhlaza kuneeseli ze-T eziveliswe ngokwasemzimbeni. Ke ngoko, ii-TCR eziveliswe ngokwasemzimbeni zinokukhuphisana nee-TCR eziguqulwe ngokwemfuza kwizigulana, nto leyo enokuchaphazela amandla onyango lomhlaza. Ukuze koyiswe le ngxaki, ukusebenzisa i-CRISPR/Cas9 ukususa i-TCR-β eveliswe ngokwasemzimbeni kwiiseli ezamkelayo kwaye emva koko kudluliselwe iiseli ze-TCR-β kwizigulana ezinomhlaza kunokubonisa iimpendulo ezingcono zokuzikhusela kumhlaza ngaphandle kwesidingo sokhuphiswano lwesondo lwe-TCR eveliswe ngokwasemzimbeni (Fan et al. 2018). Ke ngoko, ngaphandle kweeseli ze-T eziguqulwe ngokwasemzimbeni ze-CRISPR, ii-TCR zinovelwano oluphindwe kaliwaka kwii-antigen ze-tumor kuneeseli ze-T eziveliswe ngokwemfuza ze-TCR eziqhelekileyo. Ukongeza, kwii-leukemia ezahlukeneyo, iiseli ze-T eziguqulweyo eziveliswe yi-γδ TCR+ CRISPR zibonisa ukubonakaliswa okuphezulu kweeseli ze-CD4+ kunye ne-CD8+ T kunokudluliselwa kwe-TCR eqhelekileyo (Legut et al., 2018). Ngoko ke, ii-T cells eziguquliweyo eziveliswa yi-CRISPR/Cas9 zinokuba yindlela esebenzayo yonyango lwe-immunotherapy ukoyisa i-TNBC.
Ii-Integrins ziimolekyuli zokunamathela kweeseli ezikhoyo kwi-cellular transmembranes kwaye zikhuthaza ukubopha kweeseli kwi-extracellular matrix (ECM) (Hamidi kunye no-Ivaska, 2018). Ukuphazamiseka kwe-integrins kunxulunyaniswa nophuhliso lomhlaza kunye nokufuduka ngokutshintsha i-extracellular matrix, okukhokelela ekusindeni kweeseli zomhlaza kwi-circulation (Hamidi kunye no-Ivaska, 2018). I-CRISPR/Cas9-mediated integrin knockdown inciphisa ukuqhubela phambili kwe-tumor, i-metastasis, kunye ne-colonization kwi-TNBC. Ukuthotywa kwe-integrin a5 (ITGA5) kuye kwaxelwa ukuba kunciphisa ukufuduka kweeseli kunye nokuqhubela phambili kwezinye ii-cancer ezifana nomhlaza wemiphunga (Ju et al., 2017), nto leyo ebonisa ukuba i-integrin a5 inokuba yinto ephambili kwi-pathogenesis ye-TNBC.
Ukuvelisa iimpuku eziphumayo kusetyenziswa iindlela zesintu ze-embryonic stem (ES) yinkqubo enzima, ethatha ixesha, kwaye engasebenzi kakuhle. Kuthatha iinyanga neminyaka ukujolisa kwiiseli ze-ES ngokusebenzisa i-homologous recombination, ukuzalisa iimpuku ze-chimeric, uze uzidibanise neempuku ze-heterozygous ukuvelisa inzala ye-homozygous. Ukuzalanisa okuntsonkothileyo kuyadingeka ukudala iimpuku ezineenguqu ezininzi zezakhi zofuzo. Nangona kunjalo, kukho iingxaki ezininzi eziye zavela xa kusetyenziswa iimpuku eziveliswa kukususwa kweeseli ze-ES kusetyenziswa i-CRISPR/Cas9 okanye ngokufaka izinto ze-CRISPR/Cas9 kumaqanda achunyisiweyo eseli enye. Umzekelo, ukungeniswa kotshintsho kuhlala kwenzeka kwi-biallelic loci kwaye akuchazi mfuzo. Kutshanje kubikwe ukuba iiseli ze-ES ezisebenzisa iteknoloji ye-CRISPR/Cas9 zinokufaka iinguqu ze-biallelic ngaxeshanye kwiijini ezifikelela kwi-5 (Nishizono et al., 2021). Ngenxa yesi sizathu, i-Cas9 mRNA kunye nee-gRNA ezintlanu zezakhi zofuzo zatshintshelwa ngaxeshanye kwiiseli ze-ES. Oku kugxininisa isithembiso kunye nokusebenza kakuhle kwale nkqubo, nangona ezi nguqu zisenokuba zadluliselwa xa kwaveliswa imigca yabasunguli ukuze kuveliswe iimpuku ezihlanu ezigobileyo. Olu phononongo lukwabika ukufunyanwa okumangalisayo: iiseli ze-ES azisafuneki ukwenza iimpuku eziguqulwe ngokwezakhi zofuzo. Endaweni yoko, ukususa iimveliso ezithile zezakhi zofuzo, i-Cas9 mRNA kunye ne-gRNA zafakwa kwi-embryos yesigaba seseli enye. Ngenxa yoko, kuye kwenziwa imigca yabasunguli abagobileyo enokuthi isetyenziswe ngokwethiyori ukufunda iziphumo zokususwa kwezakhi zofuzo kwiimpuku (Qin et al., 2016). Ke ngoko, i-CRISPR/Cas9 ivumela ukudalwa kweimpuku eziguqulwe ngokwezakhi zofuzo ukunyanga i-TNBC ngexabiso eliphantsi kunobunjineli bezakhi zofuzo bendabuko. Kusetyenziswa inkqubo ye-CRISPR/Cas, kuye kwaveliswa imodeli entsha yempuku egobileyo enokuthi ingenise ngempumelelo utshintsho lweendawo kwizakhi zofuzo enye okanye ezingaphezulu kwi-TNBC. Ngokusekelwe kule ngcamango, iimodeli zezilwanyana ze-TNBC zingaphuhliswa kusetyenziswa ukususwa kwe-BRCA1 kunye ne-p53 nge-CRISPR/Cas9, okubangela ukulahlekelwa kukulungiswa kwe-HR, ukungazinzi kwe-genomic, kunye ne-phenotypes ye-mutant (Annunziato et al., 2020).
Okwangoku, iindlela ezahlukeneyo ezifana ne-mammography, i-magnetic resonance imaging (MRI) kunye ne-ultrasound zisetyenziselwa ukufumanisa i-TNBC. Nangona kunjalo, ezi ndlela zinemida ethile. I-Mammography isetyenziselwa ukufumanisa izicubu zebele zasekuhlaleni endaweni ye-metastases, apho iiseli zomhlaza ziye zafudukela kwamanye amalungu omzimba. I-Ultrasonography ayisiyondlela ithembekileyo yokufumanisa i-TNBC (Chen noLee-Felker, 2023). I-MRI inovakalelo oluphezulu kune-ultrasound kunye ne-mammography, kodwa ukuchaneka kwayo kokuxilongwa kunqongophele (Sha noChen, 2022). I-tissue biopsy yindlela ehlaselayo yokuchonga iiseli zomhlaza. Nangona kunjalo, kwezinye iimeko, i-biopsy inokungaphumeleli kwizicubu zomhlaza ukuba inaliti iphambuka kwindawo enomdla. Ukongeza, ibiza kakhulu kwaye inokuba yingozi kwisigulana. I-TNBC luhlobo lomhlaza olungafaniyo, ngoko ke i-biopsy ayinakubonelela ngolwazi olwaneleyo malunga nohlobo lomhlaza. Ke ngoko, indlela entsha ethembekileyo yokufumanisa i-TNBC ifuneka ngokukhawuleza. I-CRISPR/Cas9 inokusebenza njengendlela ebuthathaka kakhulu kwaye engaphantsi kakhulu yokufumanisa umhlaza webele. Ngenxa yoku, amaqhinga asekelwe kwi-CRISPR/Cas9 angasetyenziselwa ukuphucula iindlela ze-PCR zokuxilongwa kwe-TNBC. Okokuqala, iiproteni ze-Cas9 kunye ne-cpf1 zenkqubo ye-CRISPR zisetyenziselwa ukususa i-DNA engeyonyani, kwaye ke ezi proteni zimbini (i-Cas9 kunye ne-cpf1) zinokuqonda ulandelelwano lwe-PAM ngaphambi kokuba zibophelele kwi-DNA ekujoliswe kuyo (Deepak Singh et al., 2021). . Ngoko ke, i-PCR inokuchonga utshintsho olunxulumene nophuhliso lomhlaza. Izifundo ezininzi ziye zamkela esi sicwangciso esisekelwe kwi-CRISPR ukufumanisa utshintsho olwahlukeneyo kwiintlobo ezahlukeneyo zomhlaza (Safari et al., 2019). Ngoko ke, iindlela ze-PCR ezisekelwe kwi-CRISPR zinokunciphisa ukuxhomekeka kokuxilongwa kwe-TNBC kwiindlela ezihlaselayo ezifana ne-biopsy-based immunohistochemistry.
Utshintsho lwe-genotypic kulawulo lwee-hormone receptor lubonakaliswe nakwi-TNBC (Chen and Russo, 2009). Amaqhinga e-PCR asekelwe kwi-CRISPR/Cas9 asebenzisa ii-microarrays zokuxilongwa kwendawo yokhathalelo anokubeka esweni ngempumelelo ezi nguqu (Hajian et al., 2019). Ingabonelela ababoneleli beenkonzo zempilo ngolwazi olufanelekileyo malunga noguquko oluthile kwizigulana ze-TNBC, olunokunceda ukuphucula ikhosi yabo yonyango. Nangona kunjalo, le ndlela idibeneyo inemida. Ke ngoko, kufuneka imizamo yophando olubanzi ngaphambi kokuba le ndlela ye-CRISPR/Cas-PCR isetyenziswe kunyango lokufumanisa i-TNBC (Yang et al., 2019).
Ixesha leposi: Oktobha-14-2024