I-Clustered regularly interspaced short palindromic repeats (CRISPR) iyindlela yokuhlela i-genome yesizukulwane sesithathu eguqule umhlaba ngemiphumela yayo ephezulu. Isetshenziswe ekwelapheni izifo ezahlukahlukene zebhayoloji kanye nokutheleleka. Amabhaktheriya ahlukahlukene kanye namanye ama-prokaryotes (njenge-archaea) nawo anezinhlelo ze-CRISPR/Cas9 zokuzivikela kuma-phages. Kubikwe ukuthi amasu asekelwe ku-CRISPR/Cas9 angavimbela ukukhula nokuqhubeka komdlavuza webele ongemuhle kathathu (TNBC) ngokubhekisisa izakhi zofuzo zokumelana ezingase zishintshe, ukuqoshwa kwezakhi zofuzo, kanye nokulawulwa kwe-epigenetic. Lokhu kungenelela kokwelapha kungasiza ekubhekaneni nezinkinga eziyinselele ezifana nokumelana nemithi okubonwa ngisho naku-TNBC. Njengamanje, kusetshenziswa izindlela ezahlukene ukuletha i-CRISPR/Cas9 kumaseli aqondiwe, njengezindlela zomzimba (i-microinjection, i-electroporation kanye ne-hydrodynamic), igciwane (i-adeno-associated virus kanye ne-lentivirus) kanye nezinhlayiya ezingezona ezamagciwane (ama-liposomes nama-lipid nanoparticles). Nakuba kuye kwasungulwa amamodeli ahlukahlukene ukuze kufundwe izimbangela zama-molecule ze-TNBC, ukuntuleka kwezindlela ezibucayi neziqondiwe zokuletha amathuluzi okuhlela i-genome in vivo kunciphisa ukusetshenziswa kwawo kwezokwelapha. Ngamafuphi, lokhu kubuyekezwa kuhlola ngokuphelele inqubekela phambili, izinselele, imikhawulo, kanye namathuba okwelashwa kwe-CRISPR/Cas9 kwe-TNBC ngokusekelwe ebufakazini obukhona. Siphinde sigcizelele ukuthi ukuhlanganiswa kobuhlakani bokwenziwa kanye nokufunda komshini kungathuthukisa kanjani amasu e-CRISPR/Cas9 ekwelapheni i-TNBC.
Umdlavuza ubonakala ngokuhlukana kwamaseli okungalawulwa, ukuphazamiseka kwezindawo zokuhlola umjikelezo wamaseli, kanye nokuguquka kwezakhi zofuzo ezicindezela isimila (ama-TSG) (Matthews et al., 2022). Phakathi kwezinhlobo ezahlukene zomdlavuza, umdlavuza webele uhlobo lomdlavuza oluvame kakhulu kwabesifazane futhi unezinga eliphezulu lokufa emhlabeni wonke (Waks and Winer, 2019). Umdlavuza webele uyisifo esingafani esinezici ezahlukahlukene, njengezici ze-histological nezebhayoloji, ukuvezwa kwezokwelapha kanye nokuziphatha, kanye nokusabela ekwelashweni (Weigelt et al., 2010). Ukuhlukaniswa komdlavuza webele kunikeza ukuqonda okunembile kokuxilongwa komdlavuza webele kanye nokubikezela kwesimila. Ukusetshenziswa kwama-biomarker avamile kanye nezici ze-clinicopathological kube yisici esiyinhloko sokuhlukaniswa komdlavuza webele (Tsang and Tse, 2019). Ukubikezela kanye nokusabela ekwelashweni komdlavuza webele kuthonywa yizici eziningi, okuhlanganisa ukuba khona kwe-estrogen receptor (ER), i-progesterone receptor (PR), i-human epidermal growth factor receptor 2 (HER2/neu), kanye nebanga le-histological, uhlobo lwesimila, kanye nebanga. Usayizi kanye nokusabalala kwama-lymph node (Al-Tubaiti, 2020). Kutholakale izinhlobo ezinhlanu zamangqamuzana ezingaphakathi zomdlavuza webele, okuhlanganisa i-luminal A, i-luminal B, i-HER2-enriched, i-basal-like, kanye ne-claudin-low (Prat et al., 2015). I-TNBC uhlobo lwamangqamuzana omdlavuza olungavezi yonke i-ER, i-PR, kanye ne-HER2 (Yin et al., 2020). Lezi zici ze-pathological zihlotshaniswa ne-TNBC, okuqinisekisa ukuqhubekela phambili kwayo okusheshayo kanye nohlobo lwayo olunolaka kakhulu kunanoma yiluphi olunye uhlobo lomdlavuza webele (Feng et al., 2018). Ngaphezu kwalokho, uPeru et al (2000) basebenzise ubuchwepheshe be-microarray ukuhlukanisa kabusha umdlavuza webele nokuhlonza izinhlobo ezinhlanu zangaphakathi zomdlavuza webele (Cadenas, 2012). Umdlavuza webele ofana ne-basal uhlobo lwamangqamuzana omdlavuza webele olune-phenotype engemihle kathathu futhi oluhlotshaniswa nokuqhubekela phambili okusheshayo. Kufanele kuqashelwe ukuthi yonke imidlavuza yebele efana ne-basal ivame ukuhlonzwa kabi njenge-TNBC, kodwa ama-77% kuphela ayi-TNBC; Ngokuphambene nalokho, ama-71-91% e-TNBC afana ne-basal, okubonisa ukuthi lezi zinhlobo ezimbili zomdlavuza webele ziyahlangana futhi zimele ukuhlukaniswa okuhlukile (Wang D.-Y. et al., 2019). Lokhu kudala isidingo sokuchaza ukungafani kwe-TNBC ukuze kuchazwe ukubikezela futhi kutholakale izimpendulo ezingaba khona ekwelashweni kwamanje nokwesikhathi esizayo. Ngaphezu kwalokho, i-TNBC ibangela ama-15-20% azo zonke izimo zomdlavuza webele futhi ivame kakhulu kwabesifazane abangaphansi kweminyaka engama-50 ubudala. Ukuguqulwa kwe-BRCA1 noma i-BRCA2 kubikwe cishe kuma-20% amacala e-TNBC (Xie et al., 2017; Tzikas et al., 2017). , 2020). Izifundo ziphinde zabonisa ukuthi i-TNBC ine-microenvironment ehlukile yomzimba ehlanganisa amazinga aphezulu ezici zokukhula kwe-vascular endothelial, ama-macrophage ahlobene ne-tumor (TAMs), ama-lymphocyte angena ngaphakathi kwe-tumor (TILs), kanye namanye ama-molecule ahilelekile ekukhuleni nasekufudukeni kwe-tumor. Ngakho-ke, ukuqonda i-microenvironment ye-TNBC kubalulekile ekubikezelweni kwayo kanye nokwelashwa kwayo (Fan and He, 2022).
Ukuhlola ukubikezela kwe-TNBC nokuqinisekisa ukwelashwa okuphumelelayo, ukuxilongwa okunembile, okusekelwe kakhulu ku-immunohistochemistry (IHC) ukuthola i-ER, i-PR kanye ne-HER2, kanye ne-mammography ukuthola ama-neoplasms ebele, kubalulekile. Kodwa-ke, i-mammography ayikwazi ukukhombisa ngokwanele izici zangaphakathi kwesisu njenge-necrosis kanye ne-fibrosis (Deepak Singh et al., 2021). Njengoba ukubikezela okubi kanye nokuxilongwa kuvimbela odokotela ekunikezeni imithi efanele (Chaudhary, 2020), kusetshenziswa amasu ahlukahlukene ukuthuthukisa ukunakekelwa kweziguli ezine-TNBC. Njengamanje, kusetshenziswa imithi emibili, i-doxorubicin kanye ne-cyclophosphamide, ezigulini ezine-TNBC futhi ibonise imiphumela emihle. Ngaphezu kwalokho, kusetshenziswa eminye imithi ye-platinum, njenge-carboplatin kanye ne-cisplatin (Sikov et al., 2015). Ngaphezu kwalokho, ama-PARP inhibitors afana ne-Olaparib, i-Velaparib, kanye ne-PF-01367338 asetshenziswe njengemithi engaba khona ye-chemotherapy yokwelapha i-TNBC (Ishino et al., 2018). Njengoba izindlela zokubonisana ze-Wnt/b-Catenin, NOTCH, kanye ne-Hedgehog zibikwe ukuthi zihilelekile ekwakhekeni nasekuqhubekeni kwe-TNBC, ukuqondiswa kwemithi kulezi zindlela kungaba isu elibalulekile (Aysola et al., 2013). Nakuba ukuhlinzwa, ukwelashwa ngemisebe kanye ne-chemotherapy kusalokhu kuyinsika eyinhloko yokwelashwa kwe-TNBC namuhla, kuye kwenziwa intuthuko enkulu ekuthuthukiseni izindlela zokwelapha ezintsha okuhlanganisa ukwelashwa okuqondisiwe, i-immunotherapy, amathuluzi ahlukahlukene okuhlela izakhi zofuzo ahlobene ne-CRISPR njenge-Cas9n, dCas9., CRISPR/Cas12, i-prime editing, kanye ne-CRISPR/Cas9-target gene therapy. Lapha sizogxila kumathuluzi ahlukahlukene okuhlela izakhi zofuzo ahlobene ne-CRISPR asetshenziswa ekwelapheni i-TNBC. Phakathi kwawo, i-CRISPR/Cas9 ithole ukunakwa okubanzi.
I-Cas9n, eyaziwa nangokuthi i-Cas9 nickase, uhlobo oluhlelwe ngokwezakhi zofuzo lweprotheyini ye-Cas9 etholakala ohlelweni lokuhlela i-genome ye-CRISPR/Cas9 (Gupta et al., 2019). Ngendlela yayo yokuqala, iphrotheyini ye-Cas9 iqukethe izizinda ezimbili ze-nuclease: i-RuvC ne-HNH, umsebenzi wayo oyinhloko ukuqhekeka kwezintambo zombili ze-DNA. Kodwa-ke, ku-Cas9n, enye yezizinda ze-nuclease, i-HNH, iguqulwa ngokwezakhi zofuzo futhi ayisebenzi. Ngakho-ke, isizinda se-HNH se-Cas9n asisebenzi. Yisizinda se-RuvC kuphela esihlala sisebenza, okuvumela i-Cas9n ukuthi isike noma idale ukuqhekeka kuzintambo ze-DNA eyodwa (Trevino noZhang, 2014). Uma kuqhathaniswa ne-Cas9, i-Cas9n inganciphisa ngempumelelo imiphumela engeyona eyomgomo futhi ithuthukise ngokunembile izindlela zokulungisa amaseli. I-Cas9n ingasetshenziswa ukuxazulula izinkinga ezahlukahlukene, njengokudala ukuqhekeka ezindaweni ezithile ku-DNA enezintambo ezimbili. Ngale njongo, ama-molecule amabili e-Cas9n ahlanganiswa futhi asetshenziswa ndawonye (Yee, 2016). I-Mixed lineage kinase 3 (MLK3) iyi-protein kinase esebenza nge-mitogen esebenza njengomlawuli oyinhloko ngesikhathi se-TNBC metastasis (Cronan et al., 2012).
I-MLK3 ingasebenzisa izindlela eziningi zokubonisa izimpawu eziholela ekusakazekeni kwe-TNBC. Isibonelo, indlela ye-c-Jun N-terminal kinase (JNK) ilawula ukuhamba kweseli kanye nokuwohloka kwe-extracellular matrix, kanti i-MLK3 isebenzisa indlela ye-JNK, ngaleyo ndlela ithuthukise ukuhamba kweseli futhi inikeze izakhiwo ezihlaselayo (Rattanasinchai noGallo, 2016). I-MLK3 iphinde ilawule i-EMT. Ukuze yenze lokhu, isebenzisa izinto zokubhala ezingezansi njenge-Snail, Slug kanye ne-Twist. Lezi zinto zivimbela ukuvezwa kwezimpawu ze-epithelial futhi zikhuthaza ukuvezwa kwezimpawu ze-mesenchymal, okuholela ekutholakaleni kwe-metastatic phenotype (Casalino et al., 2023). I-MLK3 ibonwe idlala indima ekuguqulweni kwe-ECM kanye nokusebenza kwe-proteases njenge-matrix metalloproteinases (MMPs) ezonakalisa i-ECM. Lokhu kwenza kube lula ukuhlasela kweseli lesimila futhi kusabalale ezindaweni ezikude (Katari et al., 2019). Ngakho-ke, izifundo zangaphambilini zibonise ukuthi i-MLK3 idlala indima ebalulekile ku-TNBC. URattanasinchai noGallo basebenzise amamodeli e-TNBC ukutadisha indima ye-MLK3 futhi bathola ukuthi ikhuthaza ukuthuthukiswa komdlavuza ngezindlela ezithile zokubonisa izimpawu. Basebenzise i-CRISPR/Cas9n ukuhlela i-MLK3 futhi babona ukwehla okukhulu kokusabalala kwe-TNBC (Rattanasinchai noGallo, 2016).
I-dCas9, evame ukubizwa ngokuthi i-Cas9 engasebenzi, iwuhlobo olushintshiwe lweprotheyini ye-Cas9. Ngokungafani ne-Cas9 esebenzayo, i-dCas9 ayinawo umsebenzi we-endonuclease, okwenza ingakwazi ukubangela ukuqhekeka kwe-DNA kabili. Ngakho-ke, i-dCas9 ingasetshenziswa ukukhomba ngqo izindawo ezithile ze-genome ngaphandle kokushintsha noma ukuguqulwa kokulandelana kwe-DNA (Wang et al., 2016). Ku-dCas9, amaprotheni amabili e-endonuclease, i-RuvC kanye ne-HNH, awenziwa angasebenzi ngokucindezela izinsalela ezibalulekile ze-amino acid. (Richter et al., 2016). Naphezu kokuntuleka kwayo komsebenzi wokuqhekeka kwe-DNA, i-dCas9 idlala indima ebalulekile ocwaningweni lwezakhi zofuzo kanye ne-biotechnology. Isibonelo, ivumela ukubona ngeso lengqondo izindawo ezithile ze-genome, yenza kube lula ukulawulwa kokubhalwa kwezakhi zofuzo, futhi ikhuthaza ukuguqulwa kwe-epigenetic (Brocken et al., 2018).
I-transcription factor ZEB1 (i-zinc finger E-box binding homeobox 1) idlala indima ethile futhi ebalulekile ekulamuleni ukuguquka kwe-epithelial-mesenchymal (EMT), inqubo yeselula eyenzeka ngesikhathi sokuthuthukiswa kombungu, ukulungiswa kwezicubu, kanye nokuqhubeka komdlavuza. Ihilela ukuguqulwa kwamaseli e-epithelial abe amaseli e-mesenchymal, okuholela ekushintsheni kokwakheka kweseli, ukuhamba, ukuhlasela, njll. (Wu et al., 2020). I-ZEB1 ivimbela izimpawu eziningi ze-epithelial, njenge-E-cadherin ne-Occludin, ezibhekene nokugcina ukunamathela kweseli kanye ne-polarity yamaseli e-epithelial ku-TNBC (Moreno-Bueno et al., 2008). Ngaphezu kwalokho, i-ZEB1 isebenzisa izimpawu ezithile ezifana ne-N-cadherin, i-vimentin kanye ne-fibronectin (Konradi et al., 2014) futhi ilawula nezakhi zofuzo ezihilelekile ekuguqulweni kabusha kwe-cytoskeletal njenge-Rho GTPases kanye ne-matrix metalloproteinases (MMPs) (Huang). et al., 2014). 2022), ngaphezu kwalokho, kuthinta nezindlela ezahlukahlukene zokubonisa izimpawu njengokuguqula i-growth factor-β (TGF-β), i-Wnt signaling, njll., ngaleyo ndlela kukhuthaze ukuhlasela kwe-tumor kanye ne-metastasis ku-TNBC (Chen et al., 2016). ). Izifundo eziningi kanye nobufakazi besayensi bubonisa ukuthi i-ZEB1 inamandla amakhulu njenge-ejenti ebalulekile yokuthola nokungenelela kokwelapha kwe-TNBC. Esifundweni samuva nje, uWaryah et al. basebenzise imodeli ye-TNBC futhi bathola ukucindezela okuphelele kwe-ZEB1 yi-dCas9. Ngakho-ke, babone ukucaciswa okuphezulu kakhulu kanye nokuvinjelwa okucishe kuphelele kwe-ZEB1 ezimweni ezi-in vivo (Waryah et al., 2023).
I-CRISPR/Cas12 iyithuluzi lokuhlela izakhi zofuzo elibizwa ngokuthi i-CRISPR/Cpf1. Ithathwe ohlelweni lwe-CRISPR/Cas, lapho igama elithi Cas12 libhekisela kuphrotheni ehlobene ne-CRISPR 12 (Bharathkumar et al., 2022), efana ngokuphelele ne-Cas9. Umehluko kuphela ukuthi iqukethe iphrotheni ye-Cas12. Uma iqhathaniswa ne-Cas9, i-Cas12 inemisebenzi ethile ehlukile njengokukhiqiza iziphetho ezinamathelayo ngesikhathi senqubo yokuhlela izakhi zofuzo, kanti i-Cas9 ikhiqiza iziphetho ezibuthuntu (Wang et al., 2021). Le mpahla ye-Cas12 inegalelo kubuchwepheshe bayo bokuphatha i-DNA ethile. Njengephrotheni ekwazi ukubona nokusika i-DNA eqondiwe ngokunembile, inokuguquguquka okungavamile, okwenza ibe ithuluzi eliphumelelayo lezinhlelo zokusebenza ezahlukahlukene, okuhlanganisa nokuhlela izakhi zofuzo (Pickar-Oliver noGersbach, 2019).
Njengezinye izinhlobo ze-CRISPR, i-CRISPR/Cas12 iyakwazi futhi ukukhipha noma ukusebenzisa izakhi zofuzo ku-TNBC, ihlose izakhi zofuzo ezidlala indima ebalulekile ekuqaliseni kwayo noma ekuphenduleni ekwelashweni (uYang noZhang, 2023). Ukuze kufezwe le nqubo, kwasungulwa i-RNA eqondisayo (i-gRNA) eqondisa i-Cas12 ku-gene eqondiwe. Uma i-Cas12 isibophezele ku-target yayo, ingenisa ukuqhekeka kwe-double-strand ku-DNA futhi isebenze izindlela zokulungisa i-DNA. Ngesikhathi senqubo yokulungisa, ama-nucleotide angalungile angafakwa, okuholela ekuguqulweni kwezakhi zofuzo kanye nokulahlekelwa umsebenzi (uZhang et al., 2021a). Ngaphezu kwalokho, inguqulo ethuthukisiwe ye-enzyme ye-Cas12 (ebizwa ngokuthi i-dCas12) nayo yasetshenziswa ukwenza izakhi zofuzo zisebenze. Ngokuyihlanganisa ne-activator yokubhala, kungenzeka ukuthi kukhuthazwe izakhi zofuzo ezithile, ngaleyo ndlela zisebenze. Lobu buchwepheshe bunamandla amakhulu okukhuthaza ukusebenza kwezakhi zofuzo ezicindezela isimila (uSultan et al., 2022).
I-Prime editing ubuchwepheshe obusha obuthuthukisiwe nobungcono kakhulu bokuhlela i-genome. Iyakwazi ukuguqula i-DNA yento ephilayo ngokunembe kakhulu (Chen noLiu, 2023). I-Prime editing itholakala ngokuhlanganiswa kwezingxenye ezimbili eziyinhloko: i-enzyme ye-CRISPR/Cas9 eguquliwe kanye ne-reverse transcriptase. Indima ye-enzyme ye-CRISPR/Cas9 ukukhomba ngokukhetha izindawo eziqondile ku-genome, kuyilapho i-reverse transcriptase isiza ukuguqula ngokuphelele i-DNA endaweni eqondiwe (Hassan et al., 2021). Endleleni yokuhlela i-primer, isinyathelo sokuqala sihilela ukudalwa kwe-primer editing guide RNA (pegRNA) (Standage-Beier et al., 2021). Iqukethe ukulandelana kwe-target kanye ne-RNA template ehambisana nendawo yokuhlela oyifunayo ku-DNA eqondiwe. I-pegRNA ibe isifakwa kumaseli e-target kanye ne-primer editing nuclease (PE2). I-PE2 iyiprotheni yokuhlanganisa equkethe i-Cas9 enzyme, i-reverse transcriptase, kanye ne-primer editing adapter (Martín-Alonso et al., 2021). Ngaphakathi kweseli, i-pegRNA kanye ne-PE2 complexes zifuna i-DNA ethile ezifuna ukuyishintsha. I-enzyme ye-Cas9 inquma i-DNA futhi idale ithempulethi equkethe imicu eyodwa (Choi et al., 2022). I-Reverse transcriptase isebenzisa le thempulethi ukulungiselela i-DNA ukuze ihlelwe. Kanye nokukopisha i-DNA, imiyalelo yokuhlela ithempulethi ye-RNA nayo ifakiwe. Ekugcineni, i-DNA strand esanda kudalwa isetshenziswa njengethempulethi ukulungisa ukuphuka kwe-DNA, okuholela ekushintsheni kwe-DNA okuqukethe ukuguqulwa okufiswayo (Ochoa-Sanchez et al., 2021). Izindlela zokuhlela ze-primer zingaholela ekuguqukeni okubanzi kwezakhi zofuzo. Ingagxila ekuguqulweni kwamaphuzu, ukufakwa, ukususwa, ngisho nokufakwa esikhundleni kwezakhi zofuzo (Anzalone et al., 2019; Chen noLiu, 2023). Ukuhlela kwe-Prime kunikeza izinzuzo eziningana ngaphezu kobuchwepheshe bokuhlela i-genome bangaphambilini. Lokhu kufaka phakathi ukunemba okwandisiwe, imiphumela encishisiwe engaqondile, kanye nekhono lokuhlela i-DNA ngaphandle kokuthembela ekuqhekekeni kwe-DNA double strand (Anzalone et al., 2020).
Ubuchwepheshe be-CRISPR/Cas9 basungulwa ekuqaleni ukuvikela amabhaktheriya ekudlulisweni kwe-plasmid kanye nokutheleleka kwe-phage futhi kamuva basetshenziswa kabusha njengethuluzi elisebenzayo lokuqondisa i-DNA elisekelwe ku-RNA lokuhlela i-genome (uJiang noDoudna, 2017). Ngaphezu kwalokho, uhlelo lwe-CRISPR/Cas9 lubikwe ukuthi lukhona ku-50% kanye no-87% wama-genome amabhaktheriya kanye nama-archaeal, ngokulandelana (u-Ishino et al., 2018). I-CRISPR/Cas9 iyithuluzi elingase libe khona lokususa, ukufaka kanye nokulungisa noma yikuphi ukulandelana kwezakhi zofuzo okungajwayelekile kusetshenziswa izindlela ze-in vivo kanye ne-in vitro (uSabit et al., 2021). Ngaphezu kwalokho, i-CRISPR/Cas9 iboniswe iyingxenye yesistimu yokuzivikela yomzimba eguquguqukayo ngenxa yokucaciswa kwayo kwezakhi zofuzo ezihlosiwe (uChen noZhang, 2018).
I-CRISPR/Cas9 iqukethe i-Cas9 kanye ne-RNA eyodwa yomhlahlandlela (sgRNA). I-Cas9 iyi-endonuclease eyakhiwe izingxenye eziningi zamaprotheni. Ngaphezu kwalokho, i-Cas9 inezakhiwo ezihlukile zesakhiwo kanye nokwakheka (Pacesa et al., 2022) njengoba iqukethe ama-lobes amabili: ukuqashelwa (REC) kanye ne-nuclease (NUC). I-lobe ye-REC ihlukaniswe ngezifunda ezintathu: i-REC1, i-REC2, kanye nama-bridge helices (Cromwell et al., 2018). I-NUC iqukethe ama-lobes amathathu, okuyi-RuvC (RuvC I, RuvC II, RuvC III), i-HNH kanye nesizinda sokusebenzisana se-protospacer adjacent motif (PAM) (Isithombe 1) (Song et al., 2016). I-sgRNA iqukethe izingxenye ezimbili, kufaka phakathi i-CRISPR RNA (crRNA) kanye ne-transcoding small RNA (tracer RNA) (Isithombe 1). I-sgRNA ixhumanisa iphrotheni ye-Cas9 nama-connexin ukuze yakhe i-complex esebenzayo, eyaziwa nangokuthi i-effector complex (Richter et al., 2012). I-crRNA iyi-base pair ye-nucleotide engu-18-20 edlala indima ebalulekile ekuboneni ukulandelana kwe-DNA okuqondiwe. Ngaphezu kwalokho, i-crRNA ihambisana ne-DNA okuqondiwe, futhi i-tracrRNA isebenza njengesicafold se-Cas9 nuclease ukubopha i-DNA okuqondiwe (Manghwar et al., 2019). Ngakolunye uhlangothi, ukulandelana kwe-PAM kune-nucleotide engu-3, okuqinisekisa, kucacisa futhi kuqondise ukubopha kwe-effector complex ku-DNA. Ama-subunit e-Cas9 protein complex i-RuvC ne-HNH anomsebenzi we-catalytic (Richter et al., 2012; Asmamaw noZawdie, 2021). Isizinda se-nuclease se-HNH se-subunit ye-Cas9 sihlukanisa intambo ye-DNA ehlotshaniswa ne-crRNA. Isizinda se-RuvC nuclease sihlukanisa eminye imicu ye-DNA futhi sidale ukuqhekeka kwemicu emibili (ama-DSB), ngemva kwalokho kusebenze izindlela ezimbili ezahlukene zokulungisa ukuqhekeka kwe-DNA (uJiang noDoudna, 2017) (Isithombe 1).
Isithombe 1. Ukubuka konke kwe-CRISPR/Cas9 (A). Izingxenye zesistimu ye-CRISPR/Cas9: (i). I-Cas9 endonuclease inesibopho sokusika ukulandelana kwe-DNA okuqondiwe, (ii) i-RNA eyodwa yomhlahlandlela (sg) ephuma ekuhlanganisweni kwe-crRNA kanye ne-tra-crRNA chimeras. (ezimbili). I-Cas9 ifaka izingxenye eziningana njenge-Rec I, Rec II, NUC lobe (i-HNH kanye ne-Ruv C ziyizingxenye ezincane) kanye nesizinda sokusebenzisana kwe-PAM (C) nemisebenzi ehambisanayo. I-CRISPR/Cas9 protein complex ihlukanisa ukulandelana kwe-DNA ibe amafomu angaphelelisi futhi ahambisanayo (D). I-CRISPR/Cas9 ihlela i-genome ngezigaba ezintathu: ukuqashelwa, ukusika kanye nokulungisa. I-sg-RNA eklanyelwe iqondisa i-Cas9 futhi iqaphele ukulandelana okufiswayo nge-crRNA yengxenye ehambisanayo. I-Cas9 iqaphela ukulandelana kwe-PAM ku-5′-NGG-3′ futhi incibilikisa i-DNA, yakha i-DNA-RNA hybrid futhi isebenze ukuhlukana. Isizinda se-HNH se-Cas9 sinqamula umucu ohambisanayo, kanti isizinda se-RuvC sinqamula umucu ongeyona ehambisanayo. I-CRISPR/Cas9 ilungisa i-dsDNA ngokuyiphula ngezindlela ezimbili: ukujoyina ukuphela okungeyona ehambisanayo (i-NHEJ) kanye nokulungisa okuqondiswe ku-homology (i-HDR). I-NHEJ ilungisa i-DNA enezintambo ezimbili lapho kungekho khona i-DNA yangaphandle ehambisanayo ngenqubo ye-enzyme, indlela evame ukwenza amaphutha engafaka noma isuse ukulandelana kwe-DNA okungahleliwe. I-HDR icacile kakhulu futhi idinga amathempulethi e-DNA ehambisanayo.
Izindlela zokulungisa eziqondiswa yi-CRISPR/Cas9 zifaka phakathi izindlela zokuhlanganisa izinhlangothi ezingezona ezihlangene (i-NHEJ) kanye nezindlela zokulungisa eziqondiswa yi-homology (HDR). I-NHEJ iyindlela evame ukuba namaphutha ngoba ihilela ukufakwa noma ukususwa futhi ayidingi noma iyiphi iphethini ngesikhathi sokulungisa. Isebenzisa ama-nucleotide angahleliwe ukukhiqiza amaprotheni ajwayelekile (Abbasi et al., 2021). Lolu hlelo lokulungisa luqukethe izinhlanganisela ezine ezifana ne-KU complex, i-cross-complementing protein complex type 4 (XRCC-4), i-DNA end processing enzyme, kanye ne-protein kinase DNA-PKcs (Abbasi et al., 2021). Iphrotheni eyinkimbinkimbi ye-KU inama-subunit amabili, i-Ku 70 ne-Ku 80, futhi idlala indima ebalulekile endleleni yokusebenza ye-NHEJ, njengoba indlela yokulungisa iqalwa ngokubopha ama-subunit amabili (Ku 70 ne-Ku 80) emaphethelweni angenalutho noma acishe abe buthuntu e-DNA eqondiwe (Abbasi et al., 2021), esebenza njengesikafu sokuqoqa ezinye izici ezihlobene ne-NHEJ endaweni yokulimala (Yang et al., 2020). I-XRCC-4 ne-DNA ligase zakhiwe ama-amino acid angu-334 no-911, ngokulandelana, kanti i-XRCC4-DNA ligase IV complex ivuselela ukuhlanganiswa kwama-DNA end (Chatterjee et al., 2015). I-enzyme yokucubungula ukuphela kwe-DNA, eyaziwa nangokuthi i-polynucleotide kinase 3′-phosphate, iyi-enzyme yokucubungula ukuphela kwe-DNA. Ingasusa iqembu le-3′P ku-DNA futhi i-phosphorylate iqembu le-5′OH ngesikhathi sokulungiswa kwe-DSB. Kuhilela nokulungiswa kwama-single-strand breaks (SSBs) kusetshenziswa indlela yokulungisa i-SSB (Chatterjee et al., 2015). I-protein kinase I-DNA-PKcs iyi-protein kinase encike ku-DNA equkethe i-catalytic subunit yama-PIKK (phosphatidylinositol 3-kinase-related kinases) kanye nomndeni we-ataxia telangiectasia mutated (ATM), kanye nama-ATR ahlobene ne-ATM kanye ne-Rad3. ama-strand breaks (DSBs) kanye nama-single-strand breaks (Yue et al., 2020; Peng et al., 2016).
I-HDR iyindlela yokulungisa enembile futhi efanelekile ngoba ulwazi lukopishwa kusetshenziswa ifomu eliphelele le-homologous DNA duplex, yize idinga ukuba khona kwama-chromatids angodadewabo. Lokhu kwenzeka ngesikhathi sesigaba se-S/G2 somjikelezo weseli lezilwane ezincelisayo. I-HDR iyenzeka ikakhulukazi ezinhlotsheni zemvubelo, kodwa i-NHEJ ibalulekile ezincelisayo (Burma et al., 2006; Abbasi et al., 2021). Indlela yokulungisa ephelele iboniswe kuMfanekiso 1.
Njengoba i-TNBC ibangelwa ukukhubazeka kwezakhi zofuzo kanye ne-epigenetic, ukulungiswa kokukhubazeka okulimazayo kwezakhi zofuzo/i-epigenomic kusetshenziswa i-CRISPR/Cas9 kungaba yindlela yokwelapha enengqondo (Chen et al., 2019). Ngaphezu kwalokho, ezinye izici zokubhala ezihilelekile ekulawulweni kokubhalwa kwezakhi zofuzo oluthile lweseli zingabonisa izici ezihlukile zamaseli omdlavuza, okuphakamisa ukuthi ukulawulwa kokubhalwa kwezakhi zofuzo kungaba yindlela enhle kakhulu yokwelapha umdlavuza (Drost et al., 2017). Ukusebenzisa lezi zici zama-molecule ezimila, njengeziphambeko zezakhi zofuzo, ze-epigenetic kanye ne-transcriptional, ukuze kuthuthukiswe imithi kungathuthukisa imiphumela yezokwelapha futhi kunciphise izindleko zokuhlola. I-CRISPR iyithuluzi lokuhlela izakhi zofuzo elingagcini nje ngokuthola nokuhlonza izinhloso zezakhi zofuzo ezibangela umdlavuza, kodwa futhi lingasetshenziswa ukuhlela, ukucindezela, kanye nokuguqula i-oncogenes ngamaseli abantu (Ithebula 1) (Ahmed et al., 2021). Kuye kwenziwa izikrini eziningana ze-CRISPR ukuze kubhekwe izakhi zofuzo ezihlotshaniswa nabacindezeli bezimila, ama-oncogene, kanye nokumelana nemithi. Ukusetshenziswa kwe-CRISPR/Cas9 ukuhlela ama-oncogene ahlukahlukene e-TNBC kuboniswe kuMfanekiso 2.
Isithombe 2. Ukuhlelwa kwezakhi zofuzo ze-TNBC oncogenes ezahlukahlukene kusetshenziswa i-CRISPR/Cas9 okuholela ekukhuleni kwesimila kanye nokusabalala kwaso. Lawa ma-oncogenes afaka i-CDK7, i-NAT1, i-UBR5, i-YTHDF2, i-ITGA9, i-CXCR4 kanye ne-CXCR7, i-Crypto1, i-ROR1 kanye ne-ST8SIA, ezihilelekile ekwakhiweni nasekusabalaleni kwaso kwe-TNBC.
Ukufuduka kwamangqamuzana omdlavuza, ukuhlasela, kanye nokuguquka kwe-epithelial-mesenchymal (EMT) kuhlotshaniswa ne-ITGA9. Izifundo zangaphambilini zibonise ukuthi i-ITGA9 ingumdlali obalulekile endleleni ye-Notch futhi idlala indima ethakazelisayo ekusabalaleni kwe-rhabdomyosarcoma (Molist et al., 2020). Ngaphezu kwalokho, i-ITGA9 itholakale ihlotshaniswa eduze nokubikezela kwesiguli ezinhlotsheni eziningana zezimila, okuhlanganisa nomdlavuza webele (Wang Z. et al., 2019). Ngaphezu kwalokho, ukuhlaziywa kwe-bioinformatics kwe-ITGA9 kubonise ukuthi ukubonakaliswa kwayo ku-TNBC kwakuphakeme kakhulu kunakwezinye izinhlobo zomdlavuza webele. Amazinga aphezulu e-ITGA9 ahlotshaniswa nokusabalala kwesimila kanye nokubuyela emuva kweziguli ze-TNBC. Ukususwa kwe-ITGA9 yi-CRISPR/Cas9 kuholele ezimpahleni ezifana ne-cancer stem cell (CSC), i-tumor angiogenesis, ukukhula kwesimila, kanye nokuncipha kokusabalala ngokukhuthaza ukuwohloka kwe-β-catenin ku-TNBC (Wang Z. et al., 2019).
I-Crypto-1 iyilungu lomndeni we-TGF-β futhi ibalulekile ekukhuleni kwe-embryogenesis kwasekuqaleni, ekugcinweni kwamaseli e-stem, kanye nokukhula komdlavuza (Ishii et al., 2021). Eyaziwa nangokuthi i-Tdgf-1, iyiprotheni yokubonisana ye-GPI ebangelwa yi-oncogenic ehilelekile ekulawulweni kokwakheka kwe-primitive streak, i-mesoderm kanye ne-endoderm, kanye nokusungulwa kwe-asymmetry yesobunxele/kwesokudla ekuthuthukisweni kwezitho zomzimba ngesikhathi sokukhula kwe-embryogenesis (Zhang et al., 2021b). Ngaphezu kwalokho, i-Cripto-1 iboniswe ukuthi ihilelekile ekuguqukeni kwe-epithelial-mesenchymal (EMT) njengophawu lweseli le-stem (Zhang et al., 2021b). I-EMT ibalulekile hhayi kuphela ezinqubweni ezahlukahlukene ezifana nokukhula kwe-embryonic, i-fibrosis kanye nokuphulukiswa kwamanxeba, kodwa futhi nasekungeneni komdlavuza kanye nokukhula kwe-metastasis. Ngaphezu kwalokho, i-Cripto-1 iboniswe ukuthi isebenzisana nama-receptor amane e-Notch futhi ithuthukise ukuvuthwa kwawo ngemuva kokuhumusha (Brandstadter noMaillard, 2019). Indlela yokubonisana ye-Notch iyaziwa ukuthi ihilelekile ekugcinweni kwamaseli omdlavuza webele womuntu. Izifundo zibonise ukuthi ukuqedwa kwe-Crypto-1 okubangelwa yi-CRISPR/Cas9 kuvimbela ukukhula komdlavuza kanye nokusabalala kwawo. Ngakho-ke, i-Crypto-1 ingaba yisisulu esibalulekile sokwelapha i-TNBC (Castro et al., 2015).
Iphrotheni ye-XCL12 kanye ne-chemokine receptor yayo i-CXC (CXCR4 kanye ne-CXCR7) idlala indima ehlukahlukene ekwandeni kwamaseli omdlavuza, ekukhuleni, ekufudukeni nasekuhlaseleni (Wu et al., 2015). Lawa ma-chemoreceptor ahlotshaniswa nokuthuthukiswa kwe-TNBC ngezindlela eziningi zokubonisa izimpawu kokubili kumamodeli e-vivo kanye ne-in vitro (Wu et al., 2015). Ngaphezu kwalokho, ukusebenza kwe-CXCR4 kanye ne-CXCR7 kuhlotshaniswa nokuthambekela okukhulu kokusabalala kanye nokubikezela okubi ku-TNBC (Karn et al., 2022). Ngakho-ke, ukuphuma kwe-CXCR4 kanye ne-CXCR7 kungaba izakhi zofuzo ezihlosiwe zemithi ezisebenzayo zokwelapha umdlavuza webele, kufaka phakathi i-TNBC. Ucwaningo lukaYang et al. Yang et al (2019) lusebenzise i-CRISPR/Cas9 ukuhlanganisa izakhi zofuzo ze-CXCR4 kanye ne-CXCR7 futhi lwathola ukuthi ukwanda, ukukhula, ukufuduka kanye nokuhlasela kwe-TNBC kwakuvinjelwe kakhulu (Yang et al., 2019).
Amazinga akhuphukile e-miR-3662, i-TNBC oncogene, abonwe ezicutshini zomdlavuza webele (Yi et al., 2022). Ukuwohloka kwe-miR-3662 kuboniswe ukuthi kuvimbela ukukhula kwesimila somdlavuza webele kanye nokusabalala kwaso kokubili emzimbeni kanye nasesibelethweni (Agarwal noGupta, 2021). I-HBP-1 iyisithibi esinamandla se-Wnt/-catenin signaling futhi cishe iyimbangela yokukhula kwamaseli e-TNBC okwenziwa yi-miR-3662. Muva nje, u-Yi et al. bathole ukuthi i-miR-3662-HBP1 axis ilawula indlela ye-Wnt/-catenin signaling kumaseli e-TNBC (Yi et al., 2022). Ngenxa yokubonakaliswa kwayo okuqondene nesimila, i-miR-3662 ingaba yithagethi yokwelapha engaba khona ye-TNBC. Ngakho-ke, ukuwohloka kwe-miR-3662 okubangelwa yi-CRISPR/Cas9 kungaba yindlela enhle kakhulu yokuthuthukisa imithi emisha yokwelapha i-TNBC.
I-UBR5 iyi-nucleophosphoprotein engu-300 kDa ekhonjwe njengomlawuli oyinhloko we-tumorigenesis, i-metastasis, kanye nempendulo yomzimba emdlavuza eyahlukahlukene (Shearer et al., 2015; Fu et al., 2023). I-UBR5 ibikwe ukuthi ikhushulwe kakhulu kumasampula e-TNBC futhi ivuselela umsebenzi we-ERα ngokubangela ukwanda ngomsebenzi wayo we-ubiquitin ligase (Bolt et al., 2015). Izifundo zokulandelana kwe-exome ephelele zamasampula e-TNBC ayisisekelo nazo zibonise ukwanda kokubonakaliswa kwe-UBR5, okuphakamisa indima yayo ekuthuthukisweni kwe-TNBC. Ngaphezu kwalokho, ukususwa kwe-UBR5 okuqhutshwa yi-CRISPR/Cas9 kubonise ukuvinjelwa okukhulu kwe-metastasis ye-TNBC kanye nokukhula kumamodeli egundane okuhlola. Ngaphezu kwalokho, ukufakwa kwe-UBR5 kumodeli yegundane yohlobo lwasendle kubuyisele ukusebenza kwayo okugcwele, kanti lo mphumela awuzange ubonwe ezinhlotsheni eziguquliwe ezingasebenzi (Liao et al., 2017). Ukuntuleka kwe-UBR5 kuhlotshaniswa nokwanda kwe-apoptosis, i-necrosis, kanye nokuvinjelwa kokukhula kwesimila ku-TNBC ngenxa ye-angiogenesis embi. Ngenxa yokulahlekelwa yi-UBR5, ukusabalala kwesimila ezithweni ezikude kuyancipha, kanti i-UBR5 ibangela i-EMT engavamile ikakhulukazi ngokunciphisa ukubonakaliswa kwe-E-cadherin (Zhang noWeinberg, 2018). Muva nje, i-UBR5 iboniswe njengesici esibaluleke kakhulu ekubhalweni kwe-IFN-γ-induced PDL1 ku-TNBC ngenxa yokuntuleka kwayo komsebenzi we-E3 ubiquitination. Ukuhlaziywa kwe-RNA transcriptome kwembule ukuthi i-UBR5 ingaba nemiphumela yesistimu kuma-gene ahlobene nendlela ye-IFN-γ futhi ikhuthaze ukuguqulwa kwe-PDL1 ngokwandisa amazinga okusebenza kwe-protein kinase RNA (PKR) kanye nama-transducers ayo esignali kanye nama-activators. i-transcription 1 (STAT1) kanye ne-interferon regulatory factor 1 (IRF1). Kodwa-ke, ukususwa okuhlanganisiwe kwe-UBR5 kanye ne-PD-L1 okuqondiswa yi-CRISPR/Cas9 kunomphumela wokwelapha ohambisanayo kunanoma yikuphi ukuvimba kuphela, okunemiphumela ejulile endaweni encane yesimila (Wu et al., 2022). Ngakho-ke, i-CRISPR/Cas9 ingaba ithuluzi elibalulekile lokuvimbela umsebenzi we-UBR5, ngaleyo ndlela icindezele ukukhula kwe-TNBC kanye nokukhula kwesimila.
I-ROR1 iyiprotheni ye-transmembrane yohlobo I evezwa ngesikhathi somdlavuza kanye nokukhula kombungu futhi ikhonjwe njengeprotheni ye-oncofetal (Nicholas Borcherding, 2014). Ukuziphatha okuhlaselayo komdlavuza wabantu abahlukahlukene kuhlotshaniswa nokwenyuka kwe-ROR1. Imiphumela ethembisayo itholakale ezifundweni ze-in vivo kanye ne-in vitro ezibandakanya ama-compounds okwelapha aqondisa i-ROR1 (Chien et al., 2016). Amazinga aphezulu e-ROR1 mRNA kuma-biopsies ezicubu zesifuba nawo ahlotshaniswa nama-tumors anamandla afana ne-basal (BL) kanye nokufuduka kwawo aye kwezinye izingxenye zomzimba. Ngaphezu kwalokho, ukuvezwa ngokweqile kwe-ROR1 kukhonjwe njengophawu lokubikezela ukuthuthukiswa kwe-TNBC (Chien et al., 2016). Kodwa-ke, ukuthulisa i-ROR1 kusetshenziswa i-CRISPR/Cas9 ukucindezela ukukhula kwe-TNBC kanye nokusabalala kungaba isu eliphumelelayo.
Inqubo ye-sialylation ihilela ukungezwa kwe-sialic acid kuma-glycoconjugates, okubangelwa yi-sialyltransferases (STs). I-ST8SIA1 ingeyemndeni we-ST futhi idlala indima ebalulekile ekubangeleni izifo ezahlukahlukene ezifana ne-lymphocytic leukemia kanye nomdlavuza we-colorectal (Chang et al., 2018). Ukuhlaziywa kokulandelana kwe-RNA kukhombisa ukuthi i-ST8SIA1 ivezwa kakhulu ezicutshini zebele zeziguli ze-TNBC futhi ihlobene kahle nokuguqulwa kwezakhi zofuzo ku-tumor suppressor gene p53, okungaba negalelo ekubangeleni i-TNBC (Battula et al., 2017). Ngaphezu kwalokho, i-ST8SIA1 ihilelekile ekubangeleni i-metastasis kanye nokuphindaphinda kwe-TNBC, okuphakamisa ukuthi idlala indima ebalulekile ekuveleni nasekuthuthukisweni kwe-TNBC. Kumodeli ye-in vitro ye-TNBC, ukunqotshwa kwe-ST8SIA1 yi-CRIPSR/Cas9 kuboniswe ukuthi kuvimbela ukukhula kanye nokwanda kwe-metastasis (Battula et al., 2017). Lokhu kusikisela ukuthi i-CRISPR/Cas9 ingaba ithuluzi elibalulekile lokuvimbela ukusebenza kwe-ST8SIA1 oncogene ekwelapheni i-TNBC.
I-NAT1 iyi-enzyme ye-metabolic ekhuthaza ukwakheka kwamakhemikhali e-xenobiotic esigaba sesi-II futhi ivezwa cishe kuzo zonke izicubu zomuntu. I-NAT1 ingasebenzisa i-cofactor folate ukuqondisa i-acetyl-CoA (acetyl-CoA) ngisho noma kungekho i-substrate ye-aromatic amine (Stepp et al., 2015; Laurieri et al., 2014). Izifundo zikhombisile ukuthi i-NAT1 ilawula umsebenzi we-matrix metalloproteinase 9 (MMP9) kumamodeli wamaseli omdlavuza webele futhi ivikela izinhlobo ze-oxygen ezisebenzayo (ROS) ngesikhathi sokulamba kwe-glucose (Wang et al., 2018). Ukususwa kwe-NAT1 kuboniswe ukuthi kuvimbela i-pyruvate dehydrogenase complex, okuholela ekungasebenzi kahle kwe-mitochondrial (Wang L. et al., 2019). Ngaphezu kwalokho, eminye imibiko ehlukahlukene ikhombisile ukuthi ukuvinjelwa kwe-NAT1 kusetshenziswa ama-molecule amancane kanye nokuthulisa i-siRNA kunganciphisa ukuhlasela nokwanda kwamaseli omdlavuza webele (Stepp et al., 2018). Muva nje, i-CRISPR/Cas9 isetshenziswe ukucindezela i-NAT1 kumugqa weseli lomdlavuza webele i-MDA-MB-231, okuthinta imetabolism yamaseli kuye ngezinga lokubonakaliswa kwayo. Lolu cwaningo luphinde lwabonisa ukuthi i-NAT-1 ibalulekile ekuqhubekeni nasekusabalaleni kwe-TNBC (Carlisle et al., 2020).
Ukubhalwa kwe-oncogene okuqondiswe kahle kulawulwa yi-super enhancers, transcription factors kanye nama-cofactors (Hnisz et al., 2015). Ngaphezu kwalokho, iqembu lama-cyclin-dependent kinases (ama-CDK), njenge-CDK7, CDK8, CDK9, CDK12, kanye ne-CDK13, liyadingeka ukuze kulawulwe ukubhalwa kwe-transcription. Phakathi kwawo, i-CDK7 ihilelekile ku-phosphorylation ye-RNA polymerase II, okubaluleke kakhulu ekuqalisweni nasekukhulisweni kokubhalwa kwe-oncogene ku-pathogenesis ye-TNBC. Esifundweni esiyinhloko, ukususwa kwe-CDK7 yi-CRISPR/Cas9 kuvimbele i-TNBC, okubonisa ukuthi i-pathogenesis ye-TNBC incike ku-CDK7 (Wang Y. et al., 2015).
Izifundo zibonise ukuthi ukuguquka kwezakhi zofuzo ku-MYC kanye ne-RBP (iphrotheni ebopha i-RNA) kungaholela ekuqhekekeni, kuyilapho ukuguquka kwezakhi zofuzo olulodwa ku-MYC noma i-RBP kungathinti ukukhula kwamaseli omdlavuza (Einstein et al., 2021). Ama-RBP angaphezu kuka-1000 ku-genome yomuntu ahlolwe kusetshenziswa umtapo wolwazi osekelwe ku-CRISPR/Cas9. Phakathi kwawo, ama-RBP angu-57 atholakale ebaluleke kakhulu ekukhuleni kwamaseli omdlavuza anamazinga aphezulu e-MYC (Wheeler et al., 2020). Ngaphezu kwalokho, i-YTHDF2 ibalulekile ekugcineni ukukhula kwamaseli e-TNBC futhi inciphisa inani lemibhalo ye-methylated ngesikhathi sokubhalwa nokuhumusha kwezinga eliphezulu kumaseli omdlavuza anokubonakaliswa okuphezulu kwe-MYC. Ngaphezu kwalokho, i-YTHDF2 ayibalulekile kumaseli omdlavuza, angaxhomekile kakhulu emazingeni aphezulu e-MYC ukuze andise ukusinda kweziguli ze-TNBC (Einstein et al., 2021), okuphakamisa ukuthi ingaba yithagethi yokwelapha engaba khona yemithi enganqoba i-TNBC.
Amaprotheni eminwe ye-zinc (ama-ZNF) akha cishe u-1% we-genome yomuntu iyonke. Izifundo zibonise ukuthi i-ZNF ingalawula ukwanda kwamangqamuzana emidlavuzeni eyahlukene, njengomdlavuza wesibindi, umdlavuza webele kanye nomdlavuza we-colorectal (Zhang W. et al., 2021; Li et al., 2017). Imitapo yolwazi ekhiqizwe yi-CRISPR knockout isetshenziswe ukuhlola izakhi zofuzo ezicindezela i-tumor (ama-TSG) ezahlukahlukene emaselini omdlavuza webele (Shalem et al., 2014). Kusukela lapho, ucwaningo lwe-transcriptomic lwamaseli omdlavuza webele asuswe yi-CRISPR/Cas9 ZNF319 lubonise ukuthi i-ZNF319 iyizakhi zofuzo ezivimbela i-tumor ezinciphisa ukwanda komdlavuza webele futhi ngenxa yalokho ihilelekile ezindleleni ezahlukahlukene zokubonisa izimpawu kanye neminye imisebenzi yezinto eziphilayo (Wang L. et al., 2022).
I-Ferroptosis uhlobo lokufa kwamaseli okuhleliwe okuncike ekubeni khona kwensimbi. Kuyaziwa ukuthi ukuthuthukiswa kwe-ferroptosis kuthonywa ukuba khona kwama-lipid peroxides. Ngaphezu kwalokho, i-PKCβII ibikwe ukuthi ikhombisa i-lipid peroxidation yasekuqaleni, futhi ukwanda kwe-lipid peroxidation kuhlotshaniswa ne-ferroptosis. Ukuhlolwa komtapo wolwazi we-CRISPR/Cas9-mediated kinase inhibitors kwembule ukuthi i-PKCβII ihilelekile enkambisweni ye-lipid peroxidation, okubaluleke kakhulu kwi-ferroptosis kumaseli e-MDA-MB-231 (Zhang et al., 2022). Ngakho-ke, lokhu kusikisela ukuthi ukuphuma kwe-PKCβII yi-CRISPR/Cas9 kungaba yithagethi yezakhi zofuzo ezicindezela isimila ekwelapheni izifo ezihlobene ne-ferroptosis (Zhang et al., 2022).
Kukholakala ukuthi ukumelana nemithi kuyimbangela yokufa kwabantu abacishe babe ngu-90% ezigulini zomdlavuza futhi kungenye yezinselelo ezinkulu ekwelashweni komdlavuza (Bukowski et al., 2020). Imiphumela ikhombisa ukuthi inani elanele lezakhi zofuzo ezihlobene nokuphuma kwemithi, ukulungiswa kwe-DNA, i-apoptosis kanye nezindlela ezahlukahlukene zokubonisana kwamaseli kuhlotshaniswa nokumelana nemithi (Haider et al., 2020). Phakathi kwazo, izakhi zofuzo eziningana ziqondiswe ngamathuluzi e-CRISPR/Cas9 futhi zibonise imiphumela ethembisayo ekunciphiseni ukumelana nemithi kanye nokwandisa ukusebenza kahle kwezindlela zokwelapha umdlavuza (Vaghari-Tabari et al., 2022). Ngaphezu kwalokho, imitapo yolwazi yokuhlola izakhi zofuzo ze-CRISPR/Cas9 ephumelela kakhulu ihilelekile ekuguquleni umsebenzi wezinhloso zezakhi zofuzo ezingaba khona zokulwa nemithi (Shalem et al., 2015). Ukuze kutholakale izakhi zofuzo zokumelana ne-paclitaxel, ukulandelelana kwe-RNA okuhambisana nokuhlolwa komtapo wolwazi we-sgRNA we-genome kusetshenziswe ukuhlonza izakhi zofuzo eziyisishiyagalombili ezingaba khona, okuhlanganisa i-histone deacetylase 9 (HDAC9), ezihlotshaniswa nokumelana nemithi ezigulini ezine-TNBC ephindaphindayo (B et al., 2020). ). Kolunye ucwaningo, ukwanda kokubonakaliswa kwe-diserine/threonine kanye ne-tyrosine protein kinase (DSTYK) kwabonwa ngesikhathi sokusinda kweziguli ze-TNBC ezelashwa ngemithi yokulwa nomdlavuza. Ngaphezu kwalokho, ukunqotshwa kwe-DSTYK okubangelwa yi-CRISPR/Cas9 kuthuthukisa kakhulu i-apoptosis yamaseli omdlavuza amelana nemithi e-in vitro (amaseli e-SUM102PT namaseli e-MDA-MB-468) kanye nemodeli ye-TNBC e-in vivo (Ogbu et al., 2021).
Nakuba i-TNBC inamandla okusebenza ngendlela engajwayelekile endleleni ye-MAPK, umphumela wezokwelapha eziqondiswe ku-MEK awulungile. Isikrini somtapo wolwazi we-genomic we-CRISPR/Cas9 sembule ukuthi ukuvinjelwa kwe-PSMG2 (i-proteome assembly chaperone 2) kuthinta amaseli e-TNBC BT549 kanye ne-MB468 ku-MEK inhibitor AZD6244. Ukudilizwa kwe-PSMG2 kwe-CRISPR/Cas9 kushintshe umsebenzi ojwayelekile we-proteasome, okuholele ekuhlukaniseni i-PDPK1 okwenziwa yi-autophagy, ngaleyo ndlela kuthuthukiswe amaseli esimila kumamodeli egundane e-TNBC abangelwa yi-AZD6244 (MEK inhibitor) kanye ne-MG132 (proteasome inhibitor). Ngakho-ke, ama-proteasome kanye nama-MAP kinase (MEK) inhibitors anganikezwa ngokubambisana ukuze kuncishiswe ukwanda kwamaseli esimila (Wang X. et al., 2022).
I-CRISPR/Cas9 iphinde yasetshenziswa ukuhlola ukulahleka komsebenzi wezakhi zofuzo okuholela ekuphikisweni kwe-TNBC (Shu et al., 2020). UGe et al bachaze indlela yokumelana nemithi yamaseli omdlavuza aphathwe nge-JQ1, i-BET bromodomain inhibitor (BBDI), ku-TNBC. Besebenzisa i-CRISPR/Cas9, bathole ukuthi ukususa i-rb1 gene kubangele ukuthi i-TNBC imelane nomuthi wokulwa nomdlavuza i-JQ1. Ngakho-ke, umsebenzi we-rb1 ubalulekile ekuphenduleni imithi ye-JQ1 ku-TNBC. Babike nokuthi i-paclitaxel iyi-CDK4/6 kinase/microtubule inhibitor, futhi ukuhlanganiswa kwayo nama-BBDI njenge-JQ1 kunganikeza izimpendulo zokwelapha ezithembisayo ku-TNBC ephikiswa yimithi (Ge et al., 2020).
Isimo sokubhalwa kwe-RNA ende engabhalisi (i-lncRNA) kubikwe ukuthi sibangela ukumelana nokwelashwa kwe-neoadjuvant ku-TNBC. Lolu cwaningo lubonisa ukuthi imibhalo emihlanu ehlukene ye-MALAT1 lncRNA ivezwa kakhulu ku-TNBC. Ngaphezu kwalokho, ukususwa kwe-MALAT1 okubangelwa yi-CRISPR/Cas9 kwandisa ukuzwela kwamaseli e-TNBC BT-549 ku-paclitaxel kanye ne-doxorubicin, okuphakamisa indima engaba khona yokumelana ne-MALAT1 ku-TNBC (Shaath et al., 2021).
Ukuguqulwa kwezakhi zofuzo ku-BRCA1 (BRCA1m) kuyahlukahluka ngakho-ke kunzima ukukubona. Ukuqondisa i-PARP1 (poly(ADP-ribose) polymerase), umlingani obulalayo wokwenziwa we-BRCA1, kungandisa ukuzwela kwemithi ye-TNBC. Ukusebenzisa i-CRISPR/Cas9, ukususa i-PARP1 kwandisa ukuzwela kwemithi yokulwa nomdlavuza njenge-doxorubicin, i-gemcitabine kanye ne-docetaxel kumaseli e-TNBC aguquliwe e-mBRCA1, okuphakamisa ukuthi i-PARP1 nayo ihilelekile ekumelaneni nemithi ku-TNBC (Vaghari-Tabari et al., 2022). . Ukuguqulwa kwezakhi zofuzo ezicindezela isimila i-BRCA1 noma i-BRCA2 kwaziwa ngokwandisa amathuba okuba nomdlavuza webele kubantu. Ukwelashwa kwalezi ziguli kudinga ukusetshenziswa kwezithibi ze-PARP. Ngaphezu kwalokho, kuye kwaboniswa ukuthi ukukhishwa kwe-nucleotide salvage factor DNPH1 kusetshenziswa i-CRISPR/Cas9 kungasusa i-nucleotide enobuthi i-5-hydroxymethyldeoxyuridine (hmdU) monophosphate, ngaleyo ndlela kuthuthukiswe impendulo yamaseli angenayo i-BRCA ekuzweleni kuma-PARP inhibitors (Fugger). et al., 2021). Ngakho-ke, ama-CRISPR/Cas9 kanye nama-PARP1 inhibitors angaba isu lokwelapha elibalulekile le-TNBC.
I-gene ye-penetrant glycoprotein (P-gp) iyi-gene yokumelana nemithi eminingi evame ukwanda cishe ku-41% wayo yonke i-TNBC (Sun et al., 2020). Ukuphuma kwemithi okubangelwa yi-P-gp kuye kwabonakala njengomlawuli oyinhloko wokumelana nemithi kumdlavuza webele. Ama-inhibitors e-P-gp abonise ukuzwela okukhulu emithini yokulwa nomdlavuza kumdlavuza webele (Famta et al., 2021). Ngakho-ke, ukususwa noma ukucindezelwa kwe-P-gp kanye ne-P-gp okubangelwa yi-CRISPR/Cas9 kungaba izindlela ezibaluleke kakhulu zokunqoba ukumelana nemithi ku-TNBC.
I-ATP-binding cassette transporter G2 (ABCG2) yaziwa ngokubangela ukumelana nemithi ku-TNBC (Palasuberniam et al., 2015), yize okwamanje kungekho mibiko ngobudlelwano phakathi kwe-CRISPR/Cas9 kanye ne-ABCG2 silencing kanye nokungasebenzi kwezakhi zofuzo ezicindezela ithumba. I-PTEN Ingathuthukisa ukusebenza kwe-ABCG2 (Palasuberniam et al., 2015), (Deepak Singh et al., 2021). Ngakho-ke, ukusebenzisa i-CRISP/Ca9 ukususa i-ABCG2 nokuyihlanganisa ne-ABCG2 inhibitor kungaba ukwelashwa okufanele ukunqoba ukumelana nemithi ku-TNBC. Ithebula 2 likhuluma nge-CRISPR/Cas9 ehlose zonke izakhi zofuzo ezimelana nayo.
Ithebula 2. I-CRISPR/Cas9 ihlose izakhi zofuzo ezahlukene zokumelana ne-TNBC ukuze zenze amaseli azwele imithi yokulwa nomdlavuza.
Imitapo yolwazi ye-CRISPR/Cas9 ingamathuluzi angaba khona okuhlola ukuguquka kwezakhi zofuzo okuhlobene ne-pathogenesis yomdlavuza (Chan et al., 2022). Le ndlela yokuhlola ihilela izinyathelo ezine ezifana (a) nokwakhiwa komtapo wolwazi, (b) ukudluliselwa kwe-lentiviral, (c) ukuhlolwa kwe-phenotypic, kanye (d) nokuhlaziywa kwezakhi zofuzo okuqondiwe. Nakuba i-TNBC inomsebenzi ongavamile wendlela ye-MAPK, ukubikezela kwezokwelapha kwe-MEK okuqondiswe ezigulini ze-TNBC ezithwala ukuguquka kwezakhi zofuzo ezicindezela isimila njenge-PTEN, RB1, kanye ne-TP53 kubi kakhulu.
Ngaphezu kwalokho, isikrini sokukhipha izakhi zofuzo sisebenzisa i-CRISPR/Cas9 sihlole i-molecule enamandla kakhulu futhi ekhethayo, i-dehydrocatrol, eningi ezitshalweni zezimbali namaqabunga zaseGuatemala, ukuze kuqondwe umphumela we-dehydrocatrol ku-MDA-MB-231. Izindlela zokubulala amaseli akhethiwe. Izici ze-Mesenchymal stemness zezinhlobo ze-TNBC. Lesi sikrini esisekelwe ku-CRISPR/Cas9 siphinde sembula ukuthi i-HSD17B11, i-gene efaka i-17β-hydroxysteroid dehydrogenase uhlobo 11, ivezwa kakhulu kumaseli e-MDA-MB-231 futhi iphumela ku-dehydrofalcarinol ethize kumaseli e-MDA-MB-231 (Grant et. al., 2020). Ngakho-ke, lokhu kusikisela ukuthi ukuhlolwa kwezakhi zofuzo kwe-CRISPR/Cas9 kunamandla amakhulu ekuboneni izindlela ezingaphansi kwezakhiwo zokulwa nomdlavuza zamakhemikhali emvelo angaba khona.
Ngaphezu kwalokho, ukubekeka engcupheni kwe-TNBC kumdlavuza kwahlolwa kusetshenziswa isikrini se-CRISPR/Cas9 esingachemile, esabonisa ukuxhumana phakathi kwezindlela zomdlavuza kanye nezindlela zokucindezela isimila. Izingxenye ezibalulekile zezindlela ze-mTOR kanye ne-Hippo zibikwe ukuthi zidlala indima ebalulekile ekulawulweni kwesimila ku-TNBC. Ngaphezu kwalokho, izifundo zibonise ukuthi ukuvinjelwa kwe-mTORC1/2 kanye ne-YAP oncoprotein yezokwelapha kuvimbela ngempumelelo ukusabalala kwe-TNBC kusetshenziswa imodeli ye-in vitro drug-matrix synergy kanye ne-xenografts etholakala esigulini. Ngaphezu kwalokho, ukuvinjelwa kwe-mTORC1/2 okuqondiswa yi-Torin-1 kuthuthukisa i-macropinocytosis, kanti ukuvinjelwa kwe-YAP okubangelwa yi-vertebrorfin kuholela ekufeni kwamaseli e-TNBC. Uma kuhlanganiswa, le miphumela iqokomisa amandla kanye nokuqina kokuhlolwa kwe-CRISPR ye-genome-wide in vivo ukuhlonza ukwelashwa okusha nokusebenzayo kwe-TNBC (Dai et al., 2021).
Ukuphazamiseka kwesimiso somzimba sokuzivikela komzimba kuyisici esibalulekile ekuveleni kwesifo sofuba. Amaseli omdlavuza agwema ukuphuma kwamasosha omzimba ngokugwema izindlela zokuzivikela, okuhlanganisa nokuphazamisa umsebenzi wamaseli omzimba endaweni encane yesimila futhi abeke engcupheni isimiso somzimba sokuzivikela komzimba. Ngakho-ke, ukuthuthukisa isimiso somzimba sokuzivikela esithuthukisiwe kungaba yindlela ebalulekile yokulwa nezimila. Ukusetshenziswa kokuguqulwa kwezakhi zofuzo okusekelwe ku-CRISPR/Cas9 kubhekana nezinkinga eziningana ezihlobene nokungasebenzi kahle kwesimiso somzimba sokuzivikela komzimba ngezindlela ezahlukene. I-CRISPR/Cas9 isetshenziswe ukuthuthukisa isimiso somzimba sokuzivikela esilwa nesimila ngokumelene nomdlavuza webele ngezindlela ezilandelayo (Isithombe 3).
Umfanekiso 3. I-CRISPR/Cas9 immunotherapy ihlose amaseli e-TNBC. Ukulahlekelwa yi-CDK5 kanye nokudilizwa kwe-PDL1 kanye ne-CD155 kuthuthukisa isimiso somzimba sokuzivikela. Ngokufanayo, ukulahlekelwa yi-A2AR kanye nokudilizwa kwe-TAA njenge-HER2, i-mucin 1 kanye ne-TEM8 kwandisa ukusebenza kahle kwamaseli e-CAR-T ekubulaleni amaseli omdlavuza. Ukuhlolwa kwamaseli e-T aqhutshwa yi-CRISPR/Cas9 kukhombisile ukuthi ukuphazamiseka kwe-p38 kinase kuthuthukisa umsebenzi wokulwa nomdlavuza wamaseli e-T. Amaseli e-T aguqulwe yi-CRISPR/Cas9 akhulisa ukubonakaliswa kwe-TCR (i-T cell receptor), okuholela kumaseli e-T abe nomsebenzi wokulwa nomdlavuza.
Umgomo we-immunotherapy ukukhuthaza amasosha omzimba ukuba ahlasele amangqamuzana omdlavuza. Ukwanda ngokweqile kwamaprotheni okuhlola amasosha omzimba kuvame ukuvimbela ukusabela okuzenzakalelayo kodwa kungasiza umdlavuza ukuthi ungakugwemi (Topalian et al., 2015). Lawa maprotheni avimbela ukusabela okuzivikela ngokubopha kuma-receptor ebusweni bamangqamuzana omzimba. Amaprotheni amaningi okuhlola (njenge-CD155 kanye ne-PD-L1) aqondisa i-PD-1 receptor kumaseli omzimba futhi abonakala kumdlavuza webele, ikakhulukazi i-TNBC (Li Y.-C. et al., 2020). Ukunciphisa i-PD-L1 noma i-receptor yayo kusetshenziswa i-CRISPR/Cas9 kungakhuthaza amasosha omzimba ukuba ahlasele amathumba e-TNBC (Yahata et al., 2019). Ngaphezu kwalokho, ukwehla kokubonakaliswa kwe-PD-L1 ngokususa i-CDK5 okubangelwa yi-CRISPR/Cas9 kuye kwabonakala kuvimbela ukukhula kwesimila ngaphakathi kwe-vitro kanye nasemzimbeni (Deng et al., 2020). Ukuvinjelwa kokukhula kumamodeli omdlavuza webele e-in vitro kanye nase-vivo kusikisela ukuthi ukunciphisa i-CD155 okubangelwa yi-shRNA kungaba nomthelela wokwelapha umdlavuza webele (Gao et al., 2018).
Amaseli e-CAR T aveza ama-CAR aqaphela ama-antigen ahlobene nesimila (ama-TAA) futhi angasebenzisa ukuqedwa kwamaprotheni okuhlola ukuthuthukisa umsebenzi wawo (uLi C. et al., 2020). Kunezinhloso ezahlukahlukene ezingaba khona zokwelapha amaseli e-CAR T kumdlavuza webele, kufaka phakathi ama-TAA amaningana njenge-HER2, i-mucin1, kanye ne-TEM8 (uBajgain et al., 2018). Kunobufakazi bokuthi amaseli e-CAR T aqondisa i-mesothelin (evezwa kakhulu kumaseli e-TNBC BT-459) asebenza kangcono ekulweni nomdlavuza lapho i-PD-1 ikhishwa kusetshenziswa i-CRISPR/Cas9 (uHu et al., 2019). Kodwa-ke, ukuhlukanisa amaseli e-T ezigulini bese uwahlela ngaphandle kwe-vivo kuyinqubo edinga umsebenzi omningi futhi edla isikhathi. Nakuba amaseli e-T ajwayelekile anganciphisa izidingo zokuhlukaniswa, amaseli e-T anikelayo aveza i-human leukocyte antigen (HLA) class I kanye ne-T cell receptor (TCR) kufanele asuswe ukuze kuvinjelwe ukukhubazeka kwe-graft-versus-host (uRen et al., 2017). , 2017a). Ukusebenzisa i-CRISPR/Cas9, i-HDR ingasusa i-TCR kanye ne-HLA ngasikhathi sinye futhi isuse i-CAR efaka i-gene. Izifundo zibonise ukuthi i-CRISPR/Cas9 ingasetshenziswa ukwethula ama-CAR alwa ne-CD19 endaweni ye-TCR, ngaleyo ndlela ikhiqize ama-CAR ngempumelelo ngaphandle kokuqeda amaseli e-T (Dimitri et al., 2022). Ubuchwepheshe be-Multiplex buthuthukiswe ukukhiqiza amaseli e-CAR T allogeneic ngokuqeda i-TCR ngasikhathi sinye, i-beta-2-microglobulin (B2M), i-HLA-I subunit kanye namanye amaprotheni afana ne-PD-1 kanye ne-CTLA-4, angaba nezakhiwo ezinkulu zokulwa nomdlavuza. ngokumelene ne-TNBC (Eyquem et al., 2017; Liu et al., 2017; Ren et al., 2017b; Dimitri et al., 2022).
Ukusebenzisa i-CRISPR/Cas9 kungathuthukisa ukusebenza kahle kwamaseli e-CAR-T. I-Adenosine yaziwa ukuthi inezakhiwo zokucindezela amasosha omzimba futhi inganciphisa ukuzivikela komzimba komdlavuza ngokuvimbela ukusebenza kwamaseli e-T nokwenza kusebenze ama-receptor e-adenosine A2A (A2AR) (Vigano et al., 2019). Ukuthulisa i-A2AR kusetshenziswa i-CRISPR/Cas9 kuthuthukise kakhulu ukusebenza kahle kwamaseli e-CAR-T emzimbeni (Giuffrida et al., 2021). Amaseli e-T ayaziwa ukuthi abonisa izici ezahlukahlukene ze-phenotypic njengokwanda kwamaseli, ukuhlukanisa, ukucindezeleka kwe-oxidative kanye nokucindezeleka kwe-genomic. Ukuhlolwa kwamaseli e-T okusekelwe ku-CRISPR/Cas9 kwembule ukuphazamiseka kwama-kinase angu-25 ahlukene e-T cell receptor. Phakathi kwawo, ukususwa kwe-p38 kinase kuye kwabonakala kuthuthukisa umsebenzi wokulwa nomdlavuza wamaseli e-T, okubonisa ukuthi i-p38 kinase iyisilawuli esiyinhloko sokulawulwa kwamaseli e-CAR-T (Gurasamy et al., 2020).
Amaseli e-T angaguqulwa ngokwezakhi zofuzo kusetshenziswa i-CRISPR/Cas9 ukukhiqiza ama-TCR anamandla kakhulu. Ukudlulisela amaseli e-T aguqulwe ngokwezakhi zofuzo ezigulini kubonise umsebenzi onamandla wokulwa nomdlavuza kunamaseli e-T angokwemvelo. Ngakho-ke, ama-TCR angokwemvelo angancintisana nama-TCR aguqulwe ngokwezakhi zofuzo ezigulini, okungathinta amandla okwelashwa ngomdlavuza. Ukuze kunqotshwe le nkinga, ukusebenzisa i-CRISPR/Cas9 ukususa i-TCR-β engokwemvelo kumaseli atholakele bese kamuva kudlulisela amaseli e-TCR-β ezigulini zomdlavuza kungabonisa izimpendulo ezingcono zokuzivikela komzimba ezilwa nomdlavuza ngaphandle kwesidingo sokuncintisana ngokocansi kwe-TCR engokwemvelo (Fan et al. 2018). Ngakho-ke, ngaphandle kwamaseli e-T aguqulwe yi-CRISPR, ama-TCR azwela kakhulu kuma-antigen esimila kunamaseli e-T ajwayelekile aguqulwe yi-TCR. Ngaphezu kwalokho, kuma-leukemia ahlukahlukene, amaseli e-T ashintshiwe akhiqizwa yi-γδ TCR+ CRISPR abonisa ukuvezwa okuphezulu kwamaseli e-T e-CD4+ kanye ne-CD8+ kunokudluliselwa kwe-TCR okujwayelekile (Legut et al., 2018). Ngakho-ke, amaseli e-T aguquliwe akhiqizwa yi-CRISPR/Cas9 angaba yindlela ephumelelayo yokwelapha nge-immunotherapy yokunqoba i-TNBC.
Ama-integrin angama-molecule okunamathela kwamaseli akhona kuma-transmembrane amaseli futhi akhuthaza ukubopha kwamaseli ku-matrix engaphandle kweseli (ECM) (Hamidi no-Ivaska, 2018). Ukuphazamiseka kwama-integrin kuhlotshaniswa nokuthuthukiswa komdlavuza kanye nokufuduka ngokushintsha i-matrix engaphandle kweseli, okuholela ekusindeni kwamaseli omdlavuza ekujikelezeni kwegazi (Hamidi no-Ivaska, 2018). I-CRISPR/Cas9-mediated integrin knockdown ibambezela ukuqhubeka kwesimila, ukusabalala kwamangqamuzana, kanye nokwakheka kwamakoloni ku-TNBC. Ukushaqeka kwe-integrin a5 (ITGA5) kubikwe ukuthi kunciphisa ukufuduka kwamaseli kanye nokuqhubekela phambili kwezinye izinhlobo zomdlavuza ezifana nomdlavuza wamaphaphu (Ju et al., 2017), okuphakamisa ukuthi i-integrin a5 ingaba yisici esibalulekile ekuzalweni kwe-TNBC.
Ukukhiqiza amagundane angenawo amandla kusetshenziswa izindlela zendabuko zamaseli e-embryonic stem (ES) kuyinqubo edinga umsebenzi omningi, edla isikhathi, futhi engasebenzi kahle. Kuthatha izinyanga neminyaka ukuqondisa amaseli e-ES ngokusebenzisa i-homologous recombination, ukuzala amagundane e-chimeric, bese uwahlanganisa namagundane e-heterozygous ukukhiqiza inzalo ye-homozygous. Ukuzalanisa okuyinkimbinkimbi kuyadingeka ukudala amagundane anezinguquko eziningi zofuzo. Kodwa-ke, kuye kwavela izinkinga eziningi lapho kusetshenziswa amagundane akhiqizwa ukususwa kwamaseli e-ES kusetshenziswa i-CRISPR/Cas9 noma ngokujova izingxenye ze-CRISPR/Cas9 emaqandeni avundisiwe eseli elilodwa. Isibonelo, ukwethulwa kwezinguquko kuvame ukwenzeka endaweni ye-biallelic futhi akucaci ngofuzo. Muva nje kubikwe ukuthi amaseli e-ES asebenzisa ubuchwepheshe be-CRISPR/Cas9 angafaka izinguquko ze-biallelic ngasikhathi sinye kumajini afinyelela ku-5 (Nishizono et al., 2021). Ngale njongo, i-Cas9 mRNA kanye nama-gRNA amahlanu aqondene nezakhi zofuzo adluliselwa ngasikhathi sinye kumaseli e-ES. Lokhu kugqamisa isithembiso kanye nokusebenza kahle kwale nqubo, yize lokhu kuguqulwa kungenzeka ukuthi kwadluliselwa lapho imigqa yabasunguli ikhiqizwa ukuze kukhiqizwe amagundane amahlanu aqothukile. Ucwaningo luphinde lubike ngokutholwa okumangazayo: Amaseli e-ES awasadingeki ukudala amagundane aguquliwe ngokwezakhi zofuzo. Esikhundleni salokho, ukuze kususwe imikhiqizo ethile yezakhi zofuzo, i-Cas9 mRNA kanye ne-gRNA kwafakwa kuma-embryo esigaba seseli elilodwa. Ngenxa yalokho, kudalwa imigqa yabasunguli abaqothukile engasetshenziswa ngokomqondo ukutadisha imiphumela yokususwa kwezakhi zofuzo kumagundane (Qin et al., 2016). Ngakho-ke, i-CRISPR/Cas9 ivumela ukudalwa kwamagundane aguqulwe izakhi zofuzo ukuze aphathe i-TNBC ngezindleko eziphansi kunobunjiniyela bezakhi zofuzo bendabuko. Kusetshenziswa uhlelo lwe-CRISPR/Cas, kuye kwasungulwa imodeli entsha yegundane eliqothukile elingangenisa ngempumelelo ukuguqulwa kwamaphuzu ku-gene eyodwa noma ngaphezulu e-endogenous ku-TNBC. Ngokusekelwe kulo mqondo, amamodeli ezilwane e-TNBC angathuthukiswa futhi kusetshenziswa ukususwa kwe-BRCA1 kanye ne-p53 okubangelwa yi-CRISPR/Cas9, okuholela ekulahlekelweni kokulungiswa kwe-HR, ukungazinzi kwe-genomic, kanye ne-phenotypes eguquliwe (Annunziato et al., 2020).
Njengamanje, izindlela ezahlukahlukene ezifana ne-mammography, i-magnetic resonance imaging (MRI) kanye ne-ultrasound zisetshenziswa ukuxilonga i-TNBC. Kodwa-ke, lezi zindlela zinemikhawulo ethile. I-Mammography isetshenziselwa ukuxilonga izicubu zebele lendawo kune-metastases, lapho amangqamuzana omdlavuza esuke ethuthela kwezinye izitho zomzimba. I-Ultrasonography akuyona indlela ethembekile yokuxilonga i-TNBC (Chen noLee-Felker, 2023). I-MRI inokuzwela okuphezulu kune-ultrasound kanye ne-mammography, kodwa ukunemba kwayo kokuxilonga kunqunyelwe (Sha noChen, 2022). I-tissue biopsy iyindlela ehlaselayo yokuhlonza amangqamuzana omdlavuza. Kodwa-ke, kwezinye izimo, i-biopsy ingaphuthelwa izicubu zomdlavuza uma inaliti iduka endaweni ethakazelisayo. Ngaphezu kwalokho, ibiza kakhulu futhi ingaba yingozi esigulini. I-TNBC uhlobo lomdlavuza oluhlukile, ngakho-ke i-biopsy ingase inganikezi ulwazi olwanele ngohlobo lomdlavuza. Ngakho-ke, indlela entsha ethembekile yokuxilonga yokuthola i-TNBC iyadingeka ngokushesha. I-CRISPR/Cas9 ingasebenza njengendlela ebucayi kakhulu futhi engangenisi kakhulu yokuxilonga umdlavuza webele. Ngenxa yalokhu, amasu asekelwe ku-CRISPR/Cas9 angasetshenziswa ukuthuthukisa izindlela ze-PCR zokuxilonga i-TNBC. Okokuqala, amaprotheni e-Cas9 kanye ne-cpf1 ohlelweni lwe-CRISPR asetshenziselwa ukususa i-DNA engaqondile, bese lawa maprotheni amabili (i-Cas9 kanye ne-cpf1) ekwazi ukubona ukulandelana kwe-PAM ngaphambi kokubopha ku-DNA eqondiwe (Deepak Singh et al., 2021). Ngakho-ke, i-PCR ingabona izinguquko ezihlobene nokuthuthukiswa komdlavuza. Izifundo eziningana ziye zamukela leli qhinga elisekelwe ku-CRISPR ukuthola izinguquko ezahlukene kumdlavuza ohlukene (Safari et al., 2019). Ngakho-ke, izindlela ze-PCR ezisekelwe ku-CRISPR zinganciphisa ukuncika kokuxilongwa kwe-TNBC ezindleleni ezihlaselayo njenge-immunohistochemistry esekelwe ku-biopsy.
Izinguquko ze-genotypic ekulawulweni kwama-hormone receptor nazo ziboniswe ku-TNBC (Chen noRusso, 2009). Amasu e-PCR asekelwe ku-CRISPR/Cas9 asebenzisa ama-microarrays ukuze kuhlolwe izindawo zokunakekelwa angaqapha ngempumelelo lezi zinguquko (Hajian et al., 2019). Kungaphinde kunikeze abahlinzeki bezempilo ulwazi olufanele mayelana nezinguquko ezithile ezigulini ze-TNBC, okungasiza ekuthuthukiseni inkambo yazo yokwelashwa. Kodwa-ke, le ndlela ehlanganisiwe inemikhawulo. Ngakho-ke, kudingeka imizamo eminingi yocwaningo ngaphambi kokuba le ndlela ye-CRISPR/Cas-PCR isetshenziswe ekunakekelweni kwezempilo ukuze kutholakale i-TNBC (Yang et al., 2019).
Isikhathi sokuthunyelwe: Okthoba-14-2024