CRISPR) inzira yekugadzirisa majini echizvarwa chechitatu: CRISPR yakachinja nyika

Clustered regularly interspaced short palindromic repeats (CRISPR) inzira yekugadzirisa genome yechizvarwa chechitatu yakachinja nyika nemhedzisiro yayo yepamusoro-soro. Yakashandiswa kurapa zvirwere zvakasiyana-siyana zvebhayoloji nehutachiona. Mabhakitiriya akasiyana-siyana nemamwe maprokaryotes (akadai searchaea) anewo masisitimu eCRISPR/Cas9 ekudzivirira kubva kumaphages. Zvakataurwa kuti nzira dzeCRISPR/Cas9-based dzinogona kudzivirira kukura nekufambira mberi kwekenza yemazamu ine triple negative (TNBC) nekutarisa majini ekudzivirira anogona kuchinja, transcription, uye epigenetic regulation. Kupindira uku kwekurapa kunogona kubatsira kugadzirisa matambudziko akaoma akadai sekuramba kwemishonga kunoonekwa kunyangwe muTNBC. Parizvino, nzira dzakasiyana-siyana dzinoshandiswa kuendesa CRISPR/Cas9 kumasero akananga, akadai semuviri (microinjection, electroporation uye hydrodynamic modes), viral (adeno-associated virus uye lentivirus) uye non-viral (liposomes uye lipid nanoparticles). Kunyangwe mamodheru akasiyana-siyana akagadzirwa kuti adzidze zvikonzero zvemamorekuru eTNBC, kushaikwa kwenzira dzakanangana nedzekupa maturusi ekugadzirisa genome mumuviri kunoderedza kushandiswa kwawo mukiriniki. Mukupfupisa, ongororo iyi inoongorora zvizere kufambira mberi, matambudziko, zvipingamupinyi, uye tarisiro yekurapwa kweCRISPR/Cas9 kweTNBC zvichibva pauchapupu huripo. Tinosimbisawo kuti kusanganiswa kwehungwaru hwekugadzira nekudzidza kwemuchina kunogona sei kuvandudza nzira dzeCRISPR/Cas9 mukurapa kweTNBC.
Kenza inoratidzwa nekupatsanurwa kwemasero kusingadzoreki, kukanganiswa kwenzvimbo dzekutarisa masero, uye kushanduka kwemajini ekudzivirira tumor (TSGs) (Matthews et al., 2022). Pakati pemhando dzakasiyana dzekenza, kenza yemazamu ndiyo mhando yekenza yakajairika muvakadzi uye ine huwandu hwakawanda hwerufu pasi rose (Waks naWiner, 2019). Kenza yemazamu chirwere chakasiyana chine hunhu hwakasiyana, senge hunhu hwehistological uye hwebiological, kuratidzwa kwekiriniki uye maitiro, uye mhinduro yekurapwa (Weigelt et al., 2010). Kupatsanurwa kwekenza yemazamu kunopa ruzivo rwakajeka rwekuongororwa kwekenza yemazamu uye fungidziro yebundu. Kushandiswa kwezviratidzo zvakajairika uye hunhu hwekiriniki kwave kuri chinhu chikuru chekupatsanurwa kwekenza yemazamu (Tsang naTse, 2019). Kufungidzira uye mhinduro yekurapwa kwekenza yemazamu kunokonzerwa nezvinhu zvakawanda, kusanganisira kuvapo kweestrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2/neu), uye histological grade, mhando yebundu, uye giredhi. Kukura uye lymph node metastasis (Al-Tubaiti, 2020). Mhando shanu dzekenza yemazamu dzakawanikwa, dzinosanganisira luminal A, luminal B, HER2-enriched, basal-like, uye claudin-low (Prat et al., 2015). TNBC rudzi rwekenza yemazamu rusingaratidze ER yese, PR, uye HER2 (Yin et al., 2020). Zviratidzo izvi zvinobatanidzwa neTNBC, zvichiratidza kufambira mberi kwayo nekukurumidza uye hunhu hwayo hwehasha kupfuura chero rumwe rudzi rwekenza yemazamu (Feng et al., 2018). Pamusoro pezvo, Peru et al (2000) vakashandisa tekinoroji ye microarray kuti vagadzirezve kenza yemazamu uye vaone mhando shanu dzekenza yemazamu (Cadenas, 2012). Kenza yemazamu yakafanana neBasal rudzi rwekenza yemazamu ine phenotype ine triple-negative uye inobatanidzwa nekufambira mberi nekukurumidza. Zvinofanira kucherechedzwa kuti kenza dzese dzezamu dzakaita sebasal dzinowanzoonekwa zvisirizvo seTNBC, asi 77% chete ndiyo TNBC; Kusiyana neizvi, 71–91% yeTNBC yakafanana nebasal, zvichiratidza kuti mhando mbiri idzi dzekenza dzezamu dzinopindirana uye dzinomiririra mapoka akasiyana (Wang D.-Y. et al., 2019). Izvi zvinoita kuti pave nekudiwa kwekutarisa kusiyana kweTNBC kuti tijekese fungidziro uye kuona mhinduro dzinogona kuitika kumishonga iripo uye yeramangwana. Pamusoro pezvo, TNBC inomirira 15–20% yezvirwere zvese zvekenza yezamu uye inowanikwa zvakanyanya muvakadzi vari pasi pemakore makumi mashanu. Kuchinja kweBRCA1 kana BRCA2 kwakataurwa muinenge 20% yezviitiko zveTNBC (Xie et al., 2017; Tzikas et al., 2017). , 2020). Zvidzidzo zvakaratidzawo kuti TNBC ine immune microenvironment yakasiyana inosanganisira huwandu hwakawanda hwe vascular endothelial growth factors, tumor-associated macrophages (TAMs), tumor-infiltrating lymphocytes (TILs), uye mamwe mamorekuru ane chekuita nekukura nekufamba kwegomarara. Nokudaro, kunzwisisa microenvironment yeTNBC kwakakosha pakufanotaura kwayo nekurapa (Fan naHe, 2022).
Kuti tiongorore kufambira mberi kweTNBC uye kuona kuti chirwere ichi chinorapwa zvakanaka, kuongororwa kwakarurama, kwakavakirwa pa immunohistochemistry (IHC) kuti tione ER, PR uye HER2, uye mammography kuti tione maronda emazamu, zvakakosha. Zvisinei, mammography haigone kuratidza zvakakwana zviratidzo zvemukati memudumbu zvakaita se necrosis uye fibrosis (Deepak Singh et al., 2021). Sezvo kufambira mberi kusina kunaka uye kuongororwa kwechirwere kuchidzivirira vanachiremba kunyora mishonga yakakodzera (Chaudhary, 2020), nzira dzakasiyana-siyana dzinoshandiswa kuvandudza kutarisirwa kwevarwere vane TNBC. Parizvino, mishonga miviri, doxorubicin uye cyclophosphamide, inoshandiswa muvarwere vane TNBC uye yakaratidza mhedzisiro yakanaka. Pamusoro pezvo, mimwe mishonga yeplatinum inoshandiswa, yakaita se carboplatin uye cisplatin (Sikov et al., 2015). Pamusoro pezvo, PARP inhibitors dzakadai seOlaparib, Velaparib, uye PF-01367338 dzakashandiswa semishonga inogona kushandiswa chemotherapy yekurapa TNBC (Ishino et al., 2018). Sezvo nzira dzeWnt/b-Catenin, NOTCH, uye Hedgehog signaling pathways dzakataurwa kuti dzine chekuita nekuitika uye kufambira mberi kweTNBC, kunanga mishonga kune idzi nzira kungave nzira yakakosha (Aysola et al., 2013). Kunyangwe kuvhiyiwa, kurapwa nemwaranzi uye chemotherapy zvichiri nzira huru yekurapa kweTNBC nhasi, kufambira mberi kukuru kwakaitwa mukugadzira mishonga mitsva inosanganisira kurapwa kwakanangana, immunotherapy, maturusi akasiyana-siyana ekugadzirisa majini ane chekuita neCRISPR akadai seCas9n, dCas9., CRISPR/Cas12, prime editing, uye CRISPR/Cas9-targeted gene therapy. Pano tichatarisa pane maturusi akasiyana-siyana ekugadzirisa majini ane chekuita neCRISPR anoshandiswa mukurapa TNBC. Pakati pawo, CRISPR/Cas9 yakagamuchirwa zvakanyanya.
Cas9n, inozivikanwawo seCas9 nickase, imhando yeprotein yeCas9 yakagadzirwa nemajini inobva muCRISPR/Cas9 genome editing system (Gupta et al., 2019). Muchimiro chayo chepakutanga, protein yeCas9 ine ma domain maviri enuclease: RuvC neHNH, basa guru rayo riri kupatsanurwa kwe tambo dzese dzeDNA. Zvisinei, muCas9n, imwe yenzvimbo dzenuclease, HNH, inochinja majini uye inova isingashande. Nokudaro, nzvimbo yeHNH yeCas9n inoramba isingashande. Nzvimbo yeRuvC chete ndiyo inoramba ichishanda, zvichibvumira Cas9n kucheka kana kugadzira ma breaks pa tambo imwe chete yeDNA (Trevino naZhang, 2014). Zvichienzaniswa neCas9, Cas9n inogona kuderedza zvinobudirira mhedzisiro isiri yechinangwa uye kugadzirisa nemazvo nzira dzekugadzirisa masero. Cas9n inogona kushandiswa kugadzirisa matambudziko akasiyana-siyana, akadai sekugadzira ma breaks panzvimbo dzakatarwa muDNA ine tambo mbiri. Nechinangwa ichi, mamorekuru maviri eCas9n akabatanidzwa uye akashandiswa pamwe chete (Yee, 2016). Mixed lineage kinase 3 (MLK3) iprotein kinase inoshandiswa ne mitogen inoshanda semutongi mukuru panguva yeTNBC metastasis (Cronan et al., 2012).
MLK3 inogona kumutsa nzira dzakawanda dzekutumira zviratidzo dzinotungamira kuTNBC metastasis. Semuenzaniso, nzira ye c-Jun N-terminal kinase (JNK) inodzora kufamba kwemasero uye kuora kwe extracellular matrix, uye MLK3 inomutsa nzira yeJNK, nokudaro ichivandudza kufamba kwemasero uye inopa hunhu hwekupinda (Rattanasinchai naGallo, 2016). MLK3 inodzorawo EMT. Kuti iite izvi, inomutsa zvinhu zve transcription zvakaita seSnail, Slug naTwist. Zvinhu izvi zvinodzivisa kutaura kwe epithelial markers uye zvinokurudzira kutaura kwe mesenchymal markers, zvichikonzera kuwanikwa kwe metastatic phenotype (Casalino et al., 2023). MLK3 yakaonekwa iine basa muECM kugadzirisa uye kumutsa ma protease akadai se matrix metalloproteinases (MMPs) anoparadza ECM. Izvi zvinoita kuti tumor cell ipinde uye ipararire kunzvimbo dziri kure (Katari et al., 2019). Saka, zvidzidzo zvekare zvakaratidza kuti MLK3 ine basa rakakosha muTNBC. Rattanasinchai naGallo vakashandisa maTNBC models kuti vadzidze basa reMLK3 uye vakaona kuti inokurudzira kukura kwegomarara kuburikidza nenzira dzakatarwa dzekutumira zviratidzo. Vakashandisa CRISPR/Cas9n kugadzirisa MLK3 uye vakaona kudzikira kukuru kweTNBC metastasis (Rattanasinchai naGallo, 2016).
dCas9, inowanzonzi Cas9 isina simba, imhando yeprotein yeCas9 yakagadziriswa. Kusiyana neCas9 inoshanda, dCas9 haina basa re endonuclease, zvichiita kuti isakwanise kukonzera kuputsika kweDNA kaviri. Nokudaro, dCas9 inogona kushandiswa kunanga nzvimbo dzakatarwa dze genome pasina shanduko kana shanduko kuDNA sequence (Wang et al., 2016). Mu dCas9, mapuroteni maviri e endonuclease, RuvC neHNH, haashande nekudzvinyirira amino acid residues dzakakosha. (Richter et al., 2016). Pasinei nekushaikwa kwayo kweDNA cleavage activity, dCas9 inoita mabasa akakosha mukutsvagisa majini uye biotechnology. Semuenzaniso, inobvumira kuona nzvimbo dzakatarwa dze genome, inobatsira transcriptional regulation, uye inokurudzira epigenetic modifications (Brocken et al., 2018).
Chinhu chinonzi transcription factor ZEB1 (zinc finger E-box binding homeobox 1) chinoita basa rakakosha mukubatsira epithelial-mesenchymal transition (EMT), maitiro emasero anoitika panguva yekukura kwemwana ari mudumbu, kugadziriswa kwetishu, uye kufambira mberi kwekenza. Izvi zvinosanganisira kushandurwa kwemasero epithelial kuita masero emesenchymal, zvichikonzera shanduko muchimiro chemasero, kufamba, kupinda kwemasero, nezvimwewo. (Wu et al., 2020). ZEB1 inodzivisa zviratidzo zvakawanda zveepithelial, zvakaita seE-cadherin naOccludin, izvo zvine basa rekuchengetedza kubatana kwemasero uye polarity yemasero eepithelial muTNBC (Moreno-Bueno et al., 2008). Pamusoro pezvo, ZEB1 inomutsa zvimwe zviratidzo zvakaita seN-cadherin, vimentin uye fibronectin (Konradi et al., 2014) uye inotongawo majini anobatanidzwa mukugadzirisa cytoskeletal senge Rho GTPases uye matrix metalloproteinases (MMPs) (Huang). et al., 2014). 2022), pamusoro pezvo, zvinokanganisawo nzira dzakasiyana-siyana dzekutumira zviratidzo dzakadai sekushandura kukura kwemasero-β (TGF-β), Wnt signaling, nezvimwewo, nokudaro zvichikurudzira kupinda kwebundu uye metastasis muTNBC (Chen et al., 2016). ). Zvidzidzo zvakawanda uye humbowo hwesainzi zvinoratidza kuti ZEB1 ine mukana mukuru semushonga unokosha wekuona nekurapa TNBC. Muchidzidzo chakaitwa munguva pfupi yapfuura, Waryah et al. vakashandisa modhi yeTNBC uye vakakwanisa kudzvinyirira ZEB1 zvachose nedCas9. Saka, vakaona kunyatsojeka kwakanyanya uye kudziviswa kweZEB1 zvachose mumamiriro ezvinhu ari mumuviri (Waryah et al., 2023).
CRISPR/Cas12 chishandiso chekugadzirisa majini chinonzi CRISPR/Cpf1. Chinobva muCRISPR/Cas system, apo izwi rekuti Cas12 rinoreva protein 12 inobatanidzwa neCRISPR (Bharathkumar et al., 2022), iyo yakafanana zvachose neCas9. Musiyano chete ndewekuti ine protein yeCas12. Kana ichienzaniswa neCas9, Cas12 ine mamwe mabasa akasiyana akadai sekugadzira magumo anonamira panguva yekugadzirisa majini, nepo Cas9 ichigadzira magumo asina kujeka (Wang et al., 2021). Hunhu uhwu hweCas12 hunobatsira muhunyanzvi hwayo hwekugadzirisa DNA. Seprotein inokwanisa kuziva nekucheka DNA chaiyo, ine hunyanzvi hunoshamisa, zvichiita kuti ive chishandiso chinoshanda kune akasiyana mashandisirwo, kusanganisira kugadzirisa majini (Pickar-Oliver naGersbach, 2019).
Kufanana nedzimwe mhando dzeCRISPR, CRISPR/Cas12 inokwanisawo kubvisa kana kumutsa majini muTNBC, ichinanga majini anoita basa rakakosha mukukura kwayo kana kupindura kurapwa (Yang naZhang, 2023). Kuti izvi zviitike, gwara reRNA (gRNA) rakagadzirwa rinotungamira Cas12 kune gene rakananga. Kana Cas12 yangobatana kune chinangwa chayo, inotanga kuputsika kwetambo mbiri muDNA uye inomutsa nzira dzekugadzirisa DNA. Munguva yekugadzirisa, nucleotides dzisina kururama dzinogona kuiswa, zvichikonzera shanduko dzemajini uye kurasikirwa nebasa (Zhang et al., 2021a). Pamusoro pezvo, shanduro yakagadziridzwa yeCas12 enzyme (inonzi dCas12) yakashandiswawo kumutsa majini. Nekuisanganisa ne transcription activator, zvinokwanisika kukonzera mamwe majini, nokudaro zvichiaita kuti ashande. Iyi tekinoroji ine mukana mukuru mukusimudzira kumutswa kwemajini anodzvinyirira tumor (Sultan et al., 2022).
Prime editing itekinoroji itsva yakagadzirwa uye yepamusoro-soro yekugadzirisa genome. Inokwanisa kugadzirisa DNA yechinhu chipenyu nemazvo (Chen naLiu, 2023). Prime editing inowanikwa kuburikidza nekubatanidzwa kwezvikamu zviviri zvikuru: modified CRISPR/Cas9 enzyme uye reverse transcriptase. Basa reCRISPR/Cas9 enzyme nderekutarisa nzvimbo dzakananga mugenome, nepo reverse transcriptase ichibatsira kugadzirisa DNA zvakakwana panzvimbo yakatarwa (Hassan et al., 2021). Mu primer editing mechanism, danho rekutanga rinosanganisira kugadzirwa kwe primer editing guide RNA (pegRNA) (Standage-Beier et al., 2021). Ine target sequence uye RNA template inoenderana nenzvimbo yekugadziridza inodiwa muDNA yakatarwa. PegRNA inozopinzwa mumasero ekutarisirwa pamwe chete ne primer editing nuclease (PE2). PE2 iprotein yefusion ine Cas9 enzyme, reverse transcriptase, uye primer editing adapter (Martín-Alonso et al., 2021). Mukati mesero, pegRNA nePE2 complexes vanotsvaga DNA chaiyo yavanoda kugadzirisa. Cas9 enzyme inocheka DNA uye inogadzira template ine tambo imwe chete (Choi et al., 2022). Reverse transcriptase inoshandisa template iyi kugadzirira DNA yekugadzirisa. Pamwe chete nekukopa DNA, mirairo yekugadzirisa template yeRNA inosanganisirwawo. Pakupedzisira, tambo yeDNA ichangobva kugadzirwa inoshandiswa se template kugadzirisa break muDNA, zvichikonzera shanduko yeDNA yakachinjwa ine shanduko inodiwa (Ochoa-Sanchez et al., 2021). Maitiro ekugadzirisa ekutanga anogona kutungamira mukuchinja kwakapararira mumajini. Inogona kunanga point mutations, insertions, deletions, uye kunyange gene replacements (Anzalone et al., 2019; Chen naLiu, 2023). Prime editing inopa mabhenefiti akati wandei pane matekinoroji ekugadzirisa genome ekare. Izvi zvinosanganisira kuwedzera kururama, kudzikiswa kwemhedzisiro isina chinangwa, uye kugona kugadzirisa DNA pasina kuvimba neDNA double-strand breaks (Anzalone et al., 2020).
Tekinoroji yeCRISPR/Cas9 yakatanga kugadzirwa kudzivirira mabhakitiriya kubva mukufambiswa kweplasmid uye hutachiona hwephage uye yakazoshandiswazve senzira inoshanda yeRNA-based DNA targeting tool yekugadzirisa genome (Jiang naDoudna, 2017). Pamusoro pezvo, CRISPR/Cas9 system yakataurwa kuti iripo mu50% ne87% yemabhakitiriya nema archaeal genomes, zvichiteerana (Ishino et al., 2018). CRISPR/Cas9 chishandiso chinogona kubviswa, kuiswa uye kugadziriswa kwechero gene sequence isina kujairika uchishandisa in vivo uye in vitro modes (Sabit et al., 2021). Uyezve, CRISPR/Cas9 yakaratidzwa kuva chikamu che adaptive immune system nekuda kwekunyatso shandiswa kwayo nemajini anodiwa (Chen naZhang, 2018).
CRISPR/Cas9 ine Cas9 uye imwe chete inotungamira RNA (sgRNA). Cas9 i endonuclease inoumbwa nezvikamu zvakawanda zveprotein. Pamusoro pezvo, Cas9 ine hunhu hwakasiyana hwemaumbirwo uye hunhu (Pacesa et al., 2022) sezvo ine ma lobes maviri: kuziva (REC) uye nuclease (NUC). REC lobe yakakamurwazve kuita matunhu matatu: REC1, REC2, uye bridge helices (Cromwell et al., 2018). NUC ine ma lobes matatu, anoti RuvC (RuvC I, RuvC II, RuvC III), HNH uye protospacer adjacent motif (PAM) interaction domain (Mufananidzo 1) (Song et al., 2016). sgRNA ine zvikamu zviviri, zvinosanganisira CRISPR RNA (crRNA) uye transcoding small RNA (tracer RNA) (Mufananidzo 1). SgRNA inobatanidza Cas9 protein ne connexins kuti igadzire active complex, inozivikanwawo se effector complex (Richter et al., 2012). crRNA i 18–20 nucleotide base pair inoita basa rakakosha mukuziva target DNA sequences. Pamusoro pezvo, crRNA inobatanidza ne target DNA, uye tracrRNA inoshanda se scaffold yeCas9 nuclease kuti isunge target DNA (Manghwar et al., 2019). Kune rumwe rutivi, PAM sequence ine 3 nucleotides, iyo inosimbisa, inotsanangura uye inobatanidza kusungwa kwe effector complex kuDNA. Cas9 protein complex subunits RuvC neHNH dzine catalytic activity (Richter et al., 2012; Asmamaw naZawdie, 2021). HNH nuclease domain yeCas9 subunit inobvisa DNA strand yakabatana ne crRNA. RuvC nuclease domain inocheka dzimwe tambo dzeDNA uye inogadzira double-strand breaks (DSBs), mushure mezvo nzira mbiri dzakasiyana dzekugadzirisa breaks dzeDNA dzinotanga kushanda (Jiang naDoudna, 2017) (Mufananidzo 1).
Mufananidzo 1. Pfupiso yeCRISPR/Cas9 (A). Zvikamu zveCRISPR/Cas9 system: (i). Cas9 endonuclease ine basa rekucheka DNA sequence yakanangana nayo, (ii) gwara rimwe chete (sg) RNA rinobva mukusanganiswa kwe crRNA ne tra-crRNA chimeras. (mbiri). Cas9 inosanganisira zvikamu zvakati wandei zvakaita seRec I, Rec II, NUC lobe (HNH naRuv C zvikamu zvidiki) uye PAM interaction domain (C) ine mabasa anoenderana. Iyo CRISPR/Cas9 protein complex inopatsanura DNA sequences kuita non-complementary uye complementary forms (D). CRISPR/Cas9 inogadzirisa genome muzvikamu zvitatu: kuziva, kucheka nekugadzirisa. Iyo yakagadzirwa sg-RNA inotungamira Cas9 uye inoziva sequence yaunoda kuburikidza ne complementary component crRNA. Cas9 inoziva PAM sequence pa 5′-NGG-3′ uye inonyungudutsa DNA, ichiumba DNA-RNA hybrid uye ichimutsa cleavage. Nzvimbo yeHNH yeCas9 inocheka tambo inobatanidza, uye nzvimbo yeRuvC inocheka tambo isingasanganisire. CRISPR/Cas9 inogadzirisa dsDNA nekuipwanya nenzira mbiri: non-homologous end joining (NHEJ) uye homology-directed repair (HDR). NHEJ inogadzirisa DNA ine tambo mbiri pasina DNA yekunze inosanganisa DNA kuburikidza ne enzyme process, iyo nzira inogona kukanganisa inogona kuisa kana kubvisa DNA sequences dzisina kurongeka. HDR yakanyatsojeka uye inoda ma DNA templates akafanana.
Nzira dzekugadzirisa dzinoshandiswa neCRISPR/Cas9 dzinosanganisira nzira dzisina kubatana kwemagumo (NHEJ) uye nzira dzekugadzirisa dzakatungamirirwa nehomology (HDR). NHEJ inzira inokanganisa nekuti inosanganisira kuiswa kana kubviswa uye haidi chero pateni panguva yekugadzirisa. Inoshandisa nucleotides dzisina kurongeka kugadzira mapuroteni akajairwa (Abbasi et al., 2021). Iyi sisitimu yekugadzirisa ine macomplex mana akadai seKU complex, cross-complementing protein complex type 4 (XRCC-4), DNA end processing enzyme, uye protein kinase DNA-PKcs (Abbasi et al., 2021). Puroteni yeKU complex ine ma subunits maviri, Ku 70 naKu 80, uye inoita basa rakakosha muNHEJ mechanism, sezvo nzira yekugadzirisa ichitangwa nekusunga ma subunits maviri (Ku 70 naKu 80) kumagumo asina kujeka kana kuti akada kujeka eDNA inotarirwa (Abbasi et al., 2021), inoshanda senzvimbo yekuunganidza zvimwe zvinhu zvine chekuita neNHEJ panzvimbo yekukuvara (Yang et al., 2020). XRCC-4 neDNA ligase zvinoumbwa ne334 ne911 amino acids, zvichiteerana, uye XRCC4-DNA ligase IV complex inokurudzira ligation yemagumo eDNA (Chatterjee et al., 2015). DNA end processing enzyme, inozivikanwawo sepolynucleotide kinase 3′-phosphate, iDNA end processing enzyme. Inogona kubvisa boka re3′P muDNA uye phosphorylate boka re5′OH panguva yekugadzirisa DSB. Zvinobatanidzawo kugadziriswa kwema strand breaks (SSBs) uchishandisa nzira yekugadzirisa SSB (Chatterjee et al., 2015). Protein kinase DNA-PKcs iprotein kinase inotsamira paDNA ine catalytic subunit yePIKKs (phosphatidylinositol 3-kinase-related kinases) uye ataxia telangiectasia mutated (ATM) mhuri, pamwe chete neATM neRad3-related ATRs. strand breaks (DSBs) uye single-strand breaks (Yue et al., 2020; Peng et al., 2016).
HDR inzira yekugadzirisa yakarurama uye yakakodzera nekuti ruzivo rwacho runokopwa uchishandisa chimiro chisina kusimba cheDNA duplex yakafanana, kunyangwe ichida kuvapo kwema chromatids ehanzvadzi. Izvi zvinoitika panguva yeS/G2 phase ye mammalian cell cycle. HDR inowanikwa zvakanyanya mumhando ye yeast, asi NHEJ inokosha mu mammalian (Burma et al., 2006; Abbasi et al., 2021). Nzira yekugadzirisa yakakwana inoratidzwa muMufananidzo 1.
Sezvo TNBC ichikonzerwa nekusarongeka kwemajini uye epigenetic, kugadziriswa kwezvisina kunaka zvemajini/epigenomic uchishandisa CRISPR/Cas9 kungave nzira yakanaka yekurapa (Chen et al., 2019). Pamusoro pezvo, zvimwe zvinhu zvinonyorwa mukunyora zvinosanganisirwa mukutonga kwekunyora kwemasero zvinogona kuratidza hunhu hwakasiyana hwemasero ekenza, zvichiratidza kuti kutonga kwekunyora kunogona kuva nzira yakanaka yekurapa kenza (Drost et al., 2017). Kushandisa izvi zvinhu zvemamorekuru emamota, akadai semagene, epigenetic uye transcriptional defects, pakugadzira mishonga kunogona kuvandudza mhedzisiro yekiriniki uye kuderedza mari yekuongorora. CRISPR chishandiso chinogona kugadzirisa majini chisingangogone kuona nekuona zvinangwa zvemajini zvinokonzera kenza chete, asi chinogonawo kushandiswa kugadzirisa, kudzvinyirira, uye epigenetically kugadzirisa oncogenes mumasero evanhu (Tafura 1) (Ahmed et al., 2021). CRISPR yakaongororwa kakawanda yakaitwa kutsvaga majini ane chekuita netumor suppressors, oncogenes, uye kuramba mishonga. Kushandiswa kweCRISPR/Cas9 kugadzirisa maTNBC oncogene akasiyana-siyana kunoratidzwa muMufananidzo 2.
Mufananidzo 2. Kugadziriswa kwemajini akasiyana-siyana eTNBC oncogenes uchishandisa CRISPR/Cas9 zvichikonzera kudzikira kwekukura kwegomarara uye metastasis. Ma oncogene aya anosanganisira CDK7, NAT1, UBR5, YTHDF2, ITGA9, CXCR4 uye CXCR7, Crypto1, ROR1 uye ST8SIA, ayo ane chekuita nekuitika uye metastasis yeTNBC.
Kufamba kwemasero ekenza, kupinda mumasero, uye kuchinja kwemasero emukenza (EMT) zvinobatanidzwa neITGA9. Zvidzidzo zvekare zvakaratidza kuti ITGA9 inyanzvi huru muNotch pathway uye ine basa rinonakidza mukurasika kwemasero emukenza (Molist et al., 2020). Pamusoro pezvo, ITGA9 yakawanikwa iine hukama hwakasimba nekufanotaura kwezvirwere mumarudzi akasiyana-siyana emamota, kusanganisira kenza yemazamu (Wang Z. et al., 2019). Pamusoro pezvo, ongororo ye bioinformatics yeITGA9 yakaratidza kuti kuonekwa kwayo muTNBC kwaive kwakakwira zvakanyanya kupfuura mune mamwe marudzi ekenza yemazamu. Mazinga eITGA9 akakwira anobatanidzwa nekurasika kwemasero emamota uye kudzokerazve muvarwere veTNBC. Kubviswa kweITGA9 neCRISPR/Cas9 kwakakonzera mhedzisiro yakafanana nekenza (CSC), kuwedzera kwemasero emamota, kukura kwemasero emamota, uye kuderedza kukura kwemasero emamota nekukurudzira kuora kweβ-catenin muTNBC (Wang Z. et al., 2019).
Crypto-1 inhengo yemhuri yeTGF-β uye yakakosha pakugadzirwa kwechibereko pakutanga, kuchengetedza masero emuviri, uye kupararira kwekenza (Ishii et al., 2021). Inozivikanwawo seTdgf-1, ipuroteni yeGPI-anchored signaling protein inobatanidzwa mukugadzirisa kuumbwa kwe primitive streak, mesoderm uye endoderm, pamwe nekugadzwa kwe left/right asymmetry mukukura kwenhengo dzemuviri panguva yekugadzirwa kwechibereko (Zhang et al., 2021b). Pamusoro pezvo, Cripto-1 yakaratidzwa kuti inobatanidzwa mukuchinja kwe epithelial-mesenchymal (EMT) se stem cell marker (Zhang et al., 2021b). EMT yakakosha kwete chete kune akasiyana maitiro akadai sekukura kwechibereko, fibrosis uye kuporesa maronda, asiwo pakupinda kwekenza uye kupararira kwekenza. Pamusoro pezvo, Cripto-1 yakaratidzwa kuti inoshanda nema Notch receptors mana uye inovandudza kukura kwawo mushure mekushandurwa (Brandstadter naMaillard, 2019). Nzira yeNotch signaling inozivikanwa kuti inobatsira mukuchengetedza masero ekenza yemazamu evanhu. Zvidzidzo zvakaratidza kuti CRISPR/Cas9-mediated knockout yeCrypto-1 inoderedza kukura kwekenza uye metastasis. Nokudaro, Crypto-1 inogona kuva chinangwa chakakosha chekurapa kweTNBC (Castro et al., 2015).
Mapuroteni eXCL12 pamwe chete nekemokine receptor yayo CXC (CXCR4 neCXCR7) anoita mabasa akasiyana-siyana mukuwanda kwemasero ekenza, kukura, kufamba uye kupinda (Wu et al., 2015). Makemoreceptor aya akabatanidzwa nekukura kweTNBC kuburikidza nenzira dzakawanda dzekutumira zviratidzo mumhando dzemumuviri nedzemumuviri (Wu et al., 2015). Pamusoro pezvo, kushanda kweCXCR4 neCXCR7 kunobatanidzwa nekugona kukuru kwekutapukira uye kutadza kufambira mberi muTNBC (Karn et al., 2022). Nokudaro, kudonha kweCXCR4 neCXCR7 kunogona kuva majini anoshanda ekurapa kenza yemazamu, kusanganisira TNBC. Ongororo yakaitwa naYang et al. Yang et al (2019) yakashandisa CRISPR/Cas9 kubvisa majini eCXCR4 neCXCR7 pamwe chete uye yakawana kuti kuwanda, kukura, kufamba uye kupinda kweTNBC kwakadziviswa zvakanyanya (Yang et al., 2019).
Kuwedzera kwehuwandu hwe miR-3662, chirwere cheTNBC oncogene, kwakaonekwa mumatishu ekenza yemazamu (Yi et al., 2022). Kudzikiswa kwe miR-3662 kwakaratidzwa kuti kunodzivirira kukura kwebundu rekenza yemazamu uye kupararira kwemasero mumuviri nemumuviri (Agarwal naGupta, 2021). HBP-1 inodzivirira zvikuru Wnt/-catenin signaling uye ingangove ndiyo inokonzera kukura kwemasero eTNBC kuburikidza ne miR-3662. Munguva pfupi yapfuura, Yi et al. vakawana kuti miR-3662-HBP1 axis inotonga nzira yeWnt/-catenin signaling mumasero eTNBC (Yi et al., 2022). Nekuda kwekutaura kwayo zvakanangana nebundu, miR-3662 inogona kunge iri chinangwa chekurapa cheTNBC. Saka, kudzikiswa kwe miR-3662 kuburikidza ne CRISPR/Cas9 kunogona kunge kuri nzira yakanaka yekugadzira mishonga mitsva yekurapa TNBC.
UBR5 ipuroteni ye300 kDa nucleophosphoprotein yakaonekwa senzira huru yekugadzirisa tumorigenesis, metastasis, uye immune response mukenza dzakasiyana-siyana (Shearer et al., 2015; Fu et al., 2023). UBR5 yakataurwa kuti yakawedzera zvakanyanya muTNBC samples uye inokurudzira ERα function nekukurudzira kuwanda kuburikidza nebasa rayo reubiquitin ligase (Bolt et al., 2015). Zvidzidzo zvese zve exome sequencing zve primary TNBC samples zvakaratidzawo kuwedzera kweUBR5, zvichiratidza basa rayo mukuvandudzwa kweTNBC. Pamusoro pezvo, CRISPR/Cas9-driven deletion yeUBR5 yakaratidza kudziviswa kwakakosha kweTNBC metastasis uye kukura muma experimental mouse models. Uyezve, kuiswa kweUBR5 mu wild-type mouse model kwakadzorera kushanda kwayo kwakazara, nepo mhedzisiro iyi isina kuonekwa mu inactivated mutant strains (Liao et al., 2017). Kushaikwa kweUBR5 kunobatanidzwa nekuwedzera kweapoptosis, necrosis, uye kudziviswa kwekukura kwebundu muTNBC nekuda kwekusashanda zvakanaka kweangiogenesis. Nekuda kwekurasikirwa neUBR5, kupararira kwebundu kunhengo dziri kure kunodzikira, uye UBR5 inokonzera EMT isina kujairika kunyanya nekuderedza E-cadherin expression (Zhang naWeinberg, 2018). Munguva pfupi yapfuura, UBR5 yakaratidzwa kuva chinhu chakakosha zvikuru muIFN-γ-induced PDL1 transcription muTNBC nekuda kwekushaya kwayo E3 ubiquitination activity. RNA transcriptome analysis yakaratidza kuti UBR5 inogona kuva nemigumisiro yesystemic pamajini ane chekuita neIFN-γ pathway uye kukurudzira PDL1 transactivation nekuwedzera activation levels yeprotein kinase RNA (PKR) uye signal transducers uye activators. transcription 1 (STAT1) uye interferon regulatory factor 1 (IRF1). Zvisinei, kubviswa kweUBR5 nePD-L1 kuburikidza neCRISPR/Cas9-mediated combined ablation kune simba rekurapa rinoshanda pamwe chete kupfuura chero chipi zvacho che blockade chete, uye zvine mhedzisiro yakakura pa microenvironment yebundu (Wu et al., 2022). Saka, CRISPR/Cas9 inogona kunge iri chishandiso chakakosha chekudzivirira kushanda kweUBR5, nokudaro ichidzvinyirira metastasis yeTNBC uye kukura kwebundu.
ROR1 ipuroteni yerudzi rwekutanga inoburitswa mumasero inoonekwa panguva yekenza uye kukura kwemwana ari mudumbu uye yakaonekwa sepuroteni ye oncofetal (Nicholas Borcherding, 2014). Maitiro ekupinda kwevanhu mukenza dzakasiyana-siyana ane chekuita nekuwedzera kweROR1. Mhedzisiro yakanaka yakawanikwa kubva muzvidzidzo zve in vivo uye in vitro zvinosanganisira mishonga yekurapa inotarisa ROR1 (Chien et al., 2016). Kuwanda kweROR1 mRNA mu biopsy yemazamu kwakabatanawo ne bundu remazamu rakafanana ne basal (BL) uye kufamba kwaro kuenda kune dzimwe nhengo dzemuviri. Uyezve, kuwedzera kweROR1 kwakaonekwa sechiratidzo chekufanotaura kukura kweTNBC (Chien et al., 2016). Zvisinei, kunyaradza ROR1 uchishandisa CRISPR/Cas9 kudzvinyirira kukura kweTNBC uye metastasis kungave nzira inoshanda.
Maitiro e sialylation anosanganisira kuwedzerwa kwe sialic acid kune glycoconjugates, iyo inokonzereswa ne sialyltransferases (STs). ST8SIA1 ndeyemhuri ye ST uye inoita basa rakakosha mukukonzera zvirwere zvakasiyana-siyana zvakaita se lymphocytic leukemia uye colorectal cancer (Chang et al., 2018). Kuongororwa kweRNA sequence kunoratidza kuti ST8SIA1 inoratidzwa zvakanyanya mumatishu emazamu evarwere veTNBC uye ine hukama hwakanaka nekuchinja kuri mu tumor suppressor gene p53, izvo zvinogona kubatsira mukukonzera kweTNBC (Battula et al., 2017). Pamusoro pezvo, ST8SIA1 inobatanidzwa mukukonzera uye kudzoka kweTNBC, zvichiratidza kuti inoita basa rakakosha mukuitika nekukura kweTNBC. Mune in vitro model yeTNBC, kudonhedzwa kweST8SIA1 neCRIPSR/Cas9 kwakaratidzwa kuvharira kukura uye kupararira kwemetastasis (Battula et al., 2017). Izvi zvinoratidza kuti CRISPR/Cas9 inogona kunge iri chishandiso chakakosha chekudzivisa kushanda kweST8SIA1 oncogene mukurapa TNBC.
NAT1 ienzyme yemetabolism inokonzeresa kuumbwa kwemakemikari echikamu chechipiri chexenobiotic uye inoratidzwa munhengo dzese dzemuviri wemunhu. NAT1 inogona kushandisa cofactor folate kunanga acetyl-CoA (acetyl-CoA) kunyangwe pasina aromatic amine substrate (Stepp et al., 2015; Laurieri et al., 2014). Zvidzidzo zvakaratidza kuti NAT1 inotonga matrix metalloproteinase 9 (MMP9) inoshanda mumamodeli emasero ekenza yemazamu uye inodzivirira kubva ku reactive oxygen species (ROS) panguva yekudya glucose (Wang et al., 2018). Kubviswa kweNAT1 kwakaratidzwa kuti kunodzivisa pyruvate dehydrogenase complex, zvichikonzera kusashanda zvakanaka kwemitochondrial (Wang L. et al., 2019). Pamusoro pezvo, mimwe mishumo yakasiyana-siyana yakaratidza kuti kudziviswa kweNAT1 kushandisa mamorekuru madiki uye siRNA silencing kunogona kuderedza kupinda uye kuwanda kwemasero ekenza yemazamu (Stepp et al., 2018). Munguva pfupi yapfuura, CRISPR/Cas9 yakashandiswa kudzvinyirira NAT1 mumutsara wemasero ekenza yemazamu MDA-MB-231, zvichikanganisa metabolism yemasero zvichienderana nehukuru hwematauriro ayo. Chidzidzo ichi chakaratidzawo kuti NAT-1 yakakosha pakufambira mberi uye kutapukira kweTNBC (Carlisle et al., 2020).
Kunyorwa kwakarongeka kwe oncogenes kunodzorwa ne super enhancers, transcription factors uye cofactors (Hnisz et al., 2015). Pamusoro pezvo, boka re cyclin-dependent kinases (CDKs), dzakadai seCDK7, CDK8, CDK9, CDK12, uye CDK13, zvinodiwa pakugadziriswa kwe transcription. Pakati pavo, CDK7 inobatanidzwa mu phosphorylation yeRNA polymerase II, iyo inokosha zvikuru pakutanga nekuwedzera kwe oncogene transcription mu pathogenesis yeTNBC. Mukudzidza kwakakosha, kubviswa kweCDK7 neCRISPR/Cas9 kwakadzivisa TNBC, zvichiratidza kuti pathogenesis yeTNBC inoenderana neCDK7 (Wang Y. et al., 2015).
Zvidzidzo zvakaratidza kuti shanduko muMYC neRBP (RNA binding protein) dzinogona kutungamira kuapoptosis, nepo shanduko dzemajini rimwe chete muMYC kana RBP dzisingakanganisi kukura kwemasero ekenza (Einstein et al., 2021). MaRBP anopfuura 1000 mugenome remunhu akaongororwa achishandisa raibhurari yakavakirwa paCRISPR/Cas9. Pakati pawo, maRBP makumi mashanu nenomwe akawanikwa akakosha pakukura kwemasero ekenza ane mazinga eMYC akakwira zvakanyanya (Wheeler et al., 2020). Pamusoro pezvo, YTHDF2 yakakosha pakuchengetedza kukura kwemasero eTNBC uye inoderedza huwandu hwezvinyorwa zvine methylated panguva yekunyorwa kwepamusoro uye kushandurwa mumasero ekenza ane MYC yakakwira. Pamusoro pezvo, YTHDF2 haina kukosha kumasero ekenza, ayo asinganyanyi kuvimba nemazinga eMYC akakwira kuti varwere veTNBC vararame kwenguva refu (Einstein et al., 2021), zvichiratidza kuti inogona kunge iri chinangwa chekurapa chinogona kukunda TNBC.
Mapuroteni eminwe yeZinc (ZNFs) anoumba inenge 1% yegenome remunhu rose. Zvidzidzo zvakaratidza kuti ZNF inogona kudzora kuwanda kwemasero mukenza dzakasiyana-siyana, dzakadai sekenza yechiropa, kenza yemazamu uye kenza yemudumbu (Zhang W. et al., 2021; Li et al., 2017). Maraibhurari akagadzirwa neCRISPR knockout akashandiswa kuongorora majini akasiyana-siyana ekudzivirira tumor (TSGs) mumasero ekenza yemazamu (Shalem et al., 2014). Kubva ipapo, chidzidzo che transcriptomic chemasero ekenza yemazamu akabviswa CRISPR/Cas9 ZNF319 chakaratidza kuti ZNF319 ijini rekudzivirira tumor rinoderedza kuwanda kwekenza yemazamu uye nekudaro rinobatanidzwa mumatanho akasiyana-siyana ekutumira zviratidzo uye mamwe mabasa ehupenyu (Wang L. et al., 2022).
Ferroptosis rudzi rwekufa kwemasero kwakarongwa kunoenderana nekuvapo kwesimbi. Zvinozivikanwa kuti kukura kweferroptosis kunokonzerwa nekuvapo kwemafuta peroxides. Pamusoro pezvo, PKCβII yakataurwa kuti inoratidza peroxidation yekutanga kwemafuta, uye kuwedzera kweperoxidation yemafuta kunobatanidzwa neferroptosis. Kuongorora raibhurari yeCRISPR/Cas9-mediated kinase inhibitors kwakaratidza kuti PKCβII ine chekuita ne lipid peroxidation, iyo yakakosha pa ferroptosis mumasero eMDA-MB-231 (Zhang et al., 2022). Nokudaro, izvi zvinoratidza kuti kubviswa kwePKCβII neCRISPR/Cas9 kungave chinangwa chegomarara rekudzivirira masero emudumbu pakurapa zvirwere zvine chekuita neferroptosis (Zhang et al., 2022).
Kusakwanisa kushandisa mishonga kunofungidzirwa kuti ndiko kunokonzera kufa kwevanhu vanosvika 90% muvarwere vegomarara uye ndeimwe yematambudziko makuru mukurapa gomarara (Bukowski et al., 2020). Zvakabuda zvinoratidza kuti huwandu hwakakwana hwemajini ane chekuita nekubuda kwemishonga, kugadziriswa kweDNA, apoptosis uye nzira dzakasiyana-siyana dzekutumira zviratidzo zvemasero zvine chekuita nekusagona kushandisa mishonga (Haider et al., 2020). Pakati pavo, majini akati wandei akatariswa nezvishandiso zveCRISPR/Cas9 uye akaratidza mhedzisiro inovimbisa mukuderedza kusagona kushandisa mishonga uye kuwedzera kushanda kwemishonga yekurwisa gomarara (Vaghari-Tabari et al., 2022). Pamusoro pezvo, maraibhurari ekuongorora magene eCRISPR/Cas9 ane simba guru akabatanidzwa mukugadzirisa mashandiro ezvipingamupinyi zvemagene ekusagona kushandisa mishonga (Shalem et al., 2015). Kuti vaone majini ekudzivirira paclitaxel, RNA sequencing pamwe chete ne genome-wide sgRNA library screening yakashandiswa kuona majini masere anodiwa, anosanganisira histone deacetylase 9 (HDAC9), ayo ane chekuita nekuramba kwemishonga muvarwere vane recurrent TNBC (B et al., 2020). ). Mune imwe ongororo, kuwedzera kwekutaura kwe diserine/threonine uye tyrosine protein kinase (DSTYK) kwakaonekwa panguva yekupona kwevarwere veTNBC vakarapwa nemishonga yekurwisa kenza. Uyezve, CRISPR/Cas9-mediated knockdown yeDSTYK yakasimudzira zvakanyanya apoptosis yemasero ekenza asingaurayiwi nemishonga in vitro (SUM102PT cells and MDA-MB-468 cells) uye in vivo TNBC model (Ogbu et al., 2021).
Kunyangwe TNBC ichiita kuti nzira yeMAPK isashande zvakanaka, mhedzisiro yemishonga inotarirwa neMEK haina kunaka. Ongororo yeCRISPR/Cas9 genomic library yakaratidza kuti kudziviswa kwePSMG2 (proteome assembly chaperone 2) kunoita kuti TNBC BT549 neMB468 cells zvinzwe kune MEK inhibitor AZD6244. Kudonhedzwa kwePSMG2 neCRISPR/Cas9 kwakachinja mashandiro eproteasome, zvichikonzera autophagy-mediated disassembly yePDPK1, nokudaro zvichivandudza masero egomarara muTNBC mouse models anokonzerwa neAZD6244 (MEK inhibitor) neMG132 (proteasome inhibitor). Saka, proteasome neMAP kinase (MEK) inhibitors zvinogona kupihwa pamwe chete kuderedza kuwanda kwemasero egomarara (Wang X. et al., 2022).
CRISPR/Cas9 yakashandiswawo kuongorora kurasikirwa kwebasa remajini zvichikonzera kuramba kweTNBC (Shu et al., 2020). Ge et al vakatsanangura mashandiro ekurapwa kwemasero ekenza anorapwa neJQ1, BET bromodomain inhibitor (BBDI), muTNBC. Vachishandisa CRISPR/Cas9, vakaona kuti kubvisa gene rerb1 kwakaita kuti TNBC isarambe nemishonga inorwisa kenza JQ1. Nokudaro, basa rerb1 rakakosha mukupindura mishonga yeJQ1 muTNBC. Vakashumawo kuti paclitaxel inhibitor yeCDK4/6 kinase/microtubule, uye kusanganiswa kwayo neBBDI dzakadai seJQ1 kunogona kupa mhinduro dzakanaka dzekurapa kune TNBC isingarambe mishonga (Ge et al., 2020).
Kunyorwa kweRNA refu isingakonzeri makodhi (lncRNA) kwakataurwa kuti ndiko kunokonzera kuramba kurapwa neoadjuvant muTNBC. Chidzidzo ichi chinoratidza kuti zvinyorwa zvishanu zvakasiyana zveMALAT1 lncRNA zvinoratidzwa zvakanyanya muTNBC. Pamusoro pezvo, kubviswa kweCRISPR/Cas9-mediated kweMALAT1 kwakawedzera kunzwa kwemasero eTNBC BT-549 ku paclitaxel ne doxorubicin, zvichiratidza kuti zvinogona kuita kuti MALAT1 isarambe muTNBC (Shaath et al., 2021).
Kuchinja-chinja kwemasero muBRCA1 (BRCA1m) kwakasiyana-siyana uye nekudaro kwakaoma kuona. Kutarisa PARP1 (poly(ADP-ribose) polymerase), iyo synthetic inouraya BRCA1, kunogona kuwedzera TNBC drug chemosensitivity. Kushandisa CRISPR/Cas9, kubviswa kwePARP1 kwakawedzera kunzwa kwemishonga yekurwisa kenza yakadai sedoxorubicin, gemcitabine uye docetaxel kumasero eTNBC e mBRCA1 mutant, zvichiratidza kuti PARP1 inewo mukana wekusakwanisa kushandisa mishonga muTNBC (Vaghari-Tabari et al., 2022). . Kuchinja-chinja kwemajini ekudzivirira tumor BRCA1 kana BRCA2 kunozivikanwa kuwedzera mukana wekuva nekenza yemazamu muvanhu. Kurapwa kwevarwere ava kunoda kushandiswa kwePARP inhibitors. Pamusoro pezvo, zvakaratidzwa kuti kubviswa kwe nucleotide salvage factor DNPH1 uchishandisa CRISPR/Cas9 kunogona kubvisa nucleotide ine chepfu 5-hydroxymethyldeoxyuridine (hmdU) monophosphate, nokudaro zvichiwedzera mhinduro yemasero asina BRCA kunzwiwa nePARP inhibitors (Fugger). et al., 2021). Saka, CRISPR/Cas9 nePARP1 inhibitors zvinogona kuva nzira yakakosha yekurapa TNBC.
Jini repenetrant glycoprotein (P-gp) ijini rinodzivirira mishonga yakawanda iro rinowanzo wedzera muinenge 41% yeTNBC yese (Sun et al., 2020). Kubuda kwemishonga kunokonzerwa neP-gp kwakaonekwa senzira huru yekugadzirisa kusapindirana nemishonga mukenza yemazamu. Zvinodzivirira P-gp zvakaratidza kuwedzera kwekunzwa kwemishonga inodzivirira kenza mukenza yemazamu (Famta et al., 2021). Saka, kubvisa kana kudzvinyirira zvinodzivirira P-gp neP-gp zvinotungamirirwa neCRISPR/Cas9 zvingave nzira dzakakosha dzekukunda kusapindirana nemishonga muTNBC.
ATP-binding cassette transporter G2 (ABCG2) inozivikanwa nekukonzera kusashanda kwemishonga muTNBC (Palasuberniam et al., 2015), kunyangwe parizvino pasina mishumo pamusoro pehukama huripo pakati peCRISPR/Cas9 neABCG2 silencing uye tumor suppressor gene inactivation. PTEN Inogona kuwedzera kushanda kweABCG2 (Palasuberniam et al., 2015), (Deepak Singh et al., 2021). Saka, kushandisa CRISP/Ca9 kubvisa ABCG2 uye kuisanganisa neABCG2 inhibitor kungave mushonga wakakodzera kukunda kusashanda kwemishonga muTNBC. Tafura 2 inotaura nezveCRISPR/Cas9 ichinanga majini ese ekusashanda.
Tafura 2. CRISPR/Cas9 inotarisa majini akasiyana-siyana eTNBC resistance kuti iite kuti masero anzwe mishonga yekurwisa kenza.
Maraibhurari eCRISPR/Cas9 anogona kushandiswa pakuongorora shanduko dzemajini dzine chekuita nekenza (Chan et al., 2022). Nzira iyi yekuongorora inosanganisira matanho mana akadai se (a) kuvaka raibhurari, (b) transduction ye lentiviral, (c) kuongorora phenotypic, uye (d) kuongorora majini anotarisirwa. Kunyangwe TNBC iine activation isina kujairika yenzira yeMAPK, fungidziro yekiriniki yeMEK therapy yakanangana nevarwere veTNBC vane shanduko mumajini anodzivirira tumor akadai sePTEN, RB1, uye TP53 haina kunaka.
Pamusoro pezvo, ongororo yekubuda kwemajini ichishandisa CRISPR/Cas9 yakaedza molekyuru ine simba uye yakasarudzwa, dehydrocatrol, iyo yakawanda mumaruva nemashizha eGuatemalan, kuti inzwisise mhedzisiro ye dehydrocatrol paMDA-MB-231. Maitiro ekudzivirira masero akasarudzwa. Hunhu hweMesenchymal stemness hweTNBC subtypes. Iyi ongororo yakavakirwa paCRISPR/Cas9 yakaratidzawo kuti HSD17B11, gene rinonyora 17β-hydroxysteroid dehydrogenase type 11, rinowanikwa zvakanyanya mumasero eMDA-MB-231 uye rinoguma ne dehydrofalcarinol chaiyo yemasero eMDA-MB-231 (Grant et. al., 2020). Saka, izvi zvinoratidza kuti kuongorora majini eCRISPR/Cas9 kune mukana mukuru wekuona mashandiro ari pasi pehunhu hwemakemikari echisikigo anogona kurwisa kenza.
Pamusoro pezvo, kusakwanisa kweTNBC kubatwa nekenza kwakaongororwa pachishandiswa CRISPR/Cas9 screen isina rusaruro, iyo yakaratidza hukama pakati penzira dzinokonzera oncogenic uye dzinodzvinyirira bundu. Zvikamu zvakakosha zvenzira dzemTOR neHippo zvakanzi zvine basa rakakosha mukudzora bundu muTNBC. Pamusoro pezvo, zvidzidzo zvakaratidza kuti kudziviswa kwemishonga kwemTORC1/2 neYAP oncoprotein kunodzivirira hutachiona hweTNBC uchishandisa in vitro drug-matrix synergy model uye in vivo patient-derived xenografts. Uyezve, kudziviswa kweTorin-1-mediated mTORC1/2 kunowedzera macropinocytosis, nepo kudziviswa kweYAP kunokonzerwa ne vertebrporfin kuchitungamira kurufu rwemasero eTNBC. Kana zvabatanidzwa, izvi zvinoratidza simba uye kusimba kwekuongorora kweCRISPR ye genome-wide in vivo kuti uone mishonga mitsva uye inoshanda yeTNBC (Dai et al., 2021).
Kusashanda zvakanaka kwemasoja emuviri chinhu chakakosha mukuumbwa kwebundu. Masero egomarara anodzivisa kubviswa kwemasoja emuviri nekusashandisa nzira dzekudzivirira, kusanganisira kukanganisa mashandiro emasoja emuviri muzvipenyu zvebundu uye kukanganisa masoja emuviri. Nokudaro, kugadzira masoja emuviri akavandudzwa kungave nzira huru yekurwisa bundu. Kushandiswa kweCRISPR/Cas9-based gene modification kunotarisa nyaya dzakasiyana-siyana dzine chekuita nekusashanda zvakanaka kwemasoja emuviri kubva pamaonero akasiyana. CRISPR/Cas9 yakashandiswa kuvandudza masoja ekudzivirira gomarara remazamu kuburikidza nenzira dzinotevera (Mufananidzo 3).
Mufananidzo 3. CRISPR/Cas9 immunotherapy inonanga masero eTNBC. Kurasikirwa neCDK5 uye kudonhedzwa kwePDL1 neCD155 kunosimudzira immune system. Saizvozvowo, kurasikirwa neA2AR uye kudonhedzwa kweTAA dzakadai seHER2, mucin 1 neTEM8 zvakawedzera kushanda kwemasero eCAR-T mukuuraya masero ekenza. Kuongorora masero eT anotungamirwa neCRISPR/Cas9 kwakaratidza kuti kukanganiswa kwep38 kinase kunowedzera kushanda kwemasero eT antitumor. Masero eT akagadziriswa neCRISPR/Cas9 anowedzera kutaura kweTCR (T cell receptor), zvichikonzera kuti masero eT ave nebasa rekudzivirira tumor.
Chinangwa chekurapa nekudzivirira muviri ndechekukurudzira masoja emuviri kurwisa masero ekenza. Kuwanda kwemapuroteni ekudzivirira muviri anowanzo dzivirira maitiro ekudzivirira muviri asi kunogona kubatsira kenza kudzivirira (Topalian et al., 2015). Mapuroteni aya anodzivirira maitiro ekudzivirira muviri nekusunga kuma receptors ari pamusoro pemasero ekudzivirira muviri. Mapuroteni akawanda ekudzivirira muviri (akadai seCD155 nePD-L1) anotarisa PD-1 receptor pamasero ekudzivirira muviri uye anoratidzwa mukenza yemazamu, kunyanya TNBC (Li Y.-C. et al., 2020). Kuderedza PD-L1 kana receptor yayo uchishandisa CRISPR/Cas9 kunogona kukurudzira masoja emuviri kurwisa mabundu eTNBC (Yahata et al., 2019). Pamusoro pezvo, kudzikiswa kwePD-L1 expression kuburikidza neCRISPR/Cas9-mediated deletion yeCDK5 kwakaratidzwa kuti kunodzivirira kukura kwebundu mu vitro uye mu vivo (Deng et al., 2020). Kudziviswa kwekukura kwekenza yemazamu mumhando dzemuropa uye muropa zvinoratidza kuti kuderedzwa kweCD155 kunokonzerwa ne shRNA kunogona kuva nemigumisiro yekurapa kenza yemazamu (Gao et al., 2018).
Masero eCAR T anoratidza maCAR anoziva maantigen ane chekuita negomarara (TAAs) uye anogona kushandisa mapuroteni ekuongorora kuti awedzere kushanda kwawo (Li C. et al., 2020). Kune zvinangwa zvakasiyana-siyana zvinogona kuitwa neCAR T cell therapy mukenza yemazamu, kusanganisira maTAA akati wandei akadai seHER2, mucin1, uye TEM8 (Bajgain et al., 2018). Pane humbowo hwekuti masero eCAR T anotarisa mesothelin (akawandisa mumasero eTNBC BT-459) anoshanda zvakanyanya kurwisa kenza kana PD-1 ikabviswa uchishandisa CRISPR/Cas9 (Hu et al., 2019). Zvisinei, kuparadzanisa masero eT kubva kuvarwere uye wobva waagadzirisa ex vivo inzira inotora nguva yakawanda uye inotora nguva. Kunyangwe masero eT epasi rose achigona kuderedza zvinodiwa zvekuzviparadzanisa, masero eT anopa vanhu anotsanangura human leukocyte antigen (HLA) kirasi I uye T cell receptor (TCR) anofanira kubviswa kudzivirira kusarongeka kwegraft-versus-host (Ren et al., 2017). , 2017a). Uchishandisa CRISPR/Cas9, HDR inogona kubvisa TCR neHLA panguva imwe chete uye kubvisa gene rinogadzira CAR. Zvidzidzo zvakaratidza kuti CRISPR/Cas9 inogona kushandiswa kuisa anti-CD19 CARs munzvimbo yeTCR, nokudaro ichigadzira maCAR zvinobudirira pasina kupedza T cells (Dimitri et al., 2022). Matekinoroji eMultiplex akagadzirwa kugadzira allogeneic CAR T cells nekubvisa TCR, beta-2-microglobulin (B2M), HLA-I subunit nemamwe mapuroteni akadai sePD-1 neCTLA-4, ayo anogona kunge aine hunhu hwakakura hwekurwisa kenza. kurwisa TNBC (Eyquem et al., 2017; Liu et al., 2017; Ren et al., 2017b; Dimitri et al., 2022).
Kushandisa CRISPR/Cas9 kunogona kuvandudza kushanda kwemasero eCAR-T. Adenosine inozivikanwa nekuva nemasimba ekudzivirira immune uye inogona kuderedza dziviriro yekudzivirira kenza nekuvharira mashandiro emasero eT uye kuita kuti adenosine A2A receptors (A2AR) ishande (Vigano et al., 2019). Kunyaradza A2AR uchishandisa CRISPR/Cas9 kwakavandudza zvakanyanya kushanda kwemasero eCAR-T mumuviri (Giuffrida et al., 2021). Masero eT anozivikanwa nekuratidza hunhu hwakasiyana-siyana hwe phenotypic hwakadai sekuwedzera kwemasero, kusiyana, oxidative stress uye genomic stress. Kuongorora kwemasero eT akavakirwa paCRISPR/Cas9 kwakaratidza kukanganiswa kwemasero eT cell receptor kinases makumi maviri nemashanu akasiyana. Pakati pawo, kubviswa kwep38 kinase kwakaratidzwa kusimudzira basa remasero eT antitumor, zvichiratidza kuti p38 kinase ndiyo inodzora zvakanyanya CAR-T cell regulation (Gurasamy et al., 2020).
Masero eT anogona kugadziriswa majini uchishandisa CRISPR/Cas9 kugadzira maTCR akasimba. Kutamisa masero eT akagadziriswa majini kuvarwere kwakaratidza kushanda kwakasimba kwekurwisa kenza kupfuura masero eT endogenous. Nokudaro, maTCR endogenous anogona kukwikwidzana nemaTCR akagadziriswa majini muvarwere, izvo zvinogona kukanganisa mukana wekurapa kenza. Kuti vakunde dambudziko iri, kushandisa CRISPR/Cas9 kubvisa TCR-β endogenous mumasero anogamuchira uye kuzotamisa masero eTCR-β kuvarwere vekenza kunogona kuratidza mhinduro dziri nani dzekudzivirira kenza pasina chikonzero chemakwikwi eTCR endogenous (Fan et al. 2018). Saka, kunze kwemasero eT akagadziriswa eCRISPR, maTCR anonzwa zvakanyanya maantigen etumor kupfuura masero eT akajairika anochinjirwa eTCR. Pamusoro pezvo, mumaleukemia akasiyana-siyana, masero eT akachinjwa anogadzirwa neγδ TCR+ CRISPR anoratidza kuratidzwa kwakanyanya kwemasero eCD4+ neCD8+ T kupfuura kutamiswa kweTCR kwakajairwa (Legut et al., 2018). Saka, masero eT akagadziriswa anogadzirwa neCRISPR/Cas9 anogona kunge ari nzira inoshanda yekurapa nekudzivirira chirwere cheTNBC.
MaIntegrins ndiwo mamorekuru ekunamatira masero ari mumasero etransmembrane uye anokurudzira kusungwa kwemasero kune extracellular matrix (ECM) (Hamidi naIvaska, 2018). Kusashanda zvakanaka kwe integrins kunobatanidzwa nekukura kwekenza nekufamba nekuchinja extracellular matrix, zvichikonzera kupona kwemasero ekenza mukutenderera kweropa (Hamidi naIvaska, 2018). CRISPR/Cas9-mediated integrin knockdown inononoka kufambira mberi kwebundu, metastasis, uye colonization muTNBC. Kudonhedzwa kwe integrin a5 (ITGA5) kwakataurwa kuti kunoderedza kufamba kwemasero nekufambira mberi mune mamwe magomarara akadai sekenza yemapapu (Ju et al., 2017), zvichiratidza kuti integrin a5 inogonawo kuva chinhu chikuru mukukonzera kweTNBC.
Kugadzira mbeva dzinobuda muchibereko uchishandisa nzira dzechinyakare dzemasero emwana ari mudumbu (ES) inzira inotora nguva yakawanda, inotora nguva, uye isingashande zvakanaka. Zvinotora mwedzi nemakore kunanga masero eES kuburikidza nekubatanidza mbeva dzinofanana, kubereka mbeva dzechimeric, uye wobva wadzisanganisa nembeva dzine heterozygous kuti dzibereke vana vane homozygous. Kuberekesana kwakaoma kunodiwa kuti pave nembeva dzine majini akawanda akasiyana. Zvisinei, matambudziko mazhinji amuka pakushandisa mbeva dzinogadzirwa nekubvisa masero eES uchishandisa CRISPR/Cas9 kana nekuisa microinjection yeCRISPR/Cas9 mumazai ane fertilization mumasero ega. Semuenzaniso, kuiswa kweshanduko kunowanzoitika pa biallelic loci uye hazvina chekuita nemajini. Munguva pfupi yapfuura zvakashumwa kuti masero eES anoshandisa tekinoroji yeCRISPR/Cas9 anogona kuisa biallelic mutations panguva imwe chete mumajini anosvika mashanu (Nishizono et al., 2021). Pachinangwa ichi, Cas9 mRNA uye ma gRNA mashanu e gene-specific akachinjirwa panguva imwe chete mu ES cells. Izvi zvinoratidza vimbiso uye kushanda kwemaitiro aya, kunyangwe shanduko idzi dzingave dzakapfuudzwa apo mitsetse yekutanga yaiberekwa kuti igadzire mbeva dzine mitsetse mishanu. Chidzidzo ichi chinoshumawo kuwanikwa kunoshamisa: masero eES haachadiwi kugadzira mbeva dzakagadziriswa majini. Pane kudaro, kubvisa zvigadzirwa zvemajini chaiwo, Cas9 mRNA ne gRNA zvakapinzwa muzvikamu zvemajini zve single-cell stage. Nekuda kweizvozvo, mitsetse yekutanga yekubuda yagadzirwa iyo inogona kushandiswa mudzidziso yekudzidza mhedzisiro yekubviswa kwemajini mumakonzo (Qin et al., 2016). Saka, CRISPR/Cas9 inobvumira kugadzirwa kwemakonzo e transgenic kurapa TNBC nemutengo wakaderera pane genetic engineering yechinyakare. Uchishandisa CRISPR/Cas system, modhi itsva ye mouse yekubuda yakagadzirwa inogona kubudirira kuunza point mutations mune rimwe kana akawanda majini e endogenous muTNBC. Zvichibva papfungwa iyi, mhando dzemhuka dzeTNBC dzinogonawo kugadzirwa uchishandisa CRISPR/Cas9-mediated deletion yeBRCA1 nep53, zvichikonzera kurasikirwa neHR repair, genomic instability, uye mutant phenotypes (Annunziato et al., 2020).
Parizvino, nzira dzakasiyana-siyana dzakadai semammography, magnetic resonance imaging (MRI) uye ultrasound dzinoshandiswa kuongorora TNBC. Zvisinei, nzira idzi dzine zvimwe zvisingakwanisike. Mammography inoshandiswa kuongorora nyama dzezamu dzemuno pane kuona metastases, uko masero ekenza akatamira kune dzimwe nhengo. Ultrasonography haisi nzira yakavimbika yekuongorora TNBC (Chen naLee-Felker, 2023). MRI ine kunzwisisika kwakanyanya kupfuura ultrasound nemammography, asi kunyatsoongorora kwayo kwakaganhurirwa (Sha naChen, 2022). Kuongororwa kwetishu inzira inopindira yekuziva masero ekenza. Zvisinei, mune dzimwe nguva, biopsy inogona kushaya nyama yekenza kana tsono ikatsauka kubva panzvimbo yainofarira. Pamusoro pezvo, inodhura zvikuru uye inogona kushungurudza murwere. TNBC rudzi rwekenza rwakasiyana, saka biopsy ingasapa ruzivo rwakakwana nezverudzi rwekenza. Nokudaro, nzira itsva yakavimbika yekuongorora TNBC inodiwa nekukurumidza. CRISPR/Cas9 inogona kushanda senzira inopinza uye isingapindi zvakanyanya yekuongorora kenza yemazamu. Kuti izvi zviitike, nzira dzeCRISPR/Cas9 dzinogona kushandiswa kuvandudza nzira dzePCR dzekuongorora TNBC. Kutanga, mapuroteni eCas9 necpf1 eCRISPR system anoshandiswa kubvisa DNA isiri chaiyo, uye mapuroteni maviri aya (Cas9 necpf1) anogona kuziva PAM sequence asati abatana neDNA yakanangana nayo (Deepak Singh et al., 2021). . Saka, PCR inogona kuona shanduko dzine chekuita nekukura kwegomarara. Zvidzidzo zvakati wandei zvakatora iyi nzira yeCRISPR kuti zvione shanduko dzakasiyana mumagomarara akasiyana (Safari et al., 2019). Saka, nzira dzeCRISPR-based PCR dzinogona kuderedza kuvimba kweTNBC diagnosis nenzira dzinopinda dzakadai sebiopsy-based immunohistochemistry.
Kuchinja kwe genotypic mukudzora mahormone receptor kwakaratidzwawo muTNBC (Chen naRusso, 2009). Nzira dzeCRISPR/Cas9-based PCR dzinoshandisa microarrays dzekuongorora nzvimbo dzekutarisira dzinogona kunyatsoongorora shanduko idzi (Hajian et al., 2019). Inogonawo kupa vanopa hutano ruzivo rwakakodzera nezve shanduko dzakati muvarwere veTNBC, izvo zvinogona kubatsira kuvandudza nzira yavo yekurapa. Zvisinei, nzira iyi yakabatana ine miganhu. Nokudaro, kuedza kwakawanda kunodiwa kuti nzira iyi yeCRISPR/Cas-PCR isashandiswe muhutano hwekuongorora TNBC (Yang et al., 2019).