CRISPR) bụ usoro nhazi mkpụrụ ndụ ihe nketa nke ọgbọ nke atọ: CRISPR gbanwere ụwa

Usoro nhazi mkpụrụ ndụ ihe nketa nke a na-akpọ CRISPR (CRISPR) bụ usoro nhazi mkpụrụ ndụ ihe nketa nke ọgbọ nke atọ nke agbanweela ụwa site na nsonaazụ ya dị elu. Ejirila ya agwọ ọrịa na ọrịa dị iche iche nke nje bacteria na nje. Ọtụtụ nje bacteria na prokaryotes ndị ọzọ (dịka archaea) nwekwara sistemụ CRISPR/Cas9 iji chebe onwe ha pụọ ​​​​na phages. Akọpụtala na atụmatụ dabere na CRISPR/Cas9 nwere ike igbochi uto na ọganihu nke kansa ara atọ na-adịghị mma (TNBC) site na ilekwasị anya na mkpụrụ ndụ ihe nketa ndị nwere ike ịgbanwe agbanwe, ntụgharị, na iwu epigenetic. Usoro ọgwụgwọ ndị a nwere ike inyere aka dozie nsogbu ndị siri ike dịka iguzogide ọgwụ a na-ahụ ọbụna na TNBC. Ugbu a, a na-eji ụzọ dị iche iche iji nye CRISPR/Cas9 ohere ichebe mkpụrụ ndụ, dị ka anụ ahụ (microinjection, electroporation na hydrodynamic modes), nje virus (viral na lentivirus metụtara adeno) na non-viral (liposomes na lipid nanoparticles). )-particles). Ọ bụ ezie na e mepụtala ụdị dị iche iche iji mụọ ihe kpatara TNBC, enweghị ụzọ dị mfe na nke ezubere iche maka inye ngwaọrụ ndezi mkpụrụ ndụ ihe nketa na vivo na-egbochi ojiji ha n'ụlọ ọgwụ. Na nchịkọta, nyocha a na-enyocha ọganihu, ihe ịma aka, mmachi, na atụmanya nke ọgwụgwọ CRISPR/Cas9 maka TNBC dabere na ihe akaebe dị ugbu a. Anyị na-akọwapụtakwa otu njikọta nke ọgụgụ isi arụrụ arụ na mmụta igwe nwere ike isi melite atụmatụ CRISPR/Cas9 na ọgwụgwọ TNBC.
A na-amata ọrịa kansa site na nkewa mkpụrụ ndụ a na-achịkwaghị achịkwa, mmebi nke ebe nlele mkpụrụ ndụ, na mgbanwe na mkpụrụ ndụ ihe nketa na-egbochi etuto ahụ (TSGs) (Matthews et al., 2022). N'ime ụdị ọrịa kansa dị iche iche, kansa ara bụ ụdị ọrịa kansa kachasị na ụmụ nwanyị ma nwee oke ọnụọgụ ọnwụ n'ụwa niile (Waks na Winer, 2019). Ọrịa ara ara bụ ọrịa dị iche iche nwere njirimara dị iche iche, dịka njirimara histological na bayọlọji, ngosipụta ahụike na omume, na nzaghachi nye ọgwụgwọ (Weigelt et al., 2010). Nkewa kansa ara na-enye nghọta ziri ezi maka nchọpụta kansa ara na amụma etuto ahụ. Ojiji nke ihe ndị a na-ahụkarị na njirimara clinicologic abụrụla ihe bụ isi maka nhazi nke kansa ara (Tsang na Tse, 2019). N'ọtụtụ ihe, amụma na nzaghachi nye ọgwụgwọ kansa ara na-emetụta, gụnyere ọnụnọ nke estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2/neu), na ọkwa histological, ụdị etuto ahụ, na ọkwa. nha na metastasis nke lymph node (Al-Tubaiti, 2020). Achọpụtala ụdị kansa ara ise dị n'ime ahụ, gụnyere luminal A, luminal B, HER2-enriched, basal-like, na claudin-low (Prat et al., 2015). TNBC bụ ụdị kansa molekul nke na-anaghị egosipụta ER, PR, na HER2 niile (Yin et al., 2020). A na-ejikọta njirimara ọrịa ndị a na TNBC, na-akwado ọganihu ya ngwa ngwa na okike ike karịa ụdị kansa ara ọ bụla ọzọ (Feng et al., 2018). Na mgbakwunye, Peru et al (2000) jiri teknụzụ microarray mee ka ọrịa kansa ara dịghachi ma chọpụta ụdị kansa ara ise dị n'ime ahụ (Cadenas, 2012). Ọrịa ara dị ka basal bụ ụdị kansa ara nke nwere ụdị atọ na-adịghị mma ma jikọtara ya na ọganihu ngwa ngwa. Ọ dị mkpa ịmara na ọrịa kansa ara niile dị ka basal na-abụkarị TNBC, mana naanị 77% bụ TNBC; N'ụzọ dị iche, 71–91% nke TNBC dị ka basal, na-egosi na ụdị ọrịa kansa ara abụọ a na-ejikọta ọnụ ma na-anọchite anya nhazi dị iche iche (Wang D.-Y. et al., 2019). Nke a na-emepụta mkpa ịkọwapụta ọdịiche dị na TNBC iji kọwaa amụma ma chọpụta azịza nwere ike inye ọgwụgwọ ugbu a na nke ga-abịa. Na mgbakwunye, TNBC na-akpata 15–20% nke ọrịa kansa ara niile ma na-abụkarị nke a na-ahụkarị na ụmụ nwanyị na-erubeghị afọ 50. Akọpụtala mgbanwe BRCA1 ma ọ bụ BRCA2 n'ihe dị ka 20% nke ikpe TNBC (Xie et al., 2017; Tzikas et al., 2017)., 2020). Nnyocha egosikwala na TNBC nwere obere gburugburu ebe obibi ahụ ji alụso ọrịa ọgụ nke gụnyere nnukwu ọkwa nke ihe ndị na-akpata uto endothelial vascular, macrophages metụtara tumor (TAMs), lymphocytes na-abanye n'ime tumor (TILs), na molekul ndị ọzọ metụtara uto na mbugharị tumor. Ya mere, ịghọta obere gburugburu ebe obibi nke TNBC dị oke mkpa maka amụma na ọgwụgwọ ya (Fan na He, 2022).
Iji chọpụta amụma nke TNBC ma hụ na ọgwụgwọ dị irè, nchọpụta ziri ezi, nke dabere na immunohistochemistry (IHC) iji chọpụta ER, PR na HER2, yana mammography iji chọpụta neoplasms ara, dị mkpa. Agbanyeghị, mammography enweghị ike igosipụta njirimara intratumoral dị ka necrosis na fibrosis nke ọma (Deepak Singh et al., 2021). Ebe ọ bụ na amụma na nchọpụta adịghị mma na-egbochi ndị dọkịta ịdepụta ọgwụ kwesịrị ekwesị (Chaudhary, 2020), a na-eji usoro dị iche iche eme ihe iji melite nlekọta maka ndị ọrịa nwere TNBC. Ugbu a, a na-eji ọgwụ abụọ, doxorubicin na cyclophosphamide, na ndị ọrịa nwere TNBC ma egosila ezigbo nsonaazụ. Na mgbakwunye, a na-eji ọgwụ platinum ndị ọzọ, dị ka carboplatin na cisplatin (Sikov et al., 2015). Na mgbakwunye, ejirila ndị na-egbochi PARP dị ka Olaparib, Velaparib, na PF-01367338 dị ka ọgwụ chemotherapy nwere ike maka ọgwụgwọ TNBC (Ishino et al., 2018). Ebe ọ bụ na akọpụtala na ụzọ Wnt/b-Catenin, NOCHCH, na Hedgehog na-etinye aka na mmepe na ọganihu nke TNBC, ilekwasị anya na ọgwụ ndị a nwere ike ịbụ atụmatụ dị mkpa (Aysola et al., 2013). Ọ bụ ezie na ịwa ahụ, ọgwụgwọ radieshon na chemotherapy ka bụ isi ihe na-agwọ TNBC taa, enwere ọganihu dị ukwuu n'ịmepụta ọgwụgwọ ọhụrụ gụnyere ọgwụgwọ ezubere iche, ọgwụgwọ immunotherapy, ngwaọrụ ndezi mkpụrụ ndụ ihe nketa dị iche iche metụtara CRISPR dịka Cas9n, dCas9., CRISPR/Cas12, mmezi mbụ, na ọgwụgwọ mkpụrụ ndụ ihe nketa CRISPR/Cas9. N'ebe a, anyị ga-elekwasị anya na ngwaọrụ ndezi mkpụrụ ndụ ihe nketa dị iche iche metụtara CRISPR ejiri na ọgwụgwọ TNBC. N'ime ha, CRISPR/Cas9 enwetawo nlebara anya zuru ụwa ọnụ.
Cas9n, nke a makwaara dịka Cas9 nickase, bụ ụdị protein Cas9 e mepụtara site na sistemụ CRISPR/Cas9 nke e si na sistemụ nhazi mkpụrụ ndụ ihe nketa (Gupta et al., 2019). N'ụdị mbụ ya, protein Cas9 nwere ngalaba nuclease abụọ: RuvC na HNH, ọrụ bụ isi ya bụ nkewa nke eriri DNA abụọ ahụ. Agbanyeghị, na Cas9n, otu n'ime ngalaba nuclease, HNH, na-agbanwe mkpụrụ ndụ ihe nketa ma na-aghọ ihe na-adịghị arụ ọrụ. Ya mere, ngalaba HNH nke Cas9n ka na-adịghị arụ ọrụ. Naanị ngalaba RuvC ka na-arụ ọrụ, na-enye Cas9n ohere ịkpụ ma ọ bụ mepụta mgbawa na eriri DNA otu (Trevino na Zhang, 2014). Ma e jiri ya tụnyere Cas9, Cas9n nwere ike ibelata mmetụta ndị na-abụghị ihe mgbaru ọsọ nke ọma ma melite usoro ndozi mkpụrụ ndụ nke ọma. Enwere ike iji Cas9n dozie nsogbu dị iche iche, dị ka ịmepụta mgbawa na ebe ụfọdụ na DNA nwere eriri abụọ. Maka ebumnuche a, a chịkọtara ma jiri molekul Cas9n abụọ ọnụ (Yee, 2016). Kinase 3 nke sitere na mkpụrụ ndụ agwakọtara (MLK3) bụ protein kinase nke mitogen na-arụ ọrụ nke na-eje ozi dị ka ihe na-achịkwa isi ihe n'oge TNBC metastasis (Cronan et al., 2012).
MLK3 nwere ike ime ka ọtụtụ ụzọ mgbaàmà rụọ ọrụ nke na-eduga na metastasis nke TNBC. Dịka ọmụmaatụ, ụzọ c-Jun N-terminal kinase (JNK) na-achịkwa mmegharị sel na mmebi nke matrix extracellular, MLK3 na-emekwa ka ụzọ JNK rụọ ọrụ, si otú a na-eme ka mmegharị sel dịkwuo mma ma na-enye ihe ndị na-awakpo (Rattanasinchai na Gallo, 2016). MLK3 na-achịkwakwa EMT. Iji mee nke a, ọ na-eme ka ihe ndị dị n'okpuru dị ka Snail, Slug na Twist rụọ ọrụ. Ihe ndị a na-egbochi ngosipụta nke ihe nrịbama epithelial ma na-akwalite ngosipụta nke ihe nrịbama mesenchymal, na-eduga n'inweta ụdị metastatic (Csalino et al., 2023). A hụla MLK3 ka ọ na-arụ ọrụ na mgbanwe ECM na ịgbalite proteases dị ka matrix metalloproteinases (MMPs) nke na-emebi ECM. Nke a na-eme ka mwakpo sel tumor dị mfe ma gbasaa gaa na ebe ndị dị anya (Katari et al., 2019). Ya mere, ọmụmụ ihe ndị gara aga egosila na MLK3 na-arụ ọrụ dị mkpa na TNBC. Rattanasinchai na Gallo jiri ụdị TNBC mụọ ọrụ MLK3 ma chọpụta na ọ na-akwalite mmepe kansa site na ụzọ mgbaàmà kpọmkwem. Ha jiri CRISPR/Cas9n dezie MLK3 ma hụ mbelata dị ukwuu na metastasis TNBC (Rattanasinchai na Gallo, 2016).
dCas9, nke a na-akpọkarị Cas9 na-anaghị arụ ọrụ, bụ ụdị ihe e mepụtara nke protein Cas9. N'adịghị ka Cas9 na-arụ ọrụ, dCas9 enweghị ọrụ endonuclease, nke na-eme ka ọ ghara ịkpalite mmebi DNA okpukpu abụọ. Ya mere, enwere ike iji dCas9 lekwasị anya kpọmkwem na mpaghara ụfọdụ nke genome na-enweghị mgbanwe ọ bụla ma ọ bụ mgbanwe ọ bụla na usoro DNA (Wang et al., 2016). Na dCas9, protein endonuclease abụọ, RuvC na HNH, anaghị arụ ọrụ site na igbochi ihe fọdụrụ amino acid dị mkpa. (Richter et al., 2016). N'agbanyeghị enweghị ọrụ nkewa DNA, dCas9 na-arụ ọtụtụ ọrụ dị mkpa na nyocha mkpụrụ ndụ ihe nketa na teknụzụ bayoteknọlọji. Dịka ọmụmaatụ, ọ na-enye ohere ịhụ mpaghara ụfọdụ nke genome, na-eme ka nhazi ederede dị mfe, ma na-akwalite mgbanwe epigenetic (Brocken et al., 2018).
Ihe ntụgharị ZEB1 (zinc finger E-box binding homeobox 1) na-arụ ọrụ pụrụ iche ma dị oke mkpa n'ime mgbanwe epithelial-mesenchymal (EMT), usoro sel nke na-eme n'oge mmepe nwa ebu n'afọ, ndozi anụ ahụ, na ọganihu kansa. Ọ gụnyere mgbanwe nke sel epithelial ka ọ bụrụ sel mesenchymal, na-ebute mgbanwe na nhazi sel, mmegharị ahụ, mwakpo, wdg. (Wu et al., 2020). ZEB1 na-egbochi ọtụtụ ihe nrịbama epithelial, dị ka E-cadherin na Occludin, bụ ndị na-ahụ maka ịnọgide na-enwe njikọ sel-cell na polarity nke sel epithelial na TNBC (Moreno-Bueno et al., 2008). Na mgbakwunye, ZEB1 na-eme ka ụfọdụ ihe nrịbama dị ka N-cadherin, vimentin na fibronectin rụọ ọrụ (Konradi et al., 2014) ma na-achịkwakwa mkpụrụ ndụ ihe nketa ndị metụtara mgbanwe cytoskeletal dịka Rho GTPases na matrix metalloproteinases (MMPs) (Huang). et al., 2014). 2022), na mgbakwunye, ọ na-emetụtakwa ụzọ mgbaàmà dị iche iche dịka mgbanwe ihe na-eto eto-β (TGF-β), Wnt signaling, wdg, si otú a na-akwalite mwakpo etuto ahụ na metastasis na TNBC (Chen et al., 2016). ). Ọtụtụ ọmụmụ na ihe akaebe sayensị na-egosi na ZEB1 nwere nnukwu ikike dị ka ihe bara uru maka nchọpụta na ọgwụgwọ nke TNBC. N'ọmụmụ ihe na-adịbeghị anya, Waryah et al. jiri ụdị TNBC wee nweta mmechi zuru oke nke ZEB1 site na dCas9. Ya mere, ha hụrụ nkọwapụta dị elu na ihe fọrọ nke nta ka ọ bụrụ mgbochi zuru oke nke ZEB1 na ọnọdụ in vivo (Waryah et al., 2023).
CRISPR/Cas12 bụ ngwa ndezi mkpụrụ ndụ ihe nketa a na-akpọ CRISPR/Cpf1. E si na sistemụ CRISPR/Cas nweta ya, ebe okwu ahụ bụ Cas12 na-ezo aka na protein 12 metụtara CRISPR (Bharathkumar et al., 2022), nke yiri Cas9 kpamkpam. Nanị ihe dị iche bụ na o nwere protein Cas12. Ma e jiri ya tụnyere Cas9, Cas12 nwere ụfọdụ ọrụ pụrụ iche dịka imepụta nsọtụ na-arapara n'ahụ n'oge usoro ndezi mkpụrụ ndụ ihe nketa, ebe Cas9 na-emepụta nsọtụ na-adịghị mma (Wang et al., 2021). Njirimara a nke Cas12 na-enye aka na teknụzụ nhazi DNA ya kpọmkwem. Dịka protein nwere ike ịmata nke ọma na ibelata DNA ebumnuche, ọ nwere ọtụtụ ihe dị iche iche, na-eme ka ọ bụrụ ngwa ọrụ dị irè maka ọtụtụ ngwa, gụnyere ndezi mkpụrụ ndụ ihe nketa (Pickar-Oliver na Gersbach, 2019).
Dịka ụdị CRISPR ndị ọzọ, CRISPR/Cas12 nwekwara ike ịkụtu ma ọ bụ mee ka mkpụrụ ndụ ihe nketa dị na TNBC, na-elekwasị anya na mkpụrụ ndụ ihe nketa ndị na-arụ ọrụ dị mkpa na mmepe ya ma ọ bụ nzaghachi nye ọgwụgwọ (Yang na Zhang, 2023). Iji mezuo usoro a, e mepụtara RNA nduzi (gRNA) nke na-eduzi Cas12 na mkpụrụ ndụ ihe nketa ebumnuche. Ozugbo Cas12 jikọtara na ihe mgbaru ọsọ ya, ọ na-ewebata mmebi eriri abụọ na DNA ma na-eme ka usoro ndozi DNA rụọ ọrụ. N'oge usoro ndozi ahụ, enwere ike itinye nucleotides na-ezighi ezi, na-eduga na mgbanwe mkpụrụ ndụ ihe nketa na mfu nke ọrụ (Zhang et al., 2021a). Na mgbakwunye, ejiri ụdị enzyme Cas12 ka mma (a na-akpọ dCas12) mee ihe iji mee ka mkpụrụ ndụ ihe nketa rụọ ọrụ. Site na ijikọta ya na ihe na-eme ka mkpụrụ ndụ ihe nketa dị irè, ọ ga-ekwe omume ịkpalite mkpụrụ ndụ ihe nketa ụfọdụ, si otú a na-eme ka ha rụọ ọrụ. Teknụzụ a nwere nnukwu ikike n'ịkwalite mkpali nke mkpụrụ ndụ ihe nketa na-egbochi etuto (Sultan et al., 2022).
Mmezi Prime bụ teknụzụ nhazi mkpụrụ ndụ ihe nketa ọhụrụ ma dị elu nke ukwuu. Ọ nwere ike ịgbanwe DNA nke ihe dị ndụ kpọmkwem (Chen na Liu, 2023). A na-enweta nhazi Prime site na ijikọta ihe abụọ bụ isi: enzyme CRISPR/Cas9 agbanwere agbanwe na transcriptase azụ. Ọrụ nke enzyme CRISPR/Cas9 bụ ịhọrọ ebe ndị ziri ezi na genome, ebe transcriptase azụ na-enyere aka ịgbanwe DNA nke ọma na ebe ebumnuche (Hassan et al., 2021). Na usoro nhazi primer, nzọụkwụ mbụ gụnyere imepụta ntuziaka nhazi primer RNA (pegRNA) (Standage-Beier et al., 2021). O nwere usoro ebumnuche na template RNA nke kwekọrọ na saịtị nhazi achọrọ na DNA ebumnuche. A na-etinye pegRNA n'ime mkpụrụ ndụ ebumnuche yana nuclease nhazi primer (PE2). PE2 bụ protein njikọ nke nwere enzyme Cas9, transcriptase azụ, na ihe nkwụnye nhazi primer (Martín-Alonso et al., 2021). N'ime sel ahụ, pegRNA na mgbagwoju anya PE2 na-achọ DNA kpọmkwem ha chọrọ ịgbanwe. Enzyme Cas9 na-egbutu DNA ma mepụta template nke nwere otu eriri (Choi et al., 2022). Ndagharị transcriptase na-eji template a iji kwadebe DNA maka idezi. Tinyere idetuo DNA, ntuziaka maka idezi template RNA dịkwa. N'ikpeazụ, a na-eji eriri DNA e kere ọhụrụ dị ka template iji dozie mgbawa na DNA, na-ebute usoro DNA gbanwere nke nwere mgbanwe achọrọ (Ochoa-Sanchez et al., 2021). Usoro idezi primer nwere ike ibute mgbanwe zuru oke na mkpụrụ ndụ ihe nketa. Ọ nwere ike ilekwasị anya na mgbanwe isi, ntinye, nhichapụ, na ọbụna nnọchi mkpụrụ ndụ ihe nketa (Anzalone et al., 2019; Chen na Liu, 2023). Mmezi Prime na-enye ọtụtụ uru karịa teknụzụ ndezi genome gara aga. Ndị a gụnyere mmụba izi ezi, mbelata mmetụta na-abụghị ihe mgbaru ọsọ, na ikike idezi DNA na-adabereghị na DNA okpukpu abụọ nke eriri (Anzalone et al., 2020).
E mepụtara teknụzụ CRISPR/Cas9 na mbụ iji chebe nje bacteria site na mbufe plasmid na ọrịa phage, e mesịkwara jiri ya mee ihe dị ka ngwa ọrụ DNA dị irè nke dabeere na RNA maka idezi mkpụrụ ndụ ihe nketa (Jiang na Doudna, 2017). Na mgbakwunye, akọpụtala na sistemụ CRISPR/Cas9 dị na 50% na 87% nke mkpụrụ ndụ ihe nketa nje na archaeal, n'otu n'otu (Ishino et al., 2018). CRISPR/Cas9 bụ ngwaọrụ nwere ike maka ihichapụ, itinye na idozi usoro mkpụrụ ndụ ihe nketa ọ bụla na-adịghị mma site na iji ụdị in vivo na in vitro (Sabit et al., 2021). Ọzọkwa, egosiri na CRISPR/Cas9 bụ akụkụ nke sistemụ ahụ ji alụso ọrịa ọgụ na-agbanwe agbanwe n'ihi na ọ bụ kpọmkwem maka mkpụrụ ndụ ihe nketa ndị a na-achọ (Chen na Zhang, 2018).
CRISPR/Cas9 nwere Cas9 na otu RNA nduzi (sgRNA). Cas9 bụ endonuclease nke nwere ọtụtụ ihe protein. Na mgbakwunye, Cas9 nwere ihe pụrụ iche nke nhazi na nhazi (Pacesa et al., 2022) ebe ọ nwere lobes abụọ: nnabata (REC) na nuclease (NUC). A kewara REC lobe ọzọ n'ime mpaghara atọ: REC1, REC2, na helices bridge (Cromwell et al., 2018). NUC nwere lobes atọ, ya bụ RuvC (RuvC I, RuvC II, RuvC III), HNH na ngalaba mmekọrịta protospacer neighborhood motif (PAM) (Fig. 1) (Song et al., 2016). sgRNA nwere ihe abụọ, gụnyere CRISPR RNA (crRNA) na transcoding obere RNA (tracer RNA) (Foto 1). sgRNA na-ejikọ protein Cas9 na njikọ iji mepụta mgbagwoju anya na-arụ ọrụ, nke a makwaara dị ka mgbagwoju anya effector (Richter et al., 2012). crRNA bụ njikọ ntọala nucleotide 18-20 nke na-arụ ọrụ dị mkpa n'ịchọpụta usoro DNA ebumnuche. Na mgbakwunye, crRNA na-ejikọ na DNA ebumnuche, tracrRNA na-arụkwa ọrụ dị ka ihe ndabere maka nuclease Cas9 iji kee DNA ebumnuche (Mangwar et al., 2019). N'aka nke ọzọ, usoro PAM nwere nucleotides 3, nke na-akwado, na-akọwapụta ma na-eme ka njikọ nke mgbagwoju anya effector na DNA dị. Akụkụ mgbagwoju anya protein Cas9 bụ RuvC na HNH nwere ọrụ catalytic (Richter et al., 2012; Asmamaw na Zawdie, 2021). Ngalaba nuclease HNH nke mpaghara Cas9 na-ekewa eriri DNA nke jikọtara ya na crRNA. Ngalaba nuclease RuvC na-ekewa eriri DNA ndị ọzọ ma na-emepụta mgbawa eriri abụọ (DSBs), mgbe nke ahụ gasịrị, a na-arụ ọrụ usoro ndozi DNA abụọ dị iche iche (Jiang na Doudna, 2017) (Foto 1).
Foto 1. Nchịkọta nke CRISPR/Cas9 (A). Ihe mejupụtara sistemụ CRISPR/Cas9: (i). Cas9 endonuclease bụ ọrụ maka ịbepụ usoro DNA ebumnuche, (ii) otu ntuziaka (sg) RNA sitere na njikọta nke crRNA na tra-crRNA chimeras. (abụọ). Cas9 gụnyere ọtụtụ ihe dị iche iche dịka Rec I, Rec II, NUC lobe (HNH na Ruv C bụ ihe ndị dị n'okpuru) na ngalaba mmekọrịta PAM (C) yana ọrụ ndị kwekọrọ. Ihe mgbagwoju anya protein CRISPR/Cas9 na-ekewa usoro DNA n'ime ụdị ndị na-abụghị ndị mgbakwunye na ndị na-akwado (D). CRISPR/Cas9 na-edezi genome n'ọkwa atọ: njirimara, ịkpụ na ndozi. sg-RNA e mere na-eduzi Cas9 ma na-amata usoro achọrọ site na ihe mgbakwunye crRNA. Cas9 na-amata usoro PAM na 5′-NGG-3′ ma na-agbaze DNA ahụ, na-emepụta ngwakọ DNA-RNA ma na-eme ka nkewa rụọ ọrụ. Ngalaba HNH nke Cas9 na-ekewa eriri ndị na-akwado ya, ngalaba RuvC na-ekewa eriri ndị na-abụghị nke mgbakwunye. CRISPR/Cas9 na-edozi dsDNA site na imebi ya n'ụzọ abụọ: njikọ njedebe na-abụghị nke homologue (NHEJ) na ndozi-eduzi homology (HDR). NHEJ na-edozi DNA nwere eriri abụọ na-enweghị DNA homologue mba ọzọ site na usoro enzymatic, usoro njehie nke nwere ike itinye ma ọ bụ wepụ usoro DNA na-enweghị usoro. HDR bụ nke a kapịrị ọnụ nke ukwuu ma chọọ ndebiri DNA homologous.
Ụzọ ndozi CRISPR/Cas9 nke na-akwado gụnyere njikọ njedebe na-abụghị nke otu (NHEJ) na usoro ndozi nke otu (HDR). NHEJ bụ ụzọ njehie na-adịkarị n'ihi na ọ gụnyere itinye ma ọ bụ ihichapụ ma ọ chọghị usoro ọ bụla n'oge usoro ndozi. Ọ na-eji nucleotides na-enweghị usoro iji mepụta protein ọkọlọtọ (Abbasi et al., 2021). Sistemụ ndozi a nwere mgbagwoju anọ dịka mgbagwoju KU, mgbagwoju protein nke na-emeju ụdị 4 (XRCC-4), enzyme nhazi njedebe DNA, na protein kinase DNA-PKcs (Abbasi et al., 2021). Protein KU dị mgbagwoju anya nwere obere akụkụ abụọ, Ku 70 na Ku 80, ma na-arụ ọrụ dị mkpa na usoro NHEJ, ebe ọ bụ na usoro nrụzi ahụ na-amalite site na njikọ nke obere akụkụ abụọ (Ku 70 na Ku 80) na nsọtụ dị nkọ ma ọ bụ nke fọrọ nke nta ka ọ bụrụ nke na-adịghị nkọ nke DNA ebumnuche (Abbasi et al., 2021), na-eje ozi dị ka ihe ndabere iji nweta ihe ndị ọzọ metụtara NHEJ na ebe mmerụ ahụ mere (Yang et al., 2020). XRCC-4 na DNA ligase mejupụtara amino acid 334 na 911, n'otu n'otu, na XRCC4-DNA ligase IV complex na-akpali njikọ nke njedebe DNA (Chatterjee et al., 2015). Enzyme nhazi njedebe DNA, nke a makwaara dị ka polynucleotide kinase 3′-phosphate, bụ enzyme nhazi njedebe DNA. Ọ nwere ike iwepụ otu 3′P na DNA ma phosphorylate otu 5′OH n'oge nrụzi DSB. Ọ gụnyekwara ịrụzi nke mgbawa otu eriri (SSBs) site na iji ụzọ ndozi SSB (Chatterjee et al., 2015). Protein kinase DNA-PKcs bụ protein kinase dabere na DNA nke nwere obere ihe na-eme ka ihe dị n'ime PIKKs (phosphatidylinositol 3-kinase-related kinases) na ezinụlọ ataxia telangiectasia mutated (ATM), yana ATM na Rad3-related strand breaks (DSBs) na otu eriri breaks (Yue et al., 2020; Peng et al., 2016).
HDR bụ usoro ndozi ziri ezi ma dabara adaba karịa n'ihi na a na-eji ụdị DNA duplex nke homologous depụtaghachi ozi ahụ, ọ bụ ezie na ọ chọrọ ọnụnọ nke chromatids nwanne. Nke a na-eme n'oge usoro S/G2 nke usoro mkpụrụ ndụ anụmanụ. HDR na-emekarị na ụdị yist, mana NHEJ dị oke mkpa na anụmanụ anụmanụ (Burma et al., 2006; Abbasi et al., 2021). E gosiri usoro ndozi zuru oke na Foto 1.
Ebe ọ bụ na ọrịa mkpụrụ ndụ ihe nketa na epigenetic na-akpata TNBC, mmezi nke ọrịa genomic/epigenemic dị ize ndụ site na iji CRISPR/Cas9 nwere ike ịbụ ụzọ ọgwụgwọ ezi uche dị na ya (Chen et al., 2019). Na mgbakwunye, ụfọdụ ihe ndị metụtara nhazi mkpụrụ ndụ nwere ike igosipụta njirimara pụrụ iche nke mkpụrụ ndụ kansa, na-atụ aro na nhazi mkpụrụ ndụ ihe nketa nwere ike ịbụ ụzọ dị mma maka ọgwụgwọ kansa (Drost et al., 2017). Iji njirimara mkpụrụ ndụ ihe nketa ndị a nke etuto ahụ, dị ka nkwarụ mkpụrụ ndụ ihe nketa, epigenetic na transcriptional, maka mmepe ọgwụ nwere ike imeziwanye nsonaazụ ahụike ma belata ọnụ ahịa nyocha. CRISPR bụ ngwaọrụ nhazi mkpụrụ ndụ ihe nketa nwere ike ọ bụghị naanị ịchọpụta ma chọpụta ebumnuche genomic na-akpata kansa, kamakwa enwere ike iji ya dezie, igbochi, na gbanwee oncogenes na epigenetic na mkpụrụ ndụ mmadụ (Tebụl 1) (Ahmed et al., 2021). Emeela ọtụtụ nyocha CRISPR iji chọọ mkpụrụ ndụ ihe nketa metụtara ihe mgbochi tumor, oncogenes, na iguzogide ọgwụ. E gosiri ojiji nke CRISPR/Cas9 iji dezie ụdị oncogenes dị iche iche nke TNBC na Foto nke 2.
Foto nke 2. Nhazi mkpụrụ ndụ ihe nketa nke ụdị ọrịa kansa TNBC dị iche iche site na iji CRISPR/Cas9 na-ebute mbelata uto etuto ahụ na metastasis. Oncogenes ndị a gụnyere CDK7, NAT1, UBR5, YTHDF2, ITGA9, CXCR4 na CXCR7, Crypto1, ROR1 na ST8SIA, bụ ndị metụtara mmepe na metastasis nke TNBC.
Mbugharị mkpụrụ ndụ kansa, mbuso agha, na mgbanwe epithelial-mesenchymal (EMT) jikọtara ya na ITGA9. Ọmụmụ ihe ndị gara aga egosila na ITGA9 bụ isi ihe na-eme na ụzọ Notch ma na-arụ ọrụ dị mma na metastasis rhabdomyosarcoma (Molist et al., 2020). Na mgbakwunye, achọpụtara na ITGA9 nwere njikọ chiri anya na amụma ndị ọrịa na ọtụtụ ụdị etuto ahụ, gụnyere kansa ara (Wang Z. et al., 2019). Na mgbakwunye, nyocha bioinformatics nke ITGA9 gosiri na ngosipụta ya na TNBC dị elu karịa na ụdị kansa ara ndị ọzọ. Ọkwa ITGA9 dị elu jikọtara ya na metastasis tumor na nlọghachi na ndị ọrịa TNBC. Mkpọchi ITGA9 site na CRISPR/Cas9 butere njirimara yiri nke mkpụrụ ndụ kansa (CSC), angiogenesis tumor, uto tumor, na mbelata metastasis site na ịkwalite mbibi β-catenin na TNBC (Wang Z. et al., 2019).
Crypto-1 bụ onye otu ezinụlọ TGF-β ma dị oke mkpa maka mmalite embryogenesis, mmezi nke mkpụrụ ndụ stem, na metastasis kansa (Ishii et al., 2021). A makwaara ya dị ka Tdgf-1, ọ bụ protein mgbaàmà GPI-ancoured oncogenic nke na-etinye aka na nhazi nke nhazi nke streak primitive, mesoderm na endoderm, yana ntọala nke asymmetry aka ekpe/aka nri na mmepe nke akụkụ ahụ n'oge embryogenesis (Zhang et al., 2021b). Na mgbakwunye, egosiri na Cripto-1 na-etinye aka na mgbanwe epithelial-mesenchymal (EMT) dị ka ihe nrịbama mkpụrụ ndụ stem (Zhang et al., 2021b). EMT dị oke mkpa ọ bụghị naanị maka usoro dị iche iche dịka mmepe embryonic, fibrosis na ọgwụgwọ ọnya, kamakwa maka mwakpo kansa na metastasis. Na mgbakwunye, egosiri na Cripto-1 na-emekọrịta ihe na ndị nnata Notch anọ ma na-eme ka ntozu ha ka mma (Brandstadter na Maillard, 2019). A maara na ụzọ mgbaama Notch na-etinye aka na mmezi nke mkpụrụ ndụ kansa ara mmadụ. Ọmụmụ ihe egosila na CRISPR/Cas9 nke Crypto-1 na-egbochi uto na mmepe nke kansa. Ya mere, Crypto-1 nwere ike ịghọ ihe mgbaru ọsọ ọgwụgwọ dị mkpa maka TNBC (Castro et al., 2015).
Protein XCL12 na onye nnata chemokine ya bụ CXC (CXCR4 na CXCR7) na-arụ ọrụ dị iche iche na mmụba nke mkpụrụ ndụ kansa, uto, mbugharị na mbuso agha (Wu et al., 2015). Ejikọtara ndị na-anabata chemo a na mmepe nke TNBC site na ọtụtụ ụzọ mgbaàmà na ụdị in vivo na in vitro (Wu et al., 2015). Na mgbakwunye, ịgbalite CXCR4 na CXCR7 na-ejikọta ya na nnukwu nnabata maka metastasis na amụma na-adịghị mma na TNBC (Karn et al., 2022). Ya mere, ịkụ aka nke CXCR4 na CXCR7 nwere ike ịghọ mkpụrụ ndụ ihe nketa ọgwụ dị irè maka ọgwụgwọ nke kansa ara, gụnyere TNBC. Ọmụmụ nke Yang et al. Yang et al (2019) jiri CRISPR/Cas9 mee ihe iji kụpịa mkpụrụ ndụ ihe nketa CXCR4 na CXCR7 ma chọpụta na mmụba nke TNBC, uto, mbugharị na mbuso agha egbochiri nke ukwuu (Yang et al., 2019).
A hụrụ mmụba nke miR-3662, nke bụ oncogene TNBC, n'ime anụ ahụ kansa ara (Yi et al., 2022). Egosipụtala na mbelata nke miR-3662 na-egbochi uto nke kansa ara na metastasis ma n'ime vivo na n'ime vitro (Agarwal na Gupta, 2021). HBP-1 bụ ihe na-egbochi ike nke mgbaama Wnt/-catenin ma o yikarịrị ka ọ bụ ya kpatara uto nke mkpụrụ ndụ TNBC nke miR-3662. N'oge na-adịbeghị anya, Yi et al. chọpụtara na axis miR-3662-HBP1 na-achịkwa ụzọ mgbaama Wnt/-catenin na mkpụrụ ndụ TNBC (Yi et al., 2022). N'ihi ngosipụta ya kpọmkwem nke etuto ahụ, miR-3662 nwere ike ịbụ ihe mgbaru ọsọ ọgwụgwọ maka TNBC. Ya mere, mbelata nke miR-3662 nke CRISPR/Cas9 nwere ike ịbụ ụzọ dị mma maka ịmepụta ọgwụ ọhụrụ maka ọgwụgwọ TNBC.
UBR5 bụ protein nucleophospho 300 kDa nke achọpụtala dị ka ihe na-achịkwa etuto ahụ, metastasis, na nzaghachi mgbochi ọrịa n'ọtụtụ ọrịa kansa (Shearer et al., 2015; Fu et al., 2023). Akọpụtala na UBR5 nwere oke mmụba na ihe nlele TNBC ma na-akpali ọrụ ERα site na ịkwalite mmụba site na ọrụ ubiquitin ligase ya (Bolt et al., 2015). Ọmụmụ ihe gbasara usoro exome zuru oke nke ihe nlele TNBC mbụ gosikwara mmụba nke ngosipụta nke UBR5, na-egosi ọrụ ya na mmepe nke TNBC. Na mgbakwunye, nhichapụ CRISPR/Cas9 nke UBR5 gosipụtara nnukwu mgbochi nke metastasis TNBC na uto na ụdị òké nnwale. Ọzọkwa, itinye UBR5 na ụdị òké ụdị ọhịa weghachiri ọrụ ya zuru oke, ebe a hụghị mmetụta a na ụdị mutant na-adịghị arụ ọrụ (Liao et al., 2017). Enweghị UBR5 na-ejikọta ya na mmụba nke apoptosis, necrosis, na igbochi uto nke etuto ahụ na TNBC n'ihi adịghị mma angiogenesis. N'ihi mfu nke UBR5, mgbasa nke etuto ahụ na akụkụ ahụ dị anya na-ebelata, UBR5 na-akpalikwa EMT na-adịghị mma karịsịa site na ibelata ngosipụta E-cadherin (Zhang na Weinberg, 2018). N'oge na-adịbeghị anya, egosiri na UBR5 bụ ihe dị oke mkpa na ntụgharị PDL1 nke IFN-γ kpatara na TNBC n'ihi enweghị ọrụ ubiquitination E3. Nyocha transcriptome RNA kpughere na UBR5 nwere ike inwe mmetụta sistemụ na mkpụrụ ndụ ihe nketa jikọtara ya na ụzọ IFN-γ ma kwalite transactivation PDL1 site na ịbawanye ọkwa mkpali nke protein kinase RNA (PKR) na ndị na-agbanwe ihe mgbaàmà na ndị na-eme ka ihe rụọ ọrụ ya. transcript 1 (STAT1) na interferon regulatory factor 1 (IRF1). Agbanyeghị, njikọta nke CRISPR/Cas9 nke ngosipụta UBR5 na PD-L1 nwere mmetụta ọgwụgwọ synergistic karịa igbochi naanị ha, yana mmetụta miri emi na gburugburu ebe obibi tumor (Wu et al., 2022). Ya mere, CRISPR/Cas9 nwere ike ịbụ ngwaọrụ dị mkpa iji gbochie ọrụ UBR5, si otú a na-egbochi metastasis TNBC na uto tumor.
ROR1 bụ protein transmembrane nke ụdị I nke a na-egosipụta n'oge ọrịa kansa na mmepe nwa ebu n'afọ ma achọpụtala ya dị ka protein oncofetal (Nicholas Borcherding, 2014). Omume mwakpo nke ọrịa kansa dị iche iche nke mmadụ na-ejikọta ya na mmụba nke ROR1. Enwetara nsonaazụ dị mma site na ọmụmụ in vivo na in vitro gụnyere ngwakọta ọgwụgwọ na-elekwasị anya na ROR1 (Chien et al., 2016). Ọkwa dị elu nke ROR1 mRNA na biopsies anụ ahụ ara ejikọtawokwa ya na etuto basal-like ara (BL) siri ike na mbugharị ha gaa n'akụkụ ahụ ndị ọzọ. Ọzọkwa, a chọpụtala oke ngosipụta nke ROR1 dị ka ihe nrịbama amụma maka mmepe nke TNBC (Chien et al., 2016). Agbanyeghị, imechi ROR1 site na iji CRISPR/Cas9 iji gbochie uto na metastasis TNBC ga-abụ atụmatụ dị irè.
Usoro sialylation gụnyere itinye sialic acid na glycoconjugates, nke sialyltransferases (STs) na-akwalite. ST8SIA1 bụ nke ezinụlọ ST ma na-arụ ọrụ dị mkpa na pathogenesis nke ọrịa dị iche iche dịka lymphocytic leukemia na kansa colorectal (Chang et al., 2018). Nyocha usoro RNA na-egosi na ST8SIA1 na-apụta ìhè nke ukwuu na anụ ahụ ara nke ndị ọrịa TNBC ma na-ejikọta ya na mgbanwe dị na mkpụrụ ndụ ihe nketa nke tumor na-egbochi p53, nke nwere ike itinye aka na pathogenesis nke TNBC (Battula et al., 2017). Na mgbakwunye, ST8SIA1 na-etinye aka na metastasis na nlọghachi nke TNBC, na-egosi na ọ na-arụ ọrụ dị mkpa na mmepe na mmepe nke TNBC. Na ụdị in vitro nke TNBC, egosiri na CRIPSR/Cas9 na-egbochi uto na metastasis (Battula et al., 2017). Nke a na-egosi na CRISPR/Cas9 nwere ike ịbụ ngwaọrụ dị mkpa maka igbochi ọrụ nke oncogene ST8SIA1 n'ọgwụgwọ TNBC.
NAT1 bụ enzyme metabolic nke na-akwalite nhazi nke phase II xenobiotic compounds ma na-apụta ìhè n'ihe fọrọ nke nta ka ọ bụrụ anụ ahụ mmadụ niile. NAT1 nwere ike iji cofactor folate lekwasị anya na acetyl-CoA (acetyl-CoA) ọbụlagodi na enweghị aromatic amine substrate (Stepp et al., 2015; Laurieri et al., 2014). Ọmụmụ ihe egosila na NAT1 na-achịkwa ọrụ matrix metalloproteinase 9 (MMP9) na ụdị sel kansa ara ma na-echebe megide ụdị oxygen reactive (ROS) n'oge agụụ glucose (Wang et al., 2018). Egosila na ihichapụ NAT1 na-egbochi mgbagwoju anya pyruvate dehydrogenase, na-eduga na nsogbu mitochondrial (Wang L. et al., 2019). Na mgbakwunye, akụkọ ndị ọzọ dị iche iche egosila na mgbochi NAT1 site na iji obere molekul na ịgbachi nkịtị siRNA nwere ike ibelata mwakpo na mmụba nke mkpụrụ ndụ kansa ara (Stepp et al., 2018). N'oge na-adịbeghị anya, e jiri CRISPR/Cas9 gbochie NAT1 n'ime mkpụrụ ndụ kansa ara MDA-MB-231, na-emetụta metabolism nke mkpụrụ ndụ dabere na ọkwa ngosipụta ya. Ọmụmụ ihe a gosikwara na NAT-1 dị oke mkpa maka ọganihu na metastasis nke TNBC (Carlisle et al., 2020).
A na-achịkwa nhazi nke oncogenes site na ndị na-eme ka ihe dịkwuo mma, ihe ndị na-eme ka ihe dị iche iche pụta ìhè na ndị na-emepụta ihe (Hnisz et al., 2015). Na mgbakwunye, a chọrọ otu kinases na-adabere na cyclin (CDKs), dị ka CDK7, CDK8, CDK9, CDK12, na CDK13, maka nhazi nke akwụkwọ. N'ime ha, CDK7 na-etinye aka na phosphorylation nke RNA polymerase II, nke dị oke mkpa maka mmalite na mgbasawanye nke akwụkwọ oncogene na pathogenesis nke TNBC. N'ọmụmụ ihe dị mkpa, iwepụ CDK7 site na CRISPR/Cas9 gbochiri TNBC, na-egosi na pathogenesis nke TNBC dabere na CDK7 (Wang Y. et al., 2015).
Nnyocha egosila na mgbanwe dị na MYC na RBP (protein njikọ RNA) nwere ike ibute apoptosis, ebe mgbanwe mkpụrụ ndụ ihe nketa otu na MYC ma ọ bụ RBP anaghị emetụta uto nke mkpụrụ ndụ kansa (Einstein et al., 2021). E jirila ọbá akwụkwọ CRISPR/Cas9 nyochaa ihe karịrị RBP 1000 na mkpụrụ ndụ ihe nketa mmadụ. N'ime ha, achọpụtara na RBP 57 dị oke mkpa maka uto nke mkpụrụ ndụ kansa nwere ọkwa MYC dị elu (Wheeler et al., 2020). Na mgbakwunye, YTHDF2 dị mkpa maka ịnọgide na-enwe uto mkpụrụ ndụ TNBC ma na-ebelata ọnụọgụ nke transcripts methylated n'oge ntụgharị na ntụgharị dị elu na mkpụrụ ndụ kansa nwere ngosipụta MYC dị elu. Na mgbakwunye, YTHDF2 adịghị mkpa maka mkpụrụ ndụ kansa, nke na-adabereghị na ọkwa MYC dị elu iji mee ka ndị ọrịa TNBC dịgide ndụ (Einstein et al., 2021), na-atụ aro na ọ nwere ike ịbụ ihe mgbaru ọsọ ọgwụgwọ maka ọgwụ ndị nwere ike imeri TNBC.
Protein mkpịsị aka Zinc (ZNFs) mejupụtara ihe dị ka 1% nke mkpụrụ ndụ ihe nketa mmadụ niile. Ọmụmụ ihe egosila na ZNF nwere ike ịchịkwa mmụba mkpụrụ ndụ na ọrịa kansa dị iche iche, dịka kansa imeju, kansa ara na kansa eriri afọ (Zhang W. et al., 2021; Li et al., 2017). Ejirila ọbá akwụkwọ nke CRISPR knockout mepụtara iji nyochaa ụdị mkpụrụ ndụ ihe nketa dị iche iche nke na-egbochi etuto ahụ (TSGs) na mkpụrụ ndụ kansa ara (Shalem et al., 2014). Kemgbe ahụ, ọmụmụ ihe gbasara mkpụrụ ndụ kansa ara CRISPR/Cas9 ZNF319-ehichapụla egosila na ZNF319 bụ mkpụrụ ndụ ihe nketa na-egbochi etuto ahụ nke na-ebelata mmụba nke kansa ara ma ya mere ọ na-etinye aka na usoro mgbaàmà dị iche iche na ọrụ ndị ọzọ dị ndụ (Wang L. et al., 2022).
Ferroptosis bụ ụdị ọnwụ mkpụrụ ndụ emebere nke dabere na ọnụnọ nke ígwè. A maara na mmepe nke ferroptosis na-adabere na ọnụnọ nke lipid peroxides. Na mgbakwunye, akọpụtala na PKCβII na-egosipụta peroxidation lipid mbụ, mmụba nke lipid peroxidation na-ejikọkwa ferroptosis. Nnyocha nke CRISPR/Cas9-mediated kinase inhibitors kpughere na PKCβII na-etinye aka na usoro nke lipid peroxidation, nke dị oke mkpa maka ferroptosis na mkpụrụ ndụ MDA-MB-231 (Zhang et al., 2022). Ya mere, nke a na-egosi na ịkụ PKCβII site na CRISPR/Cas9 nwere ike ịbụ ihe nwere ike ibute mkpụrụ ndụ ihe nketa na-egbochi etuto maka ọgwụgwọ nke ọrịa ndị metụtara ferroptosis (Zhang et al., 2022).
A kwenyere na iguzogide ọgwụ bụ ihe kpatara ihe dị ka 90% nke ọnwụ ndị ọrịa kansa, ọ bụkwa otu n'ime ihe ịma aka kachasị na ọgwụgwọ kansa (Bukowski et al., 2020). Nsonaazụ ya na-egosi na ọnụọgụgụ zuru oke nke mkpụrụ ndụ ihe nketa metụtara mmụba ọgwụ, ndozi DNA, apoptosis na ụzọ dị iche iche nke mgbaàmà mkpụrụ ndụ jikọtara ya na iguzogide ọgwụ (Haider et al., 2020). N'ime ha, ọtụtụ mkpụrụ ndụ ihe nketa ejirila ngwaọrụ CRISPR/Cas9 lekwasị anya ma gosipụta nsonaazụ dị mma n'ibelata iguzogide ọgwụ na ịbawanye irè nke ọgwụgwọ mgbochi kansa (Vaghari-Tabari et al., 2022). Na mgbakwunye, ọbá akwụkwọ nyocha mkpụrụ ndụ ihe nketa CRISPR/Cas9 dị elu etinyela aka na mgbanwe ọrụ nke ebumnuche mkpụrụ ndụ ihe nketa iguzogide ọgwụ nwere ike ime (Shalem et al., 2015). Iji chọpụta mkpụrụ ndụ ihe nketa ndị na-eguzogide paclitaxel, e jiri usoro RNA yana nyocha sgRNA nke genome-wide library chọpụta mkpụrụ ndụ ihe nketa asatọ, gụnyere histone deacetylase 9 (HDAC9), nke jikọtara ya na iguzogide ọgwụ na ndị ọrịa nwere TNBC ugboro ugboro (B et al., 2020).). N'ọmụmụ ihe ọzọ, a hụrụ mmụba nke ngosipụta nke diserine/threonine na tyrosine protein kinase (DSTYK) n'oge ndụ nke ndị ọrịa TNBC ejiri ọgwụ mgbochi kansa gwọọ. Ọzọkwa, mbelata nke CRISPR/Cas9 nke DSTYK mere ka apoptosis nke mkpụrụ ndụ kansa na-eguzogide ọgwụ dịkwuo mma na vitro (mkpụrụ ndụ SUM102PT na mkpụrụ ndụ MDA-MB-468) na ụdị TNBC in vivo (Ogbu et al., 2021).
Ọ bụ ezie na TNBC nwere ọrụ na-adịghị mma nke ụzọ MAPK, mmetụta ahụike nke ọgwụgwọ ndị e lekwasịrị anya na MEK adịghị mma. Nnwale genomic nke CRISPR/Cas9 gosiri na mgbochi nke PSMG2 (proteome assembly chaperone 2) na-eme ka mkpụrụ ndụ TNBC BT549 na MB468 nwee mmetụta na ihe mgbochi MEK AZD6244. Mbelata CRISPR/Cas9 nke PSMG2 gbanwere ọrụ proteasome nkịtị, na-eduga na mwepụ nke PDPK1 site na autophagy, si otú a na-eme ka mkpụrụ ndụ tumor dịkwuo mma na ụdị òké TNBC nke AZD6244 (MEK inhibitor) na MG132 (proteasome inhibitor) kpatara. Ya mere, enwere ike inye ndị na-egbochi proteasome na MAP kinase (MEK) n'ụzọ njikọta iji belata mmụba nke mkpụrụ ndụ tumor (Wang X. et al., 2022).
E jikwa CRISPR/Cas9 chọpụta ọnwụ nke ọrụ mkpụrụ ndụ ihe nketa nke na-eduga na iguzogide TNBC (Shu et al., 2020). Ge et al. kọwara usoro nke iguzogide ọgwụ nke mkpụrụ ndụ kansa ejiri JQ1, onye na-egbochi BET bromodomain (BBDI), gwọọ na TNBC. Site na iji CRISPR/Cas9, ha chọpụtara na ihichapụ mkpụrụ ndụ ihe nketa rb1 mere ka TNBC ghara iguzogide ọgwụ mgbochi kansa JQ1. Ya mere, ọrụ rb1 dị mkpa na nzaghachi nye ọgwụ JQ1 na TNBC. Ha kọkwara na paclitaxel bụ ihe na-egbochi CDK4/6 kinase/microtubule, yana njikọta ya na BBDI dịka JQ1 nwere ike inye azịza ọgwụgwọ na-ekwe nkwa maka TNBC na-eguzogide ọgwụ (Ge et al., 2020).
Akọpụtala na ihe ndị e dere n'akwụkwọ ndekọ nke RNA ogologo na-abụghị koodu (lncRNA) bụ ihe kpatara iguzogide ọgwụgwọ neoadjuvant na TNBC. Ọmụmụ ihe a na-egosi na e gosipụtara ihe ise dị iche iche nke MALAT1 lncRNA na TNBC nke ọma. Na mgbakwunye, mwepụ nke MALAT1 nke CRISPR/Cas9 mere ka mkpụrụ ndụ TNBC BT-549 dịkwuo mma na paclitaxel na doxorubicin, na-atụ aro ọrụ nwere ike ịrụ maka iguzogide MALAT1 na TNBC (Shaath et al., 2021).
Mgbanwe dị na BRCA1 (BRCA1m) dị iche iche, ya mere ọ siri ike ịchọpụta. Iji PARP1 (poly(ADP-ribose) polymerase), onye mmekọ na-egbu egbu nke BRCA1, nwere ike ime ka mmetụta nke ọgwụ TNBC dịkwuo elu. Site na iji CRISPR/Cas9, iwepụ PARP1 mụbara mmetụta nke ọgwụ ndị na-egbochi kansa dịka doxorubicin, gemcitabine na docetaxel na mkpụrụ ndụ TNBC mutant mBRCA1, na-egosi na PARP1 na-etinyekwa aka na iguzogide ọgwụ na TNBC (Vaghari-Tabari et al., 2022). A maara mgbanwe dị na mkpụrụ ndụ ihe nketa na-egbochi etuto ahụ BRCA1 ma ọ bụ BRCA2 na-eme ka ohere nke ịmalite ọrịa kansa ara dịkwuo elu n'ime ndị mmadụ. Ọgwụgwọ nke ndị ọrịa a chọrọ iji ihe mgbochi PARP. Ọzọkwa, egosila na ịkụ aka nke nucleotide salvage factor DNPH1 site na iji CRISPR/Cas9 nwere ike iwepụ nucleotide 5-hydroxymethyldeoxyuridine (hmdU) monophosphate na-egbu egbu, si otú a na-eme ka nzaghachi nke mkpụrụ ndụ ndị na-enweghị BRCA dịkwuo mma na mmetụta nke ndị na-egbochi PARP (Fugger). et al., 2021). Ya mere, ndị na-egbochi CRISPR/Cas9 na PARP1 nwere ike ịghọ atụmatụ ọgwụgwọ dị mkpa maka TNBC.
Mkpụrụ ndụ ihe nketa glycoprotein (P-gp) bụ mkpụrụ ndụ ihe nketa nke na-eguzogide ọtụtụ ọgwụ nke a na-achịkwa n'ihe dị ka 41% nke TNBC niile (Sun et al., 2020). Achọpụtala na ọgwụ P-gp na-akpata na-achịkwa iguzogide ọgwụ na kansa ara. Ndị na-egbochi P-gp egosila mmụba na mmetụta ha na ọgwụ mgbochi kansa na kansa ara (Famta et al., 2021). Ya mere, CRISPR/Cas9 na-akwalite ablation ma ọ bụ mgbochi nke P-gp na P-gp inhibitors nwere ike ịbụ ụzọ dị oke mkpa iji merie iguzogide ọgwụ na TNBC.
A maara na ihe na-ebufe kaseti ATP-binding G2 (ABCG2) na-akpata iguzogide ọgwụ na TNBC (Palasuberniam et al., 2015), ọ bụ ezie na ugbu a enweghị akụkọ gbasara mmekọrịta dị n'etiti CRISPR/Cas9 na ABCG2 ikpuchi na mbelata mkpụrụ ndụ ihe nketa tumor. PTEN nwere ike ime ka ọrụ ABCG2 ka mma (Palasuberniam et al., 2015), (Deepak Singh et al., 2021). Ya mere, iji CRISP/Ca9 wepụ ABCG2 na ijikọta ya na ihe na-egbochi ABCG2 nwere ike ịbụ ọgwụgwọ kwesịrị ekwesị iji merie iguzogide ọgwụ na TNBC. Tebụl nke abụọ kwuru maka CRISPR/Cas9 na-elekwasị anya na mkpụrụ ndụ ihe nketa iguzogide niile.
Tebụl nke 2. CRISPR/Cas9 na-elekwasị anya na mkpụrụ ndụ ihe nketa dị iche iche nke iguzogide TNBC iji mee ka mkpụrụ ndụ dịkwuo mma maka ọgwụ mgbochi kansa.
Ọbá akwụkwọ CRISPR/Cas9 bụ ngwaọrụ nwere ike iji nyochaa mgbanwe mkpụrụ ndụ ihe nketa metụtara ọrịa kansa (Chan et al., 2022). Usoro nyocha a gụnyere usoro anọ dịka (a) owuwu ọbá akwụkwọ, (b) transduction lentiviral, (c) nyocha phenotypic, na (d) nyocha mkpụrụ ndụ ihe nketa ebumnuche. Ọ bụ ezie na TNBC nwere mmegharị na-adịghị mma nke ụzọ MAPK, amụma ahụike nke ọgwụgwọ ezubere maka MEK maka ndị ọrịa TNBC na-ebu mgbanwe na mkpụrụ ndụ ihe nketa mgbochi etuto dịka PTEN, RB1, na TP53 adịghị mma nke ukwuu.
Ọzọkwa, nyocha mkpụrụ ndụ ihe nketa nke ejiri CRISPR/Cas9 nwalee molekul kachasị ike na nke nhọrọ, dehydrocatrol, nke jupụtara na ihe ndị e si na mkpụrụ osisi na akwụkwọ Guatemala wepụta, iji ghọta mmetụta nke dehydrocatrol na MDA-MB-231. Usoro nke cytotoxicity sel nhọrọ. Àgwà Mesenchymal stemness nke ụdị TNBC. Ihuenyo dabere na CRISPR/Cas9 a kpughekwara na HSD17B11, mkpụrụ ndụ ihe nketa nke na-etinye 17β-hydroxysteroid dehydrogenase type 11, na-egosipụta nke ọma na mkpụrụ ndụ MDA-MB-231 ma na-ebute dehydrofalcarinol kpọmkwem maka mkpụrụ ndụ MDA-MB-231 (Grant et. al., 2020). Ya mere, nke a na-egosi na nyocha genomic CRISPR/Cas9 nwere nnukwu ikike n'ịchọpụta usoro ndị na-akpata ihe onwunwe anticancer nke ihe ndị sitere n'okike nwere ike ime.
Tinyere nke ahụ, a mụrụ ihe gbasara adịghị ike nke TNBC na kansa site na iji nyocha CRISPR/Cas9 nke dị n'ime ahụ, nke gosiri njikọ dị n'etiti ụzọ ndị na-egbochi oncogenic na ụzọ ndị na-egbochi etuto ahụ. Akọpụtala na akụkụ dị mkpa nke ụzọ mTOR na Hippo na-arụ ọrụ dị mkpa na nhazi etuto ahụ na TNBC. Na mgbakwunye, ọmụmụ ihe egosila na mgbochi ọgwụ nke mTORC1/2 na YAP oncoprotein na-egbochi pathogenicity nke TNBC nke ọma site na iji ụdị in vitro ọgwụ-matrix synergy na in vivo xenografts sitere na ndị ọrịa. Ọzọkwa, mgbochi mTORC1/2 nke Torin-1 na-eme ka macropinocytosis dịkwuo mma, ebe mgbochi YAP nke verteporfin kpatara na-eduga na ọnwụ mkpụrụ ndụ TNBC. A na-ejikọta ha ọnụ, nsonaazụ ndị a na-egosipụta ike na ike nke nyocha CRISPR nke dị n'ime ahụ iji chọpụta ọgwụgwọ ọhụrụ na nke dị irè maka TNBC (Dai et al., 2021).
Mmebi nke usoro ahụ ji alụso ọrịa ọgụ bụ ihe dị mkpa na mmepe nke ọrịa kansa. Mkpụrụ ndụ kansa na-ezere mwepụ nke usoro ahụ ji alụso ọrịa ọgụ site n'ịghara usoro nchebe, gụnyere igbochi ọrụ nke mkpụrụ ndụ ji alụso ọrịa ọgụ na gburugburu ebe obibi tumor na imebi usoro ahụ ji alụso ọrịa ọgụ. Ya mere, ịmepụta usoro ahụ ji alụso ọrịa ọgụ ka mma nwere ike ịbụ ụzọ dị mkpa isi lụso ọrịa kansa ọgụ. Ojiji nke mgbanwe mkpụrụ ndụ ihe nketa dabere na CRISPR/Cas9 na-edozi ọtụtụ nsogbu metụtara nsogbu ahụ ji alụso ọrịa ọgụ site n'echiche dị iche iche. Ejirila CRISPR/Cas9 melite mgbochi ọrịa megide kansa ara site na ụzọ ndị a (Foto 3).
Foto nke 3. Ọgwụ mgbochi ọrịa CRISPR/Cas9 na-elekwasị anya na mkpụrụ ndụ TNBC. Mfu nke CDK5 na mbelata nke PDL1 na CD155 na-eme ka usoro ahụ ji alụso ọrịa ọgụ ka mma. N'otu aka ahụ, mfu nke A2AR na mbelata nke TAA dịka HER2, mucin 1 na TEM8 mụbara arụmọrụ nke mkpụrụ ndụ CAR-T na igbu mkpụrụ ndụ kansa. Nnyocha mkpụrụ ndụ T nke CRISPR/Cas9 na-eduzi egosila na mmebi nke kinase p38 na-eme ka ọrụ mgbochi ọrịa T dịkwuo mma. Mkpụrụ ndụ T nke CRISPR/Cas9 gbanwere na-eme ka ngosipụta nke TCR (ihe nnata mkpụrụ ndụ T) dịkwuo elu, na-eme ka mkpụrụ ndụ T nwee ọrụ mgbochi ọrịa ahụ.
Ebumnuche nke ọgwụgwọ immunotherapy bụ ịkpali sistemu ahụ ji alụso ọrịa ọgụ iji wakpo mkpụrụ ndụ kansa. Oke nrụgide nke protein ndị na-ahụ maka nchekwa ahụ ji alụso ọrịa ọgụ na-egbochi mmeghachi omume autoimmune mana ọ nwere ike inyere kansa aka izere ha (Topalian et al., 2015). Protein ndị a na-egbochi mmeghachi omume ahụ ji alụso ọrịa ọgụ site na ijikọ na ndị na-anabata ya n'elu mkpụrụ ndụ ahụ ji alụso ọrịa ọgụ. Ọtụtụ protein ndị na-ahụ maka nchekwa (dịka CD155 na PD-L1) na-elekwasị anya na onye na-anabata PD-1 na mkpụrụ ndụ ahụ ji alụso ọrịa ọgụ ma a na-egosipụta ha na kansa ara, ọkachasị TNBC (Li Y.-C. et al., 2020). Mbelata nke PD-L1 ma ọ bụ onye na-anabata ya site na iji CRISPR/Cas9 nwere ike ịkpalite sistemu ahụ ji alụso ọrịa ọgụ iji wakpo etuto TNBC (Yahata et al., 2019). Na mgbakwunye, mbelata nke ngosipụta PD-L1 site na nhichapụ CRISPR/Cas9 nke CDK5 egosila na ọ na-egbochi uto etuto ahụ in vitro na in vivo (Deng et al., 2020). Mbelata uto nke ọrịa kansa ara na in vitro na in vivo na-egosi na mbelata nke CD155 nke shRNA na-akpata nwere ike inwe mmetụta ọgwụgwọ na kansa ara (Gao et al., 2018).
Mkpụrụ ndụ CAR T na-egosipụta CAR ndị na-amata antigens metụtara etuto ahụ (TAAs) ma nwee ike iji mbelata protein ndị a na-enyocha iji mee ka ọrụ ha ka mma (Li C. et al., 2020). E nwere ọtụtụ ebumnuche enwere ike maka ọgwụgwọ mkpụrụ ndụ CAR T na kansa ara, gụnyere ọtụtụ TAA dịka HER2, mucin1, na TEM8 (Bajgain et al., 2018). E nwere ihe akaebe na mkpụrụ ndụ CAR T na-elekwasị anya na mesothelin (nke a na-egosipụta nke ukwuu na mkpụrụ ndụ TNBC BT-459) na-arụ ọrụ nke ọma megide kansa mgbe ejiri CRISPR/Cas9 chụpụ PD-1 (Hu et al., 2019). Agbanyeghị, ikewapụ mkpụrụ ndụ T n'ebe ndị ọrịa nọ wee dezie ha ex vivo bụ usoro na-ewe oge ma na-ewe oge. Ọ bụ ezie na mkpụrụ ndụ T zuru ụwa ọnụ nwere ike ibelata ihe achọrọ maka iche, a ga-ewepụ mkpụrụ ndụ T ndị na-enye onyinye nke na-egosipụta antigen leukocyte mmadụ (HLA) klas nke mbụ na onye na-anabata mkpụrụ ndụ T (TCR) iji gbochie nsogbu graft-versus-host (Ren et al., 2017). , 2017a). Site na iji CRISPR/Cas9, HDR nwere ike iwepụ TCR na HLA n'otu oge ma chụpụ mkpụrụ ndụ ihe nketa nke na-akọwapụta CAR. Ọmụmụ ihe egosila na enwere ike iji CRISPR/Cas9 webata CARs anti-CD19 n'ime ebe TCR, si otú a na-emepụta CAR nke ọma na-enweghị ibelata mkpụrụ ndụ T (Dimitri et al., 2022). Emepụtala teknụzụ Multiplex iji mepụta mkpụrụ ndụ CAR T allogeneic site n'ibelata TCR, beta-2-microglobulin (B2M), subunit HLA-I na protein ndị ọzọ dịka PD-1 na CTLA-4, nke nwere ike inwe nnukwu ihe mgbochi kansa. megide TNBC (Eyquem et al., 2017; Liu et al., 2017; Ren et al., 2017b; Dimitri et al., 2022).
Iji CRISPR/Cas9 eme ihe nwere ike ime ka mkpụrụ ndụ CAR-T dịkwuo mma. A maara na Adenosine nwere ihe ndị na-egbochi ahụ ike ma nwee ike ibelata mgbochi ọrịa kansa site na igbochi ọrụ mkpụrụ ndụ T na ịgbalite ndị nnata adenosine A2A (A2AR) (Vigano et al., 2019). Imechi A2AR site na iji CRISPR/Cas9 eme ka arụmọrụ mkpụrụ ndụ CAR-T dịkwuo mma nke ukwuu (Giuffrida et al., 2021). A maara na mkpụrụ ndụ T na-egosipụta ọtụtụ njirimara phenotypic dị iche iche dịka mgbasawanye mkpụrụ ndụ, ọdịiche, nrụgide oxidative na nrụgide genomic. Nnyocha mkpụrụ ndụ T nke dabeere na CRISPR/Cas9 kpughere mmebi nke kinases nnata mkpụrụ ndụ T 25 dị iche iche. N'ime ha, egosipụtala na ihichapụ p38 kinase na-eme ka ọrụ antitumor nke mkpụrụ ndụ T dịkwuo mma, na-egosi na p38 kinase bụ isi ihe na-achịkwa nhazi mkpụrụ ndụ CAR-T (Gurusamy et al., 2020).
Enwere ike ịgbanwe mkpụrụ ndụ T site na iji CRISPR/Cas9 iji mepụta TCRs a na-egosipụta nke ọma. Inyefe mkpụrụ ndụ T ndị a gbanwere mkpụrụ ndụ ihe nketa nye ndị ọrịa egosila na ha na-arụ ọrụ siri ike karịa mkpụrụ ndụ T ndị a gbanwere mkpụrụ ndụ ihe nketa. Ya mere, TCR ndị a gbanwere mkpụrụ ndụ ihe nketa nwere ike ịsọ mpi na TCR ndị a gbanwere mkpụrụ ndụ ihe nketa na ndị ọrịa, nke nwere ike imetụta ikike nke ọgwụgwọ kansa. Iji merie nsogbu a, iji CRISPR/Cas9 wepụ TCR-β nke dị n'ime mkpụrụ ndụ ndị nnata wee nyefee mkpụrụ ndụ TCR-β na ndị ọrịa kansa nwere ike igosi nzaghachi mgbochi ọrịa kansa ka mma na-enweghị mkpa asọmpi mmekọahụ TCR nke dị n'ime (Fan et al. 2018). Ya mere, ewezuga mkpụrụ ndụ T ndị a gbanwere CRISPR, TCRs nwere mmetụta dị ukwuu na antigens tumor karịa mkpụrụ ndụ T ndị a gbanwere TCR. Na mgbakwunye, na ọrịa leukemia dị iche iche, mkpụrụ ndụ T ndị a gbanwere nke γδ TCR+ CRISPR mepụtara na-egosipụta ngosipụta dị elu nke mkpụrụ ndụ CD4+ na CD8+ T karịa nnyefe TCR ọkọlọtọ (Legut et al., 2018). Ya mere, mkpụrụ ndụ T ndị a gbanwere agbanwe nke CRISPR/Cas9 mepụtara nwere ike ịbụ ụzọ dị irè iji merie TNBC.
Integrins bụ molekul njikọ mkpụrụ ndụ dị na transmembranes sel ma na-akwalite njikọ nke mkpụrụ ndụ na extracellular matrix (ECM) (Hamidi na Ivaska, 2018). Nkwụsị nke integrins na-ejikọta ya na mmepe kansa na mbugharị site na ịgbanwe matrix extracellular, na-eduga na ndụ mkpụrụ ndụ kansa na mgbasa (Hamidi na Ivaska, 2018). Nkwụsị integrin nke CRISPR/Cas9 na-ebelata ọganihu tumor, metastasis, na colonization na TNBC. Akọpụtala na mbelata nke integrin a5 (ITGA5) na-ebelata mbugharị mkpụrụ ndụ na ọganihu na kansa ndị ọzọ dị ka kansa akpa ume (Ju et al., 2017), na-atụ aro na integrin a5 nwekwara ike ịbụ isi ihe na-akpata ọrịa TNBC.
Ịmepụta òké ndị na-akpọchi knockout site na iji usoro mkpụrụ ndụ embryonic stem (ES) ọdịnala bụ usoro siri ike, na-ewe oge, na-adịghịkwa arụ ọrụ nke ọma. Ọ na-ewe ọnwa na afọ iji lekwasị anya na mkpụrụ ndụ ES site na njikọta homologous, mụta òké chimeric, wee kewaa ha na òké heterozygous iji mepụta ụmụ homozygous. A chọrọ ịkpa oke mgbagwoju anya iji mepụta òké nwere ọtụtụ mgbanwe mkpụrụ ndụ ihe nketa. Agbanyeghị, ọtụtụ nsogbu apụtala mgbe ejiri òké ndị a na-ewepụ site na iji CRISPR/Cas9 ma ọ bụ site na itinye ihe ndị dị na CRISPR/Cas9 n'ime àkwá otu mkpụrụ ndụ a kpụrụ akpụ. Dịka ọmụmaatụ, iwebata mgbanwe na-emekarị na biallelic loci ma ọ bụghị kpọmkwem mkpụrụ ndụ ihe nketa. A kọrọ na nso nso a na mkpụrụ ndụ ES nke na-eji teknụzụ CRISPR/Cas9 nwere ike itinye mgbanwe biallelic n'otu oge na mkpụrụ ndụ ihe nketa ruru 5 (Nishizono et al., 2021). Maka ebumnuche a, e tinyere mRNA Cas9 na gRNA mkpụrụ ndụ ihe nketa ise kpọmkwem na mkpụrụ ndụ ihe nketa n'otu oge na mkpụrụ ndụ ES. Nke a na-egosipụta nkwa na ịdị irè nke usoro a, ọ bụ ezie na mgbanwe ndị a nwere ike ịbụ na e bufere ha mgbe e mepụtara ahịrị ndị guzobere iji mepụta òké knockout anọ. Ọmụmụ ihe ahụ na-akọkwa nchọpụta dị ịtụnanya: A chọghịzi mkpụrụ ndụ ES iji mepụta òké gbanwere mkpụrụ ndụ ihe nketa. Kama, iji wepụ ngwaahịa mkpụrụ ndụ ihe nketa ụfọdụ, a gbanyere Cas9 mRNA na gRNA n'ime embrayo otu sel. N'ihi ya, e mepụtala ahịrị ndị guzobere knockout nke enwere ike iji n'ụzọ iwu kwadoro mụọ ihe ga-esi na nhichapụ mkpụrụ ndụ ihe nketa pụta na òké (Qin et al., 2016). Ya mere, CRISPR/Cas9 na-enye ohere ka e mepụta òké transgenic iji gwọọ TNBC na ọnụ ala karịa injinịa mkpụrụ ndụ ihe nketa ọdịnala. Site na iji sistemụ CRISPR/Cas, emepụtala ụdị òké knockout ọhụrụ nke nwere ike iweta mgbanwe isi na otu ma ọ bụ karịa mkpụrụ ndụ ihe nketa endogenous na TNBC. Dabere na echiche a, enwere ike ịmepụta ụdị anụmanụ nke TNBC site na iji nhichapụ CRISPR/Cas9 nke BRCA1 na p53, nke na-ebute mfu nke ndozi HR, enweghị ike ịnọ n'ọnọdụ genomic, na ụdị mgbanwe (Annunziato et al., 2020).
Ugbu a, a na-eji ụzọ dị iche iche dịka mammography, magnet resonance imaging (MRI) na ultrasound achọpụta TNBC. Agbanyeghị, ụzọ ndị a nwere oke ụfọdụ. A na-eji mammography achọpụta anụ ahụ ara mpaghara kama metastases, ebe mkpụrụ ndụ kansa agagharịla na akụkụ ahụ ndị ọzọ. Ultrasonography abụghị ụzọ a pụrụ ịtụkwasị obi maka ịchọpụta TNBC (Chen na Lee-Felker, 2023). MRI nwere mmetụta dị elu karịa ultrasound na mammography, mana izi ezi ya dị oke (Sha na Chen, 2022). Biopsy anụ ahụ bụ ụzọ ịwakpo iji chọpụta mkpụrụ ndụ kansa. Agbanyeghị, n'ọnọdụ ụfọdụ, biopsy nwere ike ịhapụ anụ ahụ kansa ma ọ bụrụ na agịga ahụ pụọ ​​n'ebe mmasị ya dị. Na mgbakwunye, ọ dị oke ọnụ ma nwee ike ịkpatara onye ọrịa ahụ nsogbu. TNBC bụ ụdị kansa dị iche iche, yabụ biopsy nwere ike ọ gaghị enye ozi zuru oke gbasara ụdị kansa ahụ. Ya mere, a chọrọ usoro ọhụrụ a pụrụ ịdabere na ya maka ịchọpụta TNBC ngwa ngwa. CRISPR/Cas9 nwere ike ije ozi dị ka ihe ọzọ dị oke nro na nke na-adịghị emerụ ahụ maka nchọpụta kansa ara. Maka nke a, enwere ike iji usoro CRISPR/Cas9 mee ka usoro PCR ka mma maka nchọpụta TNBC. Nke mbụ, a na-eji protein Cas9 na cpf1 nke sistemụ CRISPR ewepụ DNA na-abụghị nke a kapịrị ọnụ, mgbe ahụ protein abụọ a (Cas9 na cpf1) nwere ike ịmata usoro PAM tupu ejikọta ya na DNA ebumnuche (Deepak Singh et al., 2021). . Ya mere, PCR nwere ike ịchọpụta mgbanwe ndị metụtara mmepe kansa. Ọtụtụ ọmụmụ ewerela usoro CRISPR a iji chọpụta mgbanwe dị iche iche na ọrịa kansa dị iche iche (Safari et al., 2019). Ya mere, usoro PCR nke dabeere na CRISPR nwere ike ibelata ịdabere na nchọpụta TNBC na ụzọ ndị na-awakpo dịka biopsy-based immunohistochemistry.
A gosipụtakwala mgbanwe mkpụrụ ndụ ihe nketa na nhazi nke ndị na-anabata homonụ na TNBC (Chen na Russo, 2009). Usoro PCR nke dabeere na CRISPR/Cas9 nke na-eji obere usoro maka nchọpụta ebe nlekọta nwere ike inyocha mgbanwe ndị a nke ọma (Hajian et al., 2019). Ọ nwekwara ike inye ndị na-enye nlekọta ahụike ozi dị mkpa gbasara mgbanwe kpọmkwem na ndị ọrịa TNBC, nke nwere ike inyere aka melite usoro ọgwụgwọ ha. Agbanyeghị, usoro a jikọtara ọnụ nwere oke. Ya mere, a chọrọ mgbalị nyocha sara mbara tupu ejiri usoro CRISPR/Cas-PCR a na nlekọta ahụike maka nchọpụta nke TNBC (Yang et al., 2019).