Ko te CRISPR) he tikanga whakatika ira o te whakatupuranga tuatoru: i hurihia e te CRISPR te ao

Ko te Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) he tikanga whakatika ira o te whakatupuranga tuatoru kua huri i te ao me ōna hua tere-whakaputa. Kua whakamahia hei rongoā i ngā mate koiora me ngā mate pāwera. He maha ngā huakita me ētahi atu prokaryote (pēnei i te archaea) he pūnaha CRISPR/Cas9 hei tiaki i a rātou mai i ngā phages. Kua pūrongohia ka taea e ngā rautaki e hangai ana ki te CRISPR/Cas9 te aukati i te tipu me te haere whakamua o te mate pukupuku ū triple negative (TNBC) mā te aro ki ngā ira ātete kua whakarerekētia pea, te tuhi, me te whakahaere epigenetic. Ka āwhina pea ēnei wawaotanga rongoā ki te whakatika i ngā raruraru uaua pēnei i te ātete rongoā i kitea i roto i te TNBC. I tēnei wā, he maha ngā tikanga e whakamahia ana hei tuku i te CRISPR/Cas9 ki ngā pūtau ūnga, pēnei i te tinana (ngā momo microinjection, electroporation me te hydrodynamic), te huaketo (te huaketo adeno-associated me te lentivirus) me te kore-huaketo (liposomes me ngā nanoparticles lipid). )-particles). Ahakoa kua whakawhanakehia ngā tauira maha hei ako i ngā take ngota o te TNBC, ko te kore o ngā tikanga tairongo me te whai kiko mō te tuku taputapu whakatika ira i roto i te tinana ora ka whakawhāiti i tā rātou tono haumanu. Hei whakarāpopototanga, ka tirohia whānuitia e tēnei arotake te ahunga whakamua, ngā wero, ngā herenga, me ngā tūmanakohanga o te maimoatanga CRISPR/Cas9 mō te TNBC i runga i ngā taunakitanga o nāianei. Ka whakamāramahia hoki e mātou me pēhea te whakakotahitanga o te matauranga horihori me te ako mīhini ka whakapai ake i ngā rautaki CRISPR/Cas9 i roto i te maimoatanga o te TNBC.
Ko te mate pukupuku e tohuhia ana e te wehenga pūtau kāore i te whakahaerehia, te whakararuraru i ngā ara tirotiro huringa pūtau, me ngā whakarerekētanga ira i roto i ngā ira pehi pukupuku (TSG) (Matthews et al., 2022). I roto i ngā momo mate pukupuku rerekē, ko te mate pukupuku ū te momo mate pukupuku tino noa i roto i ngā wāhine, ā, he nui te reiti mate puta noa i te ao (Waks and Winer, 2019). He mate rerekē te mate pukupuku ū me ngā āhuatanga rerekē, pērā i ngā āhuatanga histological me te koiora, te whakaaturanga haumanu me te whanonga, me te urupare ki te maimoatanga (Weigelt et al., 2010). Mā te whakarōpūtanga mate pukupuku ū ka kitea he māramatanga tika mō te tautuhi mate pukupuku ū me te matapae pukupuku. Ko te whakamahinga o ngā tohu koiora noa me ngā āhuatanga haumanu te paearu matua mō te whakarōpūtanga o te mate pukupuku ū (Tsang and Tse, 2019). Ko te matapae me te urupare ki te maimoatanga o te mate pukupuku ū ka pāngia e ngā āhuatanga maha, tae atu ki te aroaro o te kaiwhakawhiwhi estrogen (ER), te kaiwhakawhiwhi progesterone (PR), te kaiwhakawhiwhi tipu epidermal tangata 2 (HER2/neu), me te taumata histological, te momo pukupuku, me te taumata. te rahi me te metastasis o te kōpuku lymph (Al-Tubaiti, 2020). E rima ngā momo ngota ngota o roto o te mate pukupuku ū kua tautuhia, tae atu ki te luminal A, te luminal B, te HER2-enriched, te basal-like, me te claudin-low (Prat et al., 2015). Ko te TNBC he momo ngota ngota o te mate pukupuku kāore e whakaatu i ngā ER, PR, me te HER2 katoa (Yin et al., 2020). E hono ana ēnei āhuatanga mate ki te TNBC, e whakaū ana i tōna tere haere me tōna āhua kaha ake i tētahi atu momo mate pukupuku ū (Feng et al., 2018). I tua atu, i whakamahia e Peru et al (2000) te hangarau microarray hei whakarōpū anō i te mate pukupuku ū me te tautuhi i ngā momo ngota e rima o roto o te mate pukupuku ū (Cadenas, 2012). Ko te mate pukupuku ū basal-like he momo mate pukupuku ū he āhua kino-toru, ā, e hono ana ki te tere haere. Me mōhio ko ngā mate pukupuku ū basal-like katoa ka kitea he hē te tātaritanga hei TNBC, engari 77% noa iho he TNBC; He rerekē, 71–91% o ngā TNBC he rite ki te turanga, e tohu ana he taupatupatu ēnei momo mate pukupuku ū e rua, ā, he rerekē ngā whakarōpūtanga (Wang D.-Y. et al., 2019). Nā tēnei ka puta he hiahia ki te whakaahua i te rerekētanga o te TNBC hei whakamārama i te matapae me te tautuhi i ngā urupare pea ki ngā maimoatanga o nāianei me ngā maimoatanga o muri mai. Hei tāpiri, ko te TNBC te 15–20% o ngā mate pukupuku ū katoa, ā, he nui ake te kitea i roto i ngā wāhine kei raro iho i te 50 tau te pakeke. Kua pūrongohia ngā whakarerekētanga BRCA1, BRCA2 rānei i roto i te 20% o ngā take TNBC (Xie et al., 2017; Tzikas et al., 2017). , 2020). Kua whakaatuhia hoki e ngā rangahau he taiao ārai mate motuhake tō te TNBC, tae atu ki ngā taumata tiketike o ngā mea whakatipu endothelial vascular, ngā macrophage e pā ana ki te pukupuku (TAM), ngā lymphocytes e uru ana ki te pukupuku (TIL), me ētahi atu ngota e uru ana ki te tipu me te hekenga o te pukupuku. Nō reira, he mea nui te mārama ki te taiao moroiti o te TNBC mō tōna matapae me te maimoatanga (Fan and He, 2022).
Hei aromatawai i te matapae o te TNBC me te whakarite kia whai hua te maimoatanga, he mea nui te tautuhi tika, e ahu mai ana i te immunohistochemistry (IHC) hei kite i te ER, PR me te HER2, me te mammography hei kite i ngā neoplasms o te ū. Heoi, kāore e taea e te mammography te whakaatu tika i ngā āhuatanga o roto i te puku pēnei i te necrosis me te fibrosis (Deepak Singh et al., 2021). Nā te mea ko te matapae kino me te tautuhi he aukati i ngā tākuta ki te tohu i ngā rongoā tika (Chaudhary, 2020), he maha ngā rautaki e whakamahia ana hei whakapai ake i te tiaki i ngā tūroro me te TNBC. I tēnei wā, e rua ngā rongoā, ko te doxorubicin me te cyclophosphamide, e whakamahia ana i roto i ngā tūroro me te TNBC, ā, kua whakaatuhia he hua pai. Hei tāpiri, ka whakamahia ētahi atu rongoā platinum, pēnei i te carboplatin me te cisplatin (Sikov et al., 2015). Hei tāpiri, kua whakamahia ngā aukati PARP pēnei i te Olaparib, Velaparib, me te PF-01367338 hei rongoā chemotherapy pea mō te maimoatanga o te TNBC (Ishino et al., 2018). Mai i te mea kua pūrongohia ko ngā ara tohu Wnt/b-Catenin, NOTCH, me Hedgehog te takenga mai me te haere whakamua o te TNBC, ko te arotahi ki ngā rongoā ki ēnei ara he rautaki nui pea (Aysola et al., 2013). Ahakoa ko te pokanga, te rongoā irahiko me te rongoā mate pukupuku te kaupapa matua o te maimoatanga mō te TNBC i ēnei rā, he nui te ahunga whakamua kua puta i roto i te whakawhanake i ngā maimoatanga hou tae atu ki te rongoā arotahi, te rongoā ārai mate, ngā taputapu whakatika ira e pā ana ki te CRISPR pērā i te Cas9n, dCas9. , CRISPR/Cas12, te whakatika tuatahi, me te rongoā ira e pā ana ki te CRISPR/Cas9. I konei ka arotahi tātou ki ngā taputapu whakatika ira e pā ana ki te CRISPR e whakamahia ana i roto i te maimoatanga o te TNBC. I roto i ēnei, kua arohia whānuitia te CRISPR/Cas9.
Ko Cas9n, e mōhiotia ana ko Cas9 nickase, he momo ira i hangaia o te pūmua Cas9 i ahu mai i te pūnaha whakatika ira CRISPR/Cas9 (Gupta et al., 2019). I tōna āhua taketake, e rua ngā rohe nuclease kei roto i te pūmua Cas9: RuvC me HNH, ko te mahi matua ko te tapahi i ngā aho DNA e rua. Heoi, i roto i te Cas9n, ko tētahi o ngā rohe nuclease, HNH, kua whakarerekētia te ira, ā, ka kore e mahi. Nō reira, ka noho kore mahi te rohe HNH o Cas9n. Ko te rohe RuvC anake e mahi ana, e āhei ai a Cas9n ki te tapahi, ki te hanga rānei i ngā whatiwhatinga i runga i ngā aho DNA takitahi (Trevino and Zhang, 2014). Ki te whakataurite ki a Cas9n, ka taea e Cas9n te whakaiti i ngā pānga kore-taunga me te whakapai tika i ngā tikanga whakatika pūtau. Ka taea te whakamahi i a Cas9n hei whakaoti rapanga maha, pēnei i te hanga whatiwhatinga i ngā wāhi motuhake i roto i te DNA aho-rua. Mō tēnei kaupapa, e rua ngā ngota Cas9n i whakakotahihia, ā, i whakamahia ngātahitia (Yee, 2016). Ko te Mixed lineage kinase 3 (MLK3) he pūmua kinase whakahohe-mitogen e mahi ana hei kaiwhakahaere matua i te wā o te metastasis TNBC (Cronan et al., 2012).
Ka taea e MLK3 te whakahohe i ngā ara tohu maha e arahi ana ki te metastasis TNBC. Hei tauira, ko te ara c-Jun N-terminal kinase (JNK) e whakahaere ana i te nekehanga o te pūtau me te whakahekenga o te matrix extracellular, ā, ka whakahohe a MLK3 i te ara JNK, ā, ka whakarei ake i te nekehanga o te pūtau me te whakawhiwhi i ngā āhuatanga whakaeke (Rattanasinchai and Gallo, 2016). Ka whakahaere anō hoki a MLK3 i te EMT. Hei mahi i tēnei, ka whakahohehia e ia ngā take whakawhiti i raro i te mato pērā i a Snail, Slug me Twist. Ka aukatihia e ēnei take te whakaaturanga o ngā tohu epithelial me te whakatairanga i te whakaaturanga o ngā tohu mesenchymal, ka arahi ki te whiwhi i te momo metastatic (Casalino et al., 2023). Kua kitea te mahi a MLK3 ki te whakahou i te ECM me te whakahohe i ngā protease pērā i ngā matrix metalloproteinases (MMP) e whakaiti ana i te ECM. Ka āwhina tēnei i te whakaekenga o te pūtau pukupuku me te horapa ki ngā wāhi tawhiti (Katari et al., 2019). Nō reira, kua whakaatuhia e ngā rangahau o mua he mahi nui tā MLK3 i roto i te TNBC. I whakamahia e Rattanasinchai rāua ko Gallo ngā tauira TNBC hei ako i te mahi a MLK3, ā, i kitea e rātou he whakatairanga i te whanaketanga o te mate pukupuku mā roto i ngā ara tohu motuhake. I whakamahia e rāua a CRISPR/Cas9n hei whakatika i te MLK3, ā, i kitea he whakahekenga nui o te horapa o te TNBC ki te pūtau (Rattanasinchai rāua ko Gallo, 2016).
Ko te dCas9, e kiia ana ko te Cas9 kore mahi, he momo whakarerekētanga o te pūmua Cas9. Kāore i rite ki te Cas9 kaha, kāore te dCas9 i te mahi endonuclease, ā, kāore e taea te whakaputa i ngā wehenga aho rua o te DNA. Nō reira, ka taea te whakamahi i te dCas9 ki te aro tika ki ngā rohe motuhake o te ira me te kore he huringa, he whakarerekētanga rānei ki te raupapatanga DNA (Wang et al., 2016). I roto i te dCas9, e rua ngā pūmua endonuclease, ko RuvC me HNH, ka whakakorengia mā te pehi i ngā toenga waikawa amino nui. (Richter et al., 2016). Ahakoa te kore o te mahi tapahi DNA, he maha ngā mahi nui a te dCas9 i roto i te rangahau ira me te hangarau koiora. Hei tauira, ka taea te whakaatu i ngā rohe motuhake o te ira, ka āwhina i te whakahaere i te whakawhitiwhiti kōrero, ā, ka whakatairanga i ngā whakarerekētanga epigenetic (Brocken et al., 2018).
He mahi motuhake, he mahi nui hoki tā te take whakawhiti ZEB1 (te herenga o te zinc finger E-box homeobox 1) ki te whakahaere i te whakawhiti epithelial-mesenchymal (EMT), he tukanga pūtau e puta ana i te wā o te whanaketanga o te kukune, te whakatikatika kiko, me te whanaketanga o te mate pukupuku. Kei roto i tēnei ko te whakawhiti o ngā pūtau epithelial ki ngā pūtau mesenchymal, ka puta he huringa i roto i te āhua o te pūtau, te nekehanga, te whakaekenga, me ētahi atu. (Wu et al., 2020). Ka aukati a ZEB1 i ngā tohu epithelial maha, pērā i te E-cadherin me te Occludin, e pupuri ana i te piri o te pūtau-pūtau me te polarity o ngā pūtau epithelial i roto i te TNBC (Moreno-Bueno et al., 2008). Hei tāpiri, ka whakahohe a ZEB1 i ētahi tohu pērā i te N-cadherin, te vimentin me te fibronectin (Konradi et al., 2014) ā, ka whakahaere hoki i ngā ira e whai wāhi ana ki te whakahou i te cytoskeletal pērā i te Rho GTPases me te matrix metalloproteinases (MMPs) (Huang). et al., 2014). 2022), hei tāpiritanga, ka pāngia hoki e ia ngā ara tohu maha pērā i te transforming growth factor-β (TGF-β), te tohu Wnt, me ētahi atu, ā, ka whakatairanga i te whakaekenga o te pukupuku me te horapa ki roto i te TNBC (Chen et al., 2016). ). He maha ngā rangahau me ngā taunakitanga pūtaiao e whakaatu ana he nui te kaha o te ZEB1 hei kaihoko whai hua mō te kimi me te wawaotanga rongoā o te TNBC. I roto i tētahi rangahau tata nei, i whakamahia e Waryah et al. tētahi tauira TNBC, ā, i tutuki te pehitanga katoa o te ZEB1 e dCas9. Nō reira, i kitea e rātou he tino teitei te motuhaketanga me te tata katoa te aukati o te ZEB1 i roto i ngā āhuatanga in vivo (Waryah et al., 2023).
He taputapu whakatika ira a CRISPR/Cas12 e kiia nei ko CRISPR/Cpf1. I ahu mai i te pūnaha CRISPR/Cas, ko te kupu Cas12 e pā ana ki te pūmua 12 e hono ana ki a CRISPR (Bharathkumar et al., 2022), he tino rite ki a Cas9. Ko te rerekētanga anake ko te mea kei roto ko te pūmua Cas12. Ki te whakatauritea ki a Cas9, he mahi ahurei a Cas12 pēnei i te whakaputa pito piri i te wā o te tukanga whakatika ira, engari ko Cas9 e whakaputa pito pūngoi ana (Wang et al., 2021). Ka whai wāhi tēnei āhuatanga o Cas12 ki tana hangarau whakahaere DNA motuhake. Hei pūmua e kaha ana ki te kite tika me te tapahi i te DNA ūnga, he tino maha ngā mahi ka taea, ka waiho hei taputapu whai hua mō ngā momo tono, tae atu ki te whakatika ira (Pickar-Oliver me Gersbach, 2019).
He rite ki ētahi atu momo CRISPR, ka taea hoki e CRISPR/Cas12 te patu, te whakahohe rānei i ngā ira i roto i te TNBC, me te arotahi ki ngā ira e whai wāhi nui ana ki tōna pathogenesis, ki tōna urupare rānei ki te maimoatanga (Yang and Zhang, 2023). Hei whakatutuki i tēnei tukanga, i whakawhanakehia he RNA aratohu (gRNA) e tuku ana i a Cas12 ki te ira ūnga. Kia herea a Cas12 ki tana ūnga, ka whakaurua mai he wehenga aho-rua ki roto i te DNA, ā, ka whakahohe i ngā tikanga whakatika DNA. I roto i te tukanga whakatika, ka uru pea ngā nucleotides hē, ka arahi ki ngā whakarerekētanga ira me te ngaronga o te mahi (Zhang et al., 2021a). Hei tāpiri, i whakamahia hoki he putanga whakapai ake o te whākōkī Cas12 (e kiia nei ko dCas12) hei whakahohe i ngā ira. Mā te whakakotahi me te whakahohenga whakawhiti, ka taea te whakaoho i ētahi ira, ā, ka whakahohe i a rātou. He nui te kaha o tēnei hangarau ki te whakatairanga i te whakahohenga o ngā ira pehi pukupuku (Sultan et al., 2022).
He hangarau whakatika ira hou, he tino pai rawa atu hoki te Prime Editing. Ka taea e ia te whakarerekē i te DNA o tētahi rauropi me te tino tika (Chen and Liu, 2023). Ka tutuki te Prime Editing mā te whakauru i ngā wāhanga matua e rua: he whākōkī CRISPR/Cas9 kua whakarerekētia me te reverse transcriptase. Ko te mahi a te whākōkī CRISPR/Cas9 he whiriwhiri i ngā wāhi tika i roto i te ira, ko te reverse transcriptase ia e āwhina ana ki te whakarerekē i te DNA i te wāhi ūnga (Hassan et al., 2021). I roto i te tikanga whakatika ira, ko te taahiraa tuatahi ko te waihanga i tētahi RNA aratohu whakatika ira (pegRNA) (Standage-Beier et al., 2021). Kei roto ko te raupapa ūnga me te tauira RNA e rite ana ki te wāhi whakatika e hiahiatia ana i roto i te DNA ūnga. Kātahi ka whakaurua te pegRNA ki roto i ngā pūtau ūnga me tētahi primer editing nuclease (PE2). Ko te PE2 he pūmua hanumi kei roto ko tētahi whākōkī Cas9, he reverse transcriptase, me tētahi urutau whakatika ira (Martín-Alonso et al., 2021). I roto i te pūtau, ka rapu te pegRNA me ngā matatini PE2 i te DNA motuhake e hiahia ana rātou ki te whakarerekē. Ka tapahia e te whākōkī Cas9 te DNA ka hangaia he tauira kei roto ko ngā aho kotahi (Choi et al., 2022). Ka whakamahia e te transcriptase whakamuri tēnei tauira hei whakarite i te DNA mō te whakatika. Haunga te tārua i te DNA, kei roto hoki ngā tohutohu mō te whakatika i te tauira RNA. Hei whakamutunga, ka whakamahia te aho DNA hou hei tauira hei whakatika i te pakaru o te DNA, ka hua ake he raupapa DNA kua whakarerekētia kei roto te whakarerekētanga e hiahiatia ana (Ochoa-Sanchez et al., 2021). Ka taea e ngā tikanga whakatika tuatahi te arahi ki ngā whakarerekētanga whānui i roto i ngā ira. Ka taea e ia te aro ki ngā whakarerekētanga pūwāhi, ngā whakaurunga, ngā mukunga, tae noa ki ngā whakakapinga ira (Anzalone et al., 2019; Chen and Liu, 2023). He maha ngā painga o te whakatika tuatahi i ngā hangarau whakatika ira o mua. Kei roto i ēnei ko te pikinga ake o te tika, te whakaiti i ngā pānga kore-taumata, me te kaha ki te whakatika i te DNA me te kore e whakawhirinaki ki ngā pakaru aho DNA takirua (Anzalone et al., 2020).
I te tīmatanga, i whakawhanakehia te hangarau CRISPR/Cas9 hei tiaki i ngā huakita mai i te whakawhiti plasmid me te mate phage, ā, i muri mai ka whakamahia anō hei taputapu whai hua mō te arotahi DNA e hangai ana ki te RNA mō te whakatika ira (Jiang and Doudna, 2017). Hei tāpiri, kua pūrongohia te pūnaha CRISPR/Cas9 kei roto i te 50% me te 87% o ngā ira huakita me ngā ira tawhito (Ishino et al., 2018). He taputapu pea te CRISPR/Cas9 mō te muku, te whakauru me te whakatika i tētahi raupapa ira rerekē mā te whakamahi i ngā momo in vivo me in vitro (Sabit et al., 2021). I tua atu, kua whakaatuhia ko CRISPR/Cas9 he wāhanga o te pūnaha ārai mate urutau nā tōna motuhaketanga mō ngā ira whainga e hiahiatia ana (Chen and Zhang, 2018).
Kei roto i te CRISPR/Cas9 ko Cas9 me te RNA ārahi kotahi (sgRNA). He endonuclease te Cas9 i titoa mai i ngā wāhanga pūmua maha. Hei tāpiri, he āhuatanga hanganga me te āhua ahurei tō Cas9 (Pacesa et al., 2022) nā te mea e rua ngā pūwero: te mōhiotanga (REC) me te nuclease (NUC). Kua wehea anō te pūwero REC kia toru ngā rohe: REC1, REC2, me ngā porowhita piriti (Cromwell et al., 2018). E toru ngā pūwero o te NUC, arā, ko RuvC (RuvC I, RuvC II, RuvC III), HNH me te rohe taunekeneke protospacer adjacent motif (PAM) (Pikitia 1) (Song et al., 2016). E rua ngā wāhanga o te sgRNA, tae atu ki te CRISPR RNA (crRNA) me te transcode small RNA (tracer RNA) (Pikitia 1). Ka hono te sgRNA i te pūmua Cas9 ki ngā hononga honohono hei hanga i tētahi matatini hohe, e mōhiotia ana ko te matatini whaihua (Richter et al., 2012). Ko te crRNA he takirua turanga nucleotide 18–20 e whai wāhi nui ana ki te mōhio ki ngā raupapatanga DNA ūnga. Hei tāpiri, ka takirua te crRNA ki te DNA ūnga, ā, ka mahi te tracrRNA hei tūāpapa mō te Cas9 nuclease hei here i te DNA ūnga (Manghwar et al., 2019). I tētahi atu taha, e 3 ngā nucleotide o te raupapatanga PAM, e whakaū ana, e tohu ana, e takawaenga ana hoki i te herenga o te matatini whaihua ki te DNA. He mahi whakakorikori kei ngā waeine matatini pūmua Cas9, arā, ko RuvC me HNH (Richter et al., 2012; Asmamaw and Zawdie, 2021). Ka tapahia e te rohe HNH nuclease o te waeine Cas9 te aho DNA e hono ana ki te crRNA. Ka tapahia e te rohe RuvC nuclease ētahi atu aho DNA, ka whakaputa i ngā wehenga aho takirua (DSB), ā, muri iho ka whakahohehia ngā tikanga whakatika wehenga DNA rerekē e rua (Jiang and Doudna, 2017) (Pikitia 1).
Pikitia 1. Tirohanga Whānui o CRISPR/Cas9 (A). Ngā wāhanga o te pūnaha CRISPR/Cas9: (i). Ko te endonuclease Cas9 te kawenga mō te tapahi i te raupapa DNA ūnga, (ii) he RNA aratohu kotahi (sg) i puta mai i te hanumitanga o ngā chimera crRNA me ngā tra-crRNA. (rua). Kei roto i te Cas9 ētahi wāhanga maha pērā i te Rec I, Rec II, te NUC lobe (he wāhanga iti a HNH me Ruv C) me te rohe taunekeneke PAM (C) me ngā mahi e rite ana. Ka tapahia e te matatini pūmua CRISPR/Cas9 ngā raupapa DNA ki ngā āhua kore-tāpiri me ngā āhua tāpiri (D). Ka whakatikahia e te CRISPR/Cas9 te ira i roto i ngā wāhanga e toru: te mōhio, te tapahi me te whakatika. Ka ārahi te sg-RNA kua hoahoatia i a Cas9, ā, ka mōhio ki te raupapa e hiahiatia ana mā te wāhanga tāpiri crRNA. Ka mōhio a Cas9 ki te raupapa PAM i te 5′-NGG-3′, ā, ka whakarewahia te DNA, ka hanga he ranu DNA-RNA, ka whakahohe i te wehenga. Ka tapahia e te rohe HNH o Cas9 te aho tāpiri, ā, ka tapahia e te rohe RuvC te aho kore-whakapiri. Ka whakatikahia e CRISPR/Cas9 te dsDNA mā te wawahi i roto i ngā huarahi e rua: te hononga pito kore-taurite (NHEJ) me te whakatikatika homology-directed (HDR). Ka whakatikahia e NHEJ te DNA aho-rua i te korenga o te DNA taurite ke mā te tukanga whākōkī, he tikanga hapa-ngāwari ka taea te whakauru, te tango rānei i ngā raupapatanga DNA matapōkere. He tino motuhake te HDR, ā, me whai tauira DNA taurite.
Kei roto i ngā ara whakatika e whakahaerehia ana e CRISPR/Cas9 ngā tikanga hono mutunga kore-homologous (NHEJ) me ngā tikanga whakatika e ārahihia ana e te homology (HDR). He ara hapa te NHEJ nā te mea he whakaurunga, he mukunga rānei, ā, kāore e hiahiatia he tauira i te wā o te tikanga whakatika. Ka whakamahia e ia ngā nucleotides matapōkere hei whakaputa i ngā pūmua paerewa (Abbasi et al., 2021). Kei roto i tēnei pūnaha whakatika e whā ngā matatini pēnei i te matatini KU, te matatini pūmua whakawhiti-whakakī momo 4 (XRCC-4), te whākōkī tukatuka mutunga DNA, me te DNA-PKcs protein kinase (Abbasi et al., 2021). E rua ngā waeine o te pūmua matatini KU, ko Ku 70 me Ku 80, ā, he mahi nui tāna i roto i te tikanga NHEJ, nā te mea ka tīmata te tikanga whakatika mā te herea o ngā waeine e rua (Ku 70 me Ku 80) ki ngā pito pūngoi, tata pūngoi rānei o te DNA ūnga (Abbasi et al., 2021), hei tūāpapa hei whakauru atu i ētahi atu āhuatanga e pā ana ki te NHEJ ki te wāhi i whara ai (Yang et al., 2020). Ko te XRCC-4 me te DNA ligase he mea tito mai i te 334 me te 911 ngā waikawa amino, ia, ā, ko te matatini XRCC4-DNA ligase IV ka whakaoho i te herenga o ngā pito DNA (Chatterjee et al., 2015). Ko te whākōkī tukatuka mutunga DNA, e mōhiotia ana ko te polynucleotide kinase 3′-phosphate, he whākōkī tukatuka mutunga DNA. Ka taea e ia te tango i te rōpū 3′P i roto i te DNA me te whakapūtā i te rōpū 5′OH i te wā e whakatika ana i te DSB. Kei roto hoki i tēnei ko te whakatika i ngā whati aho kotahi (SSB) mā te whakamahi i te ara whakatika SSB (Chatterjee et al., 2015). Ko te protein kinase DNA-PKcs he protein kinase e whakawhirinaki ana ki te DNA e tito ana i tētahi wāhanga whakakorikori o ngā PIKK (phosphatidylinositol 3-kinase-related kinases) me te whānau ataxia telangiectasia mutated (ATM), tae atu ki ngā ATR e pā ana ki te ATM me te Rad3. Ngā whati aho (DSB) me ngā whati aho kotahi (Yue et al., 2020; Peng et al., 2016).
He tikanga whakatika tika ake, he tikanga pai ake hoki te HDR nā te mea ka tāruatia ngā mōhiohio mā te whakamahi i te āhua tuturu o te DNA takirua, ahakoa me noho mai ngā chromatids tuahine. Ka puta tēnei i te wā o te wāhanga S/G2 o te huringa pūtau o ngā kararehe. Ka puta te HDR i roto i ngā momo rewena, engari he mea nui te NHEJ i roto i ngā kararehe (Burma et al., 2006; Abbasi et al., 2021). Kei te whakaatuhia te tikanga whakatika katoa i te Pikitia 1.
I te mea ko te TNBC e puta mai ana i ngā āhuatanga ira me ngā āhuatanga epigenetic, ko te whakatikatika i ngā āhuatanga kino o te ira/epigenomic mā te whakamahi i te CRISPR/Cas9 he huarahi rongoā tika pea (Chen et al., 2019). Hei tāpiri, ko ētahi o ngā āhuatanga whakawhiti e uru ana ki te whakahaere whakawhiti motuhake-pūtau ka whakaatu pea i ngā āhuatanga ahurei o ngā pūtau matepukupuku, e kī ana ko te whakahaere whakawhiti he huarahi pai pea mō te maimoatanga matepukupuku (Drost et al., 2017). Mā te whakamahi i ēnei āhuatanga ngota ngota o ngā pukupuku, pērā i ngā hapa ira, epigenetic me te whakawhiti, mō te whanaketanga rongoā ka taea te whakapai ake i ngā putanga haumanu me te whakaiti i ngā utu tirotiro. Ko te CRISPR he taputapu whakatika ira pea e kore e taea te kite me te tautuhi i ngā whāinga ira e puta ai te matepukupuku, engari ka taea hoki te whakamahi hei whakatika, hei pehi, hei whakarerekē i ngā oncogenes i roto i ngā pūtau tangata (Ripanga 1) (Ahmed et al., 2021). He maha ngā tirotiro CRISPR kua mahia hei rapu i ngā ira e pā ana ki ngā pehi pukupuku, ngā oncogenes, me te ātete rongoā. Kei te whakaatuhia te whakamahinga o CRISPR/Cas9 hei whakatika i ngā momo oncogene TNBC i te Pikitia 2.
Pikitia 2. Te whakatika ira o ngā momo oncogene TNBC mā te whakamahi i te CRISPR/Cas9, ā, ka iti ake te tipu o te pukupuku me te horapa ki te pūtau. Kei roto i ēnei oncogene ko CDK7, NAT1, UBR5, YTHDF2, ITGA9, CXCR4 me CXCR7, Crypto1, ROR1 me ST8SIA, e whai wāhi ana ki te putanga mai me te horapa ki te pūtau o te TNBC.
Ko te hekenga o ngā pūtau matepukupuku, te whakaekenga, me te whakawhiti epithelial-mesenchymal (EMT) e pā ana ki te ITGA9. Kua whakaatuhia e ngā rangahau o mua ko te ITGA9 he kaitākaro matua i roto i te ara Notch, ā, he mahi whakamere tana i roto i te metastasis rhabdomyosarcoma (Molist et al., 2020). Hei tāpiri, i kitea he hononga tata te ITGA9 ki te matapae tūroro i roto i ētahi momo pukupuku, tae atu ki te matepukupuku ū (Wang Z. et al., 2019). Hei tāpiri, i whakaatuhia e te tātaritanga bioinformatics o te ITGA9 he nui ake te whakaaturanga i roto i te TNBC i ētahi atu momo pukupuku ū. Ko ngā taumata ITGA9 teitei e pā ana ki te metastasis pukupuku me te hokinga mai i roto i ngā tūroro TNBC. Ko te whakakorenga o te ITGA9 e CRISPR/Cas9 i hua ake ai ngā āhuatanga rite ki te pūtau take matepukupuku (CSC), te angiogenesis pukupuku, te tipu pukupuku, me te whakaiti i te metastasis mā te whakatairanga i te whakahekenga β-catenin i roto i te TNBC (Wang Z. et al., 2019).
He mema a Crypto-1 nō te whānau TGF-β, ā, he mea nui mō te whanaketanga wawe o te kukune, te tiaki i te pūtau take, me te metastasis mate pukupuku (Ishii et al., 2021). E mōhiotia ana ko Tdgf-1, he pūmua tohu GPI-anchored oncogenic e whai wāhi ana ki te whakahaere i te hanganga o te raina tuatahi, te mesoderm me te endoderm, me te whakatūnga o te kore taurite maui/matau i roto i te whanaketanga o ngā whekau tinana i te wā o te whanaketanga kukune (Zhang et al., 2021b). Hei tāpiri, kua whakaatuhia te whai wāhi a Cripto-1 ki te whakawhiti epithelial-mesenchymal (EMT) hei tohu pūtau take (Zhang et al., 2021b). He mea nui te EMT ehara i te mea mō ngā tukanga maha pēnei i te whanaketanga kukune, te fibrosis me te whakaora patunga, engari mō te whakaekenga mate pukupuku me te metastasis. Hei tāpiri, kua whakaatuhia te taunekeneke a Cripto-1 me ngā kaiwhakawhiwhi Notch e whā, ā, ka whakarei ake i tō rātou pakeketanga i muri i te whakamaoritanga (Brandstadter and Maillard, 2019). E mōhiotia ana ko te ara tohu Notch te mea e whai wāhi ana ki te pupuri i ngā pūtau matepukupuku ū tangata. Kua whakaatuhia e ngā rangahau ko te whakakorenga o Crypto-1 e whakahaerehia ana e CRISPR/Cas9 ka pehi i te tipu me te horapa o te matepukupuku. Nō reira, ka noho pea a Crypto-1 hei whāinga rongoā nui mō te TNBC (Castro et al., 2015).
He maha ngā mahi a te pūmua XCL12 me tana kaiwhakawhiwhi chemokine CXC (CXCR4 me CXCR7) i roto i te tipu haere, te tipu, te hekenga me te whakaekenga o ngā pūtau matepukupuku (Wu et al., 2015). Kua honoa ēnei chemoreceptors ki te whanaketanga o te TNBC mā roto i ngā ara tohu maha i roto i ngā tauira in vivo me in vitro (Wu et al., 2015). Hei tāpiri, ko te whakahohenga o CXCR4 me CXCR7 e honoa ana ki te ngoikore ake o te metastasis me te matapae kino i roto i te TNBC (Karn et al., 2022). Nō reira, ko te whakakorenga o CXCR4 me CXCR7 ka noho hei ira whainga rongoā whai hua mō te rongoā i te matepukupuku ū, tae atu ki te TNBC. I whakamahia e te rangahau a Yang et al. Yang et al (2019) te CRISPR/Cas9 hei whakakore tahi i ngā ira CXCR4 me CXCR7, ā, i kitea he nui te aukati i te tipu haere, te tipu, te hekenga me te whakaekenga o te TNBC (Yang et al., 2019).
I kitea te pikinga o ngā taumata o te miR-3662, he oncogene TNBC, i roto i ngā kiko mate pukupuku o te ū (Yi et al., 2022). Kua whakaaturia te whakakorenga o te miR-3662 ki te aukati i te tipu me te metastasis o te pukupuku o te ū i roto i te tinana ora me te tinana vitro (Agarwal and Gupta, 2021). He aukati kaha te HBP-1 i te tohu Wnt/-catenin, ā, ko ia pea te take o te tipu o ngā pūtau TNBC e whakahaerehia ana e te miR-3662. Nō nā tata nei, i kitea e Yi et al. ko te tuaka miR-3662-HBP1 te kaiwhakahaere i te ara tohu Wnt/-catenin i roto i ngā pūtau TNBC (Yi et al., 2022). Nā tōna whakaaturanga motuhake ki te pukupuku, ko te miR-3662 pea te whāinga rongoā pea mō te TNBC. Nō reira, ko te whakakorenga o te miR-3662 e whakahaerehia ana e te CRISPR/Cas9 he huarahi pai pea mō te whakawhanake i ngā rongoā hou mō te maimoatanga o te TNBC.
Ko te UBR5 he nucleophosphoprotein 300 kDa kua tautuhia hei kaiwhakahaere matua o te tumorigenesis, te metastasis, me te urupare ārai mate i roto i ngā momo mate pukupuku (Shearer et al., 2015; Fu et al., 2023). Kua pūrongohia te pikinga ake o te UBR5 i roto i ngā tauira TNBC, ā, ka whakaoho i te mahi ERα mā te whakaoho i te tipu haere mā roto i tana mahi ubiquitin ligase (Bolt et al., 2015). I whakaatuhia hoki e ngā rangahau raupapatanga exome katoa o ngā tauira TNBC tuatahi te pikinga ake o te whakaaturanga o te UBR5, e whakaatu ana i tana mahi i roto i te whanaketanga o te TNBC. I tua atu, ko te mukunga o te UBR5 e peia ana e CRISPR/Cas9 i whakaatu i te aukati nui o te metastasis TNBC me te tipu i roto i ngā tauira kiore whakamātautau. I tua atu, ko te whakaurunga o te UBR5 ki roto i te tauira kiore momo-mohoao i whakahoki mai i tana mahi katoa, engari kāore i kitea tēnei pānga i roto i ngā momo mutant kua whakakorea (Liao et al., 2017). Ko te korenga o te UBR5 e pā ana ki te pikinga o te apoptosis, te necrosis, me te aukati i te tipu o te pukupuku i roto i te TNBC nā te ngoikore o te angiogenesis. Nā te ngaronga o te UBR5, ka whakaitihia te horapa o te pukupuku ki ngā whekau tawhiti, ā, ka puta te EMT rerekē i roto i te UBR5 mā te whakaiti i te whakaaturanga E-cadherin (Zhang and Weinberg, 2018). Nō nā tata nei, i whakaaturia te UBR5 he mea tino nui i roto i te whakawhitiwhiti PDL1 i puta mai i te IFN-γ i roto i te TNBC nā te korenga o te mahi ubiquitination E3. I whakaatuhia e te tātaritanga transcriptome RNA he pānga pūnaha pea a UBR5 ki ngā ira e pā ana ki te ara IFN-γ me te whakatairanga i te whakawhitiwhiti PDL1 mā te whakanui ake i ngā taumata whakahohenga o te RNA kinase pūmua (PKR) me ōna kaiwhakawhiti tohu me ngā kaiwhakaoho. whakawhitiwhiti 1 (STAT1) me te interferon regulatory factor 1 (IRF1). Heoi, he pānga whakaora tahi te tangohanga tahi a te CRISPR/Cas9 i te UBR5 me te whakaaturanga PD-L1 i te aukati anake, me ngā pānga nui ki te taiao moroiti o te pukupuku (Wu et al., 2022). Nō reira, he taputapu nui pea a CRISPR/Cas9 hei aukati i te mahi a te UBR5, ā, mā reira ka pehi i te metastasis TNBC me te tipu o te pukupuku.
Ko te ROR1 he pūmua transmembrane momo I e whakaatuhia ana i te wā o te mate pukupuku me te whanaketanga o te kukune, ā, kua tautuhia hei pūmua oncofetal (Nicholas Borcherding, 2014). Ko te whanonga whakaeke o ngā momo mate pukupuku tangata e hono ana ki te pikinga ake o te ROR1. Kua puta he hua pai mai i ngā rangahau in vivo me in vitro e pā ana ki ngā matū rongoā e aro ana ki te ROR1 (Chien et al., 2016). Ko ngā taumata teitei o te mRNA ROR1 i roto i ngā koiora kiko ū kua honoa hoki ki ngā pukupuku ū rite-ki-te-papa (BL) kaha me tō rātou hekenga ki ētahi atu wāhanga o te tinana. I tua atu, kua tautuhia te nui rawa o te ROR1 hei tohu matapae mō te whanaketanga o te TNBC (Chien et al., 2016). Heoi, ko te whakamutu i te ROR1 mā te whakamahi i te CRISPR/Cas9 hei pehi i te tipu TNBC me te metastasis he rautaki whai hua.
Ko te tukanga sialylation e pā ana ki te tāpiri i te waikawa sialic ki ngā glycoconjugates, e whakakorikoria ana e ngā sialyltransferases (STs). Nō te whānau ST a ST8SIA1, ā, he mahi nui tāna i roto i te pathogenesis o ngā mate maha pērā i te lymphocytic leukemia me te mate pukupuku colorectal (Chang et al., 2018). E whakaatu ana te tātari raupapa RNA he nui te whakaaturanga o ST8SIA1 i roto i te kiko ū o ngā tūroro TNBC, ā, he hononga pai ki ngā whakarerekētanga i roto i te ira pehi pukupuku p53, ka whai wāhi pea ki te pathogenesis o te TNBC (Battula et al., 2017). Hei tāpiri, kei te whai wāhi a ST8SIA1 ki te metastasis me te hokinga mai o te TNBC, e kī ana he mahi nui tāna i roto i te putanga mai me te whanaketanga o te TNBC. I roto i tētahi tauira in vitro o TNBC, i whakaaturia te whakakorenga o ST8SIA1 e CRIPSR/Cas9 ki te aukati i te tipu me te metastasis (Battula et al., 2017). E whakaatu ana tēnei he taputapu nui pea a CRISPR/Cas9 mō te aukati i te mahi a te oncogene ST8SIA1 i roto i te maimoatanga o te TNBC.
Ko te NAT1 he whākōkī pākia e whakaoho ana i te hanganga o ngā matū xenobiotic wāhanga II, ā, e whakaatuhia ana i roto i te nuinga o ngā kiko tangata. Ka taea e te NAT1 te whakamahi i te cofactor folate hei aro ki te acetyl-CoA (acetyl-CoA) ahakoa te korenga o te matū amine kakara (Stepp et al., 2015; Laurieri et al., 2014). Kua whakaatuhia e ngā rangahau ko te NAT1 e whakahaere ana i te mahi a te matrix metalloproteinase 9 (MMP9) i roto i ngā tauira pūtau matepukupuku ū, ā, e tiaki ana i ngā momo hāora tauhohe (ROS) i te wā e matekai ana te huka (Wang et al., 2018). Kua whakaatuhia ko te mukunga o te NAT1 ka aukati i te matatini pyruvate dehydrogenase, ka arahi ki te ngoikore o te mitochondrial (Wang L. et al., 2019). Hei tāpiri, kua whakaatuhia e ētahi atu pūrongo ko te aukati NAT1 mā te whakamahi i ngā ngota iti me te whakangāwari siRNA ka taea te whakaiti i te whakaekenga me te tipu o ngā pūtau matepukupuku ū (Stepp et al., 2018). Nō nā tata nei, i whakamahia a CRISPR/Cas9 hei pehi i te NAT1 i roto i te raina pūtau matepukupuku ū MDA-MB-231, ā, ka pā ki te pākia o te pūtau i runga i tōna taumata whakaaturanga. I whakaatuhia hoki e tēnei rangahau he mea nui a NAT-1 mō te haere whakamua me te horapa o te TNBC (Carlisle et al., 2020).
Ko te whakawhitiwhiti kōrero taunga o ngā oncogenes ka whakahaeretia e ngā super enhancers, ngā transcription factors me ngā cofactors (Hnisz et al., 2015). Hei tāpiri, he rōpū o ngā cyclin-dependent kinases (CDKs), pērā i te CDK7, CDK8, CDK9, CDK12, me te CDK13, e hiahiatia ana mō te whakahaere whakawhitiwhiti kōrero. I roto i ēnei, kei roto te CDK7 i te phosphorylation o te RNA polymerase II, he mea tino nui mō te tīmatanga me te whakawhānui ake o te whakawhitiwhiti kōrero oncogene i roto i te pathogenesis o te TNBC. I roto i tētahi rangahau matua, ko te mukunga o CDK7 e CRISPR/Cas9 i aukati i te TNBC, e tohu ana ko te pathogenesis o te TNBC he CDK7 te mea e whakawhirinaki ana (Wang Y. et al., 2015).
Kua whakaatuhia e ngā rangahau ka taea e ngā whakarerekētanga ira i roto i te MYC me te RBP (pūmua here RNA) te arahi ki te apoptosis, engari ko ngā whakarerekētanga ira kotahi i roto i te MYC, i te RBP rānei, kāore e pā ki te tipu o ngā pūtau matepukupuku (Einstein et al., 2021). Neke atu i te 1000 ngā RBP i roto i te ira tangata kua tirotirohia mā te whakamahi i tētahi whare pukapuka e hangai ana ki te CRISPR/Cas9. I roto i aua mea, e 57 ngā RBP i kitea he mea nui mō te tipu o ngā pūtau matepukupuku me ngā taumata MYC teitei rawa (Wheeler et al., 2020). Hei tāpiri, he mea nui a YTHDF2 mō te pupuri i te tipu o ngā pūtau TNBC, ā, ka whakaiti i te nui o ngā tuhinga methylated i te wā o te tuhinga me te whakamaoritanga taumata teitei i roto i ngā pūtau matepukupuku me te whakaaturanga MYC teitei ake. Hei tāpiri, kāore a YTHDF2 i te mea nui mō ngā pūtau matepukupuku, he iti ake te whakawhirinaki ki ngā taumata MYC teitei hei whakaroa i te oranga o ngā tūroro TNBC (Einstein et al., 2021), e kī ana he whāinga rongoā pea tēnei mō ngā rongoā ka taea te wikitoria i te TNBC.
Ko ngā pūmua matimati konutea (ZNF) e hanga ana i te tata ki te 1% o te katoa o te ira tangata. Kua whakaatuhia e ngā rangahau ka taea e te ZNF te whakahaere i te tipu o ngā pūtau i roto i ngā momo mate pukupuku, pērā i te mate pukupuku ate, te mate pukupuku ū me te mate pukupuku koroni (Zhang W. et al., 2021; Li et al., 2017). Kua whakamahia ngā whare pukapuka i hangaia e te CRISPR knockout hei tirotiro i ngā momo ira pehi pukupuku (TSG) i roto i ngā pūtau mate pukupuku ū (Shalem et al., 2014). Mai i taua wā, kua whakaatuhia e te rangahau transcriptomic o ngā pūtau mate pukupuku ū kua mukua e CRISPR/Cas9 ZNF319 ko te ZNF319 he ira pehi pukupuku e whakaiti ana i te tipu o te mate pukupuku ū, nō reira kei roto i ngā momo tikanga tohu pea me ētahi atu mahi koiora (Wang L. et al., 2022).
Ko te Ferroptosis he momo mate pūtau kua whakaritea e whakawhirinaki ana ki te aroaro o te rino. E mōhiotia ana ko te whanaketanga o te ferroptosis ka pāngia e te aroaro o ngā lipid peroxides. Hei tāpiri, kua pūrongohia te whakaatu a PKCβII i te peroxidation lipid wawe, ā, ko te pikinga o te peroxidation lipid e pā ana ki te ferroptosis. I te tirotiro i tētahi whare pukapuka o ngā aukati kinase CRISPR/Cas9 i whakaatu ko te PKCβII kei roto i te tukanga o te peroxidation lipid, he mea nui mō te ferroptosis i roto i ngā pūtau MDA-MB-231 (Zhang et al., 2022). Nō reira, e kī ana tēnei ko te whakakorenga o PKCβII e CRISPR/Cas9 he whāinga ira pehi pukupuku pea mō te maimoatanga o ngā mate e pā ana ki te ferroptosis (Zhang et al., 2022).
E whakaponohia ana ko te ātete ki ngā rongoā te take o te tata ki te 90% o ngā matenga o ngā tūroro matepukupuku, ā, ko tētahi o ngā wero nui o te maimoatanga matepukupuku (Bukowski et al., 2020). E whakaatu ana ngā hua he nui ngā ira e pā ana ki te putanga mai o te rongoā, te whakatikatika i te DNA, te apoptosis me ngā ara tohu pūtau rerekē e pā ana ki te ātete rongoā (Haider et al., 2020). I roto i ēnei, he maha ngā ira kua arohia e ngā taputapu CRISPR/Cas9, ā, kua whakaatu i ngā hua pai ki te whakaiti i te ātete rongoā me te whakanui ake i te whai huatanga o ngā maimoatanga ārai-matepukupuku (Vaghari-Tabari et al., 2022). Hei tāpiri, kua whai wāhi ngā whare pukapuka tirotiro ira CRISPR/Cas9 kaha ki te whakarerekē i te mahi a ngā ūnga ira ātete rongoā pea (Shalem et al., 2015). Hei tautuhi i ngā ira ātete paclitaxel, i whakamahia te raupapatanga RNA me te tirotiro whare pukapuka sgRNA whānui-ira hei tautuhi i ngā ira kaitono e waru, tae atu ki te histone deacetylase 9 (HDAC9), e hono ana ki te ātete rongoā i roto i ngā tūroro me te TNBC e hokihoki ana (B et al., 2020). I roto i tētahi atu rangahau, i kitea te pikinga o te whakaaturanga o te diserine/threonine me te tyrosine protein kinase (DSTYK) i te wā e ora ana ngā tūroro TNBC i rongoatia ki ngā rongoā ārai matepukupuku. I tua atu, ko te whakakorenga o te DSTYK e whakahaerehia ana e CRISPR/Cas9 i tino whakanui ake i te apoptosis o ngā pūtau matepukupuku ātete rongoā i roto i te vitro (ngā pūtau SUM102PT me ngā pūtau MDA-MB-468) me te tauira TNBC in vivo (Ogbu et al., 2021).
Ahakoa he whakahohenga rerekē te ara MAPK i roto i te TNBC, he ngoikore te pānga haumanu o ngā maimoatanga e arohia ana e te MEK. I whakaatuhia e te tirotiro whare pukapuka ira CRISPR/Cas9 ko te aukati i te PSMG2 (proteome assembly chaperone 2) ka whakanui i te aro o ngā pūtau TNBC BT549 me te MB468 ki te aukati MEK AZD6244. Ko te hinganga CRISPR/Cas9 o te PSMG2 i whakarerekē i te mahi proteasome noa, ka arahi ki te wetewete i te PDPK1 e whakahaerehia ana e te autophagy, ā, ka whakapai ake i ngā pūtau pukupuku i roto i ngā tauira kiore TNBC i whakaohokia e te AZD6244 (MEK inhibitor) me te MG132 (proteasome inhibitor). Nō reira, ka taea te whakahaere tahi i ngā aukati proteasome me te MAP kinase (MEK) hei whakaiti i te tipu o ngā pūtau pukupuku (Wang X. et al., 2022).
Kua whakamahia hoki a CRISPR/Cas9 hei tirotiro i te ngaronga o te mahi ira e arahi ana ki te ātete ki te TNBC (Shu et al., 2020). I whakamārama a Ge et al i te tikanga o te ātete rongoā o ngā pūtau matepukupuku i rongoatia ki te JQ1, he aukati BET bromodomain (BBDI), i roto i te TNBC. Mā te whakamahi i te CRISPR/Cas9, i kitea e rātou ko te muku i te ira rb1 i meinga ai te TNBC kia ātete ki te rongoā anti-matepukupuku JQ1. Nō reira, he mea nui te mahi rb1 i roto i te urupare ki ngā rongoā JQ1 i roto i te TNBC. I kōrero anō rātou ko te paclitaxel he aukati CDK4/6 kinase/microtubule, ā, ko tōna whakakotahitanga me ngā BBDI pēnei i te JQ1 ka whakarato pea i ngā urupare rongoā whai hua ki te TNBC ātete-rongoa (Ge et al., 2020).
Kua pūrongohia ko te whenua whakawhitiwhiti o te RNA roa kore-waehere (lncRNA) te take o te ātete ki te rongoā neoadjuvant i roto i te TNBC. E whakaatu ana tēnei rangahau e rima ngā momo whakawhitiwhiti MALAT1 lncRNA rerekē e tino whakaatuhia ana i roto i te TNBC. Hei tāpiri, ko te mukunga CRISPR/Cas9 o MALAT1 i whakanui ake i te aro o ngā pūtau TNBC BT-549 ki te paclitaxel me te doxorubicin, e whakaatu ana i te tūranga pea mō te ātete MALAT1 i roto i te TNBC (Shaath et al., 2021).
He rerekē ngā whakarerekētanga ira i roto i te BRCA1 (BRCA1m), nō reira he uaua ki te kite. Mā te arotahi ki te PARP1 (poly(ADP-ribose) polymerase), te hoa whakamate hangai o te BRCA1, ka piki ake pea te aro ki te rongoā TNBC. Mā te whakamahi i te CRISPR/Cas9, i whakanui ake te mukunga o te PARP1 i te aro o ngā rongoā ārai matepukupuku pērā i te doxorubicin, gemcitabine me te docetaxel ki ngā pūtau TNBC whakarerekētanga mBRCA1, e kī ana kei te whai wāhi hoki a PARP1 ki te ātete rongoā i roto i te TNBC (Vaghari-Tabari et al., 2022). E mōhiotia ana ko ngā whakarerekētanga ira i roto i ngā ira pehi pukupuku BRCA1, BRCA2 rānei, ka piki ake te tūponotanga o te whakawhanake i te matepukupuku ū i roto i te tangata. Ko te maimoatanga o ēnei tūroro me whakamahi i ngā aukati PARP. Hei tāpiritanga, kua whakaaturia ko te whakakorenga o te nucleotide salvage factor DNPH1 mā te whakamahi i te CRISPR/Cas9 ka taea te tango i te nucleotide 5-hydroxymethyldeoxyuridine (hmdU) monophosphate paitini, ā, ka whakarei ake i te urupare a ngā pūtau ngoikore-BRCA ki te aro ki ngā aukati PARP (Fugger). et al., 2021). Nō reira, ko ngā aukati CRISPR/Cas9 me PARP1 ka noho hei rautaki maimoatanga nui mō te TNBC.
Ko te ira glycoprotein penetrant (P-gp) he ira ātete maha-rongoā e piki ake ana te mana i roto i te 41% o ngā TNBC katoa (Sun et al., 2020). Kua tautuhia te putanga mai o te rongoā i puta mai i te P-gp hei kaiwhakahaere matua o te ātete rongoā i roto i te mate pukupuku ū. Kua whakaatuhia e ngā aukati P-gp te pikinga ake o te aro ki ngā rongoā ārai mate pukupuku i roto i te mate pukupuku ū (Famta et al., 2021). Nō reira, ko te tango, te pehi rānei i ngā aukati P-gp me P-gp e whakahaerehia ana e CRISPR/Cas9, he tikanga tino nui pea hei whakakore i te ātete rongoā i roto i te TNBC.
E mōhiotia ana ko te kaikawe kaseti here-ATP G2 (ABCG2) ka puta he ātete rongoā i roto i te TNBC (Palasuberniam et al., 2015), ahakoa kāore anō kia puta he pūrongo mō te whanaungatanga i waenga i te whakamōkitotanga o CRISPR/Cas9 me te ABCG2 me te whakakorenga o te ira pehi pukupuku. Tērā pea ka whakarei ake te PTEN i te whakahohenga o te ABCG2 (Palasuberniam et al., 2015), (Deepak Singh et al., 2021). Nō reira, ko te whakamahi i te CRISP/Ca9 hei tango i te ABCG2 me te whakakotahi me te aukati ABCG2 he maimoatanga tika pea hei wikitoria i te ātete rongoā i roto i te TNBC. E whakahuatia ana i roto i te Ripanga 2 te CRISPR/Cas9 e aro ana ki ngā ira ātete katoa.
Ripanga 2. Ka aro atu a CRISPR/Cas9 ki ngā ira ātete TNBC hei whakaoho i ngā pūtau ki ngā rongoā ārai matepukupuku.
He taputapu pea ngā whare pukapuka CRISPR/Cas9 mō te tirotiro i ngā whakarerekētanga ira e pā ana ki te pathogenesis o te mate pukupuku (Chan et al., 2022). E whā ngā taahiraa o tēnei tikanga tirotiro, arā, (a) te hanga whare pukapuka, (b) te whakawhiti lentiviral, (c) te tirotiro phenotypic, me te (d) te tātari ira ūnga. Ahakoa he whakahohenga rerekē te ara MAPK i roto i te TNBC, he tino kino te matapae haumanu o te rongoā arotahi MEK mō ngā tūroro TNBC e mau ana i ngā whakarerekētanga ira i roto i ngā ira pehi pukupuku pērā i te PTEN, RB1, me te TP53.
I tua atu, i whakamātauhia e te tirotiro ira mā te whakamahi i te CRISPR/Cas9 te ngota tino kaha me te whiriwhiri, te dehydrocatrol, he nui i roto i ngā tangohanga putiputi me ngā rau o Guatemala, kia mārama ai ki te pānga o te dehydrocatrol ki te MDA-MB-231. Ngā tikanga o te paitini pūtau whiriwhiri. Ngā āhuatanga o te takenga mesenchymal o ngā momo TNBC. I whakaatuhia hoki e tēnei tirotiro e hangai ana ki te CRISPR/Cas9 ko te HSD17B11, he ira e whakamārama ana i te momo 11 o te 17β-hydroxysteroid dehydrogenase, he tino kitea i roto i ngā pūtau MDA-MB-231, ā, ka hua ake he dehydrofalcarinol motuhake mō ngā pūtau MDA-MB-231 (Grant et. al., 2020). Nō reira, e kī ana tēnei he nui te kaha o te tirotiro ira CRISPR/Cas9 ki te tautuhi i ngā tikanga e whai ana i ngā āhuatanga ārai matepukupuku o ngā pūhui taiao pea.
Hei tāpiritanga, i rangahauhia te ngoikore o te TNBC ki te mate pukupuku mā te whakamahi i tētahi tirotiro CRISPR/Cas9 in vivo whānui-ira, i whakaatu i te hononga i waenga i ngā ara oncogenic me ngā ara pehi pukupuku. Kua pūrongohia he mahi nui ngā wāhanga matua o ngā ara mTOR me Hippo ki te whakahaere i te pukupuku i roto i te TNBC. Hei tāpiritanga, kua whakaatuhia e ngā rangahau ko te aukati rongoā o te mTORC1/2 me te YAP oncoprotein ka ārai pai i te pathogenicity o te TNBC mā te whakamahi i tētahi tauira synergy rongoā-matrix in vitro me ngā xenografts i ahu mai i te tūroro in vivo. Hei tāpiritanga, ko te aukati mTORC1/2 i whakahaerehia e Torin-1 ka whakarei ake i te macropinocytosis, engari ko te aukati YAP i puta mai i te verteporfin ka arahi ki te mate pūtau TNBC. Ina huihuia, e whakaatu ana ēnei hua i te kaha me te pakari o te tirotiro CRISPR in vivo whānui-ira hei tautuhi i ngā maimoatanga hou me te whai hua mō te TNBC (Dai et al., 2021).
He mea nui te koretake o te pūnaha ārai mate i roto i te whanaketanga o te mate pukupuku. Ka karo ngā pūtau mate pukupuku i te whakakorenga o te ārai mate mā te karo i ngā tikanga tiaki, tae atu ki te whakararuraru i te mahi a ngā pūtau ārai mate i roto i te taiao moroiti o te pukupuku me te whakararu i te pūnaha ārai mate. Nō reira, ko te whakawhanake i tētahi pūnaha ārai mate kua whakapaitia pea he huarahi matua ki te whawhai ki ngā pukupuku. Ko te whakamahinga o te whakarerekētanga ira e hangai ana ki te CRISPR/Cas9 ka aro atu ki ētahi take e pā ana ki te ngoikore o te ārai mate mai i ngā tirohanga rerekē. Kua whakamahia te CRISPR/Cas9 hei whakapai ake i te ārai mate pukupuku ki te mate pukupuku ū mā roto i ngā ara e whai ake nei (Pikitia 3).
Pikitia 3. Ko te rongoā ārai mate CRISPR/Cas9 e aro ana ki ngā pūtau TNBC. Ko te ngaronga o te CDK5 me te hinganga o te PDL1 me te CD155 ka whakapakari i te pūnaha ārai mate. Waihoki, ko te ngaronga o te A2AR me te hinganga o ngā TAA pēnei i te HER2, te mucin 1 me te TEM8 i whakapiki ake i te whai huatanga o ngā pūtau CAR-T ki te patu i ngā pūtau matepukupuku. Kua whakaatuhia e te tirotiro pūtau-T e aratakina ana e te CRISPR/Cas9 ko te whakakorenga o te p38 kinase ka whakarei ake i te mahi ārai-pūtau T. Ko ngā pūtau-T i whakarerekētia e te CRISPR/Cas9 ka whakanui ake i te whakaaturanga o te TCR (kaiwhakawhiwhi pūtau-T), ka hua ake he mahi ārai-pūtau T.
Ko te whāinga o te rongoā ārai mate he whakaoho i te pūnaha ārai mate ki te whakaeke i ngā pūtau mate pukupuku. Ko te nui rawa o ngā pūmua ārai ārai mate ka ārai i ngā tauhohenga ārai mate engari ka āwhina pea i ngā mate pukupuku ki te karo i aua tauhohenga (Topalian et al., 2015). Ka ārai ēnei pūmua i ngā tauhohenga ārai mate mā te herea ki ngā kaiwhakawhiwhi i runga i te mata o ngā pūtau ārai mate. He maha ngā pūmua ārai mate (pērā i te CD155 me te PD-L1) e aro ana ki te kaiwhakawhiwhi PD-1 i runga i ngā pūtau ārai mate, ā, ka whakaatuhia i roto i te mate pukupuku ū, inā koa ko te TNBC (Li Y.-C. et al., 2020). Mā te whakakorenga o te PD-L1, o tōna kaiwhakawhiwhi rānei mā te whakamahi i te CRISPR/Cas9 ka taea te whakaoho i te pūnaha ārai mate ki te whakaeke i ngā pukupuku TNBC (Yahata et al., 2019). Hei tāpiri, kua whakaatuhia te whakahekenga o te whakaaturanga PD-L1 mā te mukunga o te CDK5 e CRISPR/Cas9 hei aukati i te tipu o te pukupuku i roto i te vitro me te in vivo (Deng et al., 2020). Ko te aukati tipu i roto i ngā tauira in vitro me in vivo o te mate pukupuku ū e kī ana ko te whakahekenga o te CD155 e whakahaerehia ana e te shRNA he pānga rongoā ki te mate pukupuku ū (Gao et al., 2018).
Ka whakaatuhia e ngā pūtau CAR T ngā CAR e mōhio ana ki ngā antigens e pā ana ki te pukupuku (TAAs) ā, ka taea te whakamahi i te whakakorenga o ngā pūmua tirotiro hei whakarei ake i tā rātou mahi (Li C. et al., 2020). He maha ngā whāinga pea mō te rongoā pūtau CAR T i roto i te mate pukupuku ū, tae atu ki ētahi TAA pēnei i te HER2, mucin1, me te TEM8 (Bajgain et al., 2018). He taunakitanga kei reira he pai ake te whai huatanga o ngā pūtau CAR T e aro ana ki te mesothelin (he nui rawa te whakapuakitanga i roto i ngā pūtau TNBC BT-459) ki te mate pukupuku ina whakakorea te PD-1 mā te whakamahi i te CRISPR/Cas9 (Hu et al., 2019). Heoi, he mahi uaua, he mahi roa hoki te wehe i ngā pūtau T mai i ngā tūroro, kātahi ka whakatika i a rātou ex vivo. Ahakoa ka taea e ngā pūtau T whānui te whakaiti i ngā whakaritenga wehenga, me tango ngā pūtau T kaituku e whakaatu ana i te antigen leukocyte tangata (HLA) akomanga I me te kaiwhiwhi pūtau T (TCR) hei ārai i ngā rerekētanga graft-versus-host (Ren et al., 2017). , 2017a). Mā te whakamahi i te CRISPR/Cas9, ka taea e te HDR te whakakore i te TCR me te HLA i te wā kotahi, me te whakakore i te ira e whakamārama ana i te CAR. Kua whakaatuhia e ngā rangahau ka taea te whakamahi i te CRISPR/Cas9 ki te whakauru i ngā CAR anti-CD19 ki roto i te rohe TCR, ā, ka whai hua te whakaputa CAR me te kore e whakaiti i ngā pūtau T (Dimitri et al., 2022). Kua whakawhanakehia ngā hangarau Multiplex hei whakaputa i ngā pūtau T CAR allogeneic mā te whakakore i te TCR, te beta-2-microglobulin (B2M), te waeine HLA-I me ētahi atu pūmua pēnei i te PD-1 me te CTLA-4, he nui ake pea ngā āhuatanga ārai-matepukupuku ki te TNBC (Eyquem et al., 2017; Liu et al., 2017; Ren et al., 2017b; Dimitri et al., 2022).
Mā te whakamahi i te CRISPR/Cas9 ka taea te whakapai ake i te whai huatanga o ngā pūtau CAR-T. E mōhiotia ana he āhuatanga ārai mate a te Adenosine, ā, ka taea te whakaiti i te ārai mate pukupuku mā te aukati i te mahi a te pūtau T me te whakahohe i ngā kaiwhakawhiwhi adenosine A2A (A2AR) (Vigano et al., 2019). Mā te whakamōkito i te A2AR mā te whakamahi i te CRISPR/Cas9 i tino whakapai ake i te whai huatanga o ngā pūtau CAR-T i roto i te tinana (Giuffrida et al., 2021). E mōhiotia ana he maha ngā āhuatanga phenotypic o ngā pūtau T pērā i te whānui o te pūtau, te rerekētanga, te ahotea oxidative me te ahotea genomic. I whakaatuhia e te tirotiro pūtau T e hangai ana ki te CRISPR/Cas9 te whakararuraru o te 25 ngā receptor kinase pūtau T rerekē. I roto i ēnei, kua whakaatuhia te mukunga o te p38 kinase hei whakarei ake i te mahi antitumor o ngā pūtau T, e tohu ana ko te p38 kinase te kaiwhakahaere matua o te whakahaere pūtau CAR-T (Gurasamy et al., 2020).
Ka taea te whakarerekē i ngā pūtau-T mā te whakamahi i te CRISPR/Cas9 hei whakaputa i ngā TCR tino kitea. Ko te whakawhiti i ngā pūtau-T kua whakarerekētia ki ngā tūroro kua whakaatu i te kaha ake o te mahi ārai matepukupuku i ngā pūtau-T taketake. Nō reira, ka whakataetae pea ngā TCR taketake ki ngā TCR kua whakarerekētia ira i roto i ngā tūroro, ka pā pea ki te kaha o te rongoā ārai matepukupuku. Hei whakakore i tēnei raruraru, mā te whakamahi i te CRISPR/Cas9 hei tango i te TCR-β taketake i roto i ngā pūtau kaiwhiwhi, ā, muri iho ka whakawhiti i ngā pūtau-T TCR-β ki ngā tūroro matepukupuku, ka whakaatu pea i ngā urupare ārai matepukupuku pai ake me te kore e hiahiatia he whakataetae moepuku TCR taketake (Fan et al. 2018). Nō reira, haunga ngā pūtau-T kua whakarerekētia e te CRISPR, he mano ngā wā ka nui ake te aro o ngā TCR ki ngā antigens pukupuku i ngā pūtau-T noa kua whakawhitia e te TCR. Hei tāpiri, i roto i ngā momo leukemia, ko ngā pūtau-T kua whakarerekētia i hangaia e te γδ TCR+ CRISPR he nui ake te whakaaturanga o ngā pūtau-T CD4+ me te CD8+ i te whakawhiti TCR paerewa (Legut et al., 2018). Nō reira, ko ngā pūtau-T kua whakarerekētia i hangaia e CRISPR/Cas9 he tikanga ārai mate whai hua pea hei patu i te TNBC.
Ko ngā integrin he ngota piri pūtau kei roto i ngā kiriuhi pūtau, ā, ka whakatairanga i te herenga o ngā pūtau ki te matrix extracellular (ECM) (Hamidi and Ivaska, 2018). Ko te koretake o ngā integrin e pā ana ki te whanaketanga me te hekenga o te mate pukupuku mā te whakarerekē i te matrix extracellular, ka arahi ki te oranga o ngā pūtau mate pukupuku i roto i te tohanga toto (Hamidi and Ivaska, 2018). Ko te whakakorenga o te integrin e whakahaerehia ana e CRISPR/Cas9 ka whakaroa i te whanaketanga o te pukupuku, te metastasis, me te koroni i roto i te TNBC. Kua pūrongohia ko te whakakorenga o te integrin a5 (ITGA5) ka whakaiti i te hekenga me te haere whakamua o ngā pūtau i roto i ētahi atu mate pukupuku pēnei i te mate pukupuku pūkahukahu (Ju et al., 2017), e kī ana ko te integrin a5 pea he take matua i roto i te pathogenesis o te TNBC.
Ko te whakaputa kiore knockout mā te whakamahi i ngā tikanga tuku iho o te pūtau take embryonic (ES) he mahi uaua, he mahi roa, he mahi koretake hoki. E hia marama, e hia tau hoki te roa ki te whai i ngā pūtau ES mā te whakakotahitanga homologous, te whakaputa i ngā kiore chimeric, kātahi ka whakawhiti i a rātou ki ngā kiore heterozygous hei whakaputa uri homozygous. Me whakawhiti uaua te whānautanga ki te hanga kiore he maha ngā whakarerekētanga ira. Heoi, he maha ngā raruraru kua puta mai i te whakamahi i ngā kiore i hangaia mā te tango i ngā pūtau ES mā te whakamahi i te CRISPR/Cas9, mā te werohanga rānei i ngā wāhanga CRISPR/Cas9 ki roto i ngā hua kua whakatōkia ki roto i te pūtau kotahi. Hei tauira, he maha ngā wā ka puta ngā whakarerekētanga ki ngā loci biallelic, ā, kāore e motuhake ki te ira. I pūrongohia tata nei ka taea e ngā pūtau ES e whakamahi ana i te hangarau CRISPR/Cas9 te whakauru i ngā whakarerekētanga biallelic ki roto i te 5 ira i te wā kotahi (Nishizono et al., 2021). Mō tēnei kaupapa, i whakawhitia ngātahitia te Cas9 mRNA me ngā gRNA motuhake ki roto i ngā pūtau ES. E whakaatu ana tēnei i te oati me te whai huatanga o tēnei tikanga, ahakoa i tukuna pea ēnei whakarerekētanga ira i te wā i whakatupuria ai ngā uri whakatupu hei whakaputa i ngā kiore knockout rima tekau. E pūrongo ana hoki te rangahau i tētahi kitenga whakamiharo: Kāore e hiahiatia ana ngā pūtau ES hei hanga i ngā kiore kua whakarerekētia te ira. Engari, hei tango i ngā hua ira motuhake, i werohia te Cas9 mRNA me te gRNA ki roto i ngā kukune wāhanga pūtau-kotahi. Nō reira, kua hangaia ngā uri whakatupu knockout ka taea te whakamahi hei ako i ngā hua o te mukunga ira i roto i ngā kiore (Qin et al., 2016). Nō reira, ka taea e CRISPR/Cas9 te hanga i ngā kiore whakawhiti ira hei rongoā i te TNBC i te utu iti iho i te hangarau ira tuku iho. Mā te whakamahi i te pūnaha CRISPR/Cas, kua whakawhanakehia he tauira kiore knockout hou ka taea te whakauru angitu i ngā whakarerekētanga ira ki roto i tētahi, neke atu rānei o ngā ira taketake i roto i te TNBC. I runga i tēnei ariā, ka taea hoki te whakawhanake i ngā tauira kararehe o TNBC mā te whakamahi i te mukunga o te BRCA1 me te p53 e CRISPR/Cas9, ka hua ake te ngaronga o te whakatikatika HR, te kore pumau o te ira, me ngā momo whakarerekētanga ira (Annunziato et al., 2020).
I tēnei wā, he maha ngā tikanga pēnei i te mammography, te whakaahua aukume (MRI) me te ultrasound e whakamahia ana hei tātaritanga i te TNBC. Heoi, he iti noa ngā herenga o ēnei tikanga. Ka whakamahia te mammography hei tātaritanga i te kiko o te ū ā-rohe, kaua ki te metastases, i ngā wāhi kua heke ngā pūtau matepukupuku ki ētahi atu okana. Ehara te Ultrasonography i te tikanga pono mō te tātaritanga i te TNBC (Chen and Lee-Felker, 2023). He nui ake te aro o te MRI i te ultrasound me te mammography, engari he iti noa te tika o te tātaritanga (Sha and Chen, 2022). He huarahi whakaeke te koiora kiko ki te tautuhi i ngā pūtau matepukupuku. Heoi, i ētahi wā, ka ngaro pea i te koiora te kiko matepukupuku mēnā ka kotiti ke te ngira i te wāhi e hiahiatia ana. Hei tāpiri, he tino utu nui, ā, ka pā kino pea ki te tūroro. He momo matepukupuku rerekē te TNBC, nō reira kāore pea te koiora e whakarato i ngā mōhiohio e ranea ana mō te momo matepukupuku. Nō reira, me tere te hiahia ki tētahi tikanga hou e pono ana te tātaritanga mō te kite i te TNBC. Tērā pea ka noho te CRISPR/Cas9 hei huarahi tino aro me te iti rawa te whakaekenga mō te tātaritanga i te matepukupuku ū. Hei whakatutuki i tēnei, ka taea te whakamahi i ngā rautaki e hangai ana ki te CRISPR/Cas9 hei whakapai ake i ngā tikanga PCR mō te tātaritanga o te TNBC. Tuatahi, ka whakamahia ngā pūmua Cas9 me te cpf1 o te pūnaha CRISPR hei tango i te DNA kore-motuhake, kātahi ka taea e ēnei pūmua e rua (Cas9 me te cpf1) te kite i te raupapa PAM i mua i te herea ki te DNA ūnga (Deepak Singh et al., 2021). Nō reira, ka taea e te PCR te tautuhi i ngā whakarerekētanga e pā ana ki te whanaketanga o te mate pukupuku. He maha ngā rangahau kua whakamahi i tēnei rautaki e hangai ana ki te CRISPR hei kite i ngā whakarerekētanga rerekē i roto i ngā mate pukupuku rerekē (Safari et al., 2019). Nō reira, ka taea e ngā tikanga PCR e hangai ana ki te CRISPR te whakaiti i te whakawhirinakitanga o te tātaritanga TNBC ki ngā tikanga whakaeke pēnei i te immunohistochemistry e hangai ana ki te biopsy.
Kua whakaaturia hoki ngā panonitanga ira i roto i te whakahaere i ngā kaiwhakawhiwhi homoni i roto i te TNBC (Chen and Russo, 2009). Ka taea e ngā rautaki PCR e hangai ana ki te CRISPR/Cas9 e whakamahi ana i ngā microarrays mō ngā tātaritanga i te wāhi tiaki te aroturuki tika i ēnei whakarerekētanga ira (Hajian et al., 2019). Ka taea hoki e ia te whakarato ki ngā kaiwhakarato hauora ngā mōhiohio e tika ana mō ngā whakarerekētanga ira motuhake i roto i ngā tūroro TNBC, ka āwhina pea ki te whakapai ake i tā rātou huarahi maimoatanga. Heoi, he iti noa iho ngā herenga o tēnei tikanga whakakotahi. Nō reira, me nui ngā mahi rangahau e hiahiatia ana i mua i te whakamahinga o tēnei tikanga CRISPR/Cas-PCR i roto i te hauora hei tātaritanga i te TNBC (Yang et al., 2019).