ʻO CRISPR) kahi ʻenehana hoʻoponopono genome hanauna ʻekolu: ua hoʻololi ʻo CRISPR i ka honua

ʻO Clustered regularly interspaced short palindromic repeats (CRISPR) kahi ʻenehana hoʻoponopono genome hanauna ʻekolu i hoʻololi i ka honua me kāna mau hopena throughput kiʻekiʻe. Ua hoʻohana ʻia e mālama i nā maʻi olaola like ʻole a me nā maʻi. Loaʻa i nā bacteria like ʻole a me nā prokaryotes ʻē aʻe (e like me archaea) nā ʻōnaehana CRISPR/Cas9 e pale aku i nā phages. Ua hōʻike ʻia e hiki i nā hoʻolālā CRISPR/Cas9 ke kāohi i ka ulu ʻana a me ka holomua o ka maʻi ʻaʻai umauma triple negative (TNBC) ma o ka hoʻopaʻa ʻana i nā genes kū'ē i hoʻololi ʻia, transcription, a me ka hoʻoponopono epigenetic. Hiki i kēia mau hana therapeutic ke kōkua i ka hoʻoponopono ʻana i nā pilikia paʻakikī e like me ke kū'ē ʻana i ka lāʻau i ʻike ʻia ma TNBC. I kēia manawa, hoʻohana ʻia nā ʻano hana like ʻole e hāʻawi iā CRISPR/Cas9 i nā cell target, e like me ke kino (microinjection, electroporation a me nā ʻano hydrodynamic), viral (adeno-associated virus a me lentivirus) a me nā non-viral (liposomes a me nā lipid nanoparticles). )-particles). ʻOiai ua hoʻomohala ʻia nā ʻano hoʻohālike like ʻole e aʻo i nā kumu molekala o TNBC, ʻo ka nele o nā ʻano hana koʻikoʻi a kuhikuhi ʻia no ka hāʻawi ʻana i nā mea hana hoʻoponopono genome in vivo e kaupalena ana i kā lākou noi lapaʻau. I ka hōʻuluʻulu manaʻo, nānā piha kēia loiloi i ka holomua, nā pilikia, nā palena, a me nā manaʻolana o ka mālama ʻana iā CRISPR/Cas9 no TNBC ma muli o nā hōʻike e kū nei. Hōʻike pū mākou pehea e hiki ai i ka hui pū ʻana o ka naʻauao hana a me ke aʻo mīkini ke hoʻomaikaʻi i nā hoʻolālā CRISPR/Cas9 i ka mālama ʻana iā TNBC.
ʻIke ʻia ke kanesa e ka mahele ʻana o ke kelepona i kāohi ʻole ʻia, ka hoʻopilikia ʻana i nā wahi nānā o ke kelepona, a me nā hoʻololi ʻana i nā genes tumor suppressor (TSGs) (Matthews et al., 2022). Ma waena o nā ʻano kanesa like ʻole, ʻo ke kanesa umauma ka ʻano maʻamau o ke kanesa i nā wahine a he kiʻekiʻe ka nui o ka make ma ka honua holoʻokoʻa (Waks lāua ʻo Winer, 2019). He maʻi heterogeneous ke kanesa umauma me nā ʻano like ʻole, e like me nā hiʻohiʻona histological a me biological, ka hōʻike ʻana o ke kauka a me ke ʻano, a me ka pane ʻana i ka lāʻau lapaʻau (Weigelt et al., 2010). Hāʻawi ka hoʻokaʻawale ʻana o ke kanesa umauma i nā ʻike pololei no ka ʻike ʻana i ke kanesa umauma a me ka prognosis tumor. ʻO ka hoʻohana ʻana i nā biomarkers maʻamau a me nā hiʻohiʻona clinicopathological ka mea nui no ka hoʻokaʻawale ʻana o ke kanesa umauma (Tsang lāua ʻo Tse, 2019). Hoʻopili ʻia ka prognosis a me ka pane ʻana i ka mālama ʻana i ke kanesa umauma e nā kumu he nui, me ke alo o ka estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2/neu), a me ka histological grade, ke ʻano tumor, a me ka grade. ka nui a me ka lymph node metastasis (Al-Tubaiti, 2020). Ua ʻike ʻia ʻelima mau ʻano molekala kūloko o ka maʻi ʻaʻai umauma, me ka luminal A, luminal B, HER2-enriched, basal-like, a me claudin-low (Prat et al., 2015). ʻO TNBC kahi ʻano molekala o ka maʻi ʻaʻai ʻaʻole e hōʻike i nā ER, PR, a me HER2 āpau (Yin et al., 2020). Pili kēia mau hiʻohiʻona pathological me TNBC, e hōʻoia ana i kona holomua wikiwiki a me ke ʻano ʻoi aku ka ikaika ma mua o nā ʻano maʻi ʻaʻai umauma ʻē aʻe (Feng et al., 2018). Eia kekahi, ua hoʻohana ʻo Peru et al (2000) i ka ʻenehana microarray e hoʻokaʻawale hou i ka maʻi ʻaʻai umauma a ʻike i ʻelima mau ʻano intrinsic o ka maʻi ʻaʻai umauma (Cadenas, 2012). ʻO ka maʻi ʻaʻai umauma basal-like kahi ʻano o ka maʻi ʻaʻai umauma nona ka phenotype triple-negative a pili pū me ka holomua wikiwiki. Pono e hoʻomaopopo ʻia ua kuhi hewa ʻia nā maʻi ʻaʻai umauma basal-like āpau he TNBC, akā he 77% wale nō ʻo TNBC; I ka hoʻohālikelike ʻana, 71-91% o TNBC he basal-like, e hōʻike ana ua pili kēia mau ʻano ʻelua o ka maʻi ʻaʻai umauma a hōʻike i nā hoʻokaʻawale like ʻole (Wang D.-Y. et al., 2019). Hoʻokumu kēia i kahi pono e wehewehe i ka heterogeneity o TNBC e wehewehe i ka prognosis a ʻike i nā pane hiki i nā lāʻau lapaʻau o kēia manawa a me ka wā e hiki mai ana. Eia kekahi, ʻo TNBC ka helu no 15-20% o nā hihia maʻi ʻaʻai umauma āpau a ʻoi aku ka maʻamau i nā wahine ma lalo o 50 mau makahiki. Ua hōʻike ʻia nā mutations BRCA1 a i ʻole BRCA2 ma kahi o 20% o nā hihia TNBC (Xie et al., 2017; Tzikas et al., 2017). , 2020). Ua hōʻike pū nā haʻawina he microenvironment pale ʻokoʻa ko TNBC e pili ana i nā pae kiʻekiʻe o nā mea ulu endothelial vascular, nā macrophages e pili ana i ka tumor (TAMs), nā lymphocytes tumor-infiltrating (TILs), a me nā molekala ʻē aʻe e pili ana i ka ulu ʻana o ka tumor a me ka neʻe ʻana. No laila, ʻo ka hoʻomaopopo ʻana i ka microenvironment o TNBC he mea koʻikoʻi ia no kona prognosis a me ka mālama ʻana (Fan lāua ʻo He, 2022).
No ka loiloi ʻana i ka wānana o TNBC a hōʻoia i ka mālama pono ʻana, he mea nui ka ʻike pololei, ma muli o ka immunohistochemistry (IHC) e ʻike ai iā ER, PR a me HER2, a me ka mammography e ʻike ai i nā neoplasms umauma. Eia nō naʻe, ʻaʻole hiki i ka mammography ke hōʻike pono i nā hiʻohiʻona intratumoral e like me ka necrosis a me ka fibrosis (Deepak Singh et al., 2021). ʻOiai ʻo ka wānana maikaʻi ʻole a me ka ʻike ʻana e pale aku i nā kauka mai ke kuhikuhi ʻana i nā lāʻau lapaʻau kūpono (Chaudhary, 2020), hoʻohana ʻia nā ʻano hana like ʻole e hoʻomaikaʻi i ka mālama ʻana i nā maʻi me TNBC. I kēia manawa, ʻelua mau lāʻau lapaʻau, ʻo doxorubicin a me cyclophosphamide, i hoʻohana ʻia i nā maʻi me TNBC a ua hōʻike i nā hopena maikaʻi. Eia kekahi, hoʻohana ʻia nā lāʻau platinum ʻē aʻe, e like me carboplatin a me cisplatin (Sikov et al., 2015). Eia kekahi, ua hoʻohana ʻia nā mea hoʻopaʻa PARP e like me Olaparib, Velaparib, a me PF-01367338 ma ke ʻano he mau lāʻau chemotherapy hiki ke mālama ʻia no TNBC (Ishino et al., 2018). ʻOiai ua hōʻike ʻia nā ala hōʻailona Wnt/b-Catenin, NOTCH, a me Hedgehog e pili ana i ka hanana a me ka holomua o TNBC, ʻo ka hoʻopaʻa ʻana i nā lāʻau lapaʻau i kēia mau ala he hoʻolālā koʻikoʻi paha (Aysola et al., 2013). ʻOiai ʻo ke ʻoki kino, ka radiation therapy a me ka chemotherapy ke kumu nui o ka mālama ʻana no TNBC i kēia lā, ua holomua nui ka hoʻomohala ʻana i nā lāʻau lapaʻau hou e pili ana i ka hoʻopaʻa ʻana i kuhikuhi ʻia, immunotherapy, nā mea hana hoʻoponopono gene e pili ana iā CRISPR e like me Cas9n, dCas9. , CRISPR/Cas12, prime editing, a me ka hoʻopaʻa ʻana i ka gene CRISPR/Cas9. Maanei e kālele mākou i nā mea hana hoʻoponopono gene e pili ana iā CRISPR i hoʻohana ʻia i ka mālama ʻana iā TNBC. I waena o lākou, ua loaʻa iā CRISPR/Cas9 ka nānā nui ʻia.
ʻO Cas9n, i ʻike ʻia hoʻi ʻo Cas9 nickase, he ʻano hoʻololi i hana ʻia ma ke ʻano genetic o ka protein Cas9 i loaʻa mai ka ʻōnaehana hoʻoponopono genome CRISPR/Cas9 (Gupta et al., 2019). Ma kona ʻano mua, aia i loko o ka protein Cas9 ʻelua mau kikowaena nuclease: ʻo RuvC a me HNH, ʻo ka hana nui o ia mea ʻo ia ka ʻoki ʻana o nā kaula DNA ʻelua. Eia nō naʻe, ma Cas9n, ua hoʻololi ʻia kekahi o nā kikowaena nuclease, ʻo HNH, a lilo i mea hana ʻole. No laila, ʻaʻole hana ka kikowaena HNH o Cas9n. ʻO ka kikowaena RuvC wale nō e hana mau ana, e ʻae ana iā Cas9n e ʻoki a hana paha i nā haki ma nā kaula DNA hoʻokahi (Trevino lāua ʻo Zhang, 2014). Ke hoʻohālikelike ʻia me Cas9, hiki iā Cas9n ke hōʻemi pono i nā hopena ma waho o ka pahuhopu a hoʻomaikaʻi pololei i nā ʻano hana hoʻoponopono cell. Hiki ke hoʻohana ʻia ʻo Cas9n e hoʻoponopono i nā pilikia like ʻole, e like me ka hana ʻana i nā haki ma nā wahi kikoʻī i loko o ka DNA kaula pālua. No kēia kumu, ua hui pū ʻia ʻelua mau molekala Cas9n a hoʻohana pū ʻia (Yee, 2016). ʻO ka Mixed lineage kinase 3 (MLK3) kahi mitogen-activated protein kinase e lawelawe ana ma ke ʻano he mea hoʻoponopono koʻikoʻi i ka wā o ka metastasis TNBC (Cronan et al., 2012).
Hiki iā MLK3 ke hoʻāla i nā ala hōʻailona he nui e alakaʻi ana i ka metastasis TNBC. No ka laʻana, hoʻoponopono ke ala c-Jun N-terminal kinase (JNK) i ka neʻe ʻana o ke kelepona a me ka hoʻohaʻahaʻa ʻana o ka matrix extracellular, a hoʻāla ʻo MLK3 i ke ala JNK, no laila e hoʻonui ai i ka neʻe ʻana o ke kelepona a hāʻawi i nā waiwai invasive (Rattanasinchai lāua ʻo Gallo, 2016). Hoʻoponopono pū ʻo MLK3 i ka EMT. No ka hana ʻana i kēia, hoʻāla ia i nā mea hoʻololi transcription ma lalo e like me Snail, Slug a me Twist. Hoʻopaʻa kēia mau mea i ka hōʻike ʻana o nā māka epithelial a hoʻolaha i ka hōʻike ʻana o nā māka mesenchymal, e alakaʻi ana i ka loaʻa ʻana o kahi phenotype metastatic (Casalino et al., 2023). Ua ʻike ʻia ʻo MLK3 e pāʻani i kahi hana i ka hana hou ʻana o ECM a me ka hoʻāla ʻana o nā proteases e like me ka matrix metalloproteinases (MMPs) e hoʻohaʻahaʻa i ka ECM. Hoʻomaʻalahi kēia i ka hoʻouka ʻana o nā cell tumor a hoʻolaha i nā wahi mamao (Katari et al., 2019). No laila, ua hōʻike nā haʻawina mua he kuleana koʻikoʻi ko MLK3 ma TNBC. Ua hoʻohana ʻo Rattanasinchai lāua ʻo Gallo i nā hiʻohiʻona TNBC e aʻo i ke kuleana o MLK3 a ua ʻike ʻo ia e hoʻolaha ana i ka ulu ʻana o ka maʻi kanesa ma o nā ala hōʻailona kikoʻī. Ua hoʻohana lākou iā CRISPR/Cas9n e hoʻoponopono iā MLK3 a ua ʻike i ka emi nui ʻana o ka metastasis TNBC (Rattanasinchai lāua ʻo Gallo, 2016).
ʻO dCas9, i kapa pinepine ʻia ʻo Cas9 hana ʻole, he ʻano derivative i hoʻololi ʻia o ka protein Cas9. ʻAʻole e like me Cas9 hana, ʻaʻohe hana endonuclease o dCas9, ʻaʻole hiki iā ia ke hoʻoulu i nā haki kaula pālua o ka DNA. No laila, hiki ke hoʻohana ʻia ʻo dCas9 e kuhikuhi pololei i nā wahi kikoʻī o ka genome me ka ʻole o nā hoʻololi a hoʻololi paha i ka moʻo DNA (Wang et al., 2016). Ma dCas9, ʻelua mau protein endonuclease, ʻo RuvC lāua ʻo HNH, ua hoʻopau ʻia ma ka hoʻopau ʻana i nā koena amino acid koʻikoʻi. (Richter et al., 2016). ʻOiai ʻaʻole i hana ʻia ka hana ʻoki DNA, he nui nā kuleana koʻikoʻi o dCas9 i ka noiʻi genetic a me ka biotechnology. No ka laʻana, hiki iā ia ke nānā i nā wahi kikoʻī o ka genome, hoʻomaʻamaʻa i ka hoʻoponopono transcriptional, a paipai i nā hoʻololi epigenetic (Brocken et al., 2018).
ʻO ka transcription factor ZEB1 (zinc finger E-box binding homeobox 1) ke kuleana koʻikoʻi a koʻikoʻi hoʻi i ka hoʻololi ʻana o ka epithelial-mesenchymal (EMT), kahi hana cellular e hana ʻia i ka wā o ka ulu ʻana o ka embryonic, ka hoʻoponopono ʻana i nā ʻiʻo, a me ka holomua o ka maʻi kanesa. Pili ia i ka hoʻololi ʻana o nā cell epithelial i nā cell mesenchymal, e hopena ana i nā loli i ke ʻano o ke cell, ka motility, ka invasiveness, a pēlā aku. (Wu et al., 2020). Hoʻopaʻa ʻo ZEB1 i nā māka epithelial he nui, e like me E-cadherin lāua ʻo Occludin, nona ke kuleana no ka mālama ʻana i ka hoʻopili ʻana o nā cell-cell a me ka polarity o nā cell epithelial ma TNBC (Moreno-Bueno et al., 2008). Eia kekahi, hoʻāla ʻo ZEB1 i kekahi mau māka e like me N-cadherin, vimentin a me fibronectin (Konradi et al., 2014) a hoʻoponopono pū i nā genes i komo i ka hana hou ʻana o cytoskeletal e like me Rho GTPases a me matrix metalloproteinases (MMPs) (Huang). et al., 2014). 2022), eia kekahi, hoʻopilikia pū ia i nā ala hōʻailona like ʻole e like me ka transforming growth factor-β (TGF-β), Wnt signaling, etc., no laila e hoʻolaha ana i ka hoʻouka kaua ʻana o ka tumor a me ka metastasis ma TNBC (Chen et al., 2016). ). Hōʻike nā haʻawina he nui a me nā hōʻike ʻepekema he nui ka hiki iā ZEB1 ke lilo i mea waiwai nui no ka ʻike ʻana a me ke komo ʻana o ka therapeutic o TNBC. Ma kahi noiʻi hou, ua hoʻohana ʻo Waryah et al. i kahi kumu hoʻohālike TNBC a ua hoʻokō i ka hoʻopau piha ʻana o ZEB1 e dCas9. No laila, ua ʻike lākou i ke kikoʻī kiʻekiʻe loa a me ka aneane hoʻopau piha ʻana o ZEB1 i nā kūlana in vivo (Waryah et al., 2023).
He mea hana hoʻoponopono gene ʻo CRISPR/Cas12 i kapa ʻia ʻo CRISPR/Cpf1. Ua loaʻa mai ia mai ka ʻōnaehana CRISPR/Cas, kahi e pili ai ka huaʻōlelo Cas12 i ka protein 12 e pili ana iā CRISPR (Bharathkumar et al., 2022), kahi i like loa me Cas9. ʻO ka ʻokoʻa wale nō, aia i loko ka protein Cas12. Ke hoʻohālikelike ʻia me Cas9, loaʻa iā Cas12 kekahi mau hana kūikawā e like me ka hoʻoulu ʻana i nā wēlau sticky i ka wā o ke kaʻina hana hoʻoponopono gene, ʻoiai ʻo Cas9 e hoʻoulu i nā wēlau blunt (Wang et al., 2021). Hāʻawi kēia waiwai o Cas12 i kāna ʻenehana hoʻoponopono DNA kikoʻī. Ma ke ʻano he protein i hiki ke ʻike pololei a ʻokiʻoki i ka DNA target, he versatility kupaianaha kona, e lilo ia i mea hana pono no nā ʻano noi like ʻole, me ka hoʻoponopono gene (Pickar-Oliver lāua ʻo Gersbach, 2019).
E like me nā ʻano CRISPR ʻē aʻe, hiki iā CRISPR/Cas12 ke hoʻopau a hoʻoulu paha i nā genes ma TNBC, e kuhikuhi ana i nā genes e pāʻani ana i kahi kuleana koʻikoʻi i kona pathogenesis a i ʻole ka pane ʻana i ka lāʻau lapaʻau (Yang lāua ʻo Zhang, 2023). No ka hoʻokō ʻana i kēia kaʻina hana, ua hoʻomohala ʻia kahi alakaʻi RNA (gRNA) e kuhikuhi ana iā Cas12 i ka gene target. Ke hoʻopaʻa ʻo Cas12 i kāna pahuhopu, hoʻolauna ia i nā haki kaula pālua i loko o ka DNA a hoʻoulu i nā ʻano hana hoʻoponopono DNA. I ka wā o ke kaʻina hana hoʻoponopono, hiki ke hoʻokomo ʻia nā nucleotides hewa, e alakaʻi ana i nā mutations gene a me ka nalowale o ka hana (Zhang et al., 2021a). Eia kekahi, ua hoʻohana ʻia kahi mana i hoʻomaikaʻi ʻia o ka enzyme Cas12 (i kapa ʻia ʻo dCas12) e hoʻoulu i nā genes. Ma ka hoʻohui ʻana me kahi mea hoʻoulu transcription, hiki ke hoʻoulu i kekahi mau genes, a laila e hoʻoulu ai iā lākou. Loaʻa i kēia ʻenehana ka hiki ke hoʻolaha i ka hoʻoulu ʻana o nā genes suppressor tumor (Sultan et al., 2022).
ʻO ka hoʻoponopono mua he ʻenehana hoʻoponopono genome hou i hoʻomohala ʻia a ʻoi loa aku ka maikaʻi. Hiki iā ia ke hoʻololi i ka DNA o kahi meaola me ka pololei loa (Chen lāua ʻo Liu, 2023). Hoʻokō ʻia ka hoʻoponopono mua ma o ka hoʻohui ʻana o ʻelua mau ʻāpana nui: kahi enzyme CRISPR/Cas9 i hoʻololi ʻia a me ka transcriptase hoʻohuli. ʻO ke kuleana o ka enzyme CRISPR/Cas9 ke koho pono i nā wahi kikoʻī i loko o ka genome, ʻoiai ʻo ka transcriptase hoʻohuli e kōkua i ka hoʻololi piha ʻana i ka DNA ma kahi i manaʻo ʻia (Hassan et al., 2021). I loko o ke ʻano hoʻoponopono mua, ʻo ka hana mua e pili ana i ka hana ʻana o kahi alakaʻi hoʻoponopono mua RNA (pegRNA) (Standage-Beier et al., 2021). Loaʻa iā ia ka moʻo pahuhopu a me ka template RNA e pili ana i ka wahi hoʻoponopono i makemake ʻia i loko o ka DNA pahuhopu. A laila hoʻokomo ʻia ka pegRNA i loko o nā cell pahuhopu me kahi nuclease hoʻoponopono mua (PE2). ʻO PE2 kahi protein fusion i haku ʻia me kahi enzyme Cas9, kahi transcriptase hoʻohuli, a me kahi mea hoʻololi hoʻoponopono mua (Martín-Alonso et al., 2021). I loko o ke kelepona, ʻimi nā hui pegRNA a me PE2 i ka DNA kikoʻī a lākou e makemake ai e hoʻololi. ʻOki ka enzyme Cas9 i ka DNA a hana i kahi template i haku ʻia me nā kaula hoʻokahi (Choi et al., 2022). Hoʻohana ka reverse transcriptase i kēia template e hoʻomākaukau i ka DNA no ka hoʻoponopono ʻana. Me ke kope ʻana i ka DNA, ua hoʻokomo pū ʻia nā kuhikuhi no ka hoʻoponopono ʻana i ka template RNA. ʻO ka hope loa, hoʻohana ʻia ke kaula DNA i hana hou ʻia ma ke ʻano he template e hoʻoponopono ai i ka haki i loko o ka DNA, e hopena ana i kahi moʻo DNA i hoʻololi ʻia me ka hoʻololi i makemake ʻia (Ochoa-Sanchez et al., 2021). Hiki i nā ʻano hana hoʻoponopono primer ke alakaʻi i nā mutations ākea i nā genes. Hiki iā ia ke kuhikuhi i nā mutations kiko, nā hoʻokomo, nā holoi ʻana, a me nā pani gene (Anzalone et al., 2019; Chen lāua ʻo Liu, 2023). Hāʻawi ka hoʻoponopono Prime i kekahi mau pono ma mua o nā ʻenehana hoʻoponopono genome ma mua. ʻO kēia mau mea e pili ana i ka hoʻonui ʻana i ka pololei, ka hoʻemi ʻana i nā hopena off-target, a me ka hiki ke hoʻoponopono i ka DNA me ka ʻole o ka hilinaʻi ʻana i nā haki kaula pālua DNA (Anzalone et al., 2020).
Ua hoʻomohala mua ʻia ka ʻenehana CRISPR/Cas9 e pale i ka bacteria mai ka hoʻoili ʻana o ka plasmid a me ka maʻi phage a ua hoʻohana hou ʻia ma hope he mea hana DNA RNA-based no ka hoʻoponopono genome (Jiang lāua ʻo Doudna, 2017). Eia kekahi, ua hōʻike ʻia ka ʻōnaehana CRISPR/Cas9 i loaʻa i 50% a me 87% o nā genomes bacteria a me archaeal, kēlā me kēia (Ishino et al., 2018). ʻO CRISPR/Cas9 kahi mea hana kūpono no ka holoi ʻana, ka hoʻokomo ʻana a me ka hoʻoponopono ʻana i kekahi ʻano genetic abnormal me ka hoʻohana ʻana i nā ʻano in vivo a me in vitro (Sabit et al., 2021). Eia kekahi, ua hōʻike ʻia ʻo CRISPR/Cas9 he ʻāpana o ka ʻōnaehana pale adaptive ma muli o kona kikoʻī no nā genes target o ka hoihoi (Chen lāua ʻo Zhang, 2018).
ʻO CRISPR/Cas9 he Cas9 a me kahi RNA alakaʻi hoʻokahi (sgRNA). He endonuclease ʻo Cas9 i haku ʻia me nā ʻāpana protein he nui. Eia kekahi, loaʻa iā Cas9 nā waiwai hoʻonohonoho a me nā conformational kūikawā (Pacesa et al., 2022) ʻoiai he ʻelua lobes: recognition (REC) a me nuclease (NUC). Ua māhele hou ʻia ka lobe REC i ʻekolu mau ʻāpana: REC1, REC2, a me nā helices alahaka (Cromwell et al., 2018). ʻO ka NUC he ʻekolu lobes, ʻo ia hoʻi ʻo RuvC (RuvC I, RuvC II, RuvC III), HNH a me ka protospacer adjacent motif (PAM) interaction domain (Fig. 1) (Song et al., 2016). ʻO sgRNA he ʻelua mau ʻāpana, me CRISPR RNA (crRNA) a me ka transcoding small RNA (tracer RNA) (Kiʻi 1). Hoʻopili ka sgRNA i ka protein Cas9 i nā connexins e hana i kahi paʻakikī hana, i ʻike ʻia hoʻi ʻo ka effector complex (Richter et al., 2012). ʻO crRNA kahi hui kumu nucleotide 18-20 e pāʻani ana i kahi kuleana koʻikoʻi i ka ʻike ʻana i nā moʻo DNA pahuhopu. Eia kekahi, hui pū ʻo crRNA me ka DNA pahuhopu, a hana ʻo tracrRNA ma ke ʻano he scaffold no ka Cas9 nuclease e hoʻopaʻa i ka DNA pahuhopu (Manghwar et al., 2019). Ma ka ʻaoʻao ʻē aʻe, loaʻa i ka moʻo PAM he 3 mau nucleotides, ka mea e hōʻoia, kuhikuhi a hoʻoponopono i ka hoʻopaʻa ʻana o ka effector complex i ka DNA. Loaʻa i nā subunits complex protein Cas9 ʻo RuvC a me HNH ka hana catalytic (Richter et al., 2012; Asmamaw lāua ʻo Zawdie, 2021). ʻO ka HNH nuclease domain o ka subunit Cas9 e ʻoki i ke kaula DNA e pili ana me ka crRNA. Hoʻopau ka kikowaena RuvC nuclease i nā kaula DNA ʻē aʻe a hoʻopuka i nā haki kaula pālua (DSB), ma hope o ia mea e hoʻāla ʻia ai ʻelua mau ʻano hana hoʻoponopono haki DNA like ʻole (Jiang lāua ʻo Doudna, 2017) (Kiʻi 1).
Kiʻi 1. ʻIke holoʻokoʻa o CRISPR/Cas9 (A). Nā ʻāpana o ka ʻōnaehana CRISPR/Cas9: (i). ʻO Cas9 endonuclease ke kuleana no ka ʻoki ʻana i ka moʻo DNA i manaʻo ʻia, (ii) kahi alakaʻi hoʻokahi (sg) RNA i loaʻa mai ka hui ʻana o crRNA a me nā tra-crRNA chimeras. (ʻelua). Loaʻa iā Cas9 kekahi mau ʻāpana e like me Rec I, Rec II, NUC lobe (ʻo HNH a me Ruv C nā ʻāpana liʻiliʻi) a me ka PAM interaction domain (C) me nā hana like. Hoʻokaʻawale ka hui protein CRISPR/Cas9 i nā moʻo DNA i nā ʻano hoʻopihapiha ʻole a me nā ʻano hoʻopihapiha (D). Hoʻoponopono ʻo CRISPR/Cas9 i ka genome i ʻekolu mau pae: ʻike, ʻoki a hoʻoponopono. Alakaʻi ka sg-RNA i hoʻolālā ʻia iā Cas9 a ʻike i ka moʻo i makemake ʻia ma o ka ʻāpana hoʻopihapiha crRNA. ʻIke ʻo Cas9 i ka moʻo PAM ma 5′-NGG-3′ a hoʻoheheʻe i ka DNA, e hana ana i kahi hybrid DNA-RNA a hoʻāla i ka cleavage. ʻO ke kikowaena HNH o Cas9 e ʻoki i ke kaula hoʻopihapiha, a ʻo ke kikowaena RuvC e ʻoki i ke kaula ʻaʻole hoʻopihapiha. Hoʻoponopono ʻo CRISPR/Cas9 i ka dsDNA ma ka uhaki ʻana iā ia ma nā ʻano ʻelua: ka hui ʻana o ka hopena non-homologous (NHEJ) a me ka hoʻoponopono homology-directed (HDR). Hoʻoponopono ʻo NHEJ i ka DNA kaula pālua i ka loaʻa ʻole o ka DNA homologous haole ma o ke kaʻina hana enzymatic, kahi ʻano hana hewa-prone e hiki ke hoʻokomo a wehe paha i nā moʻo DNA random. He kikoʻī loa ka HDR a koi i nā template DNA homologous.
ʻO nā ala hoʻoponopono i hoʻopili ʻia e CRISPR/Cas9 e komo pū me nā ʻano hana hoʻohui hope ʻole (NHEJ) a me ka hoʻoponopono homology-directed (HDR). He ala hewa ʻo NHEJ no ka mea e pili ana i ka hoʻokomo ʻana a i ʻole ka holoi ʻana a ʻaʻole pono kekahi ʻano i ka wā o ka hana hoʻoponopono. Hoʻohana ia i nā nucleotides random e hana i nā protein maʻamau (Abbasi et al., 2021). Aia kēia ʻōnaehana hoʻoponopono i ʻehā mau paʻakikī e like me ka paʻakikī KU, ka paʻakikī protein cross-complementing type 4 (XRCC-4), ka enzyme hana hopena DNA, a me ka protein kinase DNA-PKcs (Abbasi et al., 2021). ʻElua mau subunits o ka protein KU complex, ʻo Ku 70 a me Ku 80, a he kuleana koʻikoʻi kāna i loko o ke ʻano NHEJ, ʻoiai ua hoʻomaka ʻia ke ʻano hoʻoponopono e ka hoʻopaʻa ʻana o ʻelua mau subunits (Ku 70 a me Ku 80) i nā wēlau blunt a kokoke blunt paha o ka DNA target (Abbasi et al., 2021), e lawelawe ana ma ke ʻano he scaffold e hoʻopaʻa i nā mea pili ʻē aʻe o NHEJ i kahi o ka ʻeha (Yang et al., 2020). Ua haku ʻia ʻo XRCC-4 a me DNA ligase me 334 a me 911 mau amino acid, kēlā me kēia, a ʻo ka XRCC4-DNA ligase IV complex e hoʻoulu i ka ligation o nā wēlau DNA (Chatterjee et al., 2015). ʻO ka enzyme hana hopena DNA, i ʻike ʻia hoʻi ʻo polynucleotide kinase 3′-phosphate, he enzyme hana hopena DNA. Hiki iā ia ke wehe i ka hui 3′P i loko o ka DNA a phosphorylate i ka hui 5′OH i ka wā o ka hoʻoponopono DSB. Hoʻokomo pū ia i ka hoʻoponopono ʻana i nā haki kaula hoʻokahi (SSBs) me ka hoʻohana ʻana i ke ala hoʻoponopono SSB (Chatterjee et al., 2015). ʻO ka protein kinase DNA-PKcs kahi kinase protein e hilinaʻi ana i ka DNA i loaʻa i kahi subunit catalytic o nā PIKK (phosphatidylinositol 3-kinase-related kinases) a me ka ʻohana ataxia telangiectasia mutated (ATM), a me ATM a me Rad3-related ATRs. nā haki kaula (DSBs) a me nā haki kaula hoʻokahi (Yue et al., 2020; Peng et al., 2016).
ʻOi aku ka pololei a me ke kūpono o ka HDR no ka mea ua kope ʻia ka ʻike me ka hoʻohana ʻana i ke ʻano piha o ka homologous DNA duplex, ʻoiai ke koi nei i ke alo o nā chromatids kaikuahine. Hana ʻia kēia i ka wā S/G2 o ke kaʻina hana cell mammalian. Loaʻa nui ka HDR i nā ʻano hū, akā he mea koʻikoʻi ka NHEJ i nā mammals (Burma et al., 2006; Abbasi et al., 2021). Hōʻike ʻia ke ʻano hoʻoponopono piha ma ke Kiʻi 1.
ʻOiai ua hoʻokumu ʻia ʻo TNBC e nā ʻano genetic a me epigenetic, ʻo ka hoʻoponopono ʻana i nā ʻano genomic/epigenomic malignant me ka hoʻohana ʻana iā CRISPR/Cas9 paha he ala lapaʻau kūpono (Chen et al., 2019). Eia kekahi, ʻo kekahi mau mea hoʻopili i pili i ka hoʻoponopono transcriptional cell-specific e hōʻike i nā ʻano kūikawā o nā cell cancer, e hōʻike ana he ala maikaʻi loa ka hoʻoponopono transcriptional no ka mālama ʻana i ka maʻi kanesa (Drost et al., 2017). ʻO ka hoʻohana ʻana i kēia mau hiʻohiʻona molekala o nā tumors, e like me nā genetic, epigenetic a me nā transcriptional defects, no ka hoʻomohala ʻana i ka lāʻau lapaʻau hiki ke hoʻomaikaʻi i nā hopena lapaʻau a hōʻemi i nā kumukūʻai screening. ʻO CRISPR kahi mea hana hoʻoponopono gene hiki ke ʻike a ʻike i nā pahuhopu genomic e hoʻoulu ai i ka maʻi kanesa, akā hiki ke hoʻohana ʻia e hoʻoponopono, kāohi, a hoʻololi epigenetically i nā oncogenes i loko o nā cell kanaka (Papa 1) (Ahmed et al., 2021). Ua hana ʻia kekahi mau pale CRISPR e ʻimi i nā genes e pili ana me nā mea hoʻopau tumor, oncogenes, a me ke kūʻē ʻana i ka lāʻau. Ua hōʻike ʻia ka hoʻohana ʻana o CRISPR/Cas9 e hoʻoponopono i nā ʻano oncogenes TNBC like ʻole ma ke Kiʻi 2.
Kiʻi 2. Hoʻoponopono ʻana o nā genes o nā ʻano oncogenes TNBC like ʻole me ka hoʻohana ʻana iā CRISPR/Cas9 e hopena ana i ka emi ʻana o ka ulu ʻana o ka puʻupuʻu a me ka metastasis. ʻO kēia mau oncogenes e komo pū me CDK7, NAT1, UBR5, YTHDF2, ITGA9, CXCR4 a me CXCR7, Crypto1, ROR1 a me ST8SIA, ka mea i pili i ka hanana a me ka metastasis o TNBC.
ʻO ka neʻe ʻana o nā cell cancer, ka hoʻouka kaua ʻana, a me ka hoʻololi ʻana o ka epithelial-mesenchymal (EMT) e pili ana me ITGA9. Ua hōʻike ʻia nā haʻawina mua he mea nui ʻo ITGA9 ma ke ala Notch a he kuleana hoihoi i ka rhabdomyosarcoma metastasis (Molist et al., 2020). Eia kekahi, ua ʻike ʻia ʻo ITGA9 e pili pono ana me ka prognosis o ka mea maʻi ma kekahi mau ʻano tumor, me ka maʻi umauma (Wang Z. et al., 2019). Eia kekahi, ua hōʻike ka loiloi bioinformatics o ITGA9 ua ʻoi aku ka kiʻekiʻe o kona hōʻike ʻana ma TNBC ma mua o nā ʻano maʻi umauma ʻē aʻe. ʻO nā pae ITGA9 kiʻekiʻe e pili ana me ka metastasis tumor a me ka hoʻi hou ʻana i nā maʻi TNBC. ʻO ka hoʻopau ʻana o ITGA9 e CRISPR/Cas9 i hopena i nā waiwai like me ke cell stem cancer (CSC), ka angiogenesis tumor, ka ulu ʻana o ka tumor, a me ka emi ʻana o ka metastasis ma ka hoʻolaha ʻana i ka hoʻohaʻahaʻa β-catenin ma TNBC (Wang Z. et al., 2019).
He lālā ʻo Crypto-1 o ka ʻohana TGF-β a he mea koʻikoʻi ia no ka embryogenesis mua, ka mālama ʻana i nā cell stem, a me ka metastasis cancer (Ishii et al., 2021). ʻIke ʻia hoʻi ʻo Tdgf-1, he protein hōʻailona GPI-anchored oncogenic ia e pili ana i ka hoʻoponopono ʻana i ka hoʻokumu ʻana o ke kaha mua, mesoderm a me endoderm, a me ka hoʻokumu ʻana o ka asymmetry hema/ʻākau i ka ulu ʻana o nā ʻōpū kino i ka wā o ka embryogenesis (Zhang et al., 2021b). Eia kekahi, ua hōʻike ʻia ʻo Cripto-1 e pili ana i ka hoʻololi epithelial-mesenchymal (EMT) ma ke ʻano he māka cell stem (Zhang et al., 2021b). He mea koʻikoʻi ʻo EMT ʻaʻole wale no nā kaʻina hana like ʻole e like me ka ulu ʻana o ka embryonic, fibrosis a me ka ho'ōla ʻana i ka ʻeha, akā no ka hoʻouka kaua ʻana o ka maʻi kanesa a me ka metastasis. Eia kekahi, ua hōʻike ʻia ʻo Cripto-1 e launa pū me ʻehā mau mea loaʻa Notch a hoʻonui i ko lākou ulu ʻana ma hope o ka unuhi ʻana (Brandstadter lāua ʻo Maillard, 2019). Ua ʻike ʻia ke ala hōʻailona Notch e pili ana i ka mālama ʻana i nā hunaola maʻi ʻaʻai umauma kanaka. Ua hōʻike ʻia nā haʻawina e kāohi ana ka knockout CRISPR/Cas9-mediated o Crypto-1 i ka ulu ʻana o ka maʻi ʻaʻai a me ka metastasis. No laila, hiki iā Crypto-1 ke lilo i pahuhopu therapeutic koʻikoʻi no TNBC (Castro et al., 2015).
ʻO ka protein XCL12 a me kāna mea hoʻokipa chemokine CXC (CXCR4 a me CXCR7) e pāʻani i nā kuleana like ʻole i ka hoʻonui ʻana o nā cell cancer, ka ulu ʻana, ka neʻe ʻana a me ka hoʻouka kaua ʻana (Wu et al., 2015). Ua pili kēia mau chemoreceptors me ka hoʻomohala ʻana o TNBC ma o nā ala hōʻailona he nui i nā hiʻohiʻona in vivo a me in vitro (Wu et al., 2015). Eia kekahi, ʻo ka hoʻoulu ʻana o CXCR4 a me CXCR7 e pili ana me ka maʻalahi nui i ka metastasis a me ka prognosis maikaʻi ʻole ma TNBC (Karn et al., 2022). No laila, ʻo ka hoʻopau ʻana o CXCR4 a me CXCR7 e lilo paha i mau genes target lāʻau lapaʻau kūpono no ka mālama ʻana i ka maʻi ʻaʻai umauma, me TNBC. Ua hoʻohana ke aʻo ʻana e Yang et al. Yang et al (2019) iā CRISPR/Cas9 e hoʻopau pū i nā genes CXCR4 a me CXCR7 a ua ʻike ʻia ua kāohi nui ʻia ka hoʻonui ʻana o TNBC, ka ulu ʻana, ka neʻe ʻana a me ka hoʻouka kaua ʻana (Yang et al., 2019).
Ua ʻike ʻia ka piʻi ʻana o nā pae o miR-3662, kahi oncogene TNBC, i loko o nā ʻiʻo kanesa umauma (Yi et al., 2022). Ua hōʻike ʻia ka hoʻokuʻu ʻana o miR-3662 e kāohi i ka ulu ʻana o ka maʻi kanesa umauma a me ka metastasis ma vivo a me in vitro (Agarwal lāua ʻo Gupta, 2021). He mea hoʻopaʻa ikaika ʻo HBP-1 i ka hōʻailona Wnt/-catenin a he kuleana paha ia no ka ulu ʻana o nā pūnaewele TNBC i hoʻopili ʻia e miR-3662. I kēia manawa, ua ʻike ʻo Yi et al. ʻo ka axis miR-3662-HBP1 e hoʻoponopono i ke ala hōʻailona Wnt/-catenin i loko o nā pūnaewele TNBC (Yi et al., 2022). Ma muli o kona hōʻike kikoʻī ʻana i ka tumor, hiki i ka miR-3662 ke lilo i pahuhopu therapeutic no TNBC. No laila, ʻo ka hoʻokuʻu ʻana o CRISPR/Cas9 i hoʻopili ʻia e miR-3662 he ala maikaʻi loa ia no ka hoʻomohala ʻana i nā lāʻau lapaʻau hou no ka mālama ʻana iā TNBC.
ʻO UBR5 kahi nucleophosphoprotein 300 kDa i ʻike ʻia he mea hoʻoponopono koʻikoʻi o ka tumorigenesis, metastasis, a me ka pane pale i nā maʻi kanesa like ʻole (Shearer et al., 2015; Fu et al., 2023). Ua hōʻike ʻia ʻo UBR5 ua hoʻonui nui ʻia i nā laʻana TNBC a hoʻoulu i ka hana ERα ma o ka hoʻoulu ʻana i ka hoʻonui ʻana ma o kāna hana ubiquitin ligase (Bolt et al., 2015). Ua hōʻike pū nā haʻawina hoʻonohonoho exome holoʻokoʻa o nā laʻana TNBC mua i ka hoʻonui ʻia ʻana o ka hōʻike ʻana o UBR5, e hōʻike ana i kona kuleana i ka hoʻomohala ʻana o TNBC. Eia kekahi, ua hōʻike ka holoi ʻana o CRISPR/Cas9-driven o UBR5 i ka pale nui ʻana o ka metastasis TNBC a me ka ulu ʻana i nā hiʻohiʻona ʻiole hoʻokolohua. Eia kekahi, ʻo ka hoʻokomo ʻana o UBR5 i loko o kahi hiʻohiʻona ʻiole wild-type i hoʻihoʻi i kāna hana piha, ʻoiai ʻaʻole i ʻike ʻia kēia hopena i nā ʻano mutant inactivated (Liao et al., 2017). ʻO ka nele o UBR5 e pili ana me ka hoʻonui ʻia o ka apoptosis, necrosis, a me ka pale ʻana i ka ulu ʻana o ka tumor ma TNBC ma muli o ka angiogenesis maikaʻi ʻole. Ma muli o ka nalowale ʻana o UBR5, ua emi ka laha ʻana o ka tumor i nā ʻōpū mamao, a hoʻoulu ʻo UBR5 i ka EMT maʻamau ma ka hōʻemi ʻana i ka hōʻike ʻana o E-cadherin (Zhang lāua ʻo Weinberg, 2018). I kēia manawa, ua hōʻike ʻia ʻo UBR5 he mea nui loa ia i ka transcription PDL1 i hoʻokomo ʻia e IFN-γ ma TNBC ma muli o kona nele i ka hana ubiquitination E3. Ua hōʻike ka loiloi transcriptome RNA e loaʻa paha iā UBR5 nā hopena ʻōnaehana i nā genes e pili ana me ke ala IFN-γ a hoʻolaha i ka transactivation PDL1 ma ka hoʻonui ʻana i nā pae hoʻāla o ka protein kinase RNA (PKR) a me kāna mau transducers hōʻailona a me nā mea hoʻoulu. transcription 1 (STAT1) a me ka interferon regulatory factor 1 (IRF1). Eia nō naʻe, ʻo ka CRISPR/Cas9-mediated combined ablation of UBR5 and PD-L1 expression he hopena therapeutic synergistic ma mua o ka blockade wale nō, me nā hopena koʻikoʻi ma ka microenvironment tumor (Wu et al., 2022). No laila, he mea nui paha ʻo CRISPR/Cas9 e kāohi i ka hana UBR5, no laila e kāohi ana i ka metastasis TNBC a me ka ulu ʻana o ka tumor.
ʻO ROR1 kahi protein transmembrane ʻano I i hōʻike ʻia i ka wā o ke kanesa a me ka ulu ʻana o ka embryonic a ua ʻike ʻia he protein oncofetal (Nicholas Borcherding, 2014). ʻO ke ʻano invasive o nā maʻi kanesa kanaka like ʻole e pili ana me ka hoʻonui ʻia o ROR1. Ua loaʻa nā hopena hoʻohiki mai nā haʻawina in vivo a me in vitro e pili ana i nā hui therapeutic e pili ana iā ROR1 (Chien et al., 2016). Ua pili pū ʻia nā pae kiʻekiʻe o ROR1 mRNA i nā biopsies ʻiʻo umauma me nā puʻupuʻu umauma basal-like (BL) aggressive a me ko lākou neʻe ʻana i nā ʻāpana ʻē aʻe o ke kino. Eia kekahi, ua ʻike ʻia ka overexpression o ROR1 ma ke ʻano he hōʻailona prognostic no ka hoʻomohala ʻana o TNBC (Chien et al., 2016). Eia naʻe, ʻo ka hoʻopau ʻana iā ROR1 me ka hoʻohana ʻana iā CRISPR/Cas9 e kāohi i ka ulu ʻana o TNBC a me ka metastasis he hoʻolālā kūpono ia.
ʻO ke kaʻina hana sialylation e pili ana i ka hoʻohui ʻana o ka waikawa sialic i nā glycoconjugates, kahi i hoʻoulu ʻia e nā sialyltransferases (STs). No ka ʻohana ST ʻo ST8SIA1 a he kuleana koʻikoʻi kāna i ka pathogenesis o nā maʻi like ʻole e like me ka lymphocytic leukemia a me ke kanesa colorectal (Chang et al., 2018). Hōʻike ka loiloi moʻo RNA ua hōʻike nui ʻia ʻo ST8SIA1 i loko o ka ʻiʻo umauma o nā maʻi TNBC a ua pili maikaʻi ʻia me nā mutations i loko o ka tumor suppressor gene p53, kahi e hiki ai ke kōkua i ka pathogenesis o TNBC (Battula et al., 2017). Eia kekahi, pili ʻo ST8SIA1 i ka metastasis a me ka hoʻi hou ʻana o TNBC, e hōʻike ana he kuleana koʻikoʻi kāna i ka hanana a me ka hoʻomohala ʻana o TNBC. Ma kahi kumu hoʻohālike in vitro o TNBC, ua hōʻike ʻia ka hoʻokuʻu ʻana o ST8SIA1 e CRIPSR/Cas9 e ālai i ka ulu ʻana a me ka metastasis (Battula et al., 2017). Hōʻike kēia he mea nui paha ʻo CRISPR/Cas9 no ke kāohi ʻana i ka hana o ka oncogene ST8SIA1 i ka mālama ʻana iā TNBC.
ʻO NAT1 kahi enzyme metabolic e hoʻoulu ai i ka hoʻokumu ʻana o nā hui xenobiotic phase II a ua hōʻike ʻia ma aneane nā ʻiʻo kanaka āpau. Hiki iā NAT1 ke hoʻohana i ka cofactor folate e kuhikuhi i ka acetyl-CoA (acetyl-CoA) ʻoiai i ka loaʻa ʻole o ka substrate amine aromatic (Stepp et al., 2015; Laurieri et al., 2014). Ua hōʻike ʻia nā haʻawina e hoʻoponopono ʻo NAT1 i ka hana matrix metalloproteinase 9 (MMP9) i nā hiʻohiʻona cell cancer umauma a pale aku i nā reactive oxygen species (ROS) i ka wā pōloli glucose (Wang et al., 2018). Ua hōʻike ʻia ka holoi ʻana o NAT1 e kāohi i ka pyruvate dehydrogenase complex, e alakaʻi ana i ka mitochondrial dysfunction (Wang L. et al., 2019). Eia kekahi, ua hōʻike ʻia nā hōʻike like ʻole e hiki i ka NAT1 inhibition me ka hoʻohana ʻana i nā mole liʻiliʻi a me ka silencing siRNA ke hōʻemi i ka invasiveness a me ka hoʻonui ʻana o nā cell cancer umauma (Stepp et al., 2018). I kēia mau lā iho nei, ua hoʻohana ʻia ʻo CRISPR/Cas9 e kāohi iā NAT1 i loko o ka laina cell cancer umauma MDA-MB-231, e hoʻopilikia ana i ka metabolism cellular ma muli o kona pae hōʻike. Ua hōʻike pū kēia haʻawina he mea koʻikoʻi ʻo NAT-1 no ka holomua a me ka metastasis o TNBC (Carlisle et al., 2020).
Hoʻoponopono ʻia ke kākau ʻana i hoʻonohonoho ʻia o nā oncogenes e nā super enhancers, nā transcription factors a me nā cofactors (Hnisz et al., 2015). Eia kekahi, pono kahi hui o nā kinases cyclin-dependent (CDKs), e like me CDK7, CDK8, CDK9, CDK12, a me CDK13, no ka hoʻoponopono ʻana i ke kākau ʻana. I waena o lākou, pili ʻo CDK7 i ka phosphorylation o RNA polymerase II, he mea nui loa ia no ka hoʻomaka ʻana a me ka hoʻonui ʻana o ka oncogene transcription i ka pathogenesis o TNBC. Ma kahi noiʻi seminal, ua kāohi ka holoi ʻana o CDK7 e CRISPR/Cas9 iā TNBC, e hōʻike ana he hilinaʻi ka pathogenesis o TNBC iā CDK7 (Wang Y. et al., 2015).
Ua hōʻike ʻia nā haʻawina e hiki i nā mutations ma MYC a me RBP (RNA binding protein) ke alakaʻi i ka apoptosis, ʻoiai ʻo nā mutations gene hoʻokahi ma MYC a i ʻole RBP ʻaʻole ia e hoʻopilikia i ka ulu ʻana o nā cell cancer (Einstein et al., 2021). Ua nānā ʻia ma mua o 1000 RBP i loko o ka genome kanaka me ka hoʻohana ʻana i kahi waihona puke CRISPR/Cas9. I waena o lākou, ua ʻike ʻia he 57 RBP he mea koʻikoʻi no ka ulu ʻana o nā cell cancer me nā pae MYC kiʻekiʻe loa (Wheeler et al., 2020). Eia kekahi, he mea nui ʻo YTHDF2 no ka mālama ʻana i ka ulu ʻana o nā cell TNBC a hoʻemi i ka nui o nā transcripts methylated i ka wā o ka transcription kiʻekiʻe a me ka unuhi ʻana i nā cell cancer me ka hōʻike MYC kiʻekiʻe. Eia kekahi, ʻaʻole pono ʻo YTHDF2 no nā cell cancer, ʻaʻole hilinaʻi nui ʻia i nā pae MYC kiʻekiʻe e hoʻolōʻihi i ke ola o nā maʻi TNBC (Einstein et al., 2021), e hōʻike ana he hiki ke lilo i pahuhopu therapeutic no nā lāʻau lapaʻau e hiki ke lanakila ma luna o TNBC.
ʻO nā protein manamana lima zinc (ZNF) e hana ana ma kahi o 1% o ka genome kanaka holoʻokoʻa. Ua hōʻike ʻia nā haʻawina e hiki i ka ZNF ke hoʻoponopono i ka ulu ʻana o nā cell i nā maʻi kanesa like ʻole, e like me ke kanesa o ke ake, ke kanesa o ka umauma a me ke kanesa colorectal (Zhang W. et al., 2021; Li et al., 2017). Ua hoʻohana ʻia nā waihona i hana ʻia e CRISPR knockout e nānā i nā ʻano genes tumor suppressor (TSGs) like ʻole i loko o nā cell kanesa umauma (Shalem et al., 2014). Mai ia manawa, ua hōʻike ʻia kahi noiʻi transcriptomic o nā cell kanesa umauma i holoi ʻia ʻo CRISPR/Cas9 ZNF319 he gene tumor suppressor ʻo ZNF319 e hōʻemi ana i ka ulu ʻana o ke kanesa umauma a no laila ua komo i nā ʻano hana hōʻailona like ʻole a me nā hana olaola ʻē aʻe (Wang L. et al., 2022).
ʻO Ferroptosis kahi ʻano make cell i hoʻolālā ʻia e hilinaʻi ana i ke alo o ka hao. Ua ʻike ʻia ua hoʻopili ʻia ka ulu ʻana o ka ferroptosis e ke alo o nā lipid peroxides. Eia kekahi, ua hōʻike ʻia ʻo PKCβII e hōʻike ana i ka lipid peroxidation mua, a ua pili ka hoʻonui ʻia ʻana o ka lipid peroxidation me ka ferroptosis. Ua hōʻike ka nānā ʻana i kahi waihona puke o nā mea hoʻopaʻa kinase CRISPR/Cas9-mediated e pili ana ʻo PKCβII i ke kaʻina hana o ka lipid peroxidation, he mea koʻikoʻi ia no ka ferroptosis i nā cell MDA-MB-231 (Zhang et al., 2022). No laila, hōʻike kēia e hiki i ka knockout o PKCβII e CRISPR/Cas9 ke lilo i pahuhopu gene suppressor tumor no ka mālama ʻana i nā maʻi e pili ana i ka ferroptosis (Zhang et al., 2022).
Ua manaʻo ʻia ke kūʻē ʻana i ka lāʻau lapaʻau ke kuleana no kahi o 90% o nā make i nā maʻi kanesa a ʻo ia kekahi o nā pilikia nui i ka mālama ʻana i ka maʻi kanesa (Bukowski et al., 2020). Hōʻike nā hopena he nui nā genes e pili ana i ka hoʻopuka ʻana o ka lāʻau, ka hoʻoponopono ʻana o ka DNA, ka apoptosis a me nā ala hōʻailona cell like ʻole e pili ana me ke kūʻē ʻana i ka lāʻau (Haider et al., 2020). I waena o lākou, ua kuhikuhi ʻia kekahi mau genes e nā mea hana CRISPR/Cas9 a ua hōʻike i nā hopena hoʻohiki i ka hōʻemi ʻana i ke kūʻē ʻana i ka lāʻau a me ka hoʻonui ʻana i ka pono o nā lāʻau anti-cancer (Vaghari-Tabari et al., 2022). Eia kekahi, ua pili nā hale waihona puke nānā knockout gene CRISPR/Cas9 kiʻekiʻe i ka hoʻololi ʻana i ka hana o nā pahuhopu gene kūʻē lāʻau (Shalem et al., 2015). No ka ʻike ʻana i nā genes kū'ē paclitaxel, ua hoʻohana ʻia ka hoʻonohonoho ʻana o ka RNA i hui pū ʻia me ka nānā ʻana o ka waihona puke sgRNA genome-ākea e ʻike i nā genes moho ʻewalu, me ka histone deacetylase 9 (HDAC9), e pili ana i ke kū'ē ʻana i ka lāʻau lapaʻau i nā poʻe maʻi me ka TNBC hou (B et al., 2020). ). Ma kekahi noiʻi ʻē aʻe, ua ʻike ʻia ka hoʻonui ʻia ʻana o ka diserine/threonine a me ka tyrosine protein kinase (DSTYK) i ka wā o ke ola ʻana o nā poʻe maʻi TNBC i mālama ʻia me nā lāʻau anticancer. Eia kekahi, ua hoʻonui nui ka CRISPR/Cas9-mediated knockdown o DSTYK i ka apoptosis o nā cell cancer kū'ē i ka lāʻau lapaʻau ma vitro (nā cell SUM102PT a me nā cell MDA-MB-468) a me kahi kumu hoʻohālike TNBC in vivo (Ogbu et al., 2021).
ʻOiai ua hoʻāla ʻia ke ala MAPK e TNBC ma ke ʻano maʻamau, ʻaʻole maikaʻi ka hopena lapaʻau o nā lāʻau lapaʻau i kuhikuhi ʻia iā MEK. Ua hōʻike kahi pale waihona genomic CRISPR/Cas9 e hoʻonui ana ka pale ʻana o PSMG2 (proteome assembly chaperone 2) i nā pūnaewele TNBC BT549 a me MB468 i ka MEK inhibitor AZD6244. Ua hoʻololi ka CRISPR/Cas9 knockdown o PSMG2 i ka hana proteasome maʻamau, e alakaʻi ana i ka autophagy-mediated disassembly o PDPK1, a laila e hoʻomaikaʻi hou aku i nā pūnaewele tumor i nā hiʻohiʻona ʻiole TNBC i hoʻoulu ʻia e AZD6244 (MEK inhibitor) a me MG132 (proteasome inhibitor). No laila, hiki ke lawelawe ʻia nā proteasome a me MAP kinase (MEK) inhibitors synergistically e hōʻemi i ka hoʻonui ʻana o nā pūnaewele tumor (Wang X. et al., 2022).
Ua hoʻohana ʻia hoʻi ʻo CRISPR/Cas9 e nānā no ka nalowale ʻana o ka hana gene e alakaʻi ana i ke kūʻē ʻana o TNBC (Shu et al., 2020). Ua wehewehe ʻo Ge et al i ke ʻano o ke kūʻē ʻana i ka lāʻau lapaʻau o nā cell cancer i mālama ʻia me JQ1, kahi mea hoʻopaʻa bromodomain BET (BBDI), ma TNBC. Ma ka hoʻohana ʻana iā CRISPR/Cas9, ua ʻike lākou ʻo ka holoi ʻana i ka gene rb1 i hoʻolilo iā TNBC i kūʻē i ka lāʻau anti-cancer JQ1. No laila, he mea nui ka hana rb1 i ka pane ʻana i nā lāʻau JQ1 ma TNBC. Ua hōʻike pū lākou he mea hoʻopaʻa CDK4/6 kinase/microtubule ʻo paclitaxel, a ʻo kona hui pū ʻana me nā BBDI e like me JQ1 e hāʻawi paha i nā pane therapeutic hoʻohiki i ka TNBC kūʻē i ka lāʻau lapaʻau (Ge et al., 2020).
Ua hōʻike ʻia ke ʻano transcriptional o ka RNA noncoding lōʻihi (lncRNA) ke kuleana no ke kūʻē ʻana i ka lāʻau neoadjuvant ma TNBC. Hōʻike kēia haʻawina he ʻelima mau transcripts MALAT1 lncRNA like ʻole i hōʻike nui ʻia ma TNBC. Eia kekahi, ua hoʻonui ka holoi ʻana o CRISPR/Cas9 o MALAT1 i ka ʻike o nā pūnaewele TNBC BT-549 i ka paclitaxel a me doxorubicin, e hōʻike ana i kahi kuleana kūpono no ke kūʻē ʻana o MALAT1 ma TNBC (Shaath et al., 2021).
He ʻokoʻa nā hoʻololi ʻana ma BRCA1 (BRCA1m) a no laila he paʻakikī ke ʻike. ʻO ka hoʻopaʻa ʻana iā PARP1 (poly(ADP-ribose) polymerase), ka hoa make synthetic o BRCA1, hiki ke hoʻonui i ka chemosensitivity lāʻau TNBC. Me ka hoʻohana ʻana iā CRISPR/Cas9, ua hoʻonui ka holoi ʻana o PARP1 i ka ʻike o nā lāʻau anticancer e like me doxorubicin, gemcitabine a me docetaxel i nā pūnaewele TNBC mutant mBRCA1, e hōʻike ana ua pili pū ʻo PARP1 i ke kūʻē ʻana i ka lāʻau ma TNBC (Vaghari-Tabari et al., 2022). . Ua ʻike ʻia nā hoʻololi ʻana i nā genes tumor suppressor BRCA1 a i ʻole BRCA2 e hoʻonui i ka hiki ke ulu i ka maʻi ʻaʻai umauma i nā kānaka. Pono ka mālama ʻana i kēia mau maʻi i ka hoʻohana ʻana i nā mea hoʻopaʻa PARP. Eia kekahi, ua hōʻike ʻia e hiki i ka hoʻopau ʻana i ka nucleotide salvage factor DNPH1 me ka hoʻohana ʻana iā CRISPR/Cas9 ke wehe i ka nucleotide toxic 5-hydroxymethyldeoxyuridine (hmdU) monophosphate, no laila e hoʻonui ai i ka pane o nā cell BRCA-deficient i ka ʻike i nā mea hoʻopaʻa PARP (Fugger). et al., 2021). No laila, hiki i nā mea hoʻopaʻa CRISPR/Cas9 a me PARP1 ke lilo i mea lapaʻau koʻikoʻi no TNBC.
ʻO ka penetrant glycoprotein (P-gp) gene kahi gene kū'ē i nā lāʻau lapaʻau he nui i hoʻonui ʻia ma kahi o 41% o nā TNBC āpau (Sun et al., 2020). Ua ʻike ʻia ka hoʻoheheʻe ʻana o ka lāʻau i hoʻokomo ʻia e P-gp ma ke ʻano he mea hoʻoponopono koʻikoʻi o ke kū'ē ʻana i ka lāʻau lapaʻau i ka maʻi ʻaʻai umauma. Ua hōʻike nā mea hoʻopaʻa P-gp i ka hoʻonui ʻia o ka ʻike i nā lāʻau anticancer i ka maʻi ʻaʻai umauma (Famta et al., 2021). No laila, ʻo ka ablation a CRISPR/Cas9-mediated a i ʻole ka hoʻopau ʻana i nā mea hoʻopaʻa P-gp a me P-gp paha he mau ʻano hana koʻikoʻi loa ia e lanakila ai i ke kū'ē ʻana i ka lāʻau lapaʻau ma TNBC.
Ua ʻike ʻia ka mea hoʻoili cassette ATP-binding G2 (ABCG2) e hoʻoulu i ke kūʻē ʻana i ka lāʻau lapaʻau ma TNBC (Palasuberniam et al., 2015), ʻoiai ʻaʻohe hōʻike i kēia manawa e pili ana i ka pilina ma waena o CRISPR/Cas9 a me ABCG2 silencing a me ka hoʻopau ʻana o ka tumor suppressor gene. Hiki i ka PTEN ke hoʻoikaika i ka hoʻāla ʻana o ABCG2 (Palasuberniam et al., 2015), (Deepak Singh et al., 2021). No laila, ʻo ka hoʻohana ʻana iā CRISP/Ca9 e wehe iā ABCG2 a me ka hoʻohui ʻana me kahi mea hoʻopaʻa ABCG2 he lāʻau kūpono paha ia e lanakila ai i ke kūʻē ʻana i ka lāʻau lapaʻau ma TNBC. Ua ʻōlelo ka Papa 2 iā CRISPR/Cas9 e kuhikuhi ana i nā genes kūʻē āpau.
Papa 2. Hoʻopili ʻo CRISPR/Cas9 i nā ʻano genes kūʻē TNBC like ʻole e hoʻonui i ka ʻike o nā cell i nā lāʻau anticancer.
He mau mea hana kūpono nā waihona puke CRISPR/Cas9 no ka nānā ʻana i nā hoʻololi ʻana o nā gene e pili ana i ka pathogenesis o ka maʻi kanesa (Chan et al., 2022). Pili kēia ʻano nānā i ʻehā mau ʻanuʻu e like me (a) ke kūkulu ʻana i ka waihona puke, (b) ka transduction lentiviral, (c) ka nānā ʻana o ka phenotypic, a me (d) ka nānā ʻana i ka gene target. ʻOiai he hoʻāla maʻamau ʻole ʻo TNBC i ke ala MAPK, ʻo ka prognosis lapaʻau o ka MEK targeted therapy no nā maʻi TNBC e lawe ana i nā hoʻololi ʻana i nā genes tumor suppressor e like me PTEN, RB1, a me TP53 he haʻahaʻa loa ia.
Eia kekahi, ua hoʻāʻo kahi pale knockout gene e hoʻohana ana iā CRISPR/Cas9 i ka molekala ikaika loa a koho, dehydrocatrol, ka mea i nui i loko o nā ʻāpana pua a me nā lau Guatemalan, e hoʻomaopopo i ka hopena o dehydrocatrol ma MDA-MB-231. Nā ʻano hana o ka cytotoxicity cell koho. Nā ʻano stemness Mesenchymal o nā subtypes TNBC. Ua hōʻike pū kēia pale CRISPR/Cas9-based ua hōʻike nui ʻia ʻo HSD17B11, kahi gene e hoʻopili ana i ka 17β-hydroxysteroid dehydrogenase type 11, i loko o nā cell MDA-MB-231 a hopena i ka dehydrofalcarinol kūikawā no nā cell MDA-MB-231 (Grant et. al., 2020). No laila, hōʻike kēia he hiki nui ka pale genomic CRISPR/Cas9 i ka ʻike ʻana i nā ʻano hana ma lalo o nā waiwai anticancer o nā hui kūlohelohe hiki.
Eia kekahi, ua aʻo ʻia ka nāwaliwali o TNBC i ka maʻi kanesa me ka hoʻohana ʻana i kahi pale CRISPR/Cas9 in vivo genome-wide unbiased, kahi i hōʻike ai i kahi pilina ma waena o nā ala oncogenic a me nā ala suppressor tumor. Ua hōʻike ʻia nā ʻāpana koʻikoʻi o nā ala mTOR a me Hippo e pāʻani i nā kuleana koʻikoʻi i ka hoʻoponopono ʻana i ka tumor ma TNBC. Eia kekahi, ua hōʻike nā haʻawina e kāohi pono ana ka pharmacological inhibition o mTORC1/2 a me YAP oncoprotein i ka pathogenicity o TNBC me ka hoʻohana ʻana i kahi kumu hoʻohālike synergy drug-matrix in vitro a me nā xenografts i loaʻa mai i ka maʻi in vivo. Eia kekahi, hoʻonui ka inhibition mTORC1/2 i hoʻopili ʻia e Torin-1 i ka macropinocytosis, ʻoiai ʻo ka inhibition YAP i hoʻokomo ʻia e verteporfin e alakaʻi ai i ka make ʻana o nā cell TNBC. I ka hui pū ʻana, hōʻike kēia mau hopena i ka mana a me ka paʻa o ka genome-wide in vivo CRISPR screening e ʻike i nā lāʻau lapaʻau hou a maikaʻi no TNBC (Dai et al., 2021).
ʻO ka hoʻoponopono ʻole ʻana o ka ʻōnaehana pale he mea nui ia i ka tumorigenesis. Pale aku nā ʻāpana kanesa i ka hoʻomaʻemaʻe ʻana o ka pale ma ke kāpae ʻana i nā ʻano pale, me ke komo ʻana i ka hana o nā ʻāpana pale i loko o ka microenvironment tumor a me ka hoʻopilikia ʻana i ka ʻōnaehana pale. No laila, ʻo ka hoʻomohala ʻana i kahi ʻōnaehana pale i hoʻomaikaʻi ʻia he ala koʻikoʻi paha ia e hakakā ai i nā tumors. ʻO ka hoʻohana ʻana i ka hoʻololi ʻana o ka gene CRISPR/Cas9 e hoʻoponopono i kekahi mau pilikia e pili ana i ka hana ʻole o ka pale mai nā manaʻo like ʻole. Ua hoʻohana ʻia ʻo CRISPR/Cas9 e hoʻomaikaʻi i ka pale antitumor e kūʻē i ka maʻi kanesa umauma ma o nā ala aʻe (Kiʻi 3).
Kiʻi 3. Hoʻopili ka immunotherapy CRISPR/Cas9 i nā pūnaewele TNBC. ʻO ka nalowale ʻana o CDK5 a me ka hoʻohaʻahaʻa ʻana o PDL1 a me CD155 e hoʻoikaika i ka ʻōnaehana pale. Pēlā nō, ʻo ka nalowale ʻana o A2AR a me ka hoʻohaʻahaʻa ʻana o TAAs e like me HER2, mucin 1 a me TEM8 i hoʻonui i ka pono o nā pūnaewele CAR-T i ka pepehi ʻana i nā pūnaewele maʻi kanesa. Ua hōʻike ʻia e ka nānā ʻana o nā pūnaewele T i hoʻokele ʻia e CRISPR/Cas9 e hoʻonui ka hoʻohaunaele ʻana o ka p38 kinase i ka hana antitumor pūnaewele T. Hoʻonui nā pūnaewele T i hoʻololi ʻia e CRISPR/Cas9 i ka hōʻike ʻana o TCR (mea loaʻa pūnaewele T), e hopena ana i nā pūnaewele T i loaʻa ka hana antitumor.
ʻO ka pahuhopu o ka immunotherapy ka hoʻoulu ʻana i ka ʻōnaehana pale e hoʻouka i nā ʻōpū kanesa. ʻO ka hoʻonui nui ʻana o nā protein checkpoint immune e pale aku i nā hopena autoimmune akā hiki ke kōkua i nā maʻi kanesa e pale aku iā lākou (Topalian et al., 2015). Pale kēia mau protein i nā hopena immune ma o ka hoʻopaʻa ʻana i nā mea loaʻa ma ka ʻili o nā ʻōpū immune. Hoʻopili nā protein checkpoint he nui (e like me CD155 a me PD-L1) i ka mea loaʻa PD-1 ma nā ʻōpū immune a hōʻike ʻia i loko o ke kanesa umauma, ʻoiai ʻo TNBC (Li Y.-C. et al., 2020). ʻO ka hoʻokuʻu ʻana o PD-L1 a i ʻole kāna mea loaʻa me ka hoʻohana ʻana iā CRISPR/Cas9 hiki ke hoʻoulu i ka ʻōnaehana pale e hoʻouka i nā ʻōpū TNBC (Yahata et al., 2019). Eia kekahi, ua hōʻike ʻia ka downregulation o ka hōʻike ʻana o PD-L1 ma o ka holoi ʻana o CRISPR/Cas9 o CDK5 e kāohi i ka ulu ʻana o ka ʻōpū ma vitro a ma vivo (Deng et al., 2020). ʻO ka pale ʻana i ka ulu ʻana i loko o nā hiʻohiʻona in vitro a me in vivo o ka maʻi ʻaʻai umauma e hōʻike ana he hopena therapeutic paha ka hōʻemi ʻana o ka shRNA i ka CD155 i ka maʻi ʻaʻai umauma (Gao et al., 2018).
Hōʻike nā pūnaewele CAR T i nā CAR e ʻike ana i nā antigens pili i ka tumor (TAAs) a hiki ke hoʻohana i ka hoʻokuʻu ʻana o nā protein checkpoint e hoʻonui i kā lākou hana (Li C. et al., 2020). Aia kekahi mau pahuhopu kūpono no ka lāʻau lapaʻau CAR T cell i ka maʻi ʻaʻai umauma, me kekahi mau TAA e like me HER2, mucin1, a me TEM8 (Bajgain et al., 2018). Aia nā hōʻike e ʻoi aku ka maikaʻi o nā pūnaewele CAR T e kuhikuhi ana i ka mesothelin (overexpressed i loko o nā pūnaewele TNBC BT-459) e kūʻē i ka maʻi ʻaʻai ke hoʻopau ʻia ʻo PD-1 me ka hoʻohana ʻana iā CRISPR/Cas9 (Hu et al., 2019). Eia nō naʻe, ʻo ka hoʻokaʻawale ʻana i nā pūnaewele T mai nā maʻi a laila hoʻoponopono iā lākou ex vivo he hana hana nui a lōʻihi hoʻi. ʻOiai hiki i nā pūnaewele T āpau ke hōʻemi i nā koi hoʻokaʻawale, pono e wehe ʻia nā pūnaewele T hāʻawi e hōʻike ana i ka antigen leukocyte kanaka (HLA) papa I a me ka mea loaʻa pūnaewele T (TCR) e pale aku i nā ʻano graft-versus-host (Ren et al., 2017). , 2017a). Me ka hoʻohana ʻana iā CRISPR/Cas9, hiki i ka HDR ke hoʻopau i ka TCR a me HLA a hoʻopau i ka gene encoding CAR. Ua hōʻike ʻia nā haʻawina e hiki ke hoʻohana ʻia ʻo CRISPR/Cas9 e hoʻokomo i nā anti-CD19 CARs i loko o ka TCR locus, no laila e hana pono i nā CARs me ka ʻole o ka hoʻopau ʻana i nā cell T (Dimitri et al., 2022). Ua hoʻomohala ʻia nā ʻenehana Multiplex e hana i nā allogeneic CAR T cell ma ka hoʻopau ʻana iā TCR, beta-2-microglobulin (B2M), HLA-I subunit a me nā protein ʻē aʻe e like me PD-1 a me CTLA-4, nona paha nā waiwai anti-cancer nui aʻe. e kūʻē iā TNBC (Eyquem et al., 2017; Liu et al., 2017; Ren et al., 2017b; Dimitri et al., 2022).
Hiki i ka hoʻohana ʻana iā CRISPR/Cas9 ke hoʻomaikaʻi i ka pono o nā pūnaewele CAR-T. Ua ʻike ʻia ʻo Adenosine he mau waiwai immunosuppressive a hiki ke hōʻemi i ka pale ʻana i ka maʻi kanesa ma ke kāohi ʻana i ka hana o nā pūnaewele T a me ka hoʻoulu ʻana i nā mea loaʻa adenosine A2A (A2AR) (Vigano et al., 2019). ʻO ka hoʻopau ʻana iā A2AR me ka hoʻohana ʻana iā CRISPR/Cas9 ua hoʻomaikaʻi nui i ka pono o nā pūnaewele CAR-T i vivo (Giuffrida et al., 2021). Ua ʻike ʻia nā pūnaewele T e hōʻike i nā ʻano phenotypic like ʻole e like me ka hoʻonui ʻana o nā pūnaewele, ka hoʻokaʻawale ʻana, ke kaumaha oxidative a me ke kaumaha genomic. Ua hōʻike ka nānā ʻana o nā pūnaewele T e pili ana iā CRISPR/Cas9 i ka hoʻopilikia ʻana o 25 mau kinase receptor cell T like ʻole. I waena o lākou, ua hōʻike ʻia ka holoi ʻana o ka p38 kinase e hoʻoikaika i ka hana antitumor o nā pūnaewele T, e hōʻike ana he mea hoʻoponopono koʻikoʻi ka p38 kinase o ka hoʻoponopono ʻana o nā pūnaewele CAR-T (Gurasamy et al., 2020).
Hiki ke hoʻololi ʻia nā pūnaewele T me ka hoʻohana ʻana iā CRISPR/Cas9 e hana i nā TCR i hōʻike nui ʻia. ʻO ka hoʻoili ʻana i nā pūnaewele T i hoʻololi ʻia i nā maʻi ua hōʻike i ka hana anticancer ikaika aʻe ma mua o nā pūnaewele T endogenous. No laila, hiki i nā TCR endogenous ke hoʻokūkū me nā TCR i hoʻololi ʻia i nā maʻi, kahi e hoʻopilikia ai i ka hiki o ka immunotherapy kanesa. No ka lanakila ʻana i kēia pilikia, ʻo ka hoʻohana ʻana iā CRISPR/Cas9 e wehe i ka TCR-β endogenous i nā pūnaewele loaʻa a ma hope e hoʻoili i nā pūnaewele T TCR-β i nā maʻi kanesa e hōʻike paha i nā pane pale anti-cancer maikaʻi me ka ʻole o ka pono no ka hoʻokūkū moekolohe TCR endogenous (Fan et al. 2018). No laila, koe wale nō nā pūnaewele T i hoʻololi ʻia e CRISPR, ʻoi aku ka maʻalahi o nā TCR i nā antigens tumor ma mua o nā pūnaewele T i hoʻololi ʻia e TCR maʻamau. Eia kekahi, i nā leukemias like ʻole, hōʻike nā pūnaewele T i hoʻololi ʻia i hana ʻia e γδ TCR+ CRISPR i ka hōʻike kiʻekiʻe o nā pūnaewele CD4+ a me CD8+ T ma mua o ka hoʻoili TCR maʻamau (Legut et al., 2018). No laila, ʻo nā pūnaewele T i hoʻololi ʻia i hana ʻia e CRISPR/Cas9 paha ke ʻano he ala immunotherapy kūpono e lanakila ai iā TNBC.
ʻO nā integrins he mau molekala hoʻopili pūnaewele i loaʻa i loko o nā transmembranes cellular a paipai i ka hoʻopaʻa ʻana o nā pūnaewele i ka matrix extracellular (ECM) (Hamidi lāua ʻo Ivaska, 2018). ʻO ka dysregulation o nā integrins e pili ana me ka ulu ʻana o ka maʻi kanesa a me ka neʻe ʻana ma o ka hoʻololi ʻana i ka matrix extracellular, e alakaʻi ana i ke ola ʻana o nā pūnaewele kanesa i ke kaʻapuni (Hamidi lāua ʻo Ivaska, 2018). Hoʻolohi ka CRISPR/Cas9-mediated integrin knockdown i ka holomua o ka tumor, metastasis, a me ka colonization ma TNBC. Ua hōʻike ʻia ka knockdown o integrin a5 (ITGA5) e hōʻemi i ka neʻe ʻana o nā pūnaewele a me ka holomua i nā maʻi kanesa ʻē aʻe e like me ke kanesa māmā (Ju et al., 2017), e hōʻike ana he mea nui paha ka integrin a5 i ka pathogenesis o TNBC.
ʻO ka hoʻoulu ʻana i nā ʻiole knockout me ka hoʻohana ʻana i nā ʻano hana kuʻuna o ke kelepona kumu embryonic (ES) he hana nui ia, he manawa lōʻihi, a he hana pono ʻole. He mau mahina a me nā makahiki e hoʻopaʻa ai i nā pūnaewele ES ma o ka hoʻohui like ʻana, hoʻoulu i nā ʻiole chimeric, a laila e hoʻohui iā lākou me nā ʻiole heterozygous e hoʻoulu i nā keiki homozygous. Pono ka hoʻohui paʻakikī e hana i nā ʻiole me nā hoʻololi genetic he nui. Eia nō naʻe, ua nui nā pilikia i kū mai i ka wā e hoʻohana ai i nā ʻiole i hoʻoulu ʻia e ka ablation o nā pūnaewele ES me ka hoʻohana ʻana iā CRISPR/Cas9 a i ʻole ma ka microinjecting CRISPR/Cas9 i nā hua manu i hoʻoulu ʻia i hoʻokahi cell. No ka laʻana, ʻo ka hoʻolauna ʻana o nā loli e hana pinepine ʻia ma nā loci biallelic a ʻaʻole ia he gene kikoʻī. Ua hōʻike koke ʻia e hiki i nā pūnaewele ES e hoʻohana ana i ka ʻenehana CRISPR/Cas9 ke hoʻokomo i nā hoʻololi biallelic i ka manawa like a hiki i 5 mau genes (Nishizono et al., 2021). No kēia kumu, ua hoʻololi like ʻia ʻo Cas9 mRNA a me ʻelima mau gRNA gene-specific i loko o nā pūnaewele ES. Hōʻike kēia i ka ʻōlelo hoʻohiki a me ka pono o kēia kaʻina hana, ʻoiai ua hoʻoili ʻia paha kēia mau mutations i ka wā i hoʻoulu ʻia ai nā laina hoʻokumu e hana i nā ʻiole knockout quintuple. Hōʻike pū ka haʻawina i kahi ʻike kupanaha: ʻAʻole pono hou nā cell ES e hana i nā ʻiole i hoʻololi ʻia i ka genetically. Akā, no ka wehe ʻana i nā huahana gene kikoʻī, ua hoʻokomo ʻia ʻo Cas9 mRNA a me gRNA i loko o nā embryos pae cell hoʻokahi. ʻO ka hopena, ua hana ʻia nā laina hoʻokumu knockout i hiki ke hoʻohana ʻia ma ke kumumanaʻo e aʻo i nā hopena o ka holoi ʻana o ka gene i nā ʻiole (Qin et al., 2016). No laila, ʻae ʻo CRISPR/Cas9 i ka hana ʻana o nā ʻiole transgenic e mālama iā TNBC ma ke kumukūʻai haʻahaʻa ma mua o ka ʻenekinia genetic kuʻuna. Me ka hoʻohana ʻana i ka ʻōnaehana CRISPR/Cas, ua hoʻomohala ʻia kahi kumu hoʻohālike ʻiole knockout hou e hiki ai ke hoʻolauna pono i nā mutations kiko i hoʻokahi a ʻoi aku paha nā genes endogenous ma TNBC. Ma muli o kēia kumumanaʻo, hiki ke hoʻomohala ʻia nā hiʻohiʻona holoholona o TNBC me ka hoʻohana ʻana i ka holoi ʻana o BRCA1 a me p53 ma muli o CRISPR/Cas9, e hopena ana i ka nalowale o ka hoʻoponopono HR, ka paʻa ʻole o ka genomic, a me nā phenotypes mutant (Annunziato et al., 2020).
I kēia manawa, hoʻohana ʻia nā ʻano hana like ʻole e like me ka mammography, magnetic resonance imaging (MRI) a me ka ultrasound e ʻike ai i ka TNBC. Eia nō naʻe, he mau palena ko kēia mau ʻano hana. Hoʻohana ʻia ka Mammography e ʻike i nā ʻiʻo umauma kūloko ma mua o nā metastases, kahi i neʻe ai nā hunaola kanesa i nā ʻōpū ʻē aʻe. ʻAʻole hilinaʻi ka Ultrasonography no ka ʻike ʻana i ka TNBC (Chen lāua ʻo Lee-Felker, 2023). ʻOi aku ka ʻike o ka MRI ma mua o ka ultrasound a me ka mammography, akā ua kaupalena ʻia kona pololei diagnostic (Sha lāua ʻo Chen, 2022). ʻO ka biopsy ʻiʻo kahi ala invasive e ʻike ai i nā hunaola kanesa. Eia nō naʻe, i kekahi mau hihia, hiki i kahi biopsy ke nalo i nā ʻiʻo kanesa inā hele hewa ka nila mai kahi hoihoi. Eia kekahi, he pipiʻi loa ia a hiki ke lilo i mea hōʻeha no ka mea maʻi. He ʻano ʻano like ʻole ka TNBC o ke kanesa, no laila ʻaʻole hiki i ka biopsy ke hāʻawi i ka ʻike lawa e pili ana i ke ʻano kanesa. No laila, pono koke kahi ʻano diagnostically hilinaʻi hou no ka ʻike ʻana i ka TNBC. Hiki iā CRISPR/Cas9 ke lawelawe ma ke ʻano he koho ʻokoʻa loa a me ka minimally invasive no ka ʻike ʻana i ka maʻi kanesa umauma. No kēia hopena, hiki ke hoʻohana ʻia nā hoʻolālā e pili ana iā CRISPR/Cas9 e hoʻomaikaʻi i nā ʻano PCR no ka ʻike ʻana iā TNBC. ʻO ka mea mua, hoʻohana ʻia nā protein Cas9 a me cpf1 o ka ʻōnaehana CRISPR e wehe i ka DNA kikoʻī ʻole, a laila hiki i kēia mau protein ʻelua (Cas9 a me cpf1) ke ʻike i ka moʻo PAM ma mua o ka hoʻopaʻa ʻana i ka DNA i manaʻo ʻia (Deepak Singh et al., 2021). . No laila, hiki i ka PCR ke ʻike i nā mutations e pili ana i ka ulu ʻana o ka maʻi kanesa. Ua hoʻohana kekahi mau noiʻi i kēia hoʻolālā e pili ana iā CRISPR e ʻike i nā mutations like ʻole i nā maʻi kanesa like ʻole (Safari et al., 2019). No laila, hiki i nā ʻano PCR e pili ana iā CRISPR ke hōʻemi i ka hilinaʻi o ka ʻike ʻana iā TNBC ma nā ʻano invasive e like me ka immunohistochemistry e pili ana i ka biopsy.
Ua hōʻike ʻia hoʻi nā loli genotypic i ka hoʻoponopono ʻana o ka hormone receptor ma TNBC (Chen lāua ʻo Russo, 2009). Hiki i nā hoʻolālā PCR e pili ana iā CRISPR/Cas9 e hoʻohana ana i nā microarrays no nā diagnostics point-of-care ke nānā pono i kēia mau mutations (Hajian et al., 2019). Hiki iā ia ke hāʻawi i nā mea lawelawe olakino me ka ʻike pili e pili ana i nā mutations kikoʻī i nā poʻe maʻi TNBC, hiki ke kōkua i ka hoʻomaikaʻi ʻana i kā lākou papa hana. Eia nō naʻe, he mau palena ko kēia ʻenehana i hui pū ʻia. No laila, pono nā hana noiʻi nui ma mua o ka hoʻohana ʻia ʻana o kēia ʻano CRISPR/Cas-PCR i ka mālama ola no ka ʻike ʻana iā TNBC (Yang et al., 2019).