CRISPR) ndi njira yosinthira majini ya m'badwo wachitatu: CRISPR inasintha dziko lonse lapansi

Clustered regularly interspaced short palindromic repeats (CRISPR) ndi njira yosinthira majini ya m'badwo wachitatu yomwe yasintha dziko lapansi ndi zotsatira zake zapamwamba. Yagwiritsidwa ntchito pochiza matenda osiyanasiyana achilengedwe ndi matenda. Mabakiteriya osiyanasiyana ndi ma prokaryotes ena (monga archaea) alinso ndi machitidwe a CRISPR/Cas9 kuti ateteze ku ma phages. Zanenedwa kuti njira zozikidwa pa CRISPR/Cas9 zitha kuletsa kukula ndi kupita patsogolo kwa khansa ya m'mawere ya triple negative (TNBC) poyang'ana majini osinthika okana, transcription, ndi epigenetic regulation. Njira zochiritsira izi zingathandize kuthana ndi mavuto ovuta monga kukana mankhwala komwe kumawoneka ngakhale mu TNBC. Pakadali pano, njira zosiyanasiyana zimagwiritsidwa ntchito popereka CRISPR/Cas9 ku maselo, monga physical (microinjection, electroporation ndi hydrodynamic modes), viral (adeno-associated virus ndi lentivirus) ndi non-viral (liposomes ndi lipid nanoparticles). Ngakhale kuti mitundu yosiyanasiyana yapangidwa kuti iphunzire zomwe zimayambitsa mamolekyu a TNBC, kusowa kwa njira zodziwikiratu komanso zolunjika zoperekera zida zosintha majini mu vivo kumachepetsa kugwiritsa ntchito kwawo kuchipatala. Mwachidule, ndemanga iyi ikuwunika bwino kupita patsogolo, zovuta, zolepheretsa, ndi chiyembekezo cha chithandizo cha CRISPR/Cas9 cha TNBC kutengera umboni womwe ulipo. Tikuwonetsanso momwe kuphatikiza kwa luntha lochita kupanga ndi kuphunzira kwa makina kungathandizire njira za CRISPR/Cas9 pochiza TNBC.
Khansa imadziwika ndi kugawikana kosalamulirika kwa maselo, kusokonezeka kwa malo owunikira maselo, ndi kusintha kwa majini oletsa khansa (TSGs) (Matthews et al., 2022). Pakati pa mitundu yosiyanasiyana ya khansa, khansa ya m'mawere ndiyo mtundu wofala kwambiri wa khansa mwa akazi ndipo ili ndi chiwerengero chachikulu cha imfa padziko lonse lapansi (Waks and Winer, 2019). Khansa ya m'mawere ndi matenda osiyanasiyana okhala ndi makhalidwe osiyanasiyana, monga mawonekedwe a histological ndi biological, mawonekedwe azachipatala ndi khalidwe, komanso momwe mankhwala amagwiritsidwira ntchito (Weigelt et al., 2010). Kugawa khansa ya m'mawere kumapereka chidziwitso cholondola cha matenda a khansa ya m'mawere ndi momwe khansa imagwiritsidwira ntchito. Kugwiritsa ntchito zizindikiro zodziwika bwino ndi zizindikiro za clinopathological kwakhala njira yayikulu yogawa khansa ya m'mawere (Tsang and Tse, 2019). Kuchuluka kwa khansa ya m'mawere komanso momwe imagwiritsidwira ntchito kumakhudzidwa ndi zinthu zambiri, kuphatikizapo kukhalapo kwa estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2/neu), ndi histological grade, mtundu wa chotupa, ndi giredi. Kukula ndi kufalikira kwa ma lymph node (Al-Tubaiti, 2020). Mitundu isanu ya khansa ya m'mawere yapezeka, kuphatikizapo luminal A, luminal B, HER2-enriched, basal-like, ndi claudin-low (Prat et al., 2015). TNBC ndi mtundu wa khansa ya m'mawere yomwe siimasonyeza ER, PR, ndi HER2 yonse (Yin et al., 2020). Zizindikiro izi zimagwirizanitsidwa ndi TNBC, kutsimikizira kupita patsogolo kwake mwachangu komanso koopsa kuposa mtundu wina uliwonse wa khansa ya m'mawere (Feng et al., 2018). Kuphatikiza apo, Peru et al (2000) adagwiritsa ntchito ukadaulo wa microarray kuti asinthe khansa ya m'mawere ndikuzindikira mitundu isanu ya khansa ya m'mawere (Cadenas, 2012). Khansa ya m'mawere yofanana ndi basal ndi mtundu wa khansa ya m'mawere yomwe ili ndi phenotype yoyipa katatu ndipo imagwirizanitsidwa ndi kupita patsogolo mwachangu. Tiyenera kudziwa kuti khansa zonse za m'mawere zofanana ndi za basal nthawi zambiri zimazindikirika molakwika ngati TNBC, koma 77% yokha ndi TNBC; Mosiyana ndi zimenezi, 71–91% ya TNBC imakhala ngati ya basal, zomwe zikusonyeza kuti mitundu iwiriyi ya khansa ya m'mawere imafanana ndipo imayimira magulu osiyanasiyana (Wang D.-Y. et al., 2019). Izi zimapangitsa kuti pakhale kufunika kofotokoza kusiyana kwa TNBC kuti tifotokoze zomwe zingachitike ndikupeza mayankho omwe angakhalepo pamankhwala omwe alipo komanso amtsogolo. Kuphatikiza apo, TNBC imakhala ndi 15–20% ya milandu yonse ya khansa ya m'mawere ndipo imapezeka kwambiri mwa akazi osakwana zaka 50. Kusintha kwa BRCA1 kapena BRCA2 kwanenedwa pafupifupi 20% ya milandu ya TNBC (Xie et al., 2017; Tzikas et al., 2017). , 2020). Kafukufuku wasonyezanso kuti TNBC ili ndi chitetezo chamthupi chosiyana chomwe chimaphatikizapo kuchuluka kwa zinthu zomwe zimayambitsa kukula kwa mitsempha yamagazi, ma macrophages ogwirizana ndi chotupa (TAMs), ma lymphocyte olowa m'thupi (TILs), ndi mamolekyu ena omwe amakhudzidwa ndi kukula ndi kusamuka kwa chotupa. Chifukwa chake, kumvetsetsa za chilengedwe cha TNBC ndikofunikira kwambiri pakukula kwake komanso chithandizo chake (Fan and He, 2022).
Kuti muwone ngati TNBC ikupita patsogolo ndikutsimikizira chithandizo chogwira mtima, kuzindikira molondola, makamaka pogwiritsa ntchito immunohistochemistry (IHC) kuti mupeze ER, PR ndi HER2, ndi mammography kuti mupeze mabala a m'mawere, ndikofunikira. Komabe, mammography singathe kuwonetsa mokwanira mawonekedwe amkati mwa chotupa monga necrosis ndi fibrosis (Deepak Singh et al., 2021). Popeza kuti matenda osakwanira komanso kuzindikira bwino kumalepheretsa madokotala kupereka mankhwala oyenera (Chaudhary, 2020), njira zosiyanasiyana zimagwiritsidwa ntchito kuti ziwongolere chisamaliro cha odwala omwe ali ndi TNBC. Pakadali pano, mankhwala awiri, doxorubicin ndi cyclophosphamide, amagwiritsidwa ntchito mwa odwala omwe ali ndi TNBC ndipo awonetsa zotsatira zabwino. Kuphatikiza apo, mankhwala ena a platinamu amagwiritsidwa ntchito, monga carboplatin ndi cisplatin (Sikov et al., 2015). Kuphatikiza apo, zoletsa za PARP monga Olaparib, Velaparib, ndi PF-01367338 zagwiritsidwa ntchito ngati mankhwala a chemotherapy omwe angagwiritsidwe ntchito pochiza TNBC (Ishino et al., 2018). Popeza njira zolumikizirana za Wnt/b-Catenin, NOTCH, ndi Hedgehog zanenedwa kuti zikukhudzidwa ndi kuchitika ndi kupita patsogolo kwa TNBC, kuyang'ana mankhwala m'njira izi kungakhale njira yofunika kwambiri (Aysola et al., 2013). Ngakhale opaleshoni, chithandizo cha radiation ndi chemotherapy zikadali njira yayikulu yochiritsira TNBC masiku ano, kupita patsogolo kwakukulu kwachitika popanga njira zatsopano zochiritsira kuphatikizapo chithandizo cholunjika, chithandizo cha immunotherapy, zida zosiyanasiyana zosinthira majini zokhudzana ndi CRISPR monga Cas9n, dCas9., CRISPR/Cas12, prime editing, ndi chithandizo cha majini cholunjika cha CRISPR/Cas9. Apa tiyang'ana kwambiri zida zosiyanasiyana zosinthira majini zokhudzana ndi CRISPR zomwe zimagwiritsidwa ntchito pochiza TNBC. Pakati pa izi, CRISPR/Cas9 yalandiridwa kwambiri.
Cas9n, yomwe imadziwikanso kuti Cas9 nickase, ndi mtundu wa puloteni ya Cas9 yopangidwa ndi majini yochokera ku dongosolo losintha majini la CRISPR/Cas9 (Gupta et al., 2019). Mu mawonekedwe ake oyambirira, puloteni ya Cas9 ili ndi magawo awiri a nuclease: RuvC ndi HNH, ntchito yake yayikulu ndi kugawanika kwa zingwe zonse ziwiri za DNA. Komabe, mu Cas9n, imodzi mwa magawo a nuclease, HNH, imasinthidwa majini ndipo imakhala yosagwira ntchito. Chifukwa chake, gawo la HNH la Cas9n siligwira ntchito. Gawo la RuvC lokha ndi lomwe limakhalabe logwira ntchito, kulola Cas9n kudula kapena kupanga ma break pa zingwe za DNA imodzi (Trevino ndi Zhang, 2014). Poyerekeza ndi Cas9, Cas9n imatha kuchepetsa bwino zotsatira zomwe sizili pa cholinga ndikuwongolera molondola njira zokonzera maselo. Cas9n ingagwiritsidwe ntchito kuthetsa mavuto osiyanasiyana, monga kupanga ma break pamalo enaake mu DNA yokhala ndi zingwe ziwiri. Pachifukwa ichi, mamolekyu awiri a Cas9n adaphatikizidwa ndikugwiritsidwa ntchito pamodzi (Yee, 2016). Mixed lineage kinase 3 (MLK3) ndi protein kinase yomwe imagwira ntchito ngati chowongolera chofunikira panthawi ya metastasis ya TNBC (Cronan et al., 2012).
MLK3 imatha kuyambitsa njira zingapo zolumikizirana zomwe zimapangitsa kuti TNBC isagwire bwino ntchito. Mwachitsanzo, njira ya c-Jun N-terminal kinase (JNK) imayendetsa kayendedwe ka maselo ndi kuwonongeka kwa matrix akunja, ndipo MLK3 imayendetsa njira ya JNK, motero imakulitsa kayendedwe ka maselo ndikupereka zinthu zowononga (Rattanasinchai ndi Gallo, 2016). MLK3 imayendetsanso EMT. Kuti izi zitheke, imayendetsa zinthu zotsatizana monga Snail, Slug ndi Twist. Zinthuzi zimaletsa kuwonetsedwa kwa zizindikiro za epithelial ndikulimbikitsa kuwonetsedwa kwa zizindikiro za mesenchymal, zomwe zimapangitsa kuti pakhale mtundu wina wa metastatic phenotype (Casalino et al., 2023). MLK3 yawonedwa kuti imagwira ntchito pakukonzanso kwa ECM ndikuyambitsa ma protease monga matrix metalloproteinases (MMPs) omwe amawononga ECM. Izi zimathandiza kuti maselo a chotupa alowe ndikufalikira kumalo akutali (Katari et al., 2019). Chifukwa chake, maphunziro am'mbuyomu awonetsa kuti MLK3 imagwira ntchito yofunika kwambiri mu TNBC. Rattanasinchai ndi Gallo adagwiritsa ntchito mitundu ya TNBC kuti aphunzire ntchito ya MLK3 ndipo adapeza kuti imalimbikitsa kukula kwa khansa kudzera m'njira zinazake zolumikizirana. Adagwiritsa ntchito CRISPR/Cas9n kusintha MLK3 ndipo adawona kuchepa kwakukulu kwa metastasis ya TNBC (Rattanasinchai ndi Gallo, 2016).
dCas9, yomwe nthawi zambiri imatchedwa kuti Cas9 yosagwira ntchito, ndi mtundu wosinthidwa wa puloteni ya Cas9. Mosiyana ndi Cas9 yogwira ntchito, dCas9 ilibe ntchito ya endonuclease, zomwe zimapangitsa kuti isathe kuyambitsa kusweka kwa DNA kawiri. Chifukwa chake, dCas9 ingagwiritsidwe ntchito kulunjika mwachindunji madera enaake a genome popanda kusintha kulikonse kapena kusintha kwa DNA sequence (Wang et al., 2016). Mu dCas9, mapuloteni awiri a endonuclease, RuvC ndi HNH, amalepheretsedwa poletsa zotsalira zofunika za amino acid. (Richter et al., 2016). Ngakhale kuti ilibe ntchito yogawa DNA, dCas9 imagwira ntchito zingapo zofunika pakufufuza majini ndi biotechnology. Mwachitsanzo, imalola kuwona madera enaake a genome, imathandizira kulamulira kwa transcriptional, ndikulimbikitsa kusintha kwa epigenetic (Brocken et al., 2018).
Cholembera cha ZEB1 (zinc chala E-box binding homeobox 1) chimagwira ntchito yapadera komanso yofunika kwambiri pakuyendetsa kusintha kwa epithelial-mesenchymal (EMT), njira yamaselo yomwe imachitika panthawi ya kukula kwa mwana wosabadwayo, kukonza minofu, ndi kupita patsogolo kwa khansa. Zimaphatikizapo kusintha kwa maselo a epithelial kukhala maselo a mesenchymal, zomwe zimapangitsa kusintha kwa mawonekedwe a maselo, kuyenda, kulowerera, ndi zina zotero. (Wu et al., 2020). ZEB1 imaletsa zizindikiro zambiri za epithelial, monga E-cadherin ndi Occludin, zomwe zimapangitsa kuti maselo azigwirizana komanso kuti maselo a epithelial akhale ogwirizana mu TNBC (Moreno-Bueno et al., 2008). Kuphatikiza apo, ZEB1 imayambitsa zizindikiro zina monga N-cadherin, vimentin ndi fibronectin (Konradi et al., 2014) komanso imayang'anira majini omwe amakhudzidwa ndi kusintha kwa cytoskeletal monga Rho GTPases ndi matrix metalloproteinases (MMPs) (Huang). et al., 2014). 2022), kuphatikiza apo, zimakhudzanso njira zosiyanasiyana zolumikizirana monga kusintha kukula kwa chinthu-β (TGF-β), Wnt signaling, ndi zina zotero, motero kulimbikitsa kulowa kwa chotupa ndi kufalikira kwa maselo mu TNBC (Chen et al., 2016). ). Kafukufuku wambiri ndi umboni wasayansi zikusonyeza kuti ZEB1 ili ndi kuthekera kwakukulu ngati chida chofunikira pakuzindikira ndi kuchiza TNBC. Mu kafukufuku waposachedwa, Waryah et al. adagwiritsa ntchito chitsanzo cha TNBC ndipo adakwanitsa kuletsa kwathunthu ZEB1 ndi dCas9. Chifukwa chake, adawona kutsimikizika kwakukulu kwambiri komanso kuletsa kwathunthu kwa ZEB1 m'mikhalidwe ya in vivo (Waryah et al., 2023).
CRISPR/Cas12 ndi chida chosinthira majini chotchedwa CRISPR/Cpf1. Chimachokera ku dongosolo la CRISPR/Cas, pomwe mawu akuti Cas12 amatanthauza puloteni yogwirizana ndi CRISPR 12 (Bharathkumar et al., 2022), yomwe ndi yofanana kwathunthu ndi Cas9. Kusiyana kokha ndikuti ili ndi puloteni ya Cas12. Poyerekeza ndi Cas9, Cas12 ili ndi ntchito zapadera monga kupanga malekezero omata panthawi yosintha majini, pomwe Cas9 imapanga malekezero osamveka (Wang et al., 2021). Kapangidwe ka Cas12 kameneka kamathandizira ukadaulo wake wapadera wosintha DNA. Monga puloteni yomwe imatha kuzindikira ndikudula DNA yomwe ikufunidwa, ili ndi kusinthasintha kwakukulu, zomwe zimapangitsa kuti ikhale chida chothandiza pa ntchito zosiyanasiyana, kuphatikizapo kusintha majini (Pickar-Oliver ndi Gersbach, 2019).
Mofanana ndi mitundu ina ya CRISPR, CRISPR/Cas12 imathanso kuchotsa kapena kuyambitsa majini mu TNBC, kulunjika majini omwe amatenga gawo lofunikira pakukula kwake kapena kuyankha chithandizo (Yang ndi Zhang, 2023). Kuti akwaniritse njirayi, RNA yotsogolera (gRNA) idapangidwa yomwe imatsogolera Cas12 ku jini lomwe likufunidwa. Cas12 ikangolumikizana ndi cholinga chake, imayambitsa kusweka kwa zingwe ziwiri mu DNA ndikuyambitsa njira zokonzanso DNA. Panthawi yokonzanso, ma nucleotide olakwika amatha kuphatikizidwa, zomwe zimapangitsa kuti majini asinthe komanso kutayika kwa ntchito (Zhang et al., 2021a). Kuphatikiza apo, mtundu wabwino wa enzyme ya Cas12 (yotchedwa dCas12) idagwiritsidwanso ntchito kuyambitsa majini. Mwa kuphatikiza ndi choyambitsa cholembera, ndizotheka kuyambitsa majini ena, potero kuwayambitsa. Ukadaulo uwu uli ndi kuthekera kwakukulu kolimbikitsa kuyambitsa majini oletsa chotupa (Sultan et al., 2022).
Kukonza koyamba ndi ukadaulo watsopano komanso wapamwamba kwambiri wokonza majini. Umatha kusintha DNA ya chamoyo molondola kwambiri (Chen ndi Liu, 2023). Kukonza koyamba kumachitika kudzera mu kuphatikiza zigawo ziwiri zazikulu: enzyme yosinthidwa ya CRISPR/Cas9 ndi reverse transcriptase. Udindo wa enzyme ya CRISPR/Cas9 ndikusankha malo enieni mu genome, pomwe reverse transcriptase imathandizira kusintha bwino DNA pamalo omwe yapangidwira (Hassan et al., 2021). Mu njira yokonza koyamba, gawo loyamba limaphatikizapo kupanga RNA yowongolera kusintha koyamba (pegRNA) (Standage-Beier et al., 2021). Ili ndi njira yowunikira ndi template ya RNA yogwirizana ndi malo owunikira omwe mukufuna mu DNA yomwe yapangidwira. Kenako pegRNA imalowetsedwa m'maselo omwe akuyenera kugwiritsidwa ntchito limodzi ndi primer editing nuclease (PE2). PE2 ndi puloteni yosakanikirana yokhala ndi enzyme ya Cas9, reverse transcriptase, ndi primer editing adapter (Martín-Alonso et al., 2021). Mkati mwa selo, ma pegRNA ndi ma complexes a PE2 amafufuza DNA yeniyeni yomwe akufuna kusintha. Enzyme ya Cas9 imadula DNA ndikupanga template yokhala ndi zingwe chimodzi (Choi et al., 2022). Reverse transcriptase imagwiritsa ntchito template iyi kukonzekera DNA kuti isinthidwe. Pamodzi ndi kukopera DNA, malangizo osinthira template ya RNA amaphatikizidwanso. Pomaliza, chingwe cha DNA chatsopano chimagwiritsidwa ntchito ngati template kukonza kusweka kwa DNA, zomwe zimapangitsa kuti DNA isinthidwe yokhala ndi kusintha komwe kukufunika (Ochoa-Sanchez et al., 2021). Njira zosinthira za primer zitha kubweretsa kusintha kwakukulu m'majini. Itha kuyang'ana kusintha kwa mfundo, kuyika, kuchotsera, komanso kusintha majini (Anzalone et al., 2019; Chen ndi Liu, 2023). Kusintha kwa Prime kumapereka zabwino zingapo kuposa ukadaulo wakale wosinthira majini. Izi zikuphatikizapo kulondola kowonjezereka, kuchepetsa zotsatira zomwe sizili pa cholinga, komanso kuthekera kosintha DNA popanda kudalira kusweka kwa zingwe ziwiri za DNA (Anzalone et al., 2020).
Ukadaulo wa CRISPR/Cas9 unapangidwa poyamba kuti uteteze mabakiteriya ku kusamutsidwa kwa plasmid ndi matenda a phage ndipo pambuyo pake unagwiritsidwanso ntchito ngati chida chothandiza chowunikira DNA chochokera ku RNA pokonza majini (Jiang ndi Doudna, 2017). Kuphatikiza apo, dongosolo la CRISPR/Cas9 lanenedwa kuti lilipo mu 50% ndi 87% ya majini a bakiteriya ndi archaeal, motsatana (Ishino et al., 2018). CRISPR/Cas9 ndi chida chomwe chingagwiritsidwe ntchito pochotsa, kuyika ndi kukonza njira iliyonse ya majini yosazolowereka pogwiritsa ntchito njira za mu vivo ndi in vitro (Sabit et al., 2021). Kuphatikiza apo, CRISPR/Cas9 yawonetsedwa kuti ndi gawo la chitetezo chamthupi chosinthika chifukwa cha kudziwika kwake kwa majini omwe akufuna (Chen ndi Zhang, 2018).
CRISPR/Cas9 ili ndi Cas9 ndi RNA imodzi yotsogolera (sgRNA). Cas9 ndi endonuclease yopangidwa ndi zigawo zingapo za mapuloteni. Kuphatikiza apo, Cas9 ili ndi mawonekedwe apadera komanso mawonekedwe (Pacesa et al., 2022) chifukwa imakhala ndi ma lobes awiri: kuzindikira (REC) ndi nuclease (NUC). Lobe ya REC imagawidwanso m'magawo atatu: REC1, REC2, ndi ma bridge helices (Cromwell et al., 2018). NUC ili ndi ma lobes atatu, omwe ndi RuvC (RuvC I, RuvC II, RuvC III), HNH ndi protospacer adjointe motif (PAM) interaction domain (Chithunzi 1) (Song et al., 2016). sgRNA ili ndi zigawo ziwiri, kuphatikiza CRISPR RNA (crRNA) ndi transcoding small RNA (tracer RNA) (Chithunzi 1). SgRNA imalumikiza puloteni ya Cas9 ndi ma connexins kuti ipange active complex, yomwe imadziwikanso kuti effector complex (Richter et al., 2012). crRNA ndi 18-20 nucleotide base pair yomwe imagwira ntchito yofunika kwambiri pozindikira ma target DNA sequences. Kuphatikiza apo, crRNA imagwirizana ndi target DNA, ndipo tracrRNA imagwira ntchito ngati scaffold ya Cas9 nuclease kuti igwirizane ndi target DNA (Manghwar et al., 2019). Kumbali inayi, PAM sequence ili ndi ma nucleotide atatu, omwe amatsimikizira, kufotokozera ndikugwirizanitsa kumangirira kwa effector complex ku DNA. Ma Cas9 protein complex subunits RuvC ndi HNH ali ndi catalytic activity (Richter et al., 2012; Asmamaw ndi Zawdie, 2021). HNH nuclease domain ya Cas9 subunit imadula chingwe cha DNA chogwirizana ndi crRNA. Dera la RuvC nuclease limaduladula ma strands ena a DNA ndikupanga ma double-strand breaks (DSBs), pambuyo pake njira ziwiri zosiyana zokonzera ma breaks a DNA zimayatsidwa (Jiang ndi Doudna, 2017) (Chithunzi 1).
Chithunzi 1. Chidule cha CRISPR/Cas9 (A). Zigawo za dongosolo la CRISPR/Cas9: (i). Cas9 endonuclease ndi yomwe imayang'anira kudula DNA sequence, (ii) guide single (sg) RNA yomwe imachokera ku kusakanikirana kwa crRNA ndi tra-crRNA chimeras. (ziwiri). Cas9 imaphatikizapo zigawo zingapo monga Rec I, Rec II, NUC lobe (HNH ndi Ruv C ndi zigawo zazing'ono) ndi PAM interaction domain (C) yokhala ndi ntchito zofanana. CRISPR/Cas9 protein complex imagawa DNA sequences kukhala mitundu yosakwanira komanso yowonjezera (D). CRISPR/Cas9 imasintha genome m'magawo atatu: kuzindikira, kudula ndi kukonza. sg-RNA yopangidwayo imatsogolera Cas9 ndikuzindikira sequence yomwe ikufunika kudzera mu complementary component crRNA. Cas9 imazindikira PAM sequence pa 5′-NGG-3′ ndikusungunula DNA, kupanga DNA-RNA hybrid ndikuyambitsa cleavage. Gawo la HNH la Cas9 limadula chingwe chowonjezera, ndipo gawo la RuvC limadula chingwe chowonjezera. CRISPR/Cas9 imakonza dsDNA poiphwanya m'njira ziwiri: cholumikizira chakumapeto chosagwirizana (NHEJ) ndi chokonzanso cholunjika (HDR). NHEJ imakonza DNA yokhala ndi zingwe ziwiri popanda DNA yachilendo yogwirizana kudzera mu njira ya enzyme, njira yolakwika yomwe imatha kuyika kapena kuchotsa ma DNA osasinthika. HDR ndi yeniyeni kwambiri ndipo imafuna ma tempuleti a DNA ofanana.
Njira zokonzera za CRISPR/Cas9 zimaphatikizapo njira zolumikizirana za malekezero osafanana (NHEJ) ndi njira zokonzera zolumikizana (HDR). NHEJ ndi njira yolakwika chifukwa imaphatikizapo kulowetsa kapena kuchotsa ndipo sikufuna mawonekedwe aliwonse panthawi yokonza. Imagwiritsa ntchito ma nucleotide osasinthika kuti ipange mapuloteni okhazikika (Abbasi et al., 2021). Dongosolo lokonzanso ili lili ndi ma complex anayi monga KU complex, cross-complementing protein complex type 4 (XRCC-4), DNA end processing enzyme, ndi protein kinase DNA-PKcs (Abbasi et al., 2021). Puloteni ya KU complex ili ndi ma subunits awiri, Ku 70 ndi Ku 80, ndipo imagwira ntchito yofunika kwambiri mu njira ya NHEJ, popeza njira yokonzanso imayambitsidwa ndi kumangirira ma subunits awiri (Ku 70 ndi Ku 80) ku malekezero osawoneka bwino kapena pafupifupi osawoneka bwino a DNA yomwe ikufunidwa (Abbasi et al., 2021), yomwe imagwira ntchito ngati malo osungira zinthu zina zokhudzana ndi NHEJ pamalo omwe avulala (Yang et al., 2020). XRCC-4 ndi DNA ligase zimapangidwa ndi ma amino acid 334 ndi 911 motsatana, ndipo XRCC4-DNA ligase IV complex imalimbikitsa kulumikiza malekezero a DNA (Chatterjee et al., 2015). Enzyme yokonza DNA end, yomwe imadziwikanso kuti polynucleotide kinase 3′-phosphate, ndi enzyme yokonza DNA end. Imatha kuchotsa gulu la 3′P mu DNA ndikupangitsa gulu la 5′OH phosphorylate panthawi yokonza DSB. Zimaphatikizaponso kukonza ma strand breaks (SSBs) pogwiritsa ntchito njira yokonzanso ya SSB (Chatterjee et al., 2015). Ma protein kinase DNA-PKcs ndi protein kinase yodalira DNA yomwe imakhala ndi catalytic subunit ya PIKKs (phosphatidylinositol 3-kinase-related kinases) ndi ataxia telangiectasia mutated (ATM) banja, komanso ATM ndi Rad3-related ATRs. ma strand breaks (DSBs) ndi ma strand breaks (Yue et al., 2020; Peng et al., 2016).
HDR ndi njira yolondola komanso yoyenera yokonzera chifukwa chidziwitsocho chimakopedwa pogwiritsa ntchito mawonekedwe osasinthika a DNA duplex yofanana, ngakhale kuti imafuna kukhalapo kwa ma chromatids alongo. Izi zimachitika panthawi ya S/G2 gawo la kayendedwe ka maselo a mammalia. HDR imapezeka makamaka mu mitundu ya yisiti, koma NHEJ ndi yofunika kwambiri mwa nyama zoyamwitsa (Burma et al., 2006; Abbasi et al., 2021). Njira yonse yokonzera ikuwonetsedwa mu Chithunzi 1.
Popeza TNBC imayambitsidwa ndi zolakwika za majini ndi epigenetic, kukonza zolakwika za majini/epigenomic pogwiritsa ntchito CRISPR/Cas9 kungakhale njira yabwino yochiritsira (Chen et al., 2019). Kuphatikiza apo, zinthu zina zolembera zomwe zimakhudzidwa ndi kayendetsedwe ka ma cell transcriptional zitha kuwonetsa mawonekedwe apadera a maselo a khansa, zomwe zikusonyeza kuti kayendetsedwe ka ma cell transcriptional kungakhale njira yabwino kwambiri yochizira khansa (Drost et al., 2017). Kugwiritsa ntchito ma molecular features a zotupa, monga majini, epigenetic ndi transcriptional defects, pakupanga mankhwala kungawongolere zotsatira zachipatala ndikuchepetsa ndalama zowunikira. CRISPR ndi chida chosinthira majini chomwe sichingathe kungozindikira ndikuzindikira zolinga za majini zomwe zimayambitsa khansa, komanso chingagwiritsidwe ntchito kusintha, kuletsa, ndi kusintha ma oncogene m'maselo a anthu (Table 1) (Ahmed et al., 2021). Ma screen angapo a CRISPR achitika kuti ayang'ane majini okhudzana ndi zoletsa zotupa, ma oncogene, ndi kukana mankhwala. Kugwiritsa ntchito CRISPR/Cas9 kusintha ma oncogene osiyanasiyana a TNBC kwawonetsedwa pa Chithunzi 2.
Chithunzi 2. Kusintha majini a TNBC oncogenes osiyanasiyana pogwiritsa ntchito CRISPR/Cas9 zomwe zimapangitsa kuti chotupa chichepe kukula ndi kufalikira kwa maselo. Ma oncogenes awa akuphatikizapo CDK7, NAT1, UBR5, YTHDF2, ITGA9, CXCR4 ndi CXCR7, Crypto1, ROR1 ndi ST8SIA, omwe amakhudzidwa ndi kuchitika ndi kufalikira kwa maselo a TNBC.
Kusamuka kwa maselo a khansa, kulowa m'thupi, ndi kusintha kwa epithelial-mesenchymal (EMT) zimagwirizanitsidwa ndi ITGA9. Kafukufuku wakale wasonyeza kuti ITGA9 ndi gawo lofunika kwambiri pa njira ya Notch ndipo imagwira ntchito yosangalatsa pa metastasis ya rhabdomyosarcoma (Molist et al., 2020). Kuphatikiza apo, ITGA9 idapezeka kuti ikugwirizana kwambiri ndi kupezeka kwa matenda a khansa m'mitundu ingapo ya khansa, kuphatikizapo khansa ya m'mawere (Wang Z. et al., 2019). Kuphatikiza apo, kusanthula kwa bioinformatics kwa ITGA9 kunawonetsa kuti kuwonekera kwake mu TNBC kunali kokwera kwambiri kuposa m'mitundu ina ya khansa ya m'mawere. Kuchuluka kwa ITGA9 kumagwirizanitsidwa ndi metastasis ya chotupa ndikubwereranso kwa odwala a TNBC. Kuchotsedwa kwa ITGA9 ndi CRISPR/Cas9 kunapangitsa kuti maselo oyambira khansa (CSC) akhale ofanana ndi khansa, angiogenesis ya chotupa, kukula kwa chotupa, komanso kuchepa kwa metastasis polimbikitsa kuwonongeka kwa β-catenin mu TNBC (Wang Z. et al., 2019).
Crypto-1 ndi membala wa banja la TGF-β ndipo ndi yofunika kwambiri pakukula kwa embryogenesis koyambirira, kusamalira maselo oyambira, komanso kufalikira kwa khansa (Ishii et al., 2021). Imadziwikanso kuti Tdgf-1, ndi puloteni yolumikizira ya GPI yomwe imagwira ntchito yoletsa mapangidwe a primitive streak, mesoderm ndi endoderm, komanso kukhazikitsidwa kwa asymmetry yakumanzere/kumanja pakukula kwa ziwalo za thupi panthawi ya kukula kwa embryogenesis (Zhang et al., 2021b). Kuphatikiza apo, Cripto-1 yawonetsedwa kuti ikugwira ntchito mu kusintha kwa epithelial-mesenchymal (EMT) ngati chizindikiro cha maselo oyambira (Zhang et al., 2021b). EMT ndi yofunika osati kokha pazochitika zosiyanasiyana monga kukula kwa embryonic, fibrosis ndi machiritso a mabala, komanso pakulowa kwa khansa ndi kufalikira kwa khansa. Kuphatikiza apo, Cripto-1 yawonetsedwa kuti imagwirizana ndi ma Notch receptors anayi ndikuwonjezera kukula kwawo pambuyo pa kusinthika (Brandstadter ndi Maillard, 2019). Njira yolumikizirana ya Notch imadziwika kuti imagwira ntchito yosamalira maselo a khansa ya m'mawere a anthu. Kafukufuku wasonyeza kuti CRISPR/Cas9-mediated knockout ya Crypto-1 imaletsa kukula kwa khansa ndi kufalikira kwa maselo. Chifukwa chake, Crypto-1 ikhoza kukhala chandamale chofunikira cha TNBC (Castro et al., 2015).
Puloteni ya XCL12 ndi cholandirira chake cha chemokine CXC (CXCR4 ndi CXCR7) zimagwira ntchito zosiyanasiyana pakuchulukana kwa maselo a khansa, kukula, kusamuka ndi kulowa (Wu et al., 2015). Ma chemoreceptor awa akhala akugwirizana ndi chitukuko cha TNBC kudzera m'njira zambiri zolumikizirana mu zitsanzo za mu vivo ndi in vitro (Wu et al., 2015). Kuphatikiza apo, kuyambika kwa CXCR4 ndi CXCR7 kumalumikizidwa ndi chiopsezo chachikulu cha kufalikira kwa maselo komanso kutayika kwa chidziwitso mu TNBC (Karn et al., 2022). Chifukwa chake, kuchotsedwa kwa CXCR4 ndi CXCR7 kungakhale majini othandiza kwambiri pochiza khansa ya m'mawere, kuphatikiza TNBC. Kafukufuku wochitidwa ndi Yang et al. Yang et al. (2019) adagwiritsa ntchito CRISPR/Cas9 kuti athetse majini a CXCR4 ndi CXCR7 ndipo adapeza kuti kuchulukana, kukula, kusamuka ndi kulowa kwa TNBC kunali koletsedwa kwambiri (Yang et al., 2019).
Kuwonjezeka kwa miR-3662, yomwe ndi TNBC oncogene, kunawonedwa m'maselo a khansa ya m'mawere (Yi et al., 2022). Kutsika kwa miR-3662 kwawonetsedwa kuti kumaletsa kukula kwa chotupa cha khansa ya m'mawere ndi kufalikira kwa maselo onse m'thupi ndi m'thupi (Agarwal ndi Gupta, 2021). HBP-1 ndi choletsa champhamvu cha Wnt/-catenin signaling ndipo mwina ndi chomwe chimayambitsa kukula kwa maselo a TNBC komwe kumayendetsedwa ndi miR-3662. Posachedwapa, Yi et al. adapeza kuti miR-3662-HBP1 axis imayendetsa njira yolumikizirana ya Wnt/-catenin m'maselo a TNBC (Yi et al., 2022). Chifukwa cha mawonekedwe ake okhudzana ndi chotupa, miR-3662 ikhoza kukhala cholinga chochiritsira cha TNBC. Chifukwa chake, kutsika kwa miR-3662 komwe kumayendetsedwa ndi CRISPR/Cas9 kungakhale njira yabwino kwambiri yopangira mankhwala atsopano ochizira TNBC.
UBR5 ndi nucleophosphoprotein ya 300 kDa yomwe yadziwika kuti ndi yolamulira kwambiri kukula kwa tumorigenesis, metastasis, ndi chitetezo chamthupi m'makhansa osiyanasiyana (Shearer et al., 2015; Fu et al., 2023). UBR5 yanenedwa kuti imawonjezeka kwambiri mu zitsanzo za TNBC ndipo imalimbikitsa ntchito ya ERα poyambitsa kuchulukana kudzera mu ntchito yake ya ubiquitin ligase (Bolt et al., 2015). Kafukufuku wonse wa exome sequencing wa zitsanzo zoyambirira za TNBC adawonetsanso kuwonjezeka kwa kuwonetsedwa kwa UBR5, zomwe zikusonyeza kuti ndi gawo lake pakukula kwa TNBC. Kuphatikiza apo, kuchotsedwa kwa UBR5 komwe kumayambitsa CRISPR/Cas9 kunawonetsa kuletsa kwakukulu kwa metastasis ya TNBC ndi kukula kwa mitundu ya mbewa zoyesera. Kuphatikiza apo, kuphatikizidwa kwa UBR5 mu mtundu wa mbewa zakuthengo kunabwezeretsa magwiridwe antchito ake onse, pomwe izi sizinawonekere m'mitundu yosasinthika ya mutant (Liao et al., 2017). Kusowa kwa UBR5 kumakhudzana ndi kuwonjezeka kwa apoptosis, necrosis, komanso kuletsa kukula kwa chotupa mu TNBC chifukwa cha angiogenesis yoipa. Chifukwa cha kutayika kwa UBR5, kufalikira kwa chotupa ku ziwalo zakutali kumachepa, ndipo UBR5 imayambitsa EMT yosazolowereka makamaka mwa kuchepetsa E-cadherin expression (Zhang ndi Weinberg, 2018). Posachedwapa, UBR5 yawonetsedwa kuti ndi chinthu chofunikira kwambiri pa IFN-γ-induced PDL1 transcription mu TNBC chifukwa cha kusowa kwa E3 ubiquitination activity. Kusanthula kwa RNA transcriptome kwawonetsa kuti UBR5 ikhoza kukhala ndi zotsatirapo pa majini okhudzana ndi njira ya IFN-γ ndikulimbikitsa transactivation ya PDL1 mwa kuwonjezera kuchuluka kwa activation ya protein kinase RNA (PKR) ndi ma transducers ake a chizindikiro ndi ma activators. transcription 1 (STAT1) ndi interferon regulatory factor 1 (IRF1). Komabe, kuchotsedwa kwa UBR5 ndi PD-L1 komwe kumayendetsedwa ndi CRISPR/Cas9 kumathandizira kwambiri pochiza kuposa kuletsa kulikonse kokha, komwe kumakhudza kwambiri chilengedwe cha chotupa (Wu et al., 2022). Chifukwa chake, CRISPR/Cas9 ikhoza kukhala chida chofunikira choletsa ntchito ya UBR5, potero kuletsa kufalikira kwa TNBC ndi kukula kwa chotupa.
ROR1 ndi puloteni ya mtundu woyamba ya transmembrane yomwe imapezeka panthawi ya khansa ndi kukula kwa mwana wosabadwayo ndipo yadziwika kuti ndi puloteni ya oncofetal (Nicholas Borcherding, 2014). Khalidwe loipa la khansa zosiyanasiyana za anthu limagwirizanitsidwa ndi kukwera kwa ROR1. Zotsatira zabwino zapezeka kuchokera ku maphunziro a in vivo ndi in vitro okhudzana ndi mankhwala othandizira ROR1 (Chien et al., 2016). Kuchuluka kwa ROR1 mRNA m'ma biopsies a minofu ya m'mawere kwagwirizanitsidwanso ndi zotupa zamphamvu za m'mawere (BL) komanso kusamuka kwawo kupita ku ziwalo zina za thupi. Kuphatikiza apo, kuwonetsa kwambiri ROR1 kwadziwika ngati chizindikiro cha kuneneratu kukula kwa TNBC (Chien et al., 2016). Komabe, kuletsa ROR1 pogwiritsa ntchito CRISPR/Cas9 kuti tiletse kukula kwa TNBC ndi kufalikira kwa maselo kungakhale njira yothandiza.
Njira ya sialylation imaphatikizapo kuwonjezera sialic acid ku glycoconjugates, zomwe zimayambitsidwa ndi sialyltransferases (STs). ST8SIA1 ndi ya banja la ST ndipo imagwira ntchito yofunika kwambiri pakuyambitsa matenda osiyanasiyana monga lymphocytic leukemia ndi khansa ya m'matumbo (Chang et al., 2018). Kusanthula kwa RNA kukuwonetsa kuti ST8SIA1 imaonekera kwambiri m'mafupa a odwala a TNBC ndipo imagwirizana bwino ndi kusintha kwa majini a chotupa p53, zomwe zingathandize pakuyambitsa matenda a TNBC (Battula et al., 2017). Kuphatikiza apo, ST8SIA1 imagwira ntchito pakuyambitsa matenda ndi kubwereranso kwa TNBC, zomwe zikusonyeza kuti imagwira ntchito yofunika kwambiri pakubuka ndi kukula kwa TNBC. Mu chitsanzo cha TNBC cha in vitro, kugwetsa ST8SIA1 ndi CRIPSR/Cas9 kwawonetsedwa kuti kumaletsa kukula ndi kufalikira kwa matendawa (Battula et al., 2017). Izi zikusonyeza kuti CRISPR/Cas9 ikhoza kukhala chida chofunikira kwambiri choletsa ntchito ya ST8SIA1 oncogene pochiza TNBC.
NAT1 ndi enzyme ya kagayidwe kachakudya yomwe imayambitsa kupangika kwa mankhwala a xenobiotic a gawo lachiwiri ndipo imapezeka pafupifupi m'thupi lonse la munthu. NAT1 ingagwiritse ntchito cofactor folate kuti igwire acetyl-CoA (acetyl-CoA) ngakhale palibe aromatic amine substrate (Stepp et al., 2015; Laurieri et al., 2014). Kafukufuku wasonyeza kuti NAT1 imalamulira matrix metalloproteinase 9 (MMP9) ntchito m'maselo a khansa ya m'mawere ndipo imateteza ku reactive oxygen species (ROS) panthawi ya glucose fasting (Wang et al., 2018). Kuchotsedwa kwa NAT1 kwawonetsedwa kuti kumaletsa pyruvate dehydrogenase complex, zomwe zimapangitsa kuti mitochondrial isagwire bwino ntchito (Wang L. et al., 2019). Kuphatikiza apo, malipoti ena osiyanasiyana awonetsa kuti kuletsa kwa NAT1 pogwiritsa ntchito mamolekyu ang'onoang'ono ndi siRNA silencing kungachepetse kufalikira ndi kuchuluka kwa maselo a khansa ya m'mawere (Stepp et al., 2018). Posachedwapa, CRISPR/Cas9 idagwiritsidwa ntchito kuletsa NAT1 mu mzere wa maselo a khansa ya m'mawere MDA-MB-231, zomwe zimakhudza kagayidwe ka maselo kutengera kuchuluka kwa momwe amaonekera. Kafukufukuyu adawonetsanso kuti NAT-1 ndiyofunikira kwambiri pakukula ndi kufalikira kwa TNBC (Carlisle et al., 2020).
Kulemba kwa oncogene kogwirizana kumayendetsedwa ndi ma super enhancers, transcription factors ndi cofactors (Hnisz et al., 2015). Kuphatikiza apo, gulu la cyclin-dependent kinases (CDKs), monga CDK7, CDK8, CDK9, CDK12, ndi CDK13, ndizofunikira pakulamulira transcriptional. Pakati pawo, CDK7 imagwira ntchito mu phosphorylation ya RNA polymerase II, yomwe ndi yofunika kwambiri pakuyamba ndi kukulitsa transcription ya oncogene mu pathogenesis ya TNBC. Mu kafukufuku woyambirira, kuchotsedwa kwa CDK7 ndi CRISPR/Cas9 kunaletsa TNBC, zomwe zikusonyeza kuti pathogenesis ya TNBC imadalira CDK7 (Wang Y. et al., 2015).
Kafukufuku wasonyeza kuti kusintha kwa majini mu MYC ndi RBP (RNA binding protein) kungayambitse apoptosis, pomwe kusintha kwa majini amodzi mu MYC kapena RBP sikukhudza kukula kwa maselo a khansa (Einstein et al., 2021). Ma RBP opitilira 1000 mu genome ya munthu ayesedwa pogwiritsa ntchito laibulale yochokera ku CRISPR/Cas9. Pakati pawo, ma RBP 57 adapezeka kuti ndi ofunikira kwambiri pakukula kwa maselo a khansa omwe ali ndi MYC yokwera kwambiri (Wheeler et al., 2020). Kuphatikiza apo, YTHDF2 ndiyofunikira pakusunga kukula kwa maselo a TNBC ndipo imachepetsa kuchuluka kwa zolemba za methylated panthawi yolemba ndi kumasulira kwapamwamba m'maselo a khansa omwe ali ndi MYC yokwera kwambiri. Kuphatikiza apo, YTHDF2 siyofunikira kwambiri pamaselo a khansa, omwe samadalira kwambiri MYC yokwera kuti apitirize kukhala ndi moyo kwa odwala a TNBC (Einstein et al., 2021), zomwe zikusonyeza kuti ikhoza kukhala njira yochiritsira mankhwala omwe angagonjetse TNBC.
Mapuloteni a zinc finger (ZNFs) amapanga pafupifupi 1% ya majini onse a munthu. Kafukufuku wasonyeza kuti ZNF imatha kulamulira kuchulukana kwa maselo m'makhansa osiyanasiyana, monga khansa ya chiwindi, khansa ya m'mawere ndi khansa ya m'matumbo (Zhang W. et al., 2021; Li et al., 2017). Malaibulale opangidwa ndi CRISPR knockout agwiritsidwa ntchito pofufuza majini osiyanasiyana oletsa khansa (TSGs) m'maselo a khansa ya m'mawere (Shalem et al., 2014). Kuyambira pamenepo, kafukufuku wolembedwa wa maselo a khansa ya m'mawere ochotsedwa a CRISPR/Cas9 ZNF319 wasonyeza kuti ZNF319 ndi jini yoletsa khansa yomwe imachepetsa kuchulukana kwa khansa ya m'mawere ndipo motero imagwira ntchito m'njira zosiyanasiyana zolumikizirana ndi ntchito zina zamoyo (Wang L. et al., 2022).
Ferroptosis ndi mtundu wa imfa yokonzedwa mwadongosolo yomwe imadalira kukhalapo kwa chitsulo. Zimadziwika kuti kukula kwa ferroptosis kumakhudzidwa ndi kukhalapo kwa lipid peroxides. Kuphatikiza apo, PKCβII yanenedwa kuti ikuwonetsa lipid peroxidation yoyambirira, ndipo kuwonjezeka kwa lipid peroxidation kumalumikizidwa ndi ferroptosis. Kuwunika laibulale ya CRISPR/Cas9-mediated kinase inhibitors kwawonetsa kuti PKCβII imagwira ntchito mu njira ya lipid peroxidation, yomwe ndi yofunika kwambiri pa ferroptosis mu maselo a MDA-MB-231 (Zhang et al., 2022). Chifukwa chake, izi zikusonyeza kuti kuchotsedwa kwa PKCβII ndi CRISPR/Cas9 kungakhale cholinga cha majini choletsa chotupa pochiza matenda okhudzana ndi ferroptosis (Zhang et al., 2022).
Kukana mankhwala kumakhulupirira kuti ndi komwe kumayambitsa pafupifupi 90% ya imfa mwa odwala khansa ndipo ndi chimodzi mwazovuta zazikulu pochiza khansa (Bukowski et al., 2020). Zotsatirazi zikusonyeza kuti majini okwanira okhudzana ndi kutuluka kwa mankhwala, kukonza DNA, apoptosis ndi njira zosiyanasiyana zolumikizirana ndi maselo zimagwirizanitsidwa ndi kukana mankhwala (Haider et al., 2020). Pakati pawo, majini angapo agwiritsidwa ntchito ndi zida za CRISPR/Cas9 ndipo awonetsa zotsatira zabwino pochepetsa kukana mankhwala ndikuwonjezera mphamvu ya mankhwala oletsa khansa (Vaghari-Tabari et al., 2022). Kuphatikiza apo, malaibulale owunikira majini a CRISPR/Cas9 omwe ali ndi mphamvu zambiri akhala akuphatikizidwa mukusintha ntchito ya majini omwe angakhalepo okana mankhwala (Shalem et al., 2015). Kuti tizindikire majini okana paclitaxel, kusanthula kwa RNA pamodzi ndi kufufuza kwa laibulale ya sgRNA yonse kunagwiritsidwa ntchito kuzindikira majini asanu ndi atatu omwe ali ndi vuto, kuphatikizapo histone deacetylase 9 (HDAC9), omwe amagwirizanitsidwa ndi kukana mankhwala mwa odwala omwe ali ndi TNBC yobwerezabwereza (B et al., 2020). ). Mu kafukufuku wina, kuwonjezeka kwa diserine/threonine ndi tyrosine protein kinase (DSTYK) kunawonedwa panthawi yomwe odwala a TNBC adalandira mankhwala oletsa khansa anali kupulumuka. Kuphatikiza apo, kugwetsa kwa DSTYK komwe kunayambitsidwa ndi CRISPR/Cas9 kunawonjezera kwambiri apoptosis ya maselo a khansa osagonjetsedwa ndi mankhwala mu vitro (maselo a SUM102PT ndi maselo a MDA-MB-468) ndi chitsanzo cha TNBC mu vivo (Ogbu et al., 2021).
Ngakhale kuti TNBC imagwira ntchito molakwika pa njira ya MAPK, zotsatira zake zachipatala za mankhwala opangidwa ndi MEK ndi zoipa. Chithunzi cha CRISPR/Cas9 genomic library chinawonetsa kuti kuletsa kwa PSMG2 (proteome assembly chaperone 2) kumathandizira maselo a TNBC BT549 ndi MB468 ku MEK inhibitor AZD6244. Kugwetsa kwa PSMG2 kwa CRISPR/Cas9 kunasintha ntchito yachibadwa ya proteasome, zomwe zinapangitsa kuti PDPK1 ichotsedwe ndi autophagy, motero kumawonjezera bwino maselo a chotupa mu mitundu ya mbewa ya TNBC yomwe imayambitsidwa ndi AZD6244 (MEK inhibitor) ndi MG132 (proteasome inhibitor). Chifukwa chake, zoletsa za proteasome ndi MAP kinase (MEK) zitha kuperekedwa mogwirizana kuti zichepetse kuchulukana kwa maselo a chotupa (Wang X. et al., 2022).
CRISPR/Cas9 yagwiritsidwanso ntchito pofufuza ngati majini atayika zomwe zimapangitsa kuti TNBC isagwire bwino ntchito (Shu et al., 2020). Ge et al anafotokoza momwe maselo a khansa amakanira mankhwala omwe amathandizidwa ndi JQ1, BET bromodomain inhibitor (BBDI), mu TNBC. Pogwiritsa ntchito CRISPR/Cas9, adapeza kuti kuchotsa jini ya rb1 kunapangitsa kuti TNBC ikhale yosagwirizana ndi mankhwala oletsa khansa JQ1. Chifukwa chake, ntchito ya rb1 ndiyofunikira poyankha mankhwala a JQ1 mu TNBC. Adanenanso kuti paclitaxel ndi CDK4/6 kinase/microtubule inhibitor, ndipo kuphatikiza kwake ndi BBDIs monga JQ1 kungapereke mayankho odalirika ochiritsira ku TNBC yosagonjetsedwa ndi mankhwala (Ge et al., 2020).
Kulemba kwa RNA yayitali yopanda ma code (lncRNA) kwanenedwa kuti ndi komwe kumayambitsa kukana kwa neoadjuvant therapy mu TNBC. Kafukufukuyu akuwonetsa kuti zolemba zisanu zosiyana za MALAT1 lncRNA zimafotokozedwa kwambiri mu TNBC. Kuphatikiza apo, kuchotsedwa kwa MALAT1 komwe kumachitika chifukwa cha CRISPR/Cas9 kunawonjezera chidwi cha maselo a TNBC BT-549 ku paclitaxel ndi doxorubicin, zomwe zikusonyeza kuti kukana kwa MALAT1 kungakhale ndi gawo lalikulu mu TNBC (Shaath et al., 2021).
Kusintha kwa maselo mu BRCA1 (BRCA1m) n’kosiyana ndipo motero n’kovuta kuzindikira. Kuyang’ana PARP1 (poly(ADP-ribose) polymerase), yomwe ndi bwenzi lopha la BRCA1, kungawonjezere mphamvu ya mankhwala a TNBC. Pogwiritsa ntchito CRISPR/Cas9, kuchotsa PARP1 kunawonjezera mphamvu ya mankhwala oletsa khansa monga doxorubicin, gemcitabine ndi docetaxel ku maselo a TNBC a mBRCA1 mutant, zomwe zikusonyeza kuti PARP1 imagwiranso ntchito pa kukana mankhwala mu TNBC (Vaghari-Tabari et al., 2022). Kusintha kwa maselo oletsa khansa mu BRCA1 kapena BRCA2 kumadziwika kuti kumawonjezera mwayi wokhala ndi khansa ya m’mawere mwa anthu. Kuchiza odwalawa kumafuna kugwiritsa ntchito zoletsa za PARP. Kuphatikiza apo, zawonetsedwa kuti kuchotsedwa kwa nucleotide salvage factor DNPH1 pogwiritsa ntchito CRISPR/Cas9 kungachotse nucleotide 5-hydroxymethyldeoxyuridine (hmdU) monophosphate yoopsa, motero kumawonjezera kuyankha kwa maselo osowa BRCA ku kukhudzidwa ndi PARP inhibitors (Fugger). et al., 2021). Chifukwa chake, CRISPR/Cas9 ndi PARP1 inhibitors zitha kukhala njira yofunika kwambiri yothandizira TNBC.
Jini ya penetrant glycoprotein (P-gp) ndi jini yolimbana ndi mankhwala ambiri yomwe nthawi zambiri imawonjezeka pafupifupi 41% ya TNBC yonse (Sun et al., 2020). Kutuluka kwa mankhwala opangidwa ndi P-gp kwadziwika kuti ndi njira yofunika kwambiri yowongolera kukana mankhwala mu khansa ya m'mawere. Zoletsa za P-gp zawonetsa kuti zimakhala ndi mphamvu zambiri ku mankhwala oletsa khansa mu khansa ya m'mawere (Famta et al., 2021). Chifukwa chake, kuchotsedwa kapena kuletsa zoletsa za P-gp ndi P-gp zomwe zimayendetsedwa ndi CRISPR/Cas9 kungakhale njira zofunika kwambiri zothetsera kukana mankhwala mu TNBC.
Chonyamula makaseti chomangira ATP G2 (ABCG2) chimadziwika kuti chimayambitsa kukana mankhwala mu TNBC (Palasuberniam et al., 2015), ngakhale pakadali pano palibe malipoti okhudza ubale womwe ulipo pakati pa CRISPR/Cas9 ndi ABCG2 silencing ndi kuletsa majini oletsa chotupa. PTEN Ingalimbikitse kuyambitsa kwa ABCG2 (Palasuberniam et al., 2015), (Deepak Singh et al., 2021). Chifukwa chake, kugwiritsa ntchito CRISP/Ca9 kuchotsa ABCG2 ndikuyiphatikiza ndi choletsa cha ABCG2 kungakhale chithandizo choyenera chothana ndi kukana mankhwala mu TNBC. Gome 2 limatchula CRISPR/Cas9 yomwe ikuyang'ana majini onse okana.
Gome 2. CRISPR/Cas9 imayang'ana majini osiyanasiyana otsutsa TNBC kuti azitha kuzindikira maselo ku mankhwala oletsa khansa.
Malaibulale a CRISPR/Cas9 ndi zida zomwe zingagwiritsidwe ntchito poyesa kusintha kwa majini komwe kumakhudzana ndi matenda a khansa (Chan et al., 2022). Njira yowunikirayi imaphatikizapo magawo anayi monga (a) kumanga laibulale, (b) kusintha kwa lentiviral, (c) kuyesa kwa phenotypic, ndi (d) kusanthula kwa majini omwe akufuna. Ngakhale kuti TNBC ili ndi kuyambitsa kosazolowereka kwa njira ya MAPK, chiyembekezo chachipatala cha chithandizo cha MEK kwa odwala a TNBC omwe ali ndi kusintha kwa majini oletsa chotupa monga PTEN, RB1, ndi TP53 ndi choyipa kwambiri.
Kuphatikiza apo, njira yoyesera majini pogwiritsa ntchito CRISPR/Cas9 idayesa molekyulu yamphamvu kwambiri komanso yosankha, dehydrocatrol, yomwe ili ndi maluwa ndi masamba ambiri ku Guatemala, kuti imvetsetse momwe dehydrocatrol imakhudzira MDA-MB-231. Njira zopewera poizoni wa maselo osankhidwa. Makhalidwe a Mesenchymal stemness a mitundu ya TNBC. Njira yowunikirayi ya CRISPR/Cas9 idawonetsanso kuti HSD17B11, jini yomwe imalemba 17β-hydroxysteroid dehydrogenase mtundu 11, imawonetsedwa kwambiri m'maselo a MDA-MB-231 ndipo imabweretsa dehydrofalcarinol yeniyeni ya maselo a MDA-MB-231 (Grant et. al., 2020). Chifukwa chake, izi zikusonyeza kuti kuyesa majini a CRISPR/Cas9 kuli ndi kuthekera kwakukulu kozindikira njira zomwe zimayang'anira mphamvu zotsutsana ndi khansa za mankhwala achilengedwe omwe angakhalepo.
Kuphatikiza apo, kufooka kwa TNBC ku khansa kunaphunziridwa pogwiritsa ntchito jenomu yonse mu vivo CRISPR/Cas9 screen, yomwe inasonyeza kugwirizana pakati pa njira zotupa ndi zoletsa khansa. Zigawo zazikulu za njira za mTOR ndi Hippo zanenedwa kuti zimagwira ntchito yofunika kwambiri pakulamulira khansa mu TNBC. Kuphatikiza apo, kafukufuku wasonyeza kuti kuletsa kwa mankhwala kwa mTORC1/2 ndi YAP oncoprotein kumaletsa bwino matenda a TNBC pogwiritsa ntchito njira ya in vitro drug-matrix synergy model ndi xenografts zochokera kwa odwala mu vivo. Kuphatikiza apo, kuletsa kwa mTORC1/2 komwe kumayendetsedwa ndi Torin-1 kumawonjezera macropinocytosis, pomwe kuletsa kwa YAP komwe kumayambitsidwa ndi vertebrorfin kumabweretsa kufa kwa maselo a TNBC. Ponseponse, zotsatirazi zikuwonetsa mphamvu ndi kulimba kwa kuyesa kwa CRISPR mu vivo kuti adziwe mankhwala atsopano komanso ogwira mtima a TNBC (Dai et al., 2021).
Kusokonekera kwa chitetezo chamthupi ndi chinthu chofunikira kwambiri pakukula kwa khansa. Maselo a khansa amapewa chitetezo chamthupi mwa kunyalanyaza njira zodzitetezera, kuphatikizapo kusokoneza ntchito ya maselo a chitetezo chamthupi m'malo ozungulira khansa ndikuwononga chitetezo chamthupi. Chifukwa chake, kupanga chitetezo chamthupi chabwino kungakhale njira yofunika kwambiri yolimbana ndi khansa. Kugwiritsa ntchito CRISPR/Cas9-based gene modification kumathetsa mavuto angapo okhudzana ndi kusokonekera kwa chitetezo chamthupi kuchokera ku malingaliro osiyanasiyana. CRISPR/Cas9 yagwiritsidwa ntchito kukulitsa chitetezo chamthupi cholimbana ndi khansa ya m'mawere kudzera m'njira zotsatirazi (Chithunzi 3).
Chithunzi 3. Chithandizo cha CRISPR/Cas9 chimalimbana ndi maselo a TNBC. Kutayika kwa CDK5 ndi kugwetsa PDL1 ndi CD155 kumawonjezera chitetezo chamthupi. Momwemonso, kutayika kwa A2AR ndi kugwetsa kwa ma TAA monga HER2, mucin 1 ndi TEM8 kunawonjezera mphamvu ya maselo a CAR-T popha maselo a khansa. Kuwunika kwa maselo a T oyendetsedwa ndi CRISPR/Cas9 kwawonetsa kuti kusokonezeka kwa p38 kinase kumawonjezera ntchito yotsutsana ndi chotupa cha maselo a T. Maselo a T osinthidwa ndi CRISPR/Cas9 amawonjezera kuwonetsedwa kwa TCR (T cell receptor), zomwe zimapangitsa maselo a T kukhala ndi ntchito yotsutsana ndi chotupa.
Cholinga cha chithandizo cha chitetezo chamthupi ndikulimbikitsa chitetezo chamthupi kuti chiukire maselo a khansa. Kuchuluka kwa mapuloteni oteteza chitetezo chamthupi nthawi zambiri kumaletsa zochita za chitetezo chamthupi koma kungathandize khansa kupewa (Topalian et al., 2015). Mapuloteni awa amaletsa zochita za chitetezo chamthupi pomangirira ku ma receptors pamwamba pa maselo a chitetezo chamthupi. Mapuloteni angapo oteteza chitetezo chamthupi (monga CD155 ndi PD-L1) amalunjika ku PD-1 receptor pamaselo a chitetezo chamthupi ndipo amawonetsedwa mu khansa ya m'mawere, makamaka TNBC (Li Y.-C. et al., 2020). Kugwetsa PD-L1 kapena receptor yake pogwiritsa ntchito CRISPR/Cas9 kungayambitse chitetezo chamthupi kuti chiukire zotupa za TNBC (Yahata et al., 2019). Kuphatikiza apo, kuchepetsa kufalikira kwa PD-L1 kudzera mu CRISPR/Cas9-mediated deletion ya CDK5 kwawonetsedwa kuti kumaletsa kukula kwa chotupa mu vitro ndi mu vivo (Deng et al., 2020). Kuletsa kukula kwa khansa ya m'mawere m'maselo a m'thupi ndi m'thupi kumasonyeza kuti kuchepetsa kwa CD155 komwe kumayendetsedwa ndi shRNA kungakhale ndi zotsatira zochizira khansa ya m'mawere (Gao et al., 2018).
Maselo a CAR T amasonyeza ma CAR omwe amazindikira ma antigen ogwirizana ndi chotupa (TAAs) ndipo amatha kugwiritsa ntchito mapuloteni oletsa kuwononga maselo kuti awonjezere ntchito yawo (Li C. et al., 2020). Pali njira zosiyanasiyana zomwe zingagwiritsidwe ntchito pochiza maselo a CAR T mu khansa ya m'mawere, kuphatikizapo ma TAA angapo monga HER2, mucin1, ndi TEM8 (Bajgain et al., 2018). Pali umboni wakuti maselo a CAR T omwe amalimbana ndi mesothelin (yomwe imachulukitsidwa kwambiri m'maselo a TNBC BT-459) amagwira ntchito bwino polimbana ndi khansa pamene PD-1 yachotsedwa pogwiritsa ntchito CRISPR/Cas9 (Hu et al., 2019). Komabe, kupatula maselo a T kwa odwala kenako nkuwasintha ex vivo ndi njira yogwira ntchito kwambiri komanso yotenga nthawi. Ngakhale maselo a T onse amatha kuchepetsa zosowa zodzipatula, maselo a T omwe amapereka omwe akuwonetsa ma leukocyte antigen a anthu (HLA) kalasi I ndi T cell receptor (TCR) ayenera kuchotsedwa kuti apewe zolakwika za graft-versus-host (Ren et al., 2017). , 2017a). Pogwiritsa ntchito CRISPR/Cas9, HDR imatha kuchotsa TCR ndi HLA nthawi imodzi ndikuchotsa jini yomwe imalemba CAR. Kafukufuku wasonyeza kuti CRISPR/Cas9 ingagwiritsidwe ntchito kuyambitsa ma CAR otsutsana ndi CD19 m'malo mwa TCR, motero imapanga ma CAR bwino popanda kuwononga ma T cell (Dimitri et al., 2022). Maukadaulo a Multiplex apangidwa kuti apange ma cell a CAR T allogeneic pochotsa TCR, beta-2-microglobulin (B2M), HLA-I subunit ndi mapuloteni ena monga PD-1 ndi CTLA-4, omwe angakhale ndi mphamvu zambiri zotsutsana ndi khansa motsutsana ndi TNBC (Eyquem et al., 2017; Liu et al., 2017; Ren et al., 2017b; Dimitri et al., 2022).
Kugwiritsa ntchito CRISPR/Cas9 kungathandize kuti maselo a CAR-T agwire bwino ntchito. Adenosine imadziwika kuti ili ndi mphamvu zoletsa chitetezo chamthupi ndipo imatha kuchepetsa chitetezo chamthupi poletsa ntchito ya maselo a T ndikuyambitsa ma receptors a adenosine A2A (A2AR) (Vigano et al., 2019). Kuletsa A2AR pogwiritsa ntchito CRISPR/Cas9 kunathandiza kwambiri kuti maselo a CAR-T agwire bwino ntchito (Giuffrida et al., 2021). Maselo a T amadziwika kuti ali ndi makhalidwe osiyanasiyana monga kukula kwa maselo, kusiyanitsa, kupsinjika kwa okosijeni komanso kupsinjika kwa majini. Kuwunika maselo a T pogwiritsa ntchito CRISPR/Cas9 kunawonetsa kusokonezeka kwa ma kinase 25 osiyanasiyana a T cell receptor. Pakati pawo, kuchotsedwa kwa p38 kinase kwawonetsedwa kuti kumawonjezera ntchito yotsutsana ndi chotupa ya maselo a T, zomwe zikusonyeza kuti p38 kinase ndiye wolamulira wofunikira wa malamulo a maselo a CAR-T (Gurasamy et al., 2020).
Maselo a T amatha kusinthidwa majini pogwiritsa ntchito CRISPR/Cas9 kuti apange ma TCR amphamvu kwambiri. Kusamutsa maselo a T osinthidwa majini kwa odwala kwawonetsa mphamvu yolimbana ndi khansa kuposa maselo a T opangidwa ndi endogenous. Chifukwa chake, ma TCR opangidwa ndi endogenous amatha kupikisana ndi ma TCR osinthidwa majini mwa odwala, zomwe zingakhudze kuthekera kwa chithandizo cha khansa. Pofuna kuthana ndi vutoli, kugwiritsa ntchito CRISPR/Cas9 kuchotsa TCR-β yopangidwa ndi endogenous m'maselo olandira ndikusamutsa maselo a TCR-β kwa odwala khansa kungawonetse mayankho abwino olimbana ndi khansa popanda kufunikira mpikisano wogonana wa endogenous TCR (Fan et al. 2018). Chifukwa chake, kupatula maselo a T osinthidwa ndi CRISPR, ma TCR amakhala okhudzidwa kwambiri ndi ma antigen a chotupa kuposa maselo a T opangidwa ndi TCR wamba. Kuphatikiza apo, m'ma leukemia osiyanasiyana, maselo a T osinthidwa opangidwa ndi γδ TCR+ CRISPR amasonyeza kuchuluka kwa maselo a CD4+ ndi CD8+ T kuposa kusuntha kwa TCR wamba (Legut et al., 2018). Motero, maselo a T osinthidwa opangidwa ndi CRISPR/Cas9 akhoza kukhala njira yothandiza kwambiri yochiritsira matenda a TNBC.
Ma Integrins ndi mamolekyu omatira a maselo omwe amapezeka mu ma cell transmembranes ndipo amalimbikitsa kumangirira kwa maselo ku extracellular matrix (ECM) (Hamidi ndi Ivaska, 2018). Kusokonekera kwa ma integrins kumalumikizidwa ndi kukula ndi kusamuka kwa khansa mwa kusintha extracellular matrix, zomwe zimapangitsa kuti maselo a khansa apulumuke m'magazi (Hamidi ndi Ivaska, 2018). CRISPR/Cas9-mediated integrin knockdown imachedwetsa kupita patsogolo kwa chotupa, kufalikira kwa maselo, ndi kulowa kwa colon mu TNBC. Kutsika kwa integrin a5 (ITGA5) kwanenedwa kuti kumachepetsa kusamuka kwa maselo ndi kusamuka kwa khansa zina monga khansa ya m'mapapo (Ju et al., 2017), zomwe zikusonyeza kuti integrin a5 ingakhalenso chinthu chofunikira kwambiri pakukula kwa TNBC.
Kupanga mbewa zotuluka pogwiritsa ntchito njira zachikhalidwe za embryonic stem cell (ES) ndi njira yovuta, yotenga nthawi, komanso yosagwira ntchito. Zimatenga miyezi ndi zaka kuti zigwire maselo a ES kudzera mu homologous recombination, kubereka mbewa za chimeric, kenako kuziphatikiza ndi mbewa za heterozygous kuti zipange ana a homozygous. Kubereketsa kophatikizana kovuta kumafunika kuti pakhale mbewa zokhala ndi ma genetic mutations angapo. Komabe, mavuto ambiri abuka pogwiritsa ntchito mbewa zomwe zimapangidwa ndi ablation ya maselo a ES pogwiritsa ntchito CRISPR/Cas9 kapena mwa microinjection ya CRISPR/Cas9 components mu mazira opangidwa ndi selo limodzi. Mwachitsanzo, kuyambitsa kusintha nthawi zambiri kumachitika pa biallelic loci ndipo sikuti ndi gene specific. Posachedwapa zanenedwa kuti maselo a ES omwe amagwiritsa ntchito ukadaulo wa CRISPR/Cas9 amatha kuyika biallelic mutations nthawi imodzi m'majini 5 (Nishizono et al., 2021). Pachifukwa ichi, Cas9 mRNA ndi ma gRNA asanu enieni a majini adasinthidwa nthawi imodzi kukhala maselo a ES. Izi zikuwonetsa lonjezo ndi kugwira ntchito kwa njirayi, ngakhale kuti kusinthaku mwina kunaperekedwa pamene mizere yoyambira idapangidwa kuti ipange mbewa zisanu ndi chimodzi zongopeka. Kafukufukuyu akuwonetsanso zomwe zapezeka modabwitsa: maselo a ES safunikanso kupanga mbewa zosinthidwa majini. M'malo mwake, kuti achotse zinthu zinazake za majini, Cas9 mRNA ndi gRNA zidalowetsedwa m'mazira a single-cell stage. Zotsatira zake, mizere yoyambira yongopeka yapangidwa yomwe ingagwiritsidwe ntchito pophunzira zotsatira za kuchotsedwa kwa majini m'mbewa (Qin et al., 2016). Chifukwa chake, CRISPR/Cas9 imalola kupangidwa kwa mbewa zosinthika majini kuti zichiritse TNBC pamtengo wotsika kuposa ukadaulo wa majini wamba. Pogwiritsa ntchito njira ya CRISPR/Cas, chitsanzo chatsopano cha mbewa chongopeka chapangidwa chomwe chingayambitse bwino kusintha kwa mfundo mu jini imodzi kapena zingapo zamkati mu TNBC. Kutengera lingaliro ili, mitundu ya nyama ya TNBC ikhozanso kupangidwa pogwiritsa ntchito CRISPR/Cas9-mediated deletion ya BRCA1 ndi p53, zomwe zimapangitsa kuti HR repair iwonongeke, kusakhazikika kwa majini, ndi ma mutant phenotypes (Annunziato et al., 2020).
Pakadali pano, njira zosiyanasiyana monga mammography, magnetic resonance imaging (MRI) ndi ultrasound zimagwiritsidwa ntchito pozindikira TNBC. Komabe, njirazi zili ndi zofooka zina. Mammography imagwiritsidwa ntchito pozindikira minofu ya m'mawere m'malo mwa metastases, komwe maselo a khansa asamukira ku ziwalo zina. Ultrasonography si njira yodalirika yodziwira TNBC (Chen ndi Lee-Felker, 2023). MRI ili ndi mphamvu zambiri kuposa ultrasound ndi mammography, koma kulondola kwake kozindikira ndi kochepa (Sha ndi Chen, 2022). Kufufuza minofu ndi njira yowunikira maselo a khansa. Komabe, nthawi zina, kusanthula minofu kumatha kusowa minofu ya khansa ngati singano yachoka pamalo omwe akufuna. Kuphatikiza apo, ndi yokwera mtengo kwambiri ndipo ikhoza kukhala yopweteka kwa wodwalayo. TNBC ndi mtundu wosiyana wa khansa, kotero kusanthula minofu sikungapereke chidziwitso chokwanira chokhudza mtundu wa khansa. Chifukwa chake, njira yatsopano yodalirika yodziwira TNBC ikufunika mwachangu. CRISPR/Cas9 ikhoza kukhala njira ina yodziwira khansa ya m'mawere yomwe ndi yovuta kwambiri komanso yosavulaza kwenikweni. Pachifukwa ichi, njira zochokera ku CRISPR/Cas9 zingagwiritsidwe ntchito kukonza njira za PCR zodziwira matenda a TNBC. Choyamba, mapuloteni a Cas9 ndi cpf1 a dongosolo la CRISPR amagwiritsidwa ntchito kuchotsa DNA yosadziwika bwino, kenako mapuloteni awiriwa (Cas9 ndi cpf1) amatha kuzindikira njira ya PAM asanamangirire ku DNA yomwe akufuna (Deepak Singh et al., 2021). Chifukwa chake, PCR imatha kuzindikira kusintha komwe kumakhudzana ndi chitukuko cha khansa. Kafukufuku angapo agwiritsa ntchito njira iyi yochokera ku CRISPR kuti azindikire kusintha kosiyanasiyana m'ma khansa osiyanasiyana (Safari et al., 2019). Chifukwa chake, njira za PCR zochokera ku CRISPR zitha kuchepetsa kudalira kwa matenda a TNBC pa njira zowononga monga biopsy-based immunohistochemistry.
Kusintha kwa majini mu kasamalidwe ka mahomoni kwawonetsedwanso mu TNBC (Chen ndi Russo, 2009). Njira za PCR zochokera ku CRISPR/Cas9 pogwiritsa ntchito ma microarrays pozindikira matenda a point-of-care zitha kuyang'anira bwino kusintha kumeneku (Hajian et al., 2019). Zingaperekenso kwa ogwira ntchito zachipatala chidziwitso chofunikira chokhudza kusintha kwa majini mwa odwala a TNBC, zomwe zingathandize kukonza njira yawo yochizira. Komabe, njira yophatikizana iyi ili ndi zofooka. Chifukwa chake, kafukufuku wambiri amafunika kuti njira iyi ya CRISPR/Cas-PCR isagwiritsidwe ntchito pazachipatala pozindikira matenda a TNBC (Yang et al., 2019).