CRISPR) wata dabara ce ta gyaran kwayoyin halitta ta ƙarni na uku: CRISPR ta kawo sauyi a duniya

CRISPR (CrisPR) wata dabara ce ta gyaran kwayoyin halitta ta ƙarni na uku wadda ta kawo sauyi a duniya tare da sakamakonta mai yawa. An yi amfani da ita don magance cututtuka daban-daban na halittu da cututtuka. Kwayoyin cuta daban-daban da sauran prokaryotes (kamar archaea) suma suna da tsarin CRISPR/Cas9 don karewa daga phages. An ruwaito cewa dabarun da suka dogara da CRISPR/Cas9 na iya hana ci gaba da ci gaban ciwon daji na nono mai rauni sau uku (TNBC) ta hanyar niyya ga kwayoyin halittar juriya da za a iya canzawa, kwafi, da kuma tsarin epigenetic. Waɗannan hanyoyin magancewa na iya taimakawa wajen magance matsaloli masu ƙalubale kamar juriyar magunguna da aka lura ko da a cikin TNBC. A halin yanzu, ana amfani da hanyoyi daban-daban don isar da CRISPR/Cas9 ga ƙwayoyin halitta, kamar su na zahiri (ƙananan allura, electroporation da yanayin hydrodynamic), kwayar cuta (ƙwayar cuta da lentivirus da ke da alaƙa da adeno) da kuma waɗanda ba su da ƙwayoyin cuta (liposomes da lipid nanoparticles). )-barbashi). Duk da cewa an ƙirƙiro samfura daban-daban don nazarin musabbabin ƙwayoyin halitta na TNBC, rashin hanyoyin da aka yi niyya don isar da kayan aikin gyaran kwayoyin halitta a cikin jiki yana iyakance amfaninsu na asibiti. A taƙaice, wannan bita ya yi cikakken nazari kan ci gaba, ƙalubale, ƙuntatawa, da kuma yiwuwar maganin CRISPR/Cas9 ga TNBC bisa ga shaidun da ake da su. Mun kuma nuna yadda haɗin fasahar wucin gadi da koyon injina zai iya inganta dabarun CRISPR/Cas9 wajen magance TNBC.
Ana siffanta cutar kansa ta hanyar rarraba ƙwayoyin halitta ba tare da kulawa ba, katsewar wuraren binciken zagayowar ƙwayoyin halitta, da kuma maye gurbi a cikin kwayoyin halittar da ke rage ciwon daji (TSGs) (Matthews et al., 2022). Daga cikin nau'ikan ciwon daji daban-daban, ciwon nono shine nau'in ciwon daji da ya fi yawa a cikin mata kuma yana da yawan mace-mace a duk duniya (Waks da Winer, 2019). Ciwon nono cuta ce mai bambancin halaye daban-daban, kamar siffofin histological da na halitta, gabatarwar asibiti da hali, da kuma amsawa ga magani (Weigelt et al., 2010). Rarraba ciwon nono yana ba da cikakken haske game da gano cutar kansar nono da kuma hasashen ciwon daji. Amfani da alamun halittu na yau da kullun da siffofin asibiti sune babban ma'auni don rarraba ciwon nono (Tsang da Tse, 2019). Hasashen da martani ga maganin ciwon nono yana da tasiri ta hanyar abubuwa da yawa, gami da kasancewar mai karɓar estrogen (ER), mai karɓar progesterone (PR), mai karɓar haɓakar epidermal na ɗan adam 2 (HER2/neu), da matakin histological, nau'in ciwon daji, da matakin. girman da metastasis na ƙwayoyin lymph node (Al-Tubaiti, 2020). An gano nau'ikan ƙwayoyin halitta guda biyar na ciwon nono, waɗanda suka haɗa da luminal A, luminal B, HER2-enriched, basal-like, da claudin-low (Prat et al., 2015). TNBC wani nau'in ciwon daji ne na ƙwayoyin halitta wanda ba ya bayyana duk ER, PR, da HER2 (Yin et al., 2020). Waɗannan siffofin cututtuka suna da alaƙa da TNBC, suna tabbatar da ci gabansa cikin sauri da kuma yanayi mafi tsauri fiye da kowane nau'in ciwon nono (Feng et al., 2018). Bugu da ƙari, Peru et al (2000) sun yi amfani da fasahar microarray don sake rarraba ciwon nono da kuma gano nau'ikan ciwon nono guda biyar na ciki (Cadenas, 2012). Ciwon nono mai kama da basal wani nau'in ciwon nono ne wanda ke da yanayin rashin kyau sau uku kuma yana da alaƙa da ci gaba cikin sauri. Ya kamata a lura cewa duk cututtukan nono masu kama da basal yawanci ana gano su a matsayin TNBC ba daidai ba, amma kashi 77% ne kawai ke da TNBC; Sabanin haka, kashi 71–91% na TNBC suna kama da basal, wanda ke nuna cewa waɗannan nau'ikan ciwon nono guda biyu sun haɗu kuma suna wakiltar rarrabuwa daban-daban (Wang D.-Y. et al., 2019). Wannan yana haifar da buƙatar siffanta bambancin TNBC don bayyana hasashen da gano yiwuwar martani ga magunguna na yanzu da na gaba. Bugu da ƙari, TNBC yana wakiltar kashi 15–20% na duk shari'o'in ciwon nono kuma ya fi yawa a cikin mata 'yan ƙasa da shekaru 50. An ba da rahoton maye gurbi na BRCA1 ko BRCA2 a kusan kashi 20% na shari'o'in TNBC (Xie et al., 2017; Tzikas et al., 2017)., 2020). Bincike ya kuma nuna cewa TNBC tana da yanayin garkuwar jiki na musamman wanda ya haɗa da manyan matakan abubuwan da ke haifar da ci gaban jijiyoyin jini, macrophages masu alaƙa da ƙari (TAMs), lymphocytes masu shiga cikin ƙari (TILs), da sauran ƙwayoyin da ke da hannu a cikin ci gaban ƙari da ƙaura. Saboda haka, fahimtar yanayin TNBC yana da mahimmanci don hasashensa da maganinsa (Fan da He, 2022).
Domin tantance hasashen TNBC da kuma tabbatar da ingantaccen magani, ganewar asali, wanda ya dogara ne akan immunohistochemistry (IHC) don gano ER, PR da HER2, da kuma mammography don gano ƙananan ƙwayoyin nono, yana da mahimmanci. Duk da haka, mammography ba zai iya nuna siffofin ciki kamar necrosis da fibrosis ba (Deepak Singh et al., 2021). Tunda rashin kyakkyawan hasashen da ganewar asali yana hana likitoci rubuta magunguna da suka dace (Chaudhary, 2020), ana amfani da dabaru daban-daban don inganta kulawa ga marasa lafiya da TNBC. A halin yanzu, ana amfani da magunguna biyu, doxorubicin da cyclophosphamide, ga marasa lafiya da TNBC kuma sun nuna sakamako mai kyau. Bugu da ƙari, ana amfani da wasu magungunan platinum, kamar carboplatin da cisplatin (Sikov et al., 2015). Bugu da ƙari, an yi amfani da masu hana PARP kamar Olaparib, Velaparib, da PF-01367338 a matsayin magungunan chemotherapy masu yuwuwar magance TNBC (Ishino et al., 2018). Tun lokacin da aka ruwaito cewa hanyoyin siginar Wnt/b-Catenin, NOTC, da Hedgehog suna da hannu a cikin faruwar da ci gaban TNBC, yin niyya ga magunguna zuwa waɗannan hanyoyin na iya zama muhimmiyar dabara (Aysola et al., 2013). Duk da cewa tiyata, maganin radiation da chemotherapy sun kasance babban tushen magani ga TNBC a yau, an sami ci gaba mai mahimmanci wajen haɓaka sabbin magunguna ciki har da maganin da aka yi niyya, maganin rigakafi, kayan aikin gyaran kwayoyin halitta daban-daban da suka shafi CRISPR kamar Cas9n, dCas9., CRISPR/Cas12, gyaran farko, da kuma maganin kwayoyin halitta da aka yi niyya ga CRISPR/Cas9. A nan za mu mayar da hankali kan kayan aikin gyaran kwayoyin halitta daban-daban da suka shafi CRISPR da ake amfani da su wajen maganin TNBC. Daga cikinsu, CRISPR/Cas9 ya sami kulawa sosai.
Cas9n, wanda aka fi sani da Cas9 nickase, wani nau'in furotin ne da aka ƙera ta hanyar halitta wanda aka samo daga tsarin gyaran kwayoyin halitta na CRISPR/Cas9 (Gupta et al., 2019). A cikin sigarsa ta asali, furotin Cas9 ya ƙunshi yankuna biyu na nuclease: RuvC da HNH, babban aikinsa shine raba zaren DNA guda biyu. Duk da haka, a cikin Cas9n, ɗaya daga cikin yankunan nuclease, HNH, yana canzawa ta hanyar halitta kuma ya zama mara aiki. Saboda haka, yankin HNH na Cas9n ya kasance ba ya aiki. Yankin RuvC kawai ya kasance yana aiki, yana ba Cas9n damar yanke ko ƙirƙirar karyewa akan zaren DNA guda ɗaya (Trevino da Zhang, 2014). Idan aka kwatanta da Cas9, Cas9n zai iya rage tasirin da ba a yi niyya ba yadda ya kamata kuma ya inganta hanyoyin gyaran ƙwayoyin halitta daidai. Ana iya amfani da Cas9n don magance matsaloli daban-daban, kamar ƙirƙirar karyewa a takamaiman wurare a cikin DNA mai zare biyu. Don wannan dalili, an haɗa ƙwayoyin Cas9n guda biyu aka yi amfani da su tare (Yee, 2016). Haɗaɗɗen jinsi kinase 3 (MLK3) wani furotin kinase ne da ke aiki da mitogen wanda ke aiki a matsayin babban mai kula da shi yayin metastasis na TNBC (Cronan et al., 2012).
MLK3 na iya kunna hanyoyin sigina da yawa waɗanda ke haifar da metastasis na TNBC. Misali, hanyar c-Jun N-terminal kinase (JNK) tana daidaita motsi na tantanin halitta da lalacewar matrix na extracellular, kuma MLK3 tana kunna hanyar JNK, ta haka tana haɓaka motsi na tantanin halitta kuma tana ba da kaddarorin mamayewa (Rattanasinchai da Gallo, 2016). MLK3 kuma tana daidaita EMT. Don yin wannan, yana kunna abubuwan da ke haifar da rubutu kamar Snail, Slug da Twist. Waɗannan abubuwan suna hana bayyanar alamun epithelial kuma suna haɓaka bayyanar alamun mesenchymal, wanda ke haifar da samun yanayin metastatic (Csalino et al., 2023). An lura da MLK3 yana taka rawa a cikin sake fasalin ECM da kunna proteases kamar matrix metalloproteinases (MMPs) waɗanda ke lalata ECM. Wannan yana sauƙaƙa mamaye ƙwayoyin tumor kuma yana yaɗuwa zuwa wurare masu nisa (Katari et al., 2019). Don haka, binciken da aka yi a baya ya nuna cewa MLK3 yana taka muhimmiyar rawa a cikin TNBC. Rattanasinchai da Gallo sun yi amfani da samfuran TNBC don nazarin rawar da MLK3 ke takawa kuma sun gano cewa yana haɓaka ci gaban cutar kansa ta hanyar takamaiman hanyoyin sigina. Sun yi amfani da CRISPR/Cas9n don gyara MLK3 kuma sun lura da raguwa mai yawa a cikin metastasis na TNBC (Rattanasinchai da Gallo, 2016).
dCas9, wanda aka fi sani da rashin aiki Cas9, wani nau'in furotin ne da aka gyara wanda aka samo daga furotin Cas9. Ba kamar Cas9 mai aiki ba, dCas9 ba shi da aikin endonuclease, wanda hakan ya sa ba zai iya haifar da karyewar DNA mai nau'i biyu ba. Saboda haka, ana iya amfani da dCas9 don kai hari ga takamaiman yankuna na kwayar halitta ba tare da wani canji ko gyare-gyare ga jerin DNA ba (Wang et al., 2016). A cikin dCas9, sunadaran endonuclease guda biyu, RuvC da HNH, ba sa aiki ta hanyar danne muhimman ragowar amino acid. (Richter et al., 2016). Duk da rashin aikin raba DNA, dCas9 yana taka muhimmiyar rawa a binciken kwayoyin halitta da fasahar kere-kere. Misali, yana ba da damar ganin takamaiman yankuna na kwayar halitta, yana sauƙaƙe tsarin rubutu, kuma yana haɓaka gyare-gyaren epigenetic (Brocken et al., 2018).
ZEB1 (zinc finger E-box binding homeobox 1) yana taka muhimmiyar rawa wajen daidaita sauyin epithelial-mesenchymal (EMT), wani tsari na tantanin halitta wanda ke faruwa yayin ci gaban tayi, gyaran nama, da ci gaban cutar kansa. Ya ƙunshi canza ƙwayoyin epithelial zuwa ƙwayoyin mesenchymal, wanda ke haifar da canje-canje a cikin yanayin tantanin halitta, motsi, mamayewa, da sauransu (Wu et al., 2020). ZEB1 yana hana alamomin epithelial da yawa, kamar E-cadherin da Occludin, waɗanda ke da alhakin kiyaye mannewar tantanin halitta da polarity na ƙwayoyin epithelial a cikin TNBC (Moreno-Bueno et al., 2008). Bugu da ƙari, ZEB1 yana kunna wasu alamomi kamar N-cadherin, vimentin da fibronectin (Konradi et al., 2014) kuma yana daidaita kwayoyin halitta da ke da hannu a sake fasalin cytoskeletal kamar Rho GTPases da matrix metalloproteinases (MMPs) (Huang). et al., 2014). 2022), ban da haka, yana shafar hanyoyi daban-daban na sigina kamar canza yanayin girma-β (TGF-β), siginar Wnt, da sauransu, ta haka yana haɓaka mamayewar ƙari da metastasis a cikin TNBC (Chen et al., 2016). ). Nazari da yawa da shaidun kimiyya sun nuna cewa ZEB1 yana da babban iko a matsayin wakili mai mahimmanci don ganowa da magance TNBC. A cikin wani bincike na baya-bayan nan, Waryah et al. sun yi amfani da samfurin TNBC kuma sun cimma cikakken dakatar da ZEB1 ta hanyar dCas9. Don haka, sun lura da takamaiman takamaiman abubuwa da kusan cikakken hana ZEB1 a cikin yanayin in vivo (Waryah et al., 2023).
CRISPR/Cas12 kayan aikin gyaran kwayoyin halitta ne da ake kira CRISPR/Cpf1. An samo shi ne daga tsarin CRISPR/Cas, inda kalmar Cas12 ke nufin furotin 12 da ke da alaƙa da CRISPR (Bharathkumar et al., 2022), wanda yake kama da Cas9 gaba ɗaya. Bambancin kawai shine yana ɗauke da furotin Cas12. Idan aka kwatanta da Cas9, Cas12 yana da wasu ayyuka na musamman kamar samar da ƙarshen manne yayin aikin gyaran kwayoyin halitta, yayin da Cas9 ke samar da ƙarshen da ba ya da ƙarfi (Wang et al., 2021). Wannan siffa ta Cas12 tana ba da gudummawa ga takamaiman fasahar sarrafa DNA. A matsayin furotin mai iya gane daidai da yanke DNA mai manufa, yana da sauƙin amfani, wanda hakan ya sa ya zama kayan aiki mai tasiri ga aikace-aikace iri-iri, gami da gyaran kwayoyin halitta (Pickar-Oliver da Gersbach, 2019).
Kamar sauran nau'ikan CRISPR, CRISPR/Cas12 yana da ikon fitar da ko kunna kwayoyin halitta a cikin TNBC, yana mai da hankali kan kwayoyin halitta waɗanda ke taka muhimmiyar rawa a cikin yanayin cutarwa ko amsawa ga magani (Yang da Zhang, 2023). Don cimma wannan tsari, an ƙirƙiri jagorar RNA (gRNA) wanda ke jagorantar Cas12 zuwa ga kwayar halittar da aka nufa. Da zarar Cas12 ya haɗu da abin da aka nufa, yana gabatar da karyewar igiya biyu a cikin DNA kuma yana kunna hanyoyin gyaran DNA. A lokacin gyaran, ana iya haɗa nucleotides marasa daidai, wanda ke haifar da maye gurbi na kwayoyin halitta da asarar aiki (Zhang et al., 2021a). Bugu da ƙari, an kuma yi amfani da ingantaccen sigar enzyme na Cas12 (wanda ake kira dCas12) don kunna kwayoyin halitta. Ta hanyar haɗa shi da mai kunna rubutu, yana yiwuwa a haifar da wasu kwayoyin halitta, ta haka ne ake kunna su. Wannan fasaha tana da babban damar haɓaka kunna kwayoyin halittar da ke hana ciwace-ciwacen daji (Sultan et al., 2022).
Gyaran Prime sabuwar fasaha ce ta gyaran kwayoyin halitta wadda ta inganta kuma ta fi inganci. Tana da ikon gyara DNA na wani abu da ya dace (Chen da Liu, 2023). Gyaran Prime ana samunsa ne ta hanyar haɗa manyan sassa guda biyu: enzyme na CRISPR/Cas9 da aka gyara da kuma reverse transcriptase. Matsayin enzyme na CRISPR/Cas9 shine a yi niyya ga takamaiman wurare a cikin kwayar halittar, yayin da reverse transcriptase ke taimakawa wajen gyara DNA sosai a wurin da aka nufa (Hassan et al., 2021). A cikin tsarin gyaran primer, mataki na farko ya ƙunshi ƙirƙirar jagorar gyaran primer RNA (pegRNA) (Standage-Beier et al., 2021). Ya ƙunshi jerin abubuwan da aka nufa da samfurin RNA wanda ya dace da wurin gyaran da ake so a cikin DNA ɗin da aka nufa. Sannan ana shigar da pegRNA cikin ƙwayoyin da aka nufa tare da nuclease na gyaran primer (PE2). PE2 furotin ne na haɗin kai wanda ya ƙunshi enzyme na Cas9, reverse transcriptase, da adaftar gyaran primer (Martín-Alonso et al., 2021). A cikin tantanin halitta, pegRNA da hadaddun PE2 suna neman takamaiman DNA da suke son gyarawa. Enzyme na Cas9 yana yanke DNA kuma yana ƙirƙirar samfuri wanda ya ƙunshi zare ɗaya (Choi et al., 2022). Juyawar transcriptase yana amfani da wannan samfurin don shirya DNA don gyarawa. Tare da kwafin DNA, an haɗa da umarnin gyara samfurin RNA. A ƙarshe, ana amfani da sabon zaren DNA a matsayin samfuri don gyara karyewar DNA, wanda ke haifar da canjin jerin DNA wanda ke ɗauke da gyaran da ake so (Ochoa-Sanchez et al., 2021). Tsarin gyara na primer na iya haifar da sauye-sauye masu yawa a cikin kwayoyin halitta. Yana iya kai hari ga sauye-sauyen maki, sakawa, gogewa, har ma da maye gurbin kwayoyin halitta (Anzalone et al., 2019; Chen da Liu, 2023). Gyaran Prime yana ba da fa'idodi da yawa akan fasahar gyaran kwayoyin halitta ta baya. Waɗannan sun haɗa da ƙaruwar daidaito, raguwar tasirin da ba a yi niyya ba, da ikon gyara DNA ba tare da dogaro da karyewar DNA mai nau'i biyu ba (Anzalone et al., 2020).
An fara ƙirƙiro fasahar CRISPR/Cas9 don kare ƙwayoyin cuta daga canja wurin ƙwayoyin cuta na plasmid da kamuwa da cuta ta phage kuma daga baya aka sake amfani da ita azaman ingantaccen kayan aikin niyya na DNA mai tushen RNA don gyaran kwayoyin halitta (Jiang da Doudna, 2017). Bugu da ƙari, an ruwaito cewa tsarin CRISPR/Cas9 yana cikin kashi 50% da 87% na kwayoyin halittar ƙwayoyin cuta da na archaeal, bi da bi (Ishino et al., 2018). CRISPR/Cas9 kayan aiki ne mai yuwuwa don gogewa, sakawa da gyara duk wani jerin kwayoyin halitta marasa kyau ta amfani da yanayin in vivo da in vitro (Sabit et al., 2021). Bugu da ƙari, an nuna CRISPR/Cas9 a matsayin wani ɓangare na tsarin garkuwar jiki mai daidaitawa saboda takamaimansa ga kwayoyin halittar da ake so (Chen da Zhang, 2018).
CRISPR/Cas9 ya ƙunshi Cas9 da kuma RNA jagora guda ɗaya (sgRNA). Cas9 wani endonuclease ne wanda ya ƙunshi sinadaran furotin da yawa. Bugu da ƙari, Cas9 yana da halaye na musamman na tsari da tsari (Pacesa et al., 2022) saboda ya ƙunshi lobes guda biyu: ganewa (REC) da nuclease (NUC). An ƙara raba lobe na REC zuwa yankuna uku: REC1, REC2, da kuma helices na gada (Cromwell et al., 2018). NUC ya ƙunshi lobes guda uku, wato RuvC (RuvC I, RuvC II, RuvC III), HNH da yankin hulɗar protospacer (PAM) (Hoto na 1) (Song et al., 2016). sgRNA ya ƙunshi sassa biyu, gami da CRISPR RNA (crRNA) da transcoding ƙaramin RNA (tracer RNA) (Hoto na 1). SgRNA tana haɗa furotin Cas9 zuwa haɗuwa don samar da hadaddun aiki, wanda aka fi sani da hadaddun effector (Richter et al., 2012). crRNA nau'in tushe ne na nucleotide 18-20 wanda ke taka muhimmiyar rawa wajen gane jerin DNA na manufa. Bugu da ƙari, crRNA yana haɗuwa da DNA na manufa, kuma tracrRNA yana aiki a matsayin ma'auni ga nuclease Cas9 don ɗaure DNA na manufa (Manghwar et al., 2019). A gefe guda kuma, jerin PAM yana da nucleotides 3, wanda ke tabbatar, ƙayyade kuma yana daidaita ɗaurewar hadaddun effector zuwa DNA. Ƙananan rukunin hadaddun furotin Cas9 RuvC da HNH suna da aikin catalytic (Richter et al., 2012; Asmamaw da Zawdie, 2021). Yankin nuclease na HNH na sashin Cas9 yana raba zaren DNA da ke da alaƙa da crRNA. Yankin nuclease na RuvC yana raba wasu zare na DNA kuma yana haifar da karyewar zare biyu (DSBs), bayan haka ana kunna hanyoyin gyara karyewar DNA guda biyu daban-daban (Jiang da Doudna, 2017) (Hoto na 1).
Hoto na 1. Bayani game da CRISPR/Cas9 (A). Abubuwan da ke cikin tsarin CRISPR/Cas9: (i). Cas9 endonuclease yana da alhakin yanke jerin DNA da aka nufa, (ii) jagora ɗaya (sg) RNA wanda ya samo asali daga haɗuwar crRNA da tra-crRNA chimeras. (biyu). Cas9 ya haɗa da abubuwa da yawa kamar Rec I, Rec II, NUC lobe (HNH da Ruv C ƙananan abubuwa ne) da yankin hulɗar PAM (C) tare da ayyuka masu dacewa. Hadadden furotin na CRISPR/Cas9 yana raba jerin DNA zuwa siffofin da ba su da ƙarin aiki da ƙarin aiki (D). CRISPR/Cas9 yana gyara kwayar halittar a matakai uku: ganewa, yankewa da gyarawa. sg-RNA da aka tsara yana jagorantar Cas9 kuma yana gane jerin da ake so ta hanyar ɓangaren haɗin gwiwa na crRNA. Cas9 yana gane jerin PAM a 5′-NGG-3′ kuma yana narkar da DNA, yana samar da haɗin DNA-RNA kuma yana kunna rabawa. Yankin HNH na Cas9 yana raba zaren da ya dace, kuma yankin RuvC yana raba zaren da ba ya dace. CRISPR/Cas9 yana gyara dsDNA ta hanyar karya shi ta hanyoyi biyu: haɗin ƙarshen da ba na homologue ba (NHEJ) da gyaran da aka jagoranta ta homology (HDR). NHEJ yana gyara DNA mai layi biyu idan babu DNA mai layi biyu ta hanyar tsarin enzymatic, wata hanyar da ke iya haifar da kurakurai wanda zai iya saka ko cire jerin DNA bazuwar. HDR yana da takamaiman tsari kuma yana buƙatar samfuran DNA masu layi daya.
Hanyoyin gyara da CRISPR/Cas9 ke jagoranta sun haɗa da hanyoyin haɗin ƙarshen da ba na homologue ba (NHEJ) da hanyoyin gyara da aka jagoranta homology (HDR). NHEJ hanya ce da ke fuskantar kurakurai saboda tana ƙunshe da sakawa ko gogewa kuma ba ta buƙatar wani tsari yayin tsarin gyara. Tana amfani da nucleotides bazuwar don samar da furotin na yau da kullun (Abbasi et al., 2021). Wannan tsarin gyara ya ƙunshi hadaddun abubuwa guda huɗu kamar hadaddun KU, hadaddun furotin mai haɗuwa nau'in 4 (XRCC-4), enzyme na sarrafa ƙarshen DNA, da kuma protein kinase DNA-PKcs (Abbasi et al., 2021). Sinadarin KU mai hadaddun sunadaran yana da ƙananan sifofi guda biyu, Ku 70 da Ku 80, kuma yana taka muhimmiyar rawa a cikin tsarin NHEJ, tunda tsarin gyara yana farawa ne ta hanyar ɗaure ƙananan sifofi guda biyu (Ku 70 da Ku 80) zuwa ƙarshen DNA mai laushi ko kusan mai laushi (Abbasi et al., 2021), yana aiki a matsayin tushe don ɗaukar wasu abubuwan da suka shafi NHEJ zuwa wurin da rauni ya faru (Yang et al., 2020). XRCC-4 da DNA ligase sun ƙunshi amino acid 334 da 911, bi da bi, kuma XRCC4-DNA ligase IV complex yana ƙarfafa ɗaure ƙarshen DNA (Chatterjee et al., 2015). Enzyme na sarrafa ƙarshen DNA, wanda aka fi sani da polynucleotide kinase 3′-phosphate, enzyme ne na sarrafa ƙarshen DNA. Yana iya cire rukunin 3′P a cikin DNA kuma yana phosphorylate ƙungiyar 5′OH yayin gyaran DSB. Hakanan ya haɗa da gyaran karyewar zare ɗaya (SSBs) ta amfani da hanyar gyara SSB (Chatterjee et al., 2015). Protein kinase DNA-PKcs wani nau'in protein kinase ne wanda ke dogara da DNA wanda ya ƙunshi wani yanki mai catalytic na PIKKs (phosphatidylinositol 3-kinase-related kinases) da kuma ataxia telangiectasia mutated (ATM), da kuma ATM da Rad3-related strand breaks (DSBs) da kuma strand strand breaks (Yue et al., 2020; Peng et al., 2016).
HDR wata hanya ce ta gyarawa mafi inganci kuma mai dacewa domin ana kwafi bayanin ta amfani da siffa mai kama da ta DNA duplex, kodayake yana buƙatar kasancewar chromatids 'yan'uwa. Wannan yana faruwa ne a lokacin matakin S/G2 na zagayowar ƙwayoyin dabbobi masu shayarwa. HDR yana faruwa galibi a cikin nau'in yisti, amma NHEJ yana da mahimmanci a cikin dabbobi masu shayarwa (Burma et al., 2006; Abbasi et al., 2021). Cikakken tsarin gyara an nuna shi a Hoto na 1.
Tunda TNBC yana faruwa ne sakamakon rashin daidaituwar kwayoyin halitta da kuma cututtukan epigenetic, gyaran mummunan rashin daidaituwar kwayoyin halitta/epigogenomic ta amfani da CRISPR/Cas9 na iya zama hanyar magani mai ma'ana (Chen et al., 2019). Bugu da ƙari, wasu abubuwan da ke tattare da tsarin rubuta bayanai na musamman ga ƙwayoyin halitta na iya nuna halaye na musamman na ƙwayoyin cutar kansa, suna nuna cewa tsarin rubuta bayanai na iya zama hanya mai kyau don maganin ciwon daji (Drost et al., 2017). Amfani da waɗannan fasalulluka na ƙwayoyin cuta, kamar lahani na kwayoyin halitta, epigenetic da transcriptional, don haɓaka magunguna na iya inganta sakamakon asibiti da rage farashin tantancewa. CRISPR kayan aiki ne mai yuwuwar gyara kwayar halitta wanda ba wai kawai zai iya gano da gano abubuwan da ke haifar da ciwon daji ba, har ma ana iya amfani da shi don gyara, dannewa, da kuma canza oncogenes a cikin ƙwayoyin ɗan adam (Tebur 1) (Ahmed et al., 2021). An gudanar da gwaje-gwajen CRISPR da yawa don neman kwayoyin halitta da ke da alaƙa da masu hana ciwon daji, oncogenes, da juriya ga magunguna. An nuna amfani da CRISPR/Cas9 don gyara nau'ikan oncogenes na TNBC daban-daban a Hoto na 2.
Hoto na 2. Gyaran kwayoyin halitta na nau'ikan TNBC oncogenes ta amfani da CRISPR/Cas9 wanda ke haifar da raguwar girman ƙari da metastasis. Waɗannan oncogenes sun haɗa da CDK7, NAT1, UBR5, YTHDF2, ITGA9, CXCR4 da CXCR7, Crypto1, ROR1 da ST8SIA, waɗanda ke da hannu a cikin faruwar da metastasis na TNBC.
Shigowar ƙwayoyin cutar kansa, mamayewa, da kuma canjin epithelial-mesenchymal (EMT) suna da alaƙa da ITGA9. Nazarin da aka yi a baya ya nuna cewa ITGA9 muhimmin abu ne a cikin hanyar Notch kuma yana taka rawa mai ban sha'awa a cikin metastasis na rhabdomyosarcoma (Molist et al., 2020). Bugu da ƙari, an gano cewa ITGA9 yana da alaƙa da hasashen marasa lafiya a cikin nau'ikan ƙari da yawa, ciki har da ciwon nono (Wang Z. et al., 2019). Bugu da ƙari, nazarin bioinformatics na ITGA9 ya nuna cewa bayyanarsa a cikin TNBC ya fi girma fiye da sauran nau'ikan ciwon nono. Matakan ITGA9 masu tasowa suna da alaƙa da metastasis na ƙari da sake dawowa a cikin marasa lafiya na TNBC. Kashe ITGA9 ta CRISPR/Cas9 ya haifar da halayen ƙwayoyin kansa (CSC) masu kama da juna, angiogenesis na ƙari, ci gaban ƙari, da rage metastasis ta hanyar haɓaka lalacewar β-catenin a cikin TNBC (Wang Z. et al., 2019).
Crypto-1 memba ne na dangin TGF-β kuma yana da mahimmanci ga farkon haihuwa, kula da ƙwayoyin halitta, da kuma metastasis na ciwon daji (Ishii et al., 2021). Wanda kuma aka sani da Tdgf-1, furotin ne mai siginar GPI wanda ke da alaƙa da kansa wanda ke da hannu wajen daidaita samuwar streak na farko, mesoderm da endoderm, da kuma kafa rashin daidaituwar hagu/dama a cikin ci gaban gabobin jiki yayin embryogenesis (Zhang et al., 2021b). Bugu da ƙari, an nuna cewa Cripto-1 yana da hannu a cikin canjin epithelial-mesenchymal (EMT) a matsayin alamar ƙwayoyin halitta (Zhang et al., 2021b). EMT yana da mahimmanci ba kawai ga ayyuka daban-daban kamar ci gaban tayi, fibrosis da warkar da rauni ba, har ma don mamayewar ciwon daji da metastasis. Bugu da ƙari, an nuna cewa Cripto-1 yana hulɗa da masu karɓar Notch guda huɗu kuma yana haɓaka balaga bayan fassarar su (Brandstadter da Maillard, 2019). An san cewa hanyar siginar Notch tana da hannu wajen kula da ƙwayoyin cutar kansar nono na ɗan adam. Bincike ya nuna cewa ƙwanƙwasa Crypto-1 ta hanyar CRISPR/Cas9 yana hana ci gaban cutar kansa da kuma metastasis. Saboda haka, Crypto-1 na iya zama muhimmin abin da ake nufi da magani ga TNBC (Castro et al., 2015).
Protein XCL12 da mai karɓar chemokine CXC (CXCR4 da CXCR7) suna taka rawa daban-daban a cikin yaduwar ƙwayoyin cutar kansa, girma, ƙaura da mamayewa (Wu et al., 2015). Waɗannan masu karɓar chemo suna da alaƙa da haɓakar TNBC ta hanyar hanyoyin sigina da yawa a cikin samfuran in vivo da in vitro (Wu et al., 2015). Bugu da ƙari, kunna CXCR4 da CXCR7 yana da alaƙa da babban saurin kamuwa da cutar metastasis da rashin kyakkyawan hasashen cutar a cikin TNBC (Karn et al., 2022). Saboda haka, bugun CXCR4 da CXCR7 na iya zama ingantattun kwayoyin halittar magani don maganin ciwon nono, gami da TNBC. Binciken Yang et al. Yang et al (2019) sun yi amfani da CRISPR/Cas9 don haɗakar da kwayoyin halittar CXCR4 da CXCR7 kuma sun gano cewa yaduwar TNBC, girma, ƙaura da mamayewa an hana su sosai (Yang et al., 2019).
An lura da ƙaruwar matakan miR-3662, wani nau'in TNBC oncogene, a cikin kyallen kansar nono (Yi et al., 2022). An nuna cewa rage girman miR-3662 yana hana ci gaban ciwon daji na nono da kuma metastasis duka a cikin jiki da kuma a cikin vitro (Agarwal da Gupta, 2021). HBP-1 mai ƙarfi ne wajen hana siginar Wnt/-catenin kuma yana iya haifar da ci gaban ƙwayoyin TNBC da miR-3662 ke haifarwa. Kwanan nan, Yi et al. sun gano cewa axis na miR-3662-HBP1 yana daidaita hanyar siginar Wnt/-catenin a cikin ƙwayoyin TNBC (Yi et al., 2022). Saboda bayyanarsa ta musamman ga ciwon daji, miR-3662 na iya zama wata manufa ta warkewa ga TNBC. Don haka, rage girman miR-3662 da CRISPR/Cas9 ke haifarwa na iya zama hanya mai kyau don haɓaka sabbin magunguna don maganin TNBC.
UBR5 wani nau'in nucleophosphoprotein ne mai nauyin 300 kDa wanda aka gano a matsayin babban mai kula da cutar tumorigenesis, metastasis, da kuma martanin garkuwar jiki a cikin nau'ikan cututtukan daji daban-daban (Shearer et al., 2015; Fu et al., 2023). An ruwaito cewa UBR5 yana da ƙarfi sosai a cikin samfuran TNBC kuma yana ƙarfafa aikin ERα ta hanyar haifar da yaduwa ta hanyar aikin ubiquitin ligase (Bolt et al., 2015). Nazarin jerin samfuran exome na manyan samfuran TNBC suma sun nuna ƙaruwar bayyanar UBR5, yana nuna rawar da yake takawa a cikin ci gaban TNBC. Bugu da ƙari, share UBR5 da CRISPR/Cas9 ke jagoranta ya nuna babban hana tasirin TNBC da girma a cikin samfuran gwaji na linzamin kwamfuta. Bugu da ƙari, haɗa UBR5 a cikin samfurin linzamin kwamfuta na daji ya dawo da cikakken aikinsa, yayin da ba a lura da wannan tasirin a cikin nau'ikan maye gurbi marasa aiki ba (Liao et al., 2017). Rashin UBR5 yana da alaƙa da ƙaruwar apoptosis, necrosis, da hana ci gaban ƙari a cikin TNBC saboda ƙarancin angiogenesis. Saboda asarar UBR5, yaduwar ƙari zuwa ga gabobin da ke nesa ya ragu, kuma UBR5 yana haifar da rashin daidaituwar EMT galibi ta hanyar rage bayyanar E-cadherin (Zhang da Weinberg, 2018). Kwanan nan, an nuna cewa UBR5 muhimmin abu ne a cikin kwafin PDL1 na IFN-γ da aka haifar a cikin TNBC saboda rashin ayyukan ubiquitination na E3. Binciken RNA na transcriptome ya nuna cewa UBR5 na iya samun tasirin tsari akan kwayoyin halitta da ke da alaƙa da hanyar IFN-γ kuma yana haɓaka transactivation na PDL1 ta hanyar ƙara matakan kunnawa na furotin kinase RNA (PKR) da masu canza siginar sa da masu kunna shi. transcription 1 (STAT1) da interferon regulatory factor 1 (IRF1). Duk da haka, haɗin gwiwar cirewar UBR5 da PD-L1 da CRISPR/Cas9 ke jagoranta yana da tasirin maganin haɗin gwiwa fiye da toshewa kaɗai, tare da babban tasiri akan yanayin ƙwayoyin cuta (Wu et al., 2022). Don haka, CRISPR/Cas9 na iya zama kayan aiki mai mahimmanci don hana aikin UBR5, ta haka yana hana metastasis na TNBC da haɓakar ƙari.
ROR1 wani nau'in furotin ne na transmembrane wanda ake nunawa yayin ci gaban ciwon daji da tayin kuma an gano shi a matsayin furotin oncofetal (Nicholas Borcherding, 2014). Halayyar mamayewar cututtukan daji daban-daban na ɗan adam yana da alaƙa da haɓaka ROR1. An sami sakamako mai kyau daga nazarin in vivo da in vitro wanda ya shafi mahaɗan magani da ke kai hari ga ROR1 (Chien et al., 2016). Matakan ROR1 mRNA a cikin biopsies na nama na ƙirji suma an danganta su da ciwon nono mai tsanani kamar basal-like breast (BL) da ƙaura zuwa wasu sassan jiki. Bugu da ƙari, an gano yawan bayyanar ROR1 a matsayin alamar hasashen ci gaban TNBC (Chien et al., 2016). Duk da haka, rufe ROR1 ta amfani da CRISPR/Cas9 don hana ci gaban TNBC da metastasis zai zama dabara mai tasiri.
Tsarin sialylation ya ƙunshi ƙara sialic acid zuwa glycoconjugates, wanda sialyltransferases (STs) ke haifarwa. ST8SIA1 na cikin dangin ST kuma yana taka muhimmiyar rawa wajen haifar da cututtuka daban-daban kamar cutar sankarar lymphocytic da ciwon daji na hanji (Chang et al., 2018). Binciken jerin RNA ya nuna cewa ST8SIA1 yana da matuƙar tasiri a cikin ƙirjin marasa lafiya na TNBC kuma yana da alaƙa mai kyau da maye gurbi a cikin kwayar halittar da ke rage ciwon daji p53, wanda zai iya ba da gudummawa ga cutar TNBC (Battula et al., 2017). Bugu da ƙari, ST8SIA1 yana da hannu a cikin metastasis da sake dawowar TNBC, yana nuna cewa yana taka muhimmiyar rawa a cikin faruwar da ci gaban TNBC. A cikin samfurin in vitro na TNBC, an nuna cewa ST8SIA1 da CRIPSR/Cas9 ke toshe ci gaba da metastasis (Battula et al., 2017). Wannan yana nuna cewa CRISPR/Cas9 na iya zama muhimmin kayan aiki don hana aikin oncogene na ST8SIA1 wajen magance TNBC.
NAT1 wani enzyme ne na rayuwa wanda ke haɓaka samuwar mahaɗan xenobiotic na mataki na II kuma yana bayyana a kusan dukkan kyallen jikin ɗan adam. NAT1 na iya amfani da cofactor folate don kai hari ga acetyl-CoA (acetyl-CoA) koda kuwa babu substrate na aromatic amine (Stepp et al., 2015; Laurieri et al., 2014). Bincike ya nuna cewa NAT1 yana daidaita aikin matrix metalloproteinase 9 (MMP9) a cikin samfuran ƙwayoyin cutar kansar nono kuma yana kare shi daga nau'in oxygen mai amsawa (ROS) yayin yunwar glucose (Wang et al., 2018). An nuna cewa goge NAT1 yana hana hadaddun pyruvate dehydrogenase, wanda ke haifar da rashin aiki na mitochondrial (Wang L. et al., 2019). Bugu da ƙari, wasu rahotanni daban-daban sun nuna cewa hana NAT1 ta amfani da ƙananan ƙwayoyin cuta da kuma rufe siRNA na iya rage mamayewa da yaduwar ƙwayoyin cutar kansar nono (Stepp et al., 2018). Kwanan nan, an yi amfani da CRISPR/Cas9 don danne NAT1 a cikin layin ƙwayoyin cutar kansar nono MDA-MB-231, wanda ke shafar metabolism na ƙwayoyin halitta dangane da matakin bayyanarsa. Wannan binciken ya kuma nuna cewa NAT-1 yana da mahimmanci ga ci gaba da metastasis na TNBC (Carlisle et al., 2020).
Ana tsara rikodin oncogenes ta hanyar super enhancers, transcription factors da cofactors (Hnisz et al., 2015). Bugu da ƙari, ana buƙatar ƙungiyar kinases masu dogaro da cyclin (CDKs), kamar CDK7, CDK8, CDK9, CDK12, da CDK13, don daidaita rubutun. Daga cikinsu, CDK7 yana da hannu a cikin phosphorylation na RNA polymerase II, wanda yake da matukar muhimmanci ga farawa da faɗaɗa rubutun oncogene a cikin pathogenesis na TNBC. A cikin wani bincike mai zurfi, share CDK7 ta CRISPR/Cas9 ya hana TNBC, yana nuna cewa pathogenesis na TNBC ya dogara da CDK7 (Wang Y. et al., 2015).
Nazarin ya nuna cewa maye gurbi a cikin MYC da RBP (protein mai ɗaure RNA) na iya haifar da apoptosis, yayin da maye gurbi ɗaya a cikin MYC ko RBP ba ya shafar ci gaban ƙwayoyin cutar kansa (Einstein et al., 2021). An tantance fiye da RBPs 1000 a cikin kwayar halittar ɗan adam ta amfani da ɗakin karatu na CRISPR/Cas9. Daga cikinsu, an gano RBPs 57 suna da mahimmanci ga ci gaban ƙwayoyin cutar kansa tare da matakan MYC masu girma (Wheeler et al., 2020). Bugu da ƙari, YTHDF2 yana da mahimmanci don kiyaye ci gaban ƙwayoyin TNBC kuma yana rage adadin rubuce-rubucen methylated yayin babban rubutu da fassara a cikin ƙwayoyin cutar kansa tare da babban bayyanar MYC. Bugu da ƙari, YTHDF2 ba shi da mahimmanci ga ƙwayoyin cutar kansa, waɗanda ba su dogara da matakan MYC masu girma don tsawaita rayuwar marasa lafiya na TNBC ba (Einstein et al., 2021), yana nuna cewa yana iya zama babban maƙasudin magani ga magunguna waɗanda za su iya shawo kan TNBC.
Sunadaran yatsar zinc (ZNFs) sun kai kimanin kashi 1% na jimlar kwayoyin halittar dan adam. Bincike ya nuna cewa ZNF na iya daidaita yaduwar kwayoyin halitta a cikin cututtuka daban-daban, kamar ciwon hanta, ciwon nono da ciwon hanji (Zhang W. et al., 2021; Li et al., 2017). An yi amfani da ɗakunan karatu da CRISPR knockout ta samar don tantance nau'ikan kwayoyin halittar da ke rage ciwon daji (TSGs) a cikin ƙwayoyin cutar kansar nono (Shalem et al., 2014). Tun daga lokacin, wani bincike na transcriptomic na ƙwayoyin cutar kansar nono da aka goge na CRISPR/Cas9 ZNF319 ya nuna cewa ZNF319 kwayar halittar da ke rage ciwon daji ce wadda ke rage yaduwar ciwon nono kuma saboda haka tana da hannu a cikin hanyoyin sigina daban-daban da sauran ayyukan halittu (Wang L. et al., 2022).
Ferroptosis wani nau'in mutuwar ƙwayoyin halitta ne da aka tsara wanda ya dogara da kasancewar ƙarfe. An san cewa ci gaban ferroptosis yana shafar kasancewar lipid peroxides. Bugu da ƙari, an ruwaito PKCβII yana nuna farkon lipid peroxidation, kuma ƙaruwar lipid peroxidation yana da alaƙa da ferroptosis. Binciken ɗakin karatu na CRISPR/Cas9-mediated kinase inhibitors ya nuna cewa PKCβII yana da hannu a cikin tsarin lipid peroxidation, wanda yake da mahimmanci ga ferroptosis a cikin ƙwayoyin MDA-MB-231 (Zhang et al., 2022). Saboda haka, wannan yana nuna cewa ƙwanƙwasa PKCβII ta CRISPR/Cas9 na iya zama abin da zai iya zama makasudin kwayar halittar hana ƙari don magance cututtukan da ke da alaƙa da ferroptosis (Zhang et al., 2022).
Ana kyautata zaton juriya ga magunguna ne ke haifar da kusan kashi 90% na mace-macen da ake samu a cikin masu fama da cutar kansa kuma yana ɗaya daga cikin manyan ƙalubalen da ake fuskanta a fannin maganin cutar kansa (Bukowski et al., 2020). Sakamakon ya nuna cewa isassun kwayoyin halitta da suka shafi kwararar magunguna, gyaran DNA, apoptosis da hanyoyin siginar ƙwayoyin halitta daban-daban suna da alaƙa da juriya ga magunguna (Haider et al., 2020). Daga cikinsu, an yi niyya ga kwayoyin halitta da dama ta hanyar kayan aikin CRISPR/Cas9 kuma sun nuna sakamako mai kyau wajen rage juriya ga magunguna da kuma ƙara ingancin magungunan hana cutar kansa (Vaghari-Tabari et al., 2022). Bugu da ƙari, ɗakunan karatu na tantance ƙwayoyin halitta na CRISPR/Cas9 masu inganci sun kasance suna da hannu wajen gyara aikin yuwuwar tasirin kwayoyin halitta masu juriya ga magunguna (Shalem et al., 2015). Don gano kwayoyin halittar juriyar paclitaxel, an yi amfani da jerin kwayoyin halittar RNA tare da tantancewar dakin karatu na sgRNA don gano kwayoyin halitta guda takwas da suka cancanta, gami da histone deacetylase 9 (HDAC9), wadanda ke da alaƙa da juriyar magani a cikin marasa lafiya da ke fama da cutar TNBC mai maimaitawa (B et al., 2020). ). A wani binciken kuma, an lura da karuwar bayyanar diserine/threonine da tyrosine protein kinase (DSTYK) yayin da marasa lafiya na TNBC da aka yi wa magani da magungunan hana ciwon daji ke rayuwa. Bugu da ƙari, raguwar DSTYK da CRISPR/Cas9 ya haifar ya inganta apoptosis na ƙwayoyin cutar kansa masu juriya ga magani a cikin vitro (ƙwayoyin SUM102PT da ƙwayoyin MDA-MB-468) da kuma samfurin TNBC a cikin vivo (Ogbu et al., 2021).
Duk da cewa TNBC tana da kunna hanyar MAPK mara kyau, tasirin asibiti na magungunan da aka yi niyya ga MEK ba shi da kyau. Wani binciken dakin gwaje-gwaje na CRISPR/Cas9 ya nuna cewa hana PSMG2 (proteome assembly chaperone 2) yana sa ƙwayoyin TNBC BT549 da MB468 su shiga cikin mai hana MEK AZD6244. Katsewar CRISPR/Cas9 na PSMG2 ya canza aikin proteasome na yau da kullun, wanda ke haifar da wargajewar PDPK1 ta hanyar autophagy, don haka yana ƙara inganta ƙwayoyin ƙari a cikin samfuran linzamin kwamfuta na TNBC waɗanda AZD6244 (MEK inhibitor) da MG132 (proteasome inhibitor) suka haifar. Don haka, ana iya ba da masu hana proteasome da MAP kinase (MEK) haɗin gwiwa don rage yaɗuwar ƙwayoyin ƙari (Wang X. et al., 2022).
An kuma yi amfani da CRISPR/Cas9 don tantance asarar aikin kwayar halitta wanda ke haifar da juriya ga TNBC (Shu et al., 2020). Ge et al. sun bayyana tsarin juriya ga magunguna na ƙwayoyin cutar kansa da aka yi wa magani da JQ1, wani mai hana bromodomain na BET (BBDI), a cikin TNBC. Ta amfani da CRISPR/Cas9, sun gano cewa goge kwayar halittar rb1 ya sa TNBC ta zama mai juriya ga maganin hana ciwon daji JQ1. Saboda haka, aikin rb1 yana da mahimmanci a cikin martani ga magungunan JQ1 a cikin TNBC. Sun kuma ba da rahoton cewa paclitaxel shine mai hana CDK4/6 kinase/microtubule, kuma haɗuwarsa da BBDIs kamar JQ1 na iya samar da amsoshin warkewa masu kyau ga TNBC masu juriya ga magani (Ge et al., 2020).
An ruwaito cewa yanayin rubutun RNA mai tsayi wanda ba ya ƙunshe da lambar sirri (lncRNA) shine ke da alhakin juriya ga maganin neoadjuvant a cikin TNBC. Wannan binciken ya nuna cewa an bayyana rubuce-rubucen MALAT1 lncRNA guda biyar daban-daban a cikin TNBC. Bugu da ƙari, sharewar MALAT1 ta CRISPR/Cas9 ta hanyar CRISPR/Cas9 ya ƙara yawan hankalin ƙwayoyin TNBC BT-549 zuwa paclitaxel da doxorubicin, yana nuna yuwuwar rawa ga juriyar MALAT1 a cikin TNBC (Shaath et al., 2021).
Sauye-sauye a cikin BRCA1 (BRCA1m) ba su da bambanci kuma saboda haka suna da wahalar ganowa. Yin niyya ga PARP1 (poly(ADP-ribose) polymerase), abokin hulɗa mai kisa na roba na BRCA1, na iya ƙara yawan haɗarin kamuwa da cutar kansa ta TNBC. Ta amfani da CRISPR/Cas9, share PARP1 ya ƙara yawan haɗarin magungunan hana ciwon daji kamar doxorubicin, gemcitabine da docetaxel ga ƙwayoyin TNBC masu maye gurbin mBRCA1, yana nuna cewa PARP1 yana da hannu a cikin juriya ga magunguna a cikin TNBC (Vaghari-Tabari et al., 2022). . Sauye-sauye a cikin kwayoyin halittar masu rage ciwon daji BRCA1 ko BRCA2 an san suna ƙara yuwuwar kamuwa da ciwon daji a cikin mutane. Maganin waɗannan marasa lafiya yana buƙatar amfani da masu hana PARP. Bugu da ƙari, an nuna cewa ƙwanƙwasa sinadarin salvation factor DNPH1 ta amfani da CRISPR/Cas9 na iya cire gubar nucleotide 5-hydroxymethyldeoxyuridine (hmdU) monophosphate, ta haka ne ke haɓaka martanin ƙwayoyin da ba su da isasshen BRCA ga amsawar masu hana PARP (Fugger). et al., 2021). Don haka, masu hana CRISPR/Cas9 da PARP1 na iya zama muhimmiyar dabarar magani ga TNBC.
Kwayar halittar glycoprotein mai shiga jiki (P-gp) kwayar halitta ce mai juriya ga magunguna da yawa wadda gabaɗaya ake ƙara yawanta a kusan kashi 41% na dukkan TNBC (Sun et al., 2020). An gano kwararar magunguna da P-gp ke haifarwa a matsayin babban mai daidaita juriya ga magunguna a cikin ciwon nono. Masu hana P-gp sun nuna ƙaruwar jin daɗin magungunan hana ciwon daji a cikin ciwon nono (Famta et al., 2021). Don haka, cirewa ko rage masu hana P-gp da P-gp da CRISPR/Cas9 ke haifarwa na iya zama hanyoyi masu mahimmanci don shawo kan juriya ga magunguna a cikin TNBC.
An san cewa mai jigilar kaset ɗin ATP mai ɗaurewa G2 (ABCG2) yana haifar da juriya ga magunguna a cikin TNBC (Palasuberniam et al., 2015), kodayake a halin yanzu babu rahotanni kan alaƙar da ke tsakanin CRISPR/Cas9 da rufewar ABCG2 da kuma rashin kunna kwayar halittar tumor. PTEN na iya haɓaka kunna ABCG2 (Palasuberniam et al., 2015), (Deepak Singh et al., 2021). Saboda haka, amfani da CRISP/Ca9 don cire ABCG2 da haɗa shi da mai hana ABCG2 na iya zama magani mai dacewa don shawo kan juriya ga magunguna a cikin TNBC. Tebur na 2 ya ambaci CRISPR/Cas9 wanda ke nufin duk kwayoyin halittar juriya.
Tebur na 2. CRISPR/Cas9 yana kai hari ga nau'ikan kwayoyin halittar da ke jure wa TNBC don wayar da kan ƙwayoyin halitta ga magungunan hana ciwon daji.
Dakunan karatu na CRISPR/Cas9 kayan aiki ne masu yuwuwa don tantance maye gurbi na kwayoyin halitta da ke da alaƙa da cutar kansa (Chan et al., 2022). Wannan hanyar tantancewa ta ƙunshi matakai huɗu kamar (a) gina ɗakin karatu, (b) transduction na lentiviral, (c) tantancewar phenotypic, da (d) nazarin kwayoyin halitta da aka yi niyya. Kodayake TNBC tana da kunnawa mara kyau na hanyar MAPK, hasashen asibiti na maganin da aka yi niyya na MEK ga marasa lafiya na TNBC waɗanda ke ɗauke da maye gurbi a cikin kwayoyin halittar da ke hana ƙari kamar PTEN, RB1, da TP53 ba shi da kyau sosai.
Bugu da ƙari, wani gwajin ƙwayoyin halitta ta amfani da CRISPR/Cas9 ya gwada ƙwayar halittar da ta fi ƙarfi da zaɓi, dehydrocatrol, wadda take da yawa a cikin abubuwan da aka samo daga fure da ganyen Guatemala, don fahimtar tasirin dehydrocatrol akan MDA-MB-231. Hanyoyin da ke tattare da gubar ƙwayoyin halitta. Halayen ƙashi na ƙwayoyin TNBC. Wannan gwajin da aka yi bisa CRISPR/Cas9 ya kuma bayyana cewa HSD17B11, kwayar halittar da ke ɗauke da nau'in 17β-hydroxysteroid dehydrogenase 11, tana bayyana sosai a cikin ƙwayoyin MDA-MB-231 kuma tana haifar da dehydrofalcarinol na musamman ga ƙwayoyin MDA-MB-231 (Grant et. al., 2020). Don haka, wannan yana nuna cewa gwajin kwayoyin halitta na CRISPR/Cas9 yana da babban yuwuwar gano hanyoyin da ke ƙarƙashin kaddarorin hana ciwon daji na mahaɗan halitta masu yuwuwa.
Bugu da ƙari, an yi nazarin raunin da TNBC ke da shi ga ciwon daji ta amfani da allon CRISPR/Cas9 mai cikakken tsari, wanda ya nuna alaƙa tsakanin hanyoyin hana ciwon daji da hanyoyin hana ciwon daji. An ruwaito cewa manyan sassan hanyoyin mTOR da Hippo suna taka muhimmiyar rawa a cikin daidaita ciwon daji a cikin TNBC. Bugu da ƙari, bincike ya nuna cewa hana mTORC1/2 da YAP oncoprotein na maganin gargajiya yana hana cutar TNBC ta hanyar amfani da samfurin haɗin gwiwar magunguna-matrix na in vitro da kuma xenografts daga marasa lafiya a cikin vivo. Bugu da ƙari, hana mTORC1/2 da Torin-1 ke shiga tsakani yana haɓaka macropinocytosis, yayin da hana YAP da verteporfin ke haifar da mutuwar ƙwayoyin TNBC. Idan aka haɗa su, waɗannan sakamakon suna nuna ƙarfi da ƙarfi na gwajin CRISPR na genome-wide a cikin vivo don gano sabbin magunguna masu tasiri ga TNBC (Dai et al., 2021).
Rashin daidaita tsarin garkuwar jiki muhimmin abu ne a cikin cutar kansa. Kwayoyin cutar kansa suna guje wa kawar da garkuwar jiki ta hanyar kauce wa hanyoyin kariya, gami da tsoma baki ga ayyukan ƙwayoyin garkuwar jiki a cikin yanayin ƙwayoyin cuta da kuma lalata tsarin garkuwar jiki. Saboda haka, haɓaka ingantaccen tsarin garkuwar jiki na iya zama babbar hanyar yaƙi da ciwace-ciwacen. Amfani da gyaran kwayoyin halitta bisa tushen CRISPR/Cas9 yana magance matsaloli da dama da suka shafi rashin aikin garkuwar jiki daga ra'ayoyi daban-daban. An yi amfani da CRISPR/Cas9 don inganta garkuwar jiki daga ciwon nono ta hanyoyi masu zuwa (Hoto na 3).
Hoto na 3. Maganin rigakafi na CRISPR/Cas9 yana kai hari ga ƙwayoyin TNBC. Asarar CDK5 da kuma rage PDL1 da CD155 suna ƙara ƙarfin garkuwar jiki. Haka nan, asarar A2AR da kuma rage TAAs kamar HER2, mucin 1 da TEM8 sun ƙara ingancin ƙwayoyin CAR-T wajen kashe ƙwayoyin cutar kansa. Binciken ƙwayoyin T da CRISPR/Cas9 ke jagoranta ya nuna cewa katsewar kinase na p38 yana ƙara ƙarfin aikin hana ƙari na ƙwayoyin T. Kwayoyin T da CRISPR/Cas9 ya gyara suna ƙara yawan bayyanar TCR (mai karɓar ƙwayoyin T), wanda ke haifar da ƙwayoyin T suna da aikin hana ƙari.
Manufar maganin rigakafi ita ce a ƙarfafa tsarin garkuwar jiki don kai hari ga ƙwayoyin cutar kansa. Yawan fitar da sunadaran binciken garkuwar jiki yawanci yana hana halayen garkuwar jiki amma yana iya taimakawa cutar kansa ta guji su (Topalian et al., 2015). Waɗannan sunadaran suna hana halayen garkuwar jiki ta hanyar ɗaurewa ga masu karɓa a saman ƙwayoyin rigakafi. Sunadaran binciken da yawa (kamar CD155 da PD-L1) suna kai hari ga mai karɓar PD-1 akan ƙwayoyin rigakafi kuma ana bayyana su a cikin ciwon nono, musamman TNBC (Li Y.-C. et al., 2020). Rushewar PD-L1 ko mai karɓar sa ta amfani da CRISPR/Cas9 na iya ƙarfafa tsarin garkuwar jiki don kai hari ga ƙari na TNBC (Yahata et al., 2019). Bugu da ƙari, an nuna cewa raguwar bayyanar PD-L1 ta hanyar share CDK5 da CRISPR/Cas9 ke jagoranta yana hana haɓakar ƙari a cikin vitro da in vitro (Deng et al., 2020). Tsarin hana ci gaban ciwon nono a cikin in vitro da in vivo yana nuna cewa raguwar CD155 da shRNA ke haifarwa na iya yin tasiri ga ciwon nono (Gao et al., 2018).
Kwayoyin CAR T suna bayyana CARs waɗanda ke gane antigens masu alaƙa da ƙari (TAAs) kuma suna iya amfani da rage sunadaran dubawa don haɓaka ayyukansu (Li C. et al., 2020). Akwai hanyoyi daban-daban da za a iya bi don maganin ƙwayoyin CAR T a cikin ciwon nono, gami da TAAs da yawa kamar HER2, mucin1, da TEM8 (Bajgain et al., 2018). Akwai shaida cewa ƙwayoyin CAR T da ke kai hari ga mesothelin (wanda aka fi bayyanawa a cikin ƙwayoyin TNBC BT-459) sun fi tasiri a kan ciwon daji lokacin da aka kawar da PD-1 ta amfani da CRISPR/Cas9 (Hu et al., 2019). Duk da haka, ware ƙwayoyin T daga marasa lafiya sannan a gyara su ex vivo tsari ne mai ɗaukar lokaci mai tsawo. Kodayake ƙwayoyin T na duniya na iya rage buƙatun warewa, ya kamata a cire ƙwayoyin T masu bayarwa waɗanda ke bayyana antigen na leukocyte na ɗan adam (HLA) aji na I da mai karɓar ƙwayoyin T (TCR) don hana rashin daidaituwar graft-versus-host (Ren et al., 2017). , 2017a). Ta amfani da CRISPR/Cas9, HDR na iya kawar da TCR da HLA a lokaci guda kuma ya kawar da kwayar halittar da ke ɗauke da CAR. Bincike ya nuna cewa ana iya amfani da CRISPR/Cas9 don shigar da CARs masu hana CD19 cikin wurin TCR, ta haka ne za a samar da CARs cikin inganci ba tare da rage ƙwayoyin T ba (Dimitri et al., 2022). An haɓaka fasahar Multiplex don samar da ƙwayoyin CAR T na allogeneic ta hanyar kawar da TCR, beta-2-microglobulin (B2M), subunit na HLA-I da sauran sunadaran kamar PD-1 da CTLA-4, waɗanda ke iya samun manyan kaddarorin hana ciwon daji. akan TNBC (Eyquem et al., 2017; Liu et al., 2017; Ren et al., 2017b; Dimitri et al., 2022).
Amfani da CRISPR/Cas9 zai iya inganta ingancin ƙwayoyin CAR-T. An san Adenosine yana da kaddarorin rage ƙarfin garkuwar jiki kuma yana iya rage garkuwar jiki ta hanyar toshe aikin ƙwayoyin T da kunna masu karɓar adenosine A2A (A2AR) (Vigano et al., 2019). Rufe A2AR ta amfani da CRISPR/Cas9 ya inganta tasirin ƙwayoyin CAR-T a cikin jiki sosai (Giuffrida et al., 2021). An san ƙwayoyin T suna nuna halaye daban-daban na phenotypic kamar faɗaɗa ƙwayoyin halitta, bambance-bambance, damuwa ta oxidative da damuwa ta genomic. Binciken ƙwayoyin T na tushen CRISPR/Cas9 ya nuna katsewar kinases masu karɓar ƙwayoyin T guda 25 daban-daban. Daga cikinsu, an nuna cewa share p38 kinase yana haɓaka aikin hana ƙari na ƙwayoyin T, yana nuna cewa p38 kinase babban mai kula da tsarin ƙwayoyin CAR-T ne (Gurusamy et al., 2020).
Ana iya canza ƙwayoyin T ta hanyar amfani da CRISPR/Cas9 don samar da TCRs masu bayyana sosai. Canja wurin ƙwayoyin T da aka gyara ta hanyar halitta zuwa ga marasa lafiya ya nuna ƙarfin aikin hana ciwon daji fiye da ƙwayoyin T na ciki. Saboda haka, TCRs na ciki na iya yin gogayya da TCRs da aka canza ta hanyar halitta a cikin marasa lafiya, wanda zai iya shafar yuwuwar maganin rigakafi na ciwon daji. Don shawo kan wannan matsalar, amfani da CRISPR/Cas9 don cire TCR-β na ciki a cikin ƙwayoyin da aka karɓa sannan daga baya a canja wurin ƙwayoyin TCR-β zuwa ga marasa lafiya na ciwon daji na iya nuna mafi kyawun martanin rigakafi na ciwon daji ba tare da buƙatar gasar jima'i ta TCR ta ciki ba (Fan et al. 2018). Don haka, ban da ƙwayoyin T da aka gyara ta CRISPR, TCRs sun fi saurin kamuwa da ƙwayoyin T na ciki fiye da ƙwayoyin T da aka canza ta TCR. Bugu da ƙari, a cikin cututtukan leukemia daban-daban, ƙwayoyin T da aka canza ta hanyar γδ TCR+ CRISPR suna nuna mafi girman bayyanar ƙwayoyin T CD4+ da CD8+ fiye da canja wurin TCR na yau da kullun (Legut et al., 2018). Saboda haka, ƙwayoyin T da aka gyara da CRISPR/Cas9 suka samar na iya zama ingantacciyar hanyar rigakafi don shawo kan TNBC.
Integrins ƙwayoyin mannewa ne na tantanin halitta waɗanda ke cikin ƙwayoyin halitta kuma suna haɓaka ɗaure ƙwayoyin halitta zuwa matrix na extracellular (ECM) (Hamidi da Ivaska, 2018). Rashin daidaita integrins yana da alaƙa da ci gaban ciwon daji da ƙaura ta hanyar canza matrix na extracellular, wanda ke haifar da rayuwar ƙwayoyin cutar kansa a cikin zagayawar jini (Hamidi da Ivaska, 2018). Rushewar integrin mai shiga tsakani na CRISPR/Cas9 yana rage ci gaban ƙari, metastasis, da mamayewa a cikin TNBC. An ruwaito cewa Rushewar integrin a5 (ITGA5) yana rage ƙaura da ci gaban ƙwayoyin halitta a cikin wasu cututtukan daji kamar ciwon huhu (Ju et al., 2017), yana nuna cewa integrin a5 na iya zama babban abin da ke haifar da TNBC.
Samar da beraye masu ƙwanƙwasa ta amfani da hanyoyin ƙwayoyin halitta na gargajiya na tayin haihuwa (ES) tsari ne mai ɗaukar lokaci, mai ɗaukar lokaci, kuma mara inganci. Yana ɗaukar watanni da shekaru don kai hari ga ƙwayoyin ES ta hanyar haɗakar juna, haifar da beraye masu chimeric, sannan a haɗa su da beraye masu heterozygous don samar da 'ya'ya masu homozygous. Ana buƙatar haɗa beraye masu rikitarwa don ƙirƙirar beraye masu maye gurbi iri-iri. Duk da haka, matsaloli da yawa sun taso lokacin amfani da beraye da aka samar ta hanyar cire ƙwayoyin ES ta amfani da CRISPR/Cas9 ko ta hanyar allurar ƙwayoyin CRISPR/Cas9 a cikin ƙwai da aka yi da takin ta hanyar sel ɗaya. Misali, gabatar da canje-canje sau da yawa yana faruwa a biallelic loci kuma ba takamaiman kwayar halitta ba ne. Kwanan nan an ruwaito cewa ƙwayoyin ES da ke amfani da fasahar CRISPR/Cas9 za su iya saka maye gurbi na biallelic a cikin har zuwa kwayoyin halitta 5 (Nishizono et al., 2021). Don wannan dalili, an canza Cas9 mRNA da gRNAs guda biyar na takamaiman kwayar halitta zuwa ƙwayoyin ES a lokaci guda. Wannan yana nuna alƙawarin da ingancin wannan hanya, kodayake waɗannan maye gurbi na iya faruwa ne lokacin da aka haɓaka layukan kafa don samar da beraye masu bugun jini sau huɗu. Binciken ya kuma ba da rahoton wani abin mamaki: Ba a buƙatar ƙwayoyin ES su sake ƙirƙirar beraye da aka gyara kwayoyin halitta ba. Madadin haka, don cire takamaiman samfuran kwayoyin halitta, an allurar Cas9 mRNA da gRNA a cikin tayin matakin tantanin halitta ɗaya. Sakamakon haka, an ƙirƙiri layukan kafa masu bugun jini waɗanda za a iya amfani da su a ka'ida don nazarin sakamakon goge kwayoyin halitta a cikin beraye (Qin et al., 2016). Don haka, CRISPR/Cas9 yana ba da damar ƙirƙirar beraye masu canzawa don magance TNBC akan farashi mai rahusa fiye da injiniyan kwayoyin halitta na gargajiya. Ta amfani da tsarin CRISPR/Cas, an ƙirƙiri sabon samfurin linzamin kwamfuta wanda zai iya gabatar da maye gurbi a cikin kwayar halitta ɗaya ko fiye a cikin TNBC. Dangane da wannan ra'ayi, ana iya haɓaka samfuran dabbobi na TNBC ta amfani da sharewar BRCA1 da p53 ta CRISPR/Cas9, wanda ke haifar da asarar gyaran HR, rashin kwanciyar hankali na kwayoyin halitta, da kuma yanayin maye gurbi (Annunziato et al., 2020).
A halin yanzu, ana amfani da hanyoyi daban-daban kamar mammography, magnetic resonance imaging (MRI) da ultrasound don gano TNBC. Duk da haka, waɗannan hanyoyin suna da wasu iyakoki. Ana amfani da mammography don gano ƙwayar nono ta gida maimakon metastases, inda ƙwayoyin cutar kansa suka ƙaura zuwa wasu gabobin jiki. Ultrasonography ba hanya ce mai inganci don gano TNBC ba (Chen da Lee-Felker, 2023). MRI yana da mafi girman amsawa fiye da duban dan tayi da mammography, amma daidaiton ganewar sa yana da iyaka (Sha da Chen, 2022). Biopsy na nama hanya ce mai haɗari don gano ƙwayoyin cutar kansa. Koyaya, a wasu lokuta, biopsy na iya rasa nama mai ciwon daji idan allurar ta ɓace daga yankin da ake sha'awa. Bugu da ƙari, yana da tsada sosai kuma yana iya zama mai rauni ga majiyyaci. TNBC nau'in ciwon daji ne daban-daban, don haka biopsy bazai samar da isasshen bayani game da nau'in ciwon daji ba. Saboda haka, ana buƙatar sabuwar hanyar da za a iya dogara da ita don gano TNBC cikin gaggawa. CRISPR/Cas9 na iya zama madadin mai matuƙar mahimmanci kuma mai ƙarancin mamayewa don gano cutar kansar nono. Don haka, ana iya amfani da dabarun da suka dogara da CRISPR/Cas9 don inganta hanyoyin PCR don gano cutar TNBC. Da farko, ana amfani da sunadaran Cas9 da cpf1 na tsarin CRISPR don cire DNA mara takamaiman, sannan waɗannan sunadaran guda biyu (Cas9 da cpf1) zasu iya gane jerin PAM kafin su haɗu da DNA da aka yi niyya (Deepak Singh et al., 2021). . Don haka, PCR na iya gano maye gurbi da ke da alaƙa da ci gaban cutar kansa. Nazari da yawa sun ɗauki wannan dabarar da ta dogara da CRISPR don gano maye gurbi daban-daban a cikin cututtukan daji daban-daban (Safari et al., 2019). Don haka, hanyoyin PCR da suka dogara da CRISPR na iya rage dogaro da ganewar TNBC akan hanyoyin mamayewa kamar immunohistochemistry na tushen biopsy.
An kuma nuna canje-canje a cikin kwayoyin halitta a cikin ƙa'idar karɓar hormones a cikin TNBC (Chen da Russo, 2009). Dabaru na PCR na tushen CRISPR/Cas9 ta amfani da ƙananan ra'ayoyi don gano alamun kulawa na iya sa ido kan waɗannan sauye-sauye yadda ya kamata (Hajian et al., 2019). Hakanan zai iya ba wa masu samar da kiwon lafiya bayanai masu dacewa game da takamaiman sauye-sauye a cikin marasa lafiya na TNBC, wanda zai iya taimakawa wajen inganta hanyar maganin su. Duk da haka, wannan dabarar da aka haɗa tana da iyakoki. Saboda haka, ana buƙatar ƙoƙari mai yawa na bincike kafin a iya amfani da wannan hanyar CRISPR/Cas-PCR a cikin kiwon lafiya don gano TNBC (Yang et al., 2019).