CRISPR) yog ib txoj kev kho genome tiam thib peb: CRISPR tau hloov pauv lub ntiaj teb

Clustered regularly interspaced short palindromic repeats (CRISPR) yog ib txoj kev kho genome tiam peb uas tau hloov pauv lub ntiaj teb nrog nws cov txiaj ntsig zoo. Nws tau siv los kho ntau yam kab mob thiab kev kis kab mob. Ntau yam kab mob thiab lwm yam prokaryotes (xws li archaea) kuj muaj CRISPR/Cas9 systems los tiv thaiv phages. Nws tau tshaj tawm tias CRISPR/Cas9-based strategy tuaj yeem tiv thaiv kev loj hlob thiab kev nce qib ntawm triple negative mis mob cancer (TNBC) los ntawm kev tsom mus rau cov noob tiv thaiv uas hloov pauv tau, kev sau ntawv, thiab kev tswj hwm epigenetic. Cov kev kho mob no yuav pab daws cov teeb meem nyuaj xws li kev tiv thaiv tshuaj pom txawm tias hauv TNBC. Tam sim no, ntau txoj kev siv los xa CRISPR/Cas9 mus rau lub hom phiaj hlwb, xws li lub cev (microinjection, electroporation thiab hydrodynamic modes), viral (adeno-associated virus thiab lentivirus) thiab non-viral (liposomes thiab lipid nanoparticles). )-particles). Txawm hais tias muaj ntau yam qauv tau tsim los kawm txog cov ua rau muaj TNBC, qhov tsis muaj cov txheej txheem rhiab heev thiab tsom mus rau kev xa cov cuab yeej kho genome hauv vivo txwv lawv cov kev siv kho mob. Hauv kev xaus, qhov kev tshuaj xyuas no tshuaj xyuas tag nrho cov kev nce qib, cov teeb meem, cov kev txwv, thiab kev cia siab ntawm CRISPR / Cas9 kev kho mob rau TNBC raws li cov pov thawj uas twb muaj lawm. Peb kuj tseem hais txog yuav ua li cas kev sib xyaw ua ke ntawm kev txawj ntse dag thiab kev kawm tshuab tuaj yeem txhim kho CRISPR / Cas9 cov tswv yim hauv kev kho mob ntawm TNBC.
Kab mob qog noj ntshav yog tus yam ntxwv los ntawm kev faib cov cell tsis tswj tau, kev cuam tshuam ntawm cov chaw kuaj xyuas lub voj voog ntawm cell, thiab kev hloov pauv hauv cov noob caj noob ces tiv thaiv qog noj ntshav (TSGs) (Matthews et al., 2022). Ntawm ntau hom kab mob qog noj ntshav sib txawv, kab mob qog noj ntshav mis yog hom kab mob qog noj ntshav feem ntau hauv cov poj niam thiab muaj tus nqi tuag ntau thoob ntiaj teb (Waks thiab Winer, 2019). Kab mob qog noj ntshav mis yog ib hom kab mob sib txawv nrog ntau yam ntxwv, xws li cov yam ntxwv ntawm cov ntaub ntawv keeb kwm thiab kab mob, kev nthuav qhia thiab tus cwj pwm hauv kev kho mob, thiab kev teb rau kev kho mob (Weigelt et al., 2010). Kev faib tawm kab mob qog noj ntshav mis muab kev nkag siab meej rau kev kuaj mob qog noj ntshav mis thiab kev kwv yees txog qog noj ntshav. Kev siv cov cim qhia thiab cov yam ntxwv ntawm kev kho mob yog qhov tseem ceeb rau kev faib tawm kab mob qog noj ntshav mis (Tsang thiab Tse, 2019). Kev kwv yees thiab kev teb rau kev kho mob qog noj ntshav mis yog cuam tshuam los ntawm ntau yam, suav nrog qhov muaj estrogen receptor (ER), progesterone receptor (PR), tib neeg epidermal growth factor receptor 2 (HER2/neu), thiab qib histological, hom qog noj ntshav, thiab qib. qhov loj thiab cov qog ntshav hauv lub mis (Al-Tubaiti, 2020). Muaj tsib hom qog ntshav hauv lub mis uas tau txheeb xyuas, suav nrog luminal A, luminal B, HER2-enriched, basal-like, thiab claudin-low (Prat et al., 2015). TNBC yog hom qog ntshav hauv lub mis uas tsis qhia txog txhua yam ER, PR, thiab HER2 (Yin et al., 2020). Cov yam ntxwv pathological no cuam tshuam nrog TNBC, lees paub nws txoj kev loj hlob sai thiab muaj kev kub ntxhov ntau dua li lwm hom qog ntshav hauv lub mis (Feng et al., 2018). Tsis tas li ntawd, Peru et al (2000) siv cov thev naus laus zis microarray los faib cov qog ntshav hauv lub mis thiab txheeb xyuas tsib hom qog ntshav hauv lub mis (Cadenas, 2012). Mob qog ntshav hauv lub mis zoo li basal yog hom qog ntshav hauv lub mis uas muaj peb hom phenotype tsis zoo thiab cuam tshuam nrog kev loj hlob sai. Yuav tsum nco ntsoov tias txhua yam mob qog noj ntshav hauv lub mis uas zoo li basal feem ntau raug kuaj mob tsis raug raws li TNBC, tab sis tsuas yog 77% yog TNBC; Qhov sib piv, 71–91% ntawm TNBC yog basal-zoo li, qhia tias ob hom mob qog noj ntshav hauv lub mis no sib tshooj thiab sawv cev rau kev faib tawm sib txawv (Wang D.-Y. et al., 2019). Qhov no tsim kom muaj qhov xav tau los piav qhia txog qhov sib txawv ntawm TNBC los piav qhia txog kev kwv yees thiab txheeb xyuas cov lus teb rau kev kho mob tam sim no thiab yav tom ntej. Tsis tas li ntawd, TNBC suav txog 15–20% ntawm tag nrho cov mob qog noj ntshav hauv lub mis thiab feem ntau tshwm sim rau cov poj niam hnub nyoog qis dua 50 xyoo. Kev hloov pauv BRCA1 lossis BRCA2 tau tshaj tawm hauv kwv yees li 20% ntawm cov mob TNBC (Xie et al., 2017; Tzikas et al., 2017). , 2020). Cov kev tshawb fawb kuj tau qhia tias TNBC muaj ib puag ncig tiv thaiv kab mob sib txawv uas suav nrog cov qib siab ntawm cov vascular endothelial growth factors, tumor-associated macrophages (TAMs), tumor-infiltrating lymphocytes (TILs), thiab lwm yam molecules uas koom nrog kev loj hlob thiab kev tsiv teb tsaws chaw ntawm tumor. Yog li ntawd, kev nkag siab txog ib puag ncig ntawm TNBC yog qhov tseem ceeb rau nws qhov kev kwv yees thiab kev kho mob (Fan thiab He, 2022).
Txhawm rau ntsuam xyuas qhov kev kwv yees ntawm TNBC thiab xyuas kom meej tias muaj kev kho mob zoo, kev kuaj mob kom raug, feem ntau yog raws li immunohistochemistry (IHC) los ntes ER, PR thiab HER2, thiab mammography los ntes cov neoplasms mis, yog qhov tseem ceeb. Txawm li cas los xij, mammography tsis tuaj yeem piav qhia txog cov yam ntxwv intratumoral xws li necrosis thiab fibrosis (Deepak Singh et al., 2021). Txij li thaum kev kwv yees tsis zoo thiab kev kuaj mob tiv thaiv cov kws kho mob los ntawm kev sau cov tshuaj kom raug (Chaudhary, 2020), ntau txoj hauv kev siv los txhim kho kev saib xyuas rau cov neeg mob uas muaj TNBC. Tam sim no, ob hom tshuaj, doxorubicin thiab cyclophosphamide, siv rau cov neeg mob uas muaj TNBC thiab tau pom cov txiaj ntsig zoo. Tsis tas li ntawd, lwm cov tshuaj platinum siv, xws li carboplatin thiab cisplatin (Sikov et al., 2015). Tsis tas li ntawd, PARP inhibitors xws li Olaparib, Velaparib, thiab PF-01367338 tau siv ua cov tshuaj chemotherapy rau kev kho mob ntawm TNBC (Ishino et al., 2018). Txij li thaum Wnt/b-Catenin, NOTCH, thiab Hedgehog signaling pathways tau tshaj tawm tias koom nrog rau qhov tshwm sim thiab kev nce qib ntawm TNBC, kev tsom cov tshuaj rau cov txoj hauv kev no yuav yog ib qho tswv yim tseem ceeb (Aysola et al., 2013). Txawm hais tias kev phais, kev kho mob hluav taws xob thiab chemotherapy tseem yog qhov tseem ceeb ntawm kev kho mob rau TNBC niaj hnub no, kev nce qib tseem ceeb tau ua tiav hauv kev tsim cov kev kho mob tshiab suav nrog kev kho mob tsom, kev tiv thaiv kab mob, ntau yam CRISPR-cuam tshuam cov cuab yeej kho cov noob caj noob ces xws li Cas9n, dCas9., CRISPR/Cas12, kev kho thawj zaug, thiab CRISPR/Cas9-targeted gene therapy. Ntawm no peb yuav tsom mus rau ntau yam CRISPR-cuam tshuam cov cuab yeej kho cov noob caj noob ces siv hauv kev kho mob ntawm TNBC. Ntawm lawv, CRISPR/Cas9 tau txais kev saib xyuas dav dav.
Cas9n, tseem hu ua Cas9 nickase, yog ib qho kev hloov pauv caj ces ntawm Cas9 protein uas tau los ntawm CRISPR/Cas9 genome editing system (Gupta et al., 2019). Hauv nws daim ntawv qub, Cas9 protein muaj ob lub nuclease domains: RuvC thiab HNH, lub luag haujlwm tseem ceeb yog kev txiav ob txoj DNA strands. Txawm li cas los xij, hauv Cas9n, ib qho ntawm cov nuclease domains, HNH, tau hloov pauv caj ces thiab tsis ua haujlwm. Yog li ntawd, HNH domain ntawm Cas9n tseem tsis ua haujlwm. Tsuas yog RuvC domain tseem ua haujlwm, tso cai rau Cas9n txiav lossis tsim kev tawg ntawm ib txoj DNA strands (Trevino thiab Zhang, 2014). Piv nrog Cas9, Cas9n tuaj yeem txo cov teebmeem tawm ntawm lub hom phiaj thiab txhim kho cov txheej txheem kho cell kom raug. Cas9n tuaj yeem siv los daws ntau yam teeb meem, xws li tsim kev tawg ntawm cov chaw tshwj xeeb hauv ob txoj DNA. Rau lub hom phiaj no, ob lub Cas9n molecule tau sib xyaw thiab siv ua ke (Yee, 2016). Mixed lineage kinase 3 (MLK3) yog ib qho protein kinase uas ua haujlwm los ntawm mitogen uas ua haujlwm ua tus tswj hwm tseem ceeb thaum lub sijhawm TNBC metastasis (Cronan et al., 2012).
MLK3 tuaj yeem ua kom muaj ntau txoj kev xa xov uas ua rau TNBC metastasis. Piv txwv li, txoj kev c-Jun N-terminal kinase (JNK) tswj kev txav ntawm lub cell thiab kev puas tsuaj ntawm cov extracellular matrix, thiab MLK3 ua kom txoj kev JNK ua haujlwm, yog li ua kom lub cell txav tau zoo dua thiab muab cov khoom tsis zoo (Rattanasinchai thiab Gallo, 2016). MLK3 kuj tswj EMT. Txhawm rau ua qhov no, nws ua kom cov yam ntxwv transcription downstream xws li Snail, Slug thiab Twist ua haujlwm. Cov yam ntxwv no txwv tsis pub qhia cov cim epithelial thiab txhawb kev qhia cov cim mesenchymal, ua rau muaj cov yam ntxwv metastatic (Casalino et al., 2023). MLK3 tau pom tias ua lub luag haujlwm hauv ECM remodeling thiab ua kom cov proteases xws li matrix metalloproteinases (MMPs) uas ua rau ECM puas tsuaj. Qhov no ua rau cov cell mob nkag mus thiab kis mus rau qhov chaw deb (Katari et al., 2019). Yog li, cov kev tshawb fawb yav dhau los tau qhia tias MLK3 ua lub luag haujlwm tseem ceeb hauv TNBC. Rattanasinchai thiab Gallo siv cov qauv TNBC los kawm txog lub luag haujlwm ntawm MLK3 thiab pom tias nws txhawb nqa kev loj hlob ntawm mob qog noj ntshav los ntawm cov kev taw qhia tshwj xeeb. Lawv siv CRISPR / Cas9n los hloov kho MLK3 thiab pom tias muaj kev txo qis hauv TNBC metastasis (Rattanasinchai thiab Gallo, 2016).
dCas9, feem ntau hu ua Cas9 tsis ua haujlwm, yog ib daim ntawv hloov kho ntawm cov protein Cas9. Tsis zoo li Cas9 uas ua haujlwm, dCas9 tsis muaj kev ua haujlwm endonuclease, ua rau nws tsis tuaj yeem ua rau DNA ob txoj hlua tawg. Yog li ntawd, dCas9 tuaj yeem siv los tsom mus rau thaj chaw tshwj xeeb ntawm lub genome yam tsis muaj kev hloov pauv lossis kev hloov kho rau cov kab ke DNA (Wang et al., 2016). Hauv dCas9, ob lub protein endonuclease, RuvC thiab HNH, raug ua haujlwm los ntawm kev tswj cov amino acid tseem ceeb. (Richter et al., 2016). Txawm hais tias nws tsis muaj kev ua haujlwm DNA cleavage, dCas9 ua ntau lub luag haujlwm tseem ceeb hauv kev tshawb fawb txog noob caj noob ces thiab biotechnology. Piv txwv li, nws tso cai rau kev pom ntawm thaj chaw tshwj xeeb ntawm lub genome, pab txhawb kev tswj hwm transcriptional, thiab txhawb kev hloov kho epigenetic (Brocken et al., 2018).
Tus yam ntxwv sau ntawv ZEB1 (zinc ntiv tes E-box khi homeobox 1) ua lub luag haujlwm tshwj xeeb thiab tseem ceeb hauv kev sib tham epithelial-mesenchymal hloov pauv (EMT), ib qho txheej txheem cellular uas tshwm sim thaum lub sijhawm embryonic kev loj hlob, kev kho cov ntaub so ntswg, thiab kev nce qib ntawm mob qog noj ntshav. Nws cuam tshuam nrog kev hloov pauv ntawm cov hlwb epithelial mus rau hauv cov hlwb mesenchymal, ua rau muaj kev hloov pauv hauv cov qauv cell, kev txav mus los, kev nkag mus rau hauv lub cev, thiab lwm yam. (Wu et al., 2020). ZEB1 inhibits ntau yam epithelial markers, xws li E-cadherin thiab Occludin, uas yog lub luag haujlwm rau kev tswj hwm cell-cell adhesion thiab polarity ntawm epithelial cells hauv TNBC (Moreno-Bueno et al., 2008). Ntxiv rau, ZEB1 ua kom muaj qee cov cim xws li N-cadherin, vimentin thiab fibronectin (Konradi et al., 2014) thiab tseem tswj cov noob caj noob ces koom nrog hauv cytoskeletal remodeling xws li Rho GTPases thiab matrix metalloproteinases (MMPs) (Huang). et al., 2014). 2022), ntxiv rau, nws kuj cuam tshuam rau ntau txoj kev xa xov xws li kev hloov pauv kev loj hlob-β (TGF-β), Wnt signaling, thiab lwm yam, yog li txhawb kev nkag mus rau hauv qog thiab metastasis hauv TNBC (Chen et al., 2016). ). Ntau txoj kev tshawb fawb thiab cov pov thawj tshawb fawb qhia tias ZEB1 muaj peev xwm zoo ua tus neeg sawv cev muaj txiaj ntsig rau kev kuaj pom thiab kev kho mob ntawm TNBC. Hauv kev tshawb fawb tsis ntev los no, Waryah et al. siv tus qauv TNBC thiab ua tiav kev tshem tawm tag nrho ntawm ZEB1 los ntawm dCas9. Yog li, lawv tau pom qhov tshwj xeeb heev thiab yuav luag tag nrho kev txwv ntawm ZEB1 hauv cov xwm txheej hauv vivo (Waryah et al., 2023).
CRISPR/Cas12 yog ib lub cuab yeej kho cov noob caj noob ces hu ua CRISPR/Cpf1. Nws yog los ntawm CRISPR/Cas system, qhov twg lo lus Cas12 hais txog CRISPR-associated protein 12 (Bharathkumar et al., 2022), uas zoo ib yam li Cas9. Qhov txawv tsuas yog tias nws muaj cov protein Cas12. Piv nrog Cas9, Cas12 muaj qee qhov haujlwm tshwj xeeb xws li tsim cov kawg nplaum thaum lub sijhawm kho cov noob caj noob ces, thaum Cas9 tsim cov kawg blunt (Wang et al., 2021). Cov khoom no ntawm Cas12 pab txhawb rau nws cov thev naus laus zis tswj DNA tshwj xeeb. Raws li ib qho protein uas muaj peev xwm lees paub thiab txiav cov DNA lub hom phiaj, nws muaj qhov ua tau zoo heev, ua rau nws yog ib lub cuab yeej zoo rau ntau yam kev siv, suav nrog kev kho cov noob caj noob ces (Pickar-Oliver thiab Gersbach, 2019).
Zoo ib yam li lwm cov CRISPR variants, CRISPR/Cas12 kuj tseem muaj peev xwm khob tawm lossis ua kom cov noob caj noob ces hauv TNBC, tsom mus rau cov noob caj noob ces uas ua lub luag haujlwm tseem ceeb hauv nws txoj kev mob lossis kev teb rau kev kho mob (Yang thiab Zhang, 2023). Txhawm rau ua tiav cov txheej txheem no, ib daim ntawv qhia RNA (gRNA) tau tsim uas coj Cas12 mus rau lub hom phiaj noob caj noob ces. Thaum Cas12 khi rau nws lub hom phiaj, nws qhia txog ob txoj hlua tawg hauv DNA thiab ua kom cov txheej txheem kho DNA ua haujlwm. Thaum lub sijhawm kho, cov nucleotides tsis raug yuav raug suav nrog, ua rau muaj kev hloov pauv noob caj noob ces thiab poob haujlwm (Zhang et al., 2021a). Tsis tas li ntawd, ib qho version zoo dua ntawm Cas12 enzyme (hu ua dCas12) kuj tau siv los ua kom cov noob caj noob ces ua haujlwm. Los ntawm kev sib xyaw nrog nws nrog tus neeg ua haujlwm sau ntawv, nws muaj peev xwm ua rau qee cov noob caj noob ces, yog li ua kom lawv ua haujlwm. Cov thev naus laus zis no muaj peev xwm zoo hauv kev txhawb nqa kev ua haujlwm ntawm cov noob caj noob ces suppressor (Sultan et al., 2022).
Kev kho kom zoo tshaj plaws yog ib qho thev naus laus zis kho kom zoo tshaj plaws uas tau tsim tshiab thiab zoo heev. Nws muaj peev xwm hloov kho DNA ntawm ib yam khoom muaj sia kom raug heev (Chen thiab Liu, 2023). Kev kho kom zoo tshaj plaws yog ua tiav los ntawm kev koom ua ke ntawm ob yam khoom tseem ceeb: ib qho enzyme CRISPR/Cas9 uas tau hloov kho thiab reverse transcriptase. Lub luag haujlwm ntawm CRISPR/Cas9 enzyme yog xaiv cov chaw tseeb hauv genome, thaum reverse transcriptase pab hloov kho DNA ntawm qhov chaw uas xav tau (Hassan et al., 2021). Hauv lub tshuab kho kom zoo tshaj plaws, thawj kauj ruam yog kev tsim ib qho primer editing guide RNA (pegRNA) (Standage-Beier et al., 2021). Nws muaj cov kab ke thiab RNA template uas sib raug rau qhov chaw kho kom zoo tshaj plaws hauv DNA uas xav tau. Tom qab ntawd pegRNA raug coj mus rau hauv cov hlwb uas xav tau nrog rau primer editing nuclease (PE2). PE2 yog ib qho protein fusion uas muaj Cas9 enzyme, reverse transcriptase, thiab primer editing adapter (Martín-Alonso et al., 2021). Sab hauv lub cell, pegRNA thiab PE2 complexes nrhiav cov DNA tshwj xeeb uas lawv xav hloov kho. Lub Cas9 enzyme txiav DNA thiab tsim ib daim qauv uas muaj cov hlua ib leeg (Choi et al., 2022). Reverse transcriptase siv daim qauv no los npaj DNA rau kev hloov kho. Nrog rau kev theej DNA, cov lus qhia rau kev hloov kho RNA template kuj suav nrog. Thaum kawg, cov hlua DNA tshiab tsim tau siv ua daim qauv los kho qhov tawg hauv DNA, ua rau muaj cov kab ke DNA hloov pauv uas muaj qhov hloov kho xav tau (Ochoa-Sanchez et al., 2021). Cov txheej txheem hloov kho primer tuaj yeem ua rau muaj kev hloov pauv dav hauv cov noob caj noob ces. Nws tuaj yeem tsom mus rau cov ntsiab lus hloov pauv, kev ntxig, kev rho tawm, thiab txawm tias cov noob caj noob ces hloov pauv (Anzalone et al., 2019; Chen thiab Liu, 2023). Kev hloov kho prime muaj ntau qhov zoo dua li cov thev naus laus zis hloov kho genome yav dhau los. Cov no suav nrog kev raug ntau dua, txo cov teebmeem tawm ntawm lub hom phiaj, thiab lub peev xwm hloov kho DNA yam tsis tas yuav cia siab rau DNA ob chav-strand tawg (Anzalone et al., 2020).
Cov thev naus laus zis CRISPR/Cas9 tau tsim thawj zaug los tiv thaiv cov kab mob los ntawm kev hloov pauv plasmid thiab phage thiab tom qab ntawd tau rov siv dua ua lub cuab yeej siv DNA raws li RNA rau kev kho genome (Jiang thiab Doudna, 2017). Tsis tas li ntawd, lub kaw lus CRISPR/Cas9 tau tshaj tawm tias muaj nyob rau hauv 50% thiab 87% ntawm cov kab mob bacterial thiab archaeal genomes, feem (Ishino et al., 2018). CRISPR/Cas9 yog lub cuab yeej muaj peev xwm rau kev rho tawm, ntxig thiab kho cov kab mob tsis zoo ntawm cov noob caj noob ces siv cov qauv hauv vivo thiab hauv vitro (Sabit et al., 2021). Ntxiv mus, CRISPR/Cas9 tau pom tias yog ib feem ntawm lub cev tiv thaiv kab mob vim nws qhov tshwj xeeb rau cov noob caj noob ces ntawm kev txaus siab (Chen thiab Zhang, 2018).
CRISPR/Cas9 muaj Cas9 thiab ib qho RNA qhia ib leeg (sgRNA). Cas9 yog ib qho endonuclease uas muaj ntau yam khoom protein. Tsis tas li ntawd, Cas9 muaj cov yam ntxwv tshwj xeeb thiab cov qauv (Pacesa et al., 2022) vim nws muaj ob lub lobes: kev lees paub (REC) thiab nuclease (NUC). Lub REC lobe ntxiv muab faib ua peb thaj chaw: REC1, REC2, thiab bridge helices (Cromwell et al., 2018). Lub NUC muaj peb lub lobes, uas yog RuvC (RuvC I, RuvC II, RuvC III), HNH thiab protospacer adjacent motif (PAM) interaction domain (Daim Duab 1) (Song et al., 2016). sgRNA muaj ob lub Cheebtsam, suav nrog CRISPR RNA (crRNA) thiab transcoding small RNA (tracer RNA) (Daim Duab 1). Lub sgRNA txuas cov protein Cas9 rau cov connexins los tsim ib qho active complex, tseem hu ua effector complex (Richter et al., 2012). crRNA yog 18–20 nucleotide base pair uas ua lub luag haujlwm tseem ceeb hauv kev lees paub cov DNA sequences. Tsis tas li ntawd, crRNA khub nrog lub hom phiaj DNA, thiab tracrRNA ua haujlwm ua lub scaffold rau Cas9 nuclease los khi lub hom phiaj DNA (Manghwar et al., 2019). Ntawm qhov tod tes, PAM sequence muaj 3 nucleotides, uas lees paub, qhia meej thiab nruab nrab qhov kev khi ntawm effector complex rau DNA. Cas9 protein complex subunits RuvC thiab HNH muaj catalytic activity (Richter et al., 2012; Asmamaw thiab Zawdie, 2021). Lub HNH nuclease domain ntawm Cas9 subunit txiav cov DNA strand cuam tshuam nrog crRNA. Lub RuvC nuclease domain txiav lwm cov DNA strands thiab tsim ob-strand breaks (DSBs), tom qab ntawd ob lub DNA break kho mechanisms sib txawv raug qhib (Jiang thiab Doudna, 2017) (Daim Duab 1).
Daim Duab 1. Txheej txheem cej luam ntawm CRISPR/Cas9 (A). Cov Cheebtsam ntawm CRISPR/Cas9 system: (i). Cas9 endonuclease yog lub luag haujlwm rau kev txiav cov DNA sequence, (ii) ib qho kev qhia ib leeg (sg) RNA uas yog los ntawm kev sib xyaw ntawm crRNA thiab tra-crRNA chimeras. (ob). Cas9 suav nrog ntau yam xws li Rec I, Rec II, NUC lobe (HNH thiab Ruv C yog cov khoom me me) thiab PAM interaction domain (C) nrog cov haujlwm sib xws. CRISPR/Cas9 protein complex txiav cov DNA sequences rau hauv cov ntawv tsis sib txuas thiab cov ntawv sib txuas (D). CRISPR/Cas9 hloov kho cov genome hauv peb theem: kev lees paub, txiav thiab kho. Tus tsim sg-RNA coj Cas9 thiab lees paub cov sequence xav tau los ntawm cov khoom sib txuas crRNA. Cas9 lees paub cov PAM sequence ntawm 5′-NGG-3′ thiab yaj cov DNA, tsim cov DNA-RNA hybrid thiab ua kom muaj kev sib cais. Lub HNH domain ntawm Cas9 txiav cov strand complementary, thiab lub RuvC domain txiav cov strand uas tsis yog complementary. CRISPR/Cas9 kho dsDNA los ntawm kev rhuav nws ua ob txoj kev: kev sib koom ua ke tsis sib xws (NHEJ) thiab kev kho homology-directed (HDR). NHEJ kho ob txoj DNA uas tsis muaj DNA txawv teb chaws los ntawm cov txheej txheem enzymatic, ib qho mechanism uas ua rau yuam kev uas tuaj yeem ntxig lossis tshem tawm cov DNA random. HDR yog qhov tshwj xeeb heev thiab xav tau cov qauv DNA sib xws.
Cov kev kho uas siv CRISPR/Cas9 suav nrog kev sib koom ua ke tsis sib xws (NHEJ) thiab cov txheej txheem kho uas coj los ntawm homology (HDR). NHEJ yog txoj kev uas yooj yim ua yuam kev vim nws cuam tshuam nrog kev ntxig lossis rho tawm thiab tsis xav tau cov qauv thaum lub sijhawm kho. Nws siv cov nucleotides random los tsim cov protein txheem (Abbasi et al., 2021). Lub kaw lus kho no muaj plaub lub complex xws li KU complex, cross-complementing protein complex hom 4 (XRCC-4), DNA end processing enzyme, thiab protein kinase DNA-PKcs (Abbasi et al., 2021). Cov protein KU complex muaj ob lub subunits, Ku 70 thiab Ku 80, thiab ua lub luag haujlwm tseem ceeb hauv NHEJ mechanism, txij li thaum lub mechanism kho yog pib los ntawm kev khi ntawm ob lub subunits (Ku 70 thiab Ku 80) rau qhov kawg ntawm lub hom phiaj DNA (Abbasi et al., 2021), ua haujlwm ua lub scaffold los nrhiav lwm yam NHEJ-cuam tshuam rau qhov chaw raug mob (Yang et al., 2020). XRCC-4 thiab DNA ligase yog tsim los ntawm 334 thiab 911 amino acids, feem, thiab XRCC4-DNA ligase IV complex txhawb kev ligation ntawm DNA kawg (Chatterjee et al., 2015). DNA kawg ua enzyme, tseem hu ua polynucleotide kinase 3′-phosphate, yog DNA kawg ua enzyme. Nws tuaj yeem tshem tawm 3′P pawg hauv DNA thiab phosphorylate 5′OH pawg thaum lub sijhawm kho DSB. Nws kuj tseem cuam tshuam txog kev kho cov kev tawg ntawm ib txoj hlua (SSBs) siv txoj kev kho SSB (Chatterjee et al., 2015). Protein kinase DNA-PKcs yog DNA-dependent protein kinase uas muaj cov catalytic subunit ntawm PIKKs (phosphatidylinositol 3-kinase-related kinases) thiab ataxia telangiectasia mutated (ATM) tsev neeg, nrog rau ATM thiab Rad3-related ATRs. strand breaks (DSBs) thiab ib txoj hlua breaks (Yue et al., 2020; Peng et al., 2016).
HDR yog ib qho kev kho kom raug dua thiab tsim nyog dua vim tias cov ntaub ntawv raug theej siv cov qauv zoo sib xws ntawm cov DNA duplex, txawm hais tias nws xav tau qhov muaj cov chromatids muam. Qhov no tshwm sim thaum lub sijhawm S/G2 ntawm lub voj voog ntawm cov tsiaj txhu. HDR tshwm sim feem ntau hauv cov hom poov xab, tab sis NHEJ tseem ceeb heev rau cov tsiaj txhu (Burma et al., 2006; Abbasi et al., 2021). Lub tshuab kho kom tiav tau qhia hauv Daim Duab 1.
Vim tias TNBC yog tshwm sim los ntawm kev hloov pauv caj ces thiab epigenetic, kev kho cov teeb meem malignant genomic/epigenomic siv CRISPR/Cas9 tej zaum yuav yog txoj hauv kev kho mob tsim nyog (Chen et al., 2019). Tsis tas li ntawd, qee cov yam ntxwv transcription uas koom nrog kev tswj hwm transcriptional tshwj xeeb ntawm cell yuav ua rau pom cov yam ntxwv tshwj xeeb ntawm cov hlwb qog noj ntshav, qhia tias kev tswj hwm transcriptional tej zaum yuav yog txoj hauv kev zoo rau kev kho mob qog noj ntshav (Drost et al., 2017). Kev siv cov yam ntxwv molecular ntawm cov qog noj ntshav, xws li cov teeb meem genetic, epigenetic thiab transcriptional, rau kev tsim tshuaj tuaj yeem txhim kho cov txiaj ntsig kho mob thiab txo cov nqi tshuaj ntsuam. CRISPR yog lub cuab yeej kho cov noob caj ces uas tsis tsuas yog tuaj yeem nrhiav thiab txheeb xyuas cov hom phiaj genomic ua rau mob qog noj ntshav, tab sis kuj tuaj yeem siv los kho, tswj, thiab hloov kho epigenetically oncogenes hauv tib neeg lub hlwb (Rooj 1) (Ahmed et al., 2021). Ntau qhov kev tshuaj ntsuam CRISPR tau ua los nrhiav cov noob caj ces cuam tshuam nrog cov tshuaj tiv thaiv qog noj ntshav, oncogenes, thiab kev tiv thaiv tshuaj. Kev siv CRISPR/Cas9 los hloov kho ntau yam TNBC oncogenes tau qhia nyob rau hauv Daim Duab 2.
Daim Duab 2. Kev kho cov noob caj noob ces ntawm ntau hom TNBC oncogenes siv CRISPR/Cas9 ua rau txo qhov mob qog noj ntshav thiab metastasis. Cov oncogenes no suav nrog CDK7, NAT1, UBR5, YTHDF2, ITGA9, CXCR4 thiab CXCR7, Crypto1, ROR1 thiab ST8SIA, uas koom nrog rau qhov tshwm sim thiab metastasis ntawm TNBC.
Kev tsiv teb tsaws chaw ntawm cov qog nqaij hlav, kev nkag mus rau hauv, thiab kev hloov pauv epithelial-mesenchymal (EMT) yog txuam nrog ITGA9. Cov kev tshawb fawb yav dhau los tau qhia tias ITGA9 yog tus neeg ua si tseem ceeb hauv Notch txoj kev thiab ua lub luag haujlwm nthuav hauv rhabdomyosarcoma metastasis (Molist et al., 2020). Tsis tas li ntawd, ITGA9 tau pom tias muaj feem cuam tshuam nrog kev kwv yees ntawm tus neeg mob hauv ntau hom qog nqaij hlav, suav nrog mob qog nqaij hlav mis (Wang Z. et al., 2019). Tsis tas li ntawd, kev tshuaj xyuas bioinformatics ntawm ITGA9 tau qhia tias nws qhov kev qhia tawm hauv TNBC yog siab dua li lwm cov qog nqaij hlav mis. Cov qib ITGA9 siab dua cuam tshuam nrog mob qog nqaij hlav metastasis thiab rov qab los hauv cov neeg mob TNBC. Kev tshem tawm ntawm ITGA9 los ntawm CRISPR / Cas9 ua rau muaj cov khoom zoo li qog nqaij hlav (CSC), qog nqaij hlav angiogenesis, qog nqaij hlav loj hlob, thiab txo qis metastasis los ntawm kev txhawb nqa β-catenin degradation hauv TNBC (Wang Z. et al., 2019).
Crypto-1 yog ib tug tswv cuab ntawm tsev neeg TGF-β thiab nws tseem ceeb heev rau kev tsim cov qe menyuam thaum ntxov, kev saib xyuas cov qe menyuam, thiab kev kis tus kab mob qog nqaij hlav (Ishii et al., 2021). Kuj tseem hu ua Tdgf-1, nws yog ib qho oncogenic GPI-anchored signaling protein uas koom nrog kev tswj hwm kev tsim cov kab mob primitive, mesoderm thiab endoderm, nrog rau kev tsim kom muaj sab laug / sab xis asymmetry hauv kev loj hlob ntawm cov kab mob hauv lub cev thaum lub sijhawm embryogenesis (Zhang et al., 2021b). Tsis tas li ntawd, Cripto-1 tau pom tias koom nrog epithelial-mesenchymal transition (EMT) ua ib qho cim qe menyuam (Zhang et al., 2021b). EMT tsis yog qhov tseem ceeb rau ntau yam txheej txheem xws li kev loj hlob ntawm cov qe menyuam, fibrosis thiab qhov txhab kho, tab sis kuj rau kev nkag mus rau qog nqaij hlav thiab metastasis. Tsis tas li ntawd, Cripto-1 tau pom tias cuam tshuam nrog plaub Notch receptors thiab txhim kho lawv cov kev loj hlob tom qab txhais lus (Brandstadter thiab Maillard, 2019). Txoj kev taw qhia Notch paub tias muaj feem cuam tshuam rau kev tswj hwm cov hlwb mob qog noj ntshav hauv lub mis. Cov kev tshawb fawb tau qhia tias CRISPR/Cas9-mediated knockout ntawm Crypto-1 tiv thaiv kev loj hlob ntawm mob qog noj ntshav thiab metastasis. Yog li ntawd, Crypto-1 yuav dhau los ua lub hom phiaj kho mob tseem ceeb rau TNBC (Castro et al., 2015).
XCL12 protein thiab nws cov chemokine receptor CXC (CXCR4 thiab CXCR7) ua ntau yam haujlwm hauv kev loj hlob ntawm cov hlwb qog noj ntshav, kev loj hlob, kev tsiv teb tsaws chaw thiab kev nkag mus (Wu et al., 2015). Cov chemoreceptors no tau cuam tshuam nrog kev txhim kho ntawm TNBC los ntawm ntau txoj hauv kev signaling hauv ob qho tib si hauv vivo thiab hauv vitro qauv (Wu et al., 2015). Tsis tas li ntawd, kev ua haujlwm ntawm CXCR4 thiab CXCR7 yog cuam tshuam nrog kev cuam tshuam ntau dua rau metastasis thiab kev kwv yees tsis zoo hauv TNBC (Karn et al., 2022). Yog li ntawd, knockout ntawm CXCR4 thiab CXCR7 yuav dhau los ua cov tshuaj zoo rau kev kho mob qog noj ntshav mis, suav nrog TNBC. Kev tshawb fawb los ntawm Yang et al. Yang et al (2019) siv CRISPR / Cas9 los sib koom ua ke CXCR4 thiab CXCR7 noob thiab pom tias TNBC proliferation, kev loj hlob, kev tsiv teb tsaws chaw thiab kev nkag mus tau raug txwv tsis pub ua (Yang et al., 2019).
Cov theem siab ntawm miR-3662, uas yog TNBC oncogene, tau pom nyob rau hauv cov nqaij mob cancer mis (Yi et al., 2022). Kev rhuav tshem ntawm miR-3662 tau pom tias yuav tiv thaiv kev loj hlob ntawm mob cancer mis thiab metastasis ob qho tib si hauv vivo thiab hauv vitro (Agarwal thiab Gupta, 2021). HBP-1 yog ib qho tshuaj inhibitor muaj zog ntawm Wnt/-catenin signaling thiab tej zaum yuav yog lub luag haujlwm rau kev loj hlob ntawm miR-3662-mediated ntawm TNBC hlwb. Tsis ntev los no, Yi et al. pom tias miR-3662-HBP1 axis tswj hwm Wnt/-catenin signaling pathway hauv TNBC hlwb (Yi et al., 2022). Vim nws qhov kev qhia tshwj xeeb rau mob cancer, miR-3662 yuav yog lub hom phiaj kho rau TNBC. Yog li, CRISPR/Cas9-mediated knockdown ntawm miR-3662 yuav yog ib txoj hauv kev zoo heev rau kev tsim cov tshuaj tshiab rau kev kho mob TNBC.
UBR5 yog 300 kDa nucleophosphoprotein uas tau txheeb xyuas tias yog tus tswj hwm tseem ceeb ntawm tumorigenesis, metastasis, thiab kev tiv thaiv kab mob hauv ntau yam mob qog noj ntshav (Shearer et al., 2015; Fu et al., 2023). UBR5 tau tshaj tawm tias tau nce siab heev hauv cov qauv TNBC thiab txhawb nqa ERα ua haujlwm los ntawm kev ua kom muaj kev loj hlob los ntawm nws cov ubiquitin ligase kev ua haujlwm (Bolt et al., 2015). Tag nrho cov kev tshawb fawb exome sequencing ntawm cov qauv TNBC thawj zaug kuj tau qhia txog kev nce ntxiv ntawm UBR5, qhia txog nws lub luag haujlwm hauv kev txhim kho TNBC. Tsis tas li ntawd, CRISPR / Cas9-tsav deletion ntawm UBR5 tau qhia txog kev txwv tsis pub TNBC metastasis thiab kev loj hlob hauv cov qauv nas sim. Ntxiv mus, kev suav nrog UBR5 hauv cov qauv nas qus tau rov qab kho nws cov haujlwm tag nrho, thaum cov nyhuv no tsis tau pom hauv cov kab mob tsis ua haujlwm (Liao et al., 2017). Qhov tsis muaj UBR5 cuam tshuam nrog kev nce apoptosis, necrosis, thiab kev txwv tsis pub muaj qog loj hlob hauv TNBC vim yog angiogenesis tsis zoo. Vim yog qhov poob ntawm UBR5, qog kis mus rau cov kabmob deb tau txo qis, thiab UBR5 ua rau EMT tsis zoo feem ntau los ntawm kev txo E-cadherin kev qhia (Zhang thiab Weinberg, 2018). Tsis ntev los no, UBR5 tau pom tias yog ib qho tseem ceeb heev hauv IFN-γ-induced PDL1 transcription hauv TNBC vim nws tsis muaj E3 ubiquitination kev ua ub no. RNA transcriptome kev tshuaj xyuas tau qhia tias UBR5 yuav muaj cov teebmeem systemic rau cov noob caj noob ces cuam tshuam nrog IFN-γ txoj kev thiab txhawb PDL1 transactivation los ntawm kev nce qib kev ua haujlwm ntawm protein kinase RNA (PKR) thiab nws cov teeb liab transducers thiab activators. transcription 1 (STAT1) thiab interferon regulatory factor 1 (IRF1). Txawm li cas los xij, CRISPR/Cas9-mediated ua ke ablation ntawm UBR5 thiab PD-L1 kev qhia tawm muaj kev cuam tshuam synergistic kho mob dua li kev thaiv ib leeg, nrog rau cov teebmeem tob rau qhov chaw ib puag ncig ntawm qog nqaij hlav (Wu et al., 2022). Yog li, CRISPR/Cas9 tej zaum yuav yog ib qho cuab yeej tseem ceeb los tiv thaiv UBR5 kev ua haujlwm, yog li ntawd suppressing TNBC metastasis thiab qog nqaij hlav loj hlob.
ROR1 yog hom I transmembrane protein uas tau qhia tawm thaum lub sijhawm mob qog noj ntshav thiab kev loj hlob ntawm lub cev thiab tau txheeb xyuas tias yog oncofetal protein (Nicholas Borcherding, 2014). Tus cwj pwm invasive ntawm ntau yam mob qog noj ntshav tib neeg yog txuam nrog kev nce ntawm ROR1. Cov txiaj ntsig zoo tau txais los ntawm kev tshawb fawb hauv vivo thiab hauv vitro uas muaj cov tshuaj kho mob tsom mus rau ROR1 (Chien et al., 2016). Cov theem siab ntawm ROR1 mRNA hauv cov ntaub so ntswg mis kuj tau cuam tshuam nrog cov qog nqaij hlav zoo li lub mis (BL) thiab lawv txoj kev tsiv teb tsaws chaw mus rau lwm qhov ntawm lub cev. Ntxiv mus, overexpression ntawm ROR1 tau txheeb xyuas tias yog lub cim qhia txog kev loj hlob ntawm TNBC (Chien et al., 2016). Txawm li cas los xij, kev ntsiag to ROR1 siv CRISPR / Cas9 los tswj TNBC kev loj hlob thiab metastasis yuav yog ib qho tswv yim zoo.
Cov txheej txheem sialylation cuam tshuam nrog kev ntxiv cov sialic acid rau hauv glycoconjugates, uas yog catalyzed los ntawm sialyltransferases (STs). ST8SIA1 koom nrog tsev neeg ST thiab ua lub luag haujlwm tseem ceeb hauv pathogenesis ntawm ntau yam kab mob xws li lymphocytic leukemia thiab colorectal cancer (Chang et al., 2018). RNA sequence analysis qhia tau hais tias ST8SIA1 tau qhia ntau heev hauv cov ntaub so ntswg ntawm lub mis ntawm cov neeg mob TNBC thiab muaj feem cuam tshuam zoo nrog kev hloov pauv hauv cov noob caj noob ces suppressor p53, uas yuav pab txhawb rau pathogenesis ntawm TNBC (Battula et al., 2017). Tsis tas li ntawd, ST8SIA1 koom nrog hauv metastasis thiab rov tshwm sim ntawm TNBC, qhia tias nws ua lub luag haujlwm tseem ceeb hauv qhov tshwm sim thiab kev loj hlob ntawm TNBC. Hauv tus qauv hauv vitro ntawm TNBC, knockdown ntawm ST8SIA1 los ntawm CRIPSR / Cas9 tau pom tias thaiv kev loj hlob thiab metastasis (Battula et al., 2017). Qhov no qhia tau hais tias CRISPR/Cas9 tej zaum yuav yog ib qho cuab yeej tseem ceeb rau kev txwv tsis pub ua haujlwm ntawm ST8SIA1 oncogene hauv kev kho mob TNBC.
NAT1 yog ib qho enzyme metabolic uas ua rau muaj kev tsim cov tshuaj xenobiotic theem II thiab qhia tawm hauv yuav luag txhua cov nqaij ntawm tib neeg. NAT1 tuaj yeem siv cov cofactor folate los tsom acetyl-CoA (acetyl-CoA) txawm tias tsis muaj cov aromatic amine substrate (Stepp et al., 2015; Laurieri et al., 2014). Cov kev tshawb fawb tau qhia tias NAT1 tswj hwm matrix metalloproteinase 9 (MMP9) ua haujlwm hauv cov qauv qog nqaij hlav mis thiab tiv thaiv cov tshuaj reactive oxygen (ROS) thaum lub sijhawm glucose starvation (Wang et al., 2018). Kev rho tawm ntawm NAT1 tau pom tias inhibit pyruvate dehydrogenase complex, ua rau mitochondrial dysfunction (Wang L. et al., 2019). Tsis tas li ntawd, ntau lwm cov ntawv tshaj tawm tau qhia tias NAT1 inhibition siv cov molecules me me thiab siRNA silencing tuaj yeem txo qhov kev nkag mus thiab kev loj hlob ntawm cov qog nqaij hlav mis (Stepp et al., 2018). Tsis ntev los no, CRISPR/Cas9 tau siv los tswj NAT1 hauv cov kab mob qog noj ntshav mis MDA-MB-231, cuam tshuam rau kev ua haujlwm ntawm lub cev nyob ntawm nws qib kev qhia tawm. Kev tshawb fawb no kuj qhia tau tias NAT-1 yog qhov tseem ceeb rau kev nce qib thiab metastasis ntawm TNBC (Carlisle et al., 2020).
Kev sib koom ua ke ntawm cov oncogenes yog tswj hwm los ntawm super enhancers, transcription factors thiab cofactors (Hnisz et al., 2015). Tsis tas li ntawd, ib pawg ntawm cyclin-dependent kinases (CDKs), xws li CDK7, CDK8, CDK9, CDK12, thiab CDK13, yog qhov yuav tsum tau rau kev tswj hwm transcriptional. Ntawm lawv, CDK7 koom nrog hauv phosphorylation ntawm RNA polymerase II, uas yog qhov tseem ceeb heev rau kev pib thiab nthuav dav ntawm oncogene transcription hauv pathogenesis ntawm TNBC. Hauv kev tshawb fawb seminal, kev rho tawm ntawm CDK7 los ntawm CRISPR / Cas9 inhibited TNBC, qhia tias pathogenesis ntawm TNBC yog CDK7 nyob ntawm (Wang Y. et al., 2015).
Cov kev tshawb fawb tau qhia tias kev hloov pauv hauv MYC thiab RBP (RNA binding protein) tuaj yeem ua rau apoptosis, thaum cov kev hloov pauv ib qho gene hauv MYC lossis RBP tsis cuam tshuam rau kev loj hlob ntawm cov hlwb qog noj ntshav (Einstein et al., 2021). Ntau tshaj 1000 RBPs hauv tib neeg genome tau raug tshuaj xyuas siv lub tsev qiv ntawv CRISPR / Cas9. Ntawm lawv, 57 RBPs tau pom tias tseem ceeb rau kev loj hlob ntawm cov hlwb qog noj ntshav nrog cov theem MYC siab heev (Wheeler et al., 2020). Tsis tas li ntawd, YTHDF2 yog qhov tseem ceeb rau kev tswj hwm kev loj hlob ntawm TNBC cell thiab txo cov nyiaj ntawm cov ntawv sau methylated thaum lub sijhawm sau ntawv thiab txhais lus siab hauv cov hlwb qog noj ntshav nrog MYC qhia siab dua. Tsis tas li ntawd, YTHDF2 tsis tseem ceeb rau cov hlwb qog noj ntshav, uas tsis tshua nyob ntawm cov theem MYC siab kom ntev lub neej ntawm cov neeg mob TNBC (Einstein et al., 2021), qhia tias nws yuav yog lub hom phiaj kho mob rau cov tshuaj uas tuaj yeem kov yeej TNBC.
Cov protein ntiv tes zinc (ZNFs) muaj li ntawm 1% ntawm tag nrho cov noob caj noob ces tib neeg. Cov kev tshawb fawb tau qhia tias ZNF tuaj yeem tswj hwm kev loj hlob ntawm cov cell hauv ntau yam mob qog noj ntshav, xws li mob qog noj ntshav siab, mob qog noj ntshav mis thiab mob qog noj ntshav colorectal (Zhang W. et al., 2021; Li et al., 2017). Cov tsev qiv ntawv tsim los ntawm CRISPR knockout tau siv los tshuaj xyuas ntau yam noob caj noob ces tiv thaiv qog noj ntshav (TSGs) hauv cov hlwb qog noj ntshav mis (Shalem et al., 2014). Txij thaum ntawd los, kev tshawb fawb transcriptomic ntawm CRISPR/Cas9 ZNF319-deleted mis qog noj ntshav tau qhia tias ZNF319 yog noob caj noob ces tiv thaiv qog noj ntshav uas txo cov qog noj ntshav mis thiab yog li ntawd koom nrog ntau yam kev ua haujlwm signaling thiab lwm yam haujlwm biological (Wang L. et al., 2022).
Ferroptosis yog ib hom kev tuag ntawm cov hlwb uas tau teeb tsa los ntawm kev siv hlau los ua lub hauv paus. Nws paub tias kev loj hlob ntawm ferroptosis yog cuam tshuam los ntawm qhov muaj lipid peroxides. Tsis tas li ntawd, PKCβII tau tshaj tawm tias qhia txog lipid peroxidation thaum ntxov, thiab kev nce ntxiv ntawm lipid peroxidation yog txuam nrog ferroptosis. Kev tshuaj xyuas lub tsev qiv ntawv ntawm CRISPR / Cas9-mediated kinase inhibitors tau qhia tias PKCβII koom nrog hauv cov txheej txheem ntawm lipid peroxidation, uas yog qhov tseem ceeb rau ferroptosis hauv MDA-MB-231 hlwb (Zhang et al., 2022). Yog li ntawd, qhov no qhia tau hais tias kev tshem tawm ntawm PKCβII los ntawm CRISPR / Cas9 tej zaum yuav yog lub hom phiaj tiv thaiv qog noj ntshav rau kev kho cov kab mob ntsig txog ferroptosis (Zhang et al., 2022).
Kev tiv thaiv tshuaj ntseeg tau tias yog lub luag haujlwm rau kwv yees li 90% ntawm cov neeg mob qog noj ntshav tuag thiab yog ib qho ntawm cov teeb meem loj hauv kev kho mob qog noj ntshav (Bukowski et al., 2020). Cov txiaj ntsig qhia tau tias muaj ntau cov noob caj noob ces uas cuam tshuam nrog kev tso tshuaj tawm, kho DNA, apoptosis thiab ntau txoj hauv kev xa xov ntawm lub hlwb muaj feem cuam tshuam nrog kev tiv thaiv tshuaj (Haider et al., 2020). Ntawm lawv, ntau cov noob caj noob ces tau raug tsom los ntawm CRISPR / Cas9 cov cuab yeej thiab tau pom cov txiaj ntsig zoo hauv kev txo qis kev tiv thaiv tshuaj thiab ua kom muaj txiaj ntsig zoo ntawm kev kho mob qog noj ntshav (Vaghari-Tabari et al., 2022). Tsis tas li ntawd, cov tsev qiv ntawv tshuaj ntsuam xyuas CRISPR / Cas9 gene knockout uas muaj txiaj ntsig zoo tau raug cuam tshuam hauv kev hloov pauv kev ua haujlwm ntawm cov hom phiaj noob caj noob ces tiv thaiv tshuaj (Shalem et al., 2015). Txhawm rau txheeb xyuas cov noob caj noob ces tiv thaiv paclitaxel, RNA sequencing ua ke nrog kev tshuaj ntsuam xyuas sgRNA thoob plaws genome tau siv los txheeb xyuas yim tus neeg sib tw noob caj noob ces, suav nrog histone deacetylase 9 (HDAC9), uas cuam tshuam nrog kev tiv thaiv tshuaj hauv cov neeg mob uas muaj TNBC rov tshwm sim (B et al., 2020). ). Hauv lwm txoj kev tshawb fawb, kev qhia tawm ntawm diserine / threonine thiab tyrosine protein kinase (DSTYK) tau pom thaum lub sijhawm muaj sia nyob ntawm cov neeg mob TNBC kho nrog cov tshuaj tiv thaiv kab mob qog noj ntshav. Ntxiv mus, CRISPR / Cas9-mediated knockdown ntawm DSTYK tau txhim kho apoptosis ntawm cov hlwb qog noj ntshav tiv thaiv tshuaj hauv vitro (SUM102PT hlwb thiab MDA-MB-468 hlwb) thiab tus qauv TNBC hauv vivo (Ogbu et al., 2021).
Txawm hais tias TNBC muaj kev ua haujlwm tsis zoo ntawm txoj kev MAPK, cov txiaj ntsig kho mob ntawm MEK-targeted therapies tsis zoo. Ib qho kev tshuaj ntsuam CRISPR/Cas9 genomic library tau qhia tias kev txwv ntawm PSMG2 (proteome assembly chaperone 2) ua rau TNBC BT549 thiab MB468 cell rhiab rau MEK inhibitor AZD6244. CRISPR/Cas9 knockdown ntawm PSMG2 hloov pauv kev ua haujlwm proteasome ib txwm muaj, ua rau autophagy-mediated disassembly ntawm PDPK1, yog li ntawd txhim kho cov hlwb qog hauv TNBC nas qauv uas tshwm sim los ntawm AZD6244 (MEK inhibitor) thiab MG132 (proteasome inhibitor). Yog li, proteasome thiab MAP kinase (MEK) inhibitors tuaj yeem tswj tau synergistically kom txo cov hlwb qog proliferation (Wang X. et al., 2022).
CRISPR/Cas9 kuj tau siv los tshuaj xyuas qhov poob ntawm cov noob caj noob ces ua rau TNBC tsis kam (Shu et al., 2020). Ge et al piav qhia txog txoj hauv kev ntawm kev tsis kam tshuaj ntawm cov hlwb qog noj ntshav kho nrog JQ1, ib qho BET bromodomain inhibitor (BBDI), hauv TNBC. Siv CRISPR/Cas9, lawv pom tias kev rho tawm cov noob caj noob ces rb1 ua rau TNBC tsis kam rau cov tshuaj tiv thaiv qog noj ntshav JQ1. Yog li ntawd, rb1 ua haujlwm tseem ceeb hauv kev teb rau cov tshuaj JQ1 hauv TNBC. Lawv kuj tau tshaj tawm tias paclitaxel yog CDK4/6 kinase/microtubule inhibitor, thiab nws txoj kev sib xyaw nrog BBDIs xws li JQ1 yuav muab cov lus teb zoo rau TNBC uas tsis kam tshuaj (Ge et al., 2020).
Cov toj roob hauv pes transcriptional ntawm RNA ntev noncoding (lncRNA) tau tshaj tawm tias yog lub luag haujlwm rau kev tiv thaiv rau neoadjuvant therapy hauv TNBC. Kev tshawb fawb no qhia tau hais tias tsib qhov sib txawv MALAT1 lncRNA transcripts tau qhia meej heev hauv TNBC. Tsis tas li ntawd, CRISPR / Cas9-mediated deletion ntawm MALAT1 ua rau muaj kev nkag siab ntawm TNBC BT-549 hlwb rau paclitaxel thiab doxorubicin, qhia txog lub luag haujlwm rau MALAT1 tsis kam hauv TNBC (Shaath et al., 2021).
Kev hloov pauv hauv BRCA1 (BRCA1m) yog heterogeneous thiab yog li ntawd nyuaj rau nrhiav tau. Kev tsom mus rau PARP1 (poly(ADP-ribose) polymerase), tus khub ua rau tuag taus ntawm BRCA1, yuav ua rau TNBC tshuaj chemosensitivity. Siv CRISPR/Cas9, kev tshem tawm ntawm PARP1 ua rau muaj kev rhiab heev ntawm cov tshuaj tiv thaiv kab mob qog noj ntshav xws li doxorubicin, gemcitabine thiab docetaxel rau mBRCA1 mutant TNBC hlwb, qhia tias PARP1 kuj koom nrog kev tiv thaiv tshuaj hauv TNBC (Vaghari-Tabari et al., 2022) . . Kev hloov pauv hauv cov noob caj noob ces suppressor BRCA1 lossis BRCA2 paub tias ua rau muaj feem yuav mob qog noj ntshav hauv tib neeg. Kev kho cov neeg mob no yuav tsum tau siv cov tshuaj PARP inhibitors. Tsis tas li ntawd xwb, nws tau pom tias kev tshem tawm cov nucleotide salvage factor DNPH1 siv CRISPR/Cas9 tuaj yeem tshem tawm cov tshuaj lom nucleotide 5-hydroxymethyldeoxyuridine (hmdU) monophosphate, yog li ua kom cov hlwb BRCA-deficient teb rau qhov rhiab heev rau PARP inhibitors (Fugger). et al., 2021). Yog li, CRISPR/Cas9 thiab PARP1 inhibitors yuav dhau los ua ib qho tseem ceeb rau kev kho mob rau TNBC.
Cov noob glycoprotein nkag mus (P-gp) yog cov noob tiv thaiv ntau yam tshuaj uas feem ntau nce siab hauv kwv yees li 41% ntawm tag nrho TNBC (Sun et al., 2020). P-gp-induced tshuaj efflux tau raug txheeb xyuas tias yog tus tswj hwm tseem ceeb ntawm kev tiv thaiv tshuaj hauv mob qog noj ntshav mis. P-gp inhibitors tau ua pov thawj tias muaj kev nkag siab ntau ntxiv rau cov tshuaj tiv thaiv mob qog noj ntshav hauv mob qog noj ntshav mis (Famta et al., 2021). Yog li, CRISPR/Cas9-mediated ablation lossis kev txwv ntawm P-gp thiab P-gp inhibitors tej zaum yuav yog cov txheej txheem tseem ceeb heev los kov yeej kev tiv thaiv tshuaj hauv TNBC.
ATP-binding cassette transporter G2 (ABCG2) paub tias ua rau muaj kev tiv thaiv tshuaj hauv TNBC (Palasuferniam et al., 2015), txawm hais tias tam sim no tsis muaj kev tshaj tawm txog kev sib raug zoo ntawm CRISPR/Cas9 thiab ABCG2 silencing thiab tumor suppressor gene inactivation. PTEN Tej zaum yuav ua rau ABCG2 ua haujlwm zoo dua (Palasuferniam et al., 2015), (Deepak Singh et al., 2021). Yog li ntawd, siv CRISP/Ca9 los tshem tawm ABCG2 thiab sib xyaw nrog ABCG2 inhibitor tej zaum yuav yog kev kho mob tsim nyog los kov yeej kev tiv thaiv tshuaj hauv TNBC. Rooj 2 hais txog CRISPR/Cas9 tsom mus rau txhua hom noob tiv thaiv.
Rooj 2. CRISPR/Cas9 tsom mus rau ntau yam noob caj noob ces TNBC kom ua rau cov hlwb rhiab rau cov tshuaj tiv thaiv kab mob qog noj ntshav.
Cov tsev qiv ntawv CRISPR/Cas9 yog cov cuab yeej siv tau rau kev tshuaj xyuas cov kev hloov pauv ntawm cov noob caj noob ces uas cuam tshuam nrog kev mob qog noj ntshav (Chan et al., 2022). Txoj kev tshuaj xyuas no suav nrog plaub kauj ruam xws li (a) kev tsim tsev qiv ntawv, (b) kev hloov pauv lentiviral, (c) kev tshuaj xyuas phenotypic, thiab (d) kev tshuaj xyuas cov noob caj noob ces. Txawm hais tias TNBC muaj kev ua haujlwm tsis zoo ntawm txoj kev MAPK, qhov kev kwv yees kho mob ntawm MEK kev kho mob rau cov neeg mob TNBC uas nqa cov kev hloov pauv hauv cov noob caj noob ces suppressor xws li PTEN, RB1, thiab TP53 tsis zoo heev.
Tsis tas li ntawd xwb, kev tshuaj ntsuam xyuas gene knockout siv CRISPR/Cas9 tau sim cov molecule muaj zog tshaj plaws thiab xaiv tau, dehydrocatrol, uas muaj ntau nyob rau hauv Guatemalan paj thiab nplooj extracts, kom nkag siab txog qhov cuam tshuam ntawm dehydrocatrol rau MDA-MB-231. Cov txheej txheem ntawm kev xaiv cytotoxicity. Mesenchymal stemness yam ntxwv ntawm TNBC subtypes. Qhov kev tshuaj ntsuam CRISPR/Cas9-raws li no kuj tau qhia tias HSD17B11, ib qho gene encoding 17β-hydroxysteroid dehydrogenase hom 11, tau qhia meej heev hauv MDA-MB-231 hlwb thiab ua rau dehydrofalcarinol tshwj xeeb rau MDA-MB-231 hlwb (Grant et. al., 2020). Yog li, qhov no qhia tau hais tias CRISPR/Cas9 genomic kev tshuaj ntsuam muaj peev xwm zoo hauv kev txheeb xyuas cov txheej txheem hauv qab cov khoom anticancer ntawm cov tshuaj ntuj tsim.
Ntxiv mus, qhov tsis muaj zog ntawm TNBC rau mob qog noj ntshav tau kawm siv kev tshuaj ntsuam genome-wide hauv vivo CRISPR / Cas9, uas qhia txog kev sib txuas ntawm oncogenic thiab tumor suppressor pathways. Cov khoom tseem ceeb ntawm mTOR thiab Hippo pathways tau tshaj tawm tias ua lub luag haujlwm tseem ceeb hauv kev tswj hwm qog noj ntshav hauv TNBC. Ntxiv mus, kev tshawb fawb tau qhia tias kev txwv tsis pub siv tshuaj ntawm mTORC1 / 2 thiab YAP oncoprotein zoo tiv thaiv qhov pathogenicity ntawm TNBC siv cov qauv tshuaj hauv vitro thiab cov neeg mob hauv vivo xenografts. Ntxiv mus, Torin-1-mediated mTORC1 / 2 inhibition txhim kho macropinocytosis, thaum verteporfin-induced YAP inhibition ua rau TNBC cell tuag. Thaum muab ua ke, cov txiaj ntsig no qhia txog lub zog thiab kev ua haujlwm ntawm genome-wide hauv vivo CRISPR kev tshuaj ntsuam kom paub txog cov kev kho mob tshiab thiab zoo rau TNBC (Dai et al., 2021).
Kev tsis ua haujlwm ntawm lub cev tiv thaiv kab mob yog ib qho tseem ceeb hauv kev loj hlob ntawm cov qog nqaij hlav. Cov qe ntshav dawb zam kev tshem tawm ntawm lub cev tiv thaiv kab mob los ntawm kev hla dhau cov txheej txheem tiv thaiv, suav nrog kev cuam tshuam rau kev ua haujlwm ntawm cov qe ntshav dawb hauv qhov chaw ib puag ncig ntawm cov qog nqaij hlav thiab ua rau lub cev tiv thaiv kab mob puas tsuaj. Yog li ntawd, kev tsim kom muaj lub cev tiv thaiv kab mob zoo dua yuav yog ib txoj hauv kev tseem ceeb los tawm tsam cov qog nqaij hlav. Kev siv CRISPR/Cas9-raws li kev hloov pauv noob caj noob ces hais txog ntau yam teeb meem ntsig txog kev ua haujlwm tsis zoo ntawm lub cev tiv thaiv kab mob los ntawm ntau qhov kev xav sib txawv. CRISPR/Cas9 tau siv los txhim kho kev tiv thaiv kab mob tiv thaiv kab mob tiv thaiv kab mob qog nqaij hlav mis los ntawm cov hauv qab no (Daim Duab 3).
Daim Duab 3. CRISPR/Cas9 immunotherapy tsom mus rau TNBC cell. Kev poob ntawm CDK5 thiab kev rhuav tshem ntawm PDL1 thiab CD155 ua rau lub cev tiv thaiv kab mob zoo dua. Ib yam li ntawd, kev poob ntawm A2AR thiab kev rhuav tshem ntawm TAAs xws li HER2, mucin 1 thiab TEM8 ua rau CAR-T cell ua haujlwm tau zoo dua hauv kev tua cov cell mob qog noj ntshav. CRISPR/Cas9-tsav T cell kev tshuaj ntsuam tau qhia tias kev cuam tshuam ntawm p38 kinase ua rau T cell ua haujlwm tiv thaiv qog noj ntshav zoo dua. T cell hloov kho los ntawm CRISPR/Cas9 ua rau TCR (T cell receptor) ua haujlwm ntau dua, ua rau T cell muaj kev ua haujlwm tiv thaiv qog noj ntshav.
Lub hom phiaj ntawm kev kho mob tiv thaiv kab mob yog txhawb kom lub cev tiv thaiv kab mob tawm tsam cov hlwb qog noj ntshav. Kev ua ntau dhau ntawm cov protein kuaj xyuas kab mob feem ntau tiv thaiv cov tshuaj tiv thaiv kab mob autoimmune tab sis yuav pab cov qog noj ntshav zam lawv (Topalian et al., 2015). Cov protein no tiv thaiv cov tshuaj tiv thaiv kab mob los ntawm kev khi rau cov receptors ntawm qhov chaw ntawm cov hlwb tiv thaiv kab mob. Ntau cov protein kuaj xyuas (xws li CD155 thiab PD-L1) tsom mus rau PD-1 receptor ntawm cov hlwb tiv thaiv kab mob thiab tau qhia tawm hauv mob qog noj ntshav mis, tshwj xeeb tshaj yog TNBC (Li Y.-C. et al., 2020). Kev rhuav tshem ntawm PD-L1 lossis nws cov receptor siv CRISPR / Cas9 tuaj yeem txhawb kom lub cev tiv thaiv kab mob tawm tsam cov qog TNBC (Yahata et al., 2019). Tsis tas li ntawd, kev txo qis ntawm PD-L1 kev qhia tawm los ntawm CRISPR / Cas9-mediated deletion ntawm CDK5 tau pom tias inhibit qog loj hlob hauv vitro thiab hauv vivo (Deng et al., 2020). Kev txwv tsis pub loj hlob hauv cov qauv hauv vitro thiab hauv vivo ntawm mob qog noj ntshav hauv lub mis qhia tias shRNA-mediated txo ntawm CD155 yuav muaj cov txiaj ntsig kho mob rau mob qog noj ntshav hauv lub mis (Gao et al., 2018).
Cov qe ntshav CAR T qhia txog CARs uas paub txog cov antigens cuam tshuam nrog qog nqaij hlav (TAAs) thiab tuaj yeem siv kev rhuav tshem ntawm cov protein checkpoint los txhim kho lawv cov haujlwm (Li C. et al., 2020). Muaj ntau lub hom phiaj rau kev kho mob CAR T cell hauv mob qog noj ntshav mis, suav nrog ntau TAAs xws li HER2, mucin1, thiab TEM8 (Bajgain et al., 2018). Muaj pov thawj tias CAR T cell tsom mus rau mesothelin (overexpressed hauv TNBC BT-459 cell) muaj txiaj ntsig zoo dua tiv thaiv mob qog noj ntshav thaum PD-1 raug rhuav tshem siv CRISPR/Cas9 (Hu et al., 2019). Txawm li cas los xij, kev cais cov qe ntshav T ntawm cov neeg mob thiab tom qab ntawd kho lawv ex vivo yog ib qho txheej txheem siv zog thiab siv sijhawm ntau. Txawm hais tias cov qe ntshav T thoob ntiaj teb tuaj yeem txo qhov yuav tsum tau cais tawm, cov qe ntshav T pub dawb uas qhia txog tib neeg leukocyte antigen (HLA) chav kawm I thiab T cell receptor (TCR) yuav tsum tau tshem tawm kom tiv thaiv kev hloov pauv ntawm cov qe ntshav-versus-host (Ren et al., 2017). , 2017a). Siv CRISPR/Cas9, HDR tuaj yeem tshem tawm TCR thiab HLA tib lub sijhawm thiab rhuav tshem cov gene encoding CAR. Cov kev tshawb fawb tau qhia tias CRISPR/Cas9 tuaj yeem siv los qhia cov anti-CD19 CARs rau hauv TCR locus, yog li ntawd tsim cov CARs zoo yam tsis ua rau cov T cell puas tsuaj (Dimitri et al., 2022). Cov thev naus laus zis Multiplex tau tsim los tsim cov allogeneic CAR T cells los ntawm kev tshem tawm TCR, beta-2-microglobulin (B2M), HLA-I subunit thiab lwm cov protein xws li PD-1 thiab CTLA-4, uas yuav muaj cov khoom tiv thaiv kab mob qog noj ntshav ntau dua. tawm tsam TNBC (Eyquem et al., 2017; Liu et al., 2017; Ren et al., 2017b; Dimitri et al., 2022).
Siv CRISPR/Cas9 tuaj yeem txhim kho qhov ua tau zoo ntawm CAR-T hlwb. Adenosine paub tias muaj cov khoom tiv thaiv kab mob thiab tuaj yeem txo qhov kev tiv thaiv kab mob qog noj ntshav los ntawm kev thaiv T hlwb ua haujlwm thiab ua kom adenosine A2A receptors (A2AR) ua haujlwm (Vigano et al., 2019). Kev ntsiag to A2AR siv CRISPR/Cas9 tau txhim kho qhov ua tau zoo ntawm CAR-T hlwb hauv vivo (Giuffrida et al., 2021). T hlwb paub tias muaj ntau yam ntxwv phenotypic xws li kev nthuav dav ntawm tes, kev sib txawv, kev ntxhov siab oxidative thiab kev ntxhov siab genomic. CRISPR/Cas9-raws li T hlwb tshuaj ntsuam tau qhia txog kev cuam tshuam ntawm 25 hom T hlwb receptor kinases sib txawv. Ntawm lawv, kev tshem tawm ntawm p38 kinase tau pom tias txhim kho qhov ua haujlwm tiv thaiv kab mob ntawm T hlwb, qhia tias p38 kinase yog tus tswj hwm tseem ceeb ntawm CAR-T hlwb tswj (Gurusamy et al., 2020).
Cov qe ntshav T tuaj yeem hloov kho caj ces siv CRISPR/Cas9 los tsim cov TCRs uas qhia tau zoo heev. Kev hloov cov qe ntshav T uas hloov kho caj ces mus rau cov neeg mob tau qhia tias muaj zog tiv thaiv kab mob qog noj ntshav dua li cov qe ntshav T endogenous. Yog li ntawd, cov TCRs endogenous yuav sib tw nrog cov TCRs uas hloov kho caj ces hauv cov neeg mob, uas yuav cuam tshuam rau lub peev xwm ntawm kev kho mob qog noj ntshav. Txhawm rau kov yeej qhov teeb meem no, kev siv CRISPR/Cas9 los tshem tawm cov TCR-β endogenous hauv cov qe ntshav tau txais thiab tom qab ntawd hloov cov qe ntshav TCR-β T mus rau cov neeg mob qog noj ntshav yuav ua rau muaj kev tiv thaiv kab mob qog noj ntshav zoo dua yam tsis tas yuav muaj kev sib tw sib deev TCR endogenous (Fan et al. 2018). Yog li, tshwj tsis yog cov qe ntshav CRISPR-modified T, TCRs muaj kev rhiab heev rau cov antigens qog noj ntshav ntau dua li cov qe ntshav TCR-transduced ib txwm muaj. Tsis tas li ntawd, hauv ntau yam kab mob leukemias, cov qe ntshav T hloov kho uas tsim los ntawm γδ TCR+ CRISPR qhia txog kev qhia ntau dua ntawm CD4+ thiab CD8+ T qe ntshav dua li kev hloov pauv TCR tus qauv (Legut et al., 2018). Yog li, cov qe ntshav T hloov kho uas tsim los ntawm CRISPR / Cas9 yuav yog ib txoj kev kho mob zoo los kov yeej TNBC.
Cov Integrins yog cov molecules adhesion ntawm cell uas muaj nyob rau hauv cellular transmembranes thiab txhawb kev khi ntawm cov cell rau extracellular matrix (ECM) (Hamidi thiab Ivaska, 2018). Kev tsis sib haum xeeb ntawm integrins yog txuam nrog kev loj hlob ntawm mob qog noj ntshav thiab kev tsiv teb tsaws chaw los ntawm kev hloov pauv ntawm extracellular matrix, ua rau cov cell mob qog noj ntshav muaj sia nyob hauv kev ncig (Hamidi thiab Ivaska, 2018). CRISPR/Cas9-mediated integrin knockdown ua rau qeeb qeeb ntawm qog noj ntshav, metastasis, thiab colonization hauv TNBC. Knockdown ntawm integrin a5 (ITGA5) tau tshaj tawm tias txo cov cell migration thiab kev nce qib hauv lwm yam mob qog noj ntshav xws li mob qog noj ntshav ntsws (Ju et al., 2017), qhia tias integrin a5 kuj tseem yuav yog qhov tseem ceeb hauv pathogenesis ntawm TNBC.
Kev tsim cov nas knockout siv cov txheej txheem embryonic stem (ES) cell ib txwm yog ib qho txheej txheem siv zog ntau, siv sijhawm ntau, thiab tsis ua haujlwm zoo. Nws siv sijhawm ntau lub hlis thiab xyoo los tsom mus rau ES cell los ntawm kev sib xyaw ua ke, yug cov nas chimeric, thiab tom qab ntawd hla lawv nrog cov nas heterozygous los tsim cov xeeb ntxwv homozygous. Kev sib xyaw ua ke nyuaj yog qhov yuav tsum tau los tsim cov nas nrog ntau qhov kev hloov pauv caj ces. Txawm li cas los xij, ntau yam teeb meem tau tshwm sim thaum siv cov nas tsim los ntawm kev tshem tawm ntawm ES cell siv CRISPR / Cas9 lossis los ntawm microinjecting CRISPR / Cas9 Cheebtsam rau hauv ib lub qe fertilized ib lub cell. Piv txwv li, kev qhia txog kev hloov pauv feem ntau tshwm sim ntawm biallelic loci thiab tsis yog gene tshwj xeeb. Tsis ntev los no nws tau tshaj tawm tias ES cell siv CRISPR / Cas9 thev naus laus zis tuaj yeem ntxig biallelic mutations hauv txog li 5 gene (Nishizono et al., 2021). Rau lub hom phiaj no, Cas9 mRNA thiab tsib gRNAs tshwj xeeb gene tau transfected tib lub sijhawm rau hauv ES cell. Qhov no qhia txog qhov kev cog lus thiab kev ua haujlwm ntawm cov txheej txheem no, txawm hais tias cov kev hloov pauv no tej zaum yuav tau dhau los thaum cov kab tsim tau yug los tsim cov nas knockout quintuple. Kev tshawb fawb kuj tshaj tawm txog kev tshawb pom xav tsis thoob: ES hlwb tsis tas yuav tsim cov nas hloov pauv caj ces. Hloov chaw, kom tshem tawm cov khoom tshwj xeeb gene, Cas9 mRNA thiab gRNA tau txhaj rau hauv cov embryos theem ib-cell. Yog li ntawd, cov kab tsim knockout tau tsim uas tuaj yeem siv los kawm txog cov txiaj ntsig ntawm kev rho tawm gene hauv nas (Qin et al., 2016). Yog li, CRISPR / Cas9 tso cai rau kev tsim cov nas transgenic los kho TNBC ntawm tus nqi qis dua li kev tsim kho caj ces ib txwm muaj. Siv CRISPR / Cas system, tus qauv nas knockout tshiab tau tsim uas tuaj yeem ua tiav cov kev hloov pauv hauv ib lossis ntau cov noob endogenous hauv TNBC. Raws li lub tswv yim no, cov qauv tsiaj ntawm TNBC kuj tseem tuaj yeem tsim tau siv CRISPR / Cas9-mediated deletion ntawm BRCA1 thiab p53, ua rau poob ntawm HR kho, genomic tsis ruaj khov, thiab mutant phenotypes (Annunziato et al., 2020).
Tam sim no, ntau txoj kev xws li mammography, magnetic resonance imaging (MRI) thiab ultrasound siv los kuaj mob TNBC. Txawm li cas los xij, cov txheej txheem no muaj qee qhov kev txwv. Mammography siv los kuaj mob cov nqaij mos hauv zos es tsis yog metastases, qhov twg cov hlwb qog nqaij hlav tau tsiv mus rau lwm yam kabmob. Ultrasonography tsis yog txoj kev txhim khu kev qha rau kev kuaj mob TNBC (Chen thiab Lee-Felker, 2023). MRI muaj kev nkag siab ntau dua li ultrasound thiab mammography, tab sis nws qhov kev kuaj mob raug txwv (Sha thiab Chen, 2022). Kev kuaj mob nqaij yog ib txoj hauv kev nkag mus rau hauv kev txheeb xyuas cov hlwb qog nqaij hlav. Txawm li cas los xij, qee zaum, kev kuaj mob nqaij yuav plam cov nqaij qog nqaij hlav yog tias lub koob mus deb ntawm thaj chaw uas xav tau. Tsis tas li ntawd, nws kim heev thiab tuaj yeem ua rau tus neeg mob raug mob. TNBC yog hom mob qog nqaij hlav sib txawv, yog li kev kuaj mob nqaij yuav tsis muab cov ntaub ntawv txaus txog hom mob qog nqaij hlav. Yog li ntawd, txoj kev kuaj mob tshiab txhim khu kev qha rau kev kuaj mob TNBC yog qhov xav tau sai. CRISPR/Cas9 tej zaum yuav ua haujlwm ua lwm txoj hauv kev uas muaj kev nkag siab zoo thiab tsawg kawg nkaus rau kev kuaj mob qog noj ntshav hauv lub mis. Txog qhov kawg no, CRISPR/Cas9-raws li cov tswv yim tuaj yeem siv los txhim kho PCR txoj hauv kev rau kev kuaj mob TNBC. Ua ntej, cov protein Cas9 thiab cpf1 ntawm CRISPR system yog siv los tshem tawm cov DNA tsis tshwj xeeb, thiab tom qab ntawd ob cov protein no (Cas9 thiab cpf1) tuaj yeem paub txog PAM kab ke ua ntej khi rau lub hom phiaj DNA (Deepak Singh et al., 2021). Yog li, PCR tuaj yeem txheeb xyuas cov kev hloov pauv cuam tshuam nrog kev loj hlob ntawm qog noj ntshav. Ntau txoj kev tshawb fawb tau siv txoj hauv kev CRISPR no los nrhiav cov kev hloov pauv sib txawv hauv cov qog noj ntshav sib txawv (Safari et al., 2019). Yog li, CRISPR-raws li PCR txoj hauv kev tuaj yeem txo qhov kev vam khom ntawm TNBC kev kuaj mob ntawm cov txheej txheem nkag mus xws li biopsy-based immunohistochemistry.
Kev hloov pauv genotypic hauv kev tswj hwm cov tshuaj hormones receptor kuj tau pom hauv TNBC (Chen thiab Russo, 2009). CRISPR/Cas9-raws li PCR cov tswv yim siv microarrays rau qhov chaw kuaj mob tuaj yeem saib xyuas cov kev hloov pauv no tau zoo (Hajian et al., 2019). Nws kuj tseem tuaj yeem muab cov kws kho mob nrog cov ntaub ntawv tseem ceeb txog kev hloov pauv tshwj xeeb hauv cov neeg mob TNBC, uas tuaj yeem pab txhim kho lawv txoj kev kho mob. Txawm li cas los xij, cov txheej txheem ua ke no muaj kev txwv. Yog li ntawd, kev tshawb fawb ntau yam yuav tsum tau ua ntej txoj kev CRISPR/Cas-PCR no tuaj yeem siv rau hauv kev kho mob rau kev kuaj mob TNBC (Yang et al., 2019).