Qhov sib txawv tseem ceeb ntawm crRNA tracrRNA thiab gRNA yog tias crRNA yog ib qho ntawm ob hom RNA ntawm CRISPR, uas yog ntxiv rau lub hom phiaj DNA ib ntus, thaum tracrRNA yog hom thib ob ntawm RNA ntawm CRISPR, uas ua haujlwm raws li Ib qho kev sib txuas scaffold rau Cas nuclease thiab gRNA yog ib qho ntawm ob lub ntsiab ntawm CRISPR-Cas9 system ntawm cov kab mob thiab archaea uas paub txog lub hom phiaj DNA thiab coj cov Cas proteins kom ua ob txoj hlua tawg hauv lub hom phiaj DNA.
CRISPR sawv cev rau kev sib txuas tsis tu ncua luv luv palindromic repeats. Nws yog ib qho ntawm cov kab mob hauv cov kab mob thiab archaea. Cov kab mob thiab archaea siv cov kab ke no los tiv thaiv lawv tus kheej los ntawm cov kab mob kis. Nws yog ib qho ntawm cov txheej txheem zoo tshaj plaws uas cuam tshuam nrog kev hloov kho caj ces zoo. Cov txheej txheem no (CRISPR-Cas9) yog siv los ua cov cuab yeej tshiab hloov kho noob hauv Biotechnology. Nws tau coj cov kev hloov tshiab tshiab rau biotechnology, tshwj xeeb tshaj yog nyob rau hauv kev lag luam ntawm noob caj noob ces.
Muaj ob lub ntsiab lus tseem ceeb ntawm CRISPR-Cas9 system. Lawv yog cov qhia RNA (gRNA) thiab CRISPR-associated (Cas) nucleases. Qhia RNA yog ib qho RNA ib ntus uas coj Cas cov proteins kom tshem tawm cov hom phiaj DNA. Qhia RNA muaj ob hom RNA. Lawv yog crispr RNA (crRNA) thiab tracrRNA. CRISPR-associated (Cas) nucleases yog non-specific endonucleases uas ua rau ob txoj hlua tawg hauv lub hom phiaj DNA. Los ntawm kev ua ob txoj hlua tawg hauv lub hom phiaj DNA, cov kab mob thiab archaea coj lawv cov txheej txheem kho endogenous los ua kom tsis muaj kab mob DNA.
CrRNA yog dab tsi?
crRNA lossis Crispr RNA yog ib qho ntawm ob hom kev qhia RNA. Raws li txoj cai, nws yog 17-20 nucleotide ib ntus. Qhov tseem ceeb tshaj plaws ntawm crRNA yog tias nws ua tiav rau lub hom phiaj DNA. Yog li ntawd, crRNA phim nrog cov kab mob DNA sib lawv liag. Qhov tshwj xeeb ntawm CRISPR-Cas 9 system nyob ntawm crRNA. Hauv cov kab mob, crRNA muaj fused rau tracrRNA ib ntus, uas yog hom thib ob ntawm CRISPR RNA. crRNA ntau lawm yog tshwm sim los ntawm qhov rov kis tau tus kab mob mus rau tus kab mob. Thaum pom, transcription ntawm cov noob uas yog coding rau crRNA tshwm sim. Tom qab ntawd, cov txheej txheem tiv thaiv pib tawm tsam tus kabmob.
Daim duab 01: CRISPR-Cas9 System
tracrRNA yog dab tsi?
Trans-activating crRNA lossis tracrRNA yog qhov thib ob ntawm guideRNA lossis CRISPR RNA. Nws yog pronounced li tracer RNA. tracrRNA ua hauj lwm raws li kev khi scaffold rau endonuclease Cas 9 protein. Hauv lwm lo lus, tracrRNA ua haujlwm ua tus kov coj Cas9 mus rau lub hom phiaj DNA. Raws li txoj cai, tracrRNA muaj 42 nucleotides. Nws tshwm sim ua ke nrog crRNA.
gRNA yog dab tsi?
Guide RNA lossis gRNA yog ib qho ntawm ob qhov tseem ceeb ntawm CRISPR-Cas9 lub cev tiv thaiv kab mob. Nws yog ib qho RNA ib ntus uas muaj ob lub ntsiab lus: crRNA thiab tracrRNA. gRNA lees paub lub hom phiaj DNA thiab coj Cas cov proteins kom ua ob txoj hlua tawg hauv lub hom phiaj DNA. Txhawm rau ua nws, crRNA muaj qhov sib ntxiv ntawm lub hom phiaj DNA thaum tracrRNA qhia Cas cov protein ua haujlwm ua tus kov.
Daim duab 02: gRNA
Tsim qhov tseeb gRNA yog ib kauj ruam tseem ceeb hauv CRISPR-Cas9 gene-editing tool. Yog li ntawd, kev ua tiav thiab kev kho kom zoo ntawm CRISPR system nyob ntawm seb qhov tseeb ntawm gRNA. gRNA tuaj yeem nthuav tawm hauv cov hlwb los ntawm kev hloov pauv plasmid. Thaum cloned plasmids tau nkag mus rau hauv cov hlwb, cov hlwb ua haujlwm tsim gRNA. Qhov feem ntau tsim gRNA suav nrog 100 lub hauv paus khub.
Dab tsi yog qhov zoo sib xws ntawm crRNA tracrRNA thiab gRNA?
- crRNA, tracrRNA thiab gRNA yog RNA sequences pom nyob rau hauv cov kab mob thiab archaea.
- Lawv txhua tus koom nrog CRISPR system.
- gRNA yog tsim los ntawm crRNA thiab tracrRNA.
- Lawv koom nrog kev lees paub cov kab mob bacteriophage DNA thiab coj cov endonucleases rau lub hom phiaj DNA.
- Vim li no, tag nrho peb hom RNA tau koom nrog hauv kev coj ua Cas9 nuclease kom ua ob txoj hlua tawg hauv kev cuam tshuam cov khoom siv txawv teb chaws.
- Kev vam meej ntawm CRISPR kev sim yog nyob ntawm txhua peb hom RNA.
Qhov txawv ntawm crRNA tracrRNA thiab gRNA yog dab tsi?
crRNA yog ib feem ntawm gRNA ntawm CRISPR uas yog ntxiv rau lub hom phiaj DNA, thaum tracrRNA yog qhov thib ob ntawm gRNA, uas ua haujlwm los ua ib qho kev khi rau Cas nuclease.gRNA yog kev sib xyaw ua ke ntawm crRNA thiab tracrRNA, thiab nws yog ib qho ntawm ob feem ntawm CRISPR-Cas9 system, uas lees paub lub hom phiaj DNA thiab coj cov nucleases los ua ob txoj hlua tawg hauv lub hom phiaj DNA. Yog li, qhov no yog qhov sib txawv tseem ceeb ntawm crRNA tracrRNA thiab gRNA.
Cov lus hauv qab no qhia txog qhov sib txawv ntawm crRNA tracrRNA thiab gRNA.
Summary – crRNA vs tracrRNA vs gRNA
CRISPR-Cas9 system yog ib qho kev tiv thaiv kab mob ntawm cov kab mob thiab archaea. Cov txheej txheem no yog siv los ua cov cuab yeej hloov kho noob hauv Biotechnology. CRISPR muaj ob hom RNA, uas yog CRISPR RNA (crRNA) thiab transactivating CRISPR RNA (tracrRNA). Lawv raug hu ua guideRNA lossis gRNA. Qhov RNA no lees paub lub hom phiaj ua ntu zus ntawm cov kab mob nkag mus thiab coj cov endonucleases kom ua ob txoj hlua khi hauv lub hom phiaj DNA. Thaum ob-strand so yog ua nyob rau hauv txawv teb chaws genetic khoom, endogenous kho mechanism (nonhomologous kawg koom; NHEJ) inactivates txawv teb chaws DNA los ntawm kev qhia kev hloov. Yog li, qhov no qhia txog qhov sib txawv ntawm crRNA tracrRNA thiab gRNA.
