ä¸é¡¹DNAç¼è¾ææ¯æ¯å¦æä¼å¿æ¶åå°æ¹æ¹é¢é¢ãä»ç®åçæ
åµæ¥çï¼NgAgoçä¼å¿å¾ææ¾ï¼ä¸»è¦ä½ç°å¨
1ï¼ååºæçä¸ä½ï¼ä¸è¯´é«ï¼ä½ä¸ä½ï¼
2ï¼ç¹å¼æ§è¾å¥½ï¼ä½æ人å¼ç¨è¯´å 为gDNAæ¯Cas9çgRNA é¿5ntï¼æ以精确度æé«1024åï¼è¿ä¸ªâç论ä¸âç4^5ä¸å¾ä¸è¯´æ¯å±å¤´ï¼å 为è±é¶çï¼ç²¾åº¦ï¼ä¸æ¯è¿ä¸ªææï¼è¿æ ·å¤è¡ç计ç®æ¯ä¸è¯¥çï¼
3ï¼ä¸ä¾èµPAM
4ï¼NgAgoæ¬èº«ååéä¸å¤§
å¦å¤è¿æä¸äºæç« ä¸ä½ä¸ºä¼å¿ï¼ä½ä¹å¯è½æ¯å£å¿çæ§è´¨ï¼æ¯å¦ä½¿ç¨ssDNAä»å¯¼ï¼æé«ç¹å¼æ§åæ¶ä¼å¸¦æ¥å¾å¤é®é¢ï¼ä¸è®ºå¦ä½ä¼å¿æ¯ææ¾çï¼ä½æ¯ä»
ä»
è¿äºæ¹é¢å¹¶ä¸å¤ï¼è¿éè¦ç 究æ´å¤é®é¢ï¼æ¯å¦ç¹å¼æ§å¥½æ¯æéå®éªåºç¡ä¸çç论ç»è®ºï¼å®é
åºç¨è±é¶çå¦ä½è¦çå
·ä½æ
åµã
ç®åæ¥çæå 项äºå
³è¿é¡¹ææ¯æªæ¥çãä¼å
å¾
éªè¯çé®é¢æ¯ï¼
1ï¼ç³»ç»æ£äº¤æ§
2ï¼å¤ä½ç¹ç¼è¾è½å
3ï¼NgAgoèç½ç模ååç¨åº¦ï¼å·¥ç¨åæ¹é çæ½åï¼
4ï¼ssDNAçéç¨æ§å缺ç¹
ä¸è¦å°çä¸è¿°é®é¢ï¼æ¡æ¡é½æ¯å¡èåçï¼ä¸æ¡ä¸ç»åå°±ä¼è¢«CRISPRæ¯ä¸å»ãå¾å¤äººç¥éCRISPRå¾ç«ï¼ä¹ç¥éCRISPRæ¯å¾å¥½çåºå ç»ç¼è¾å·¥å
·ï¼å®¹æ以为åºå ç¼è¾å·¥å
·æ¬èº«æ¯å¾ççï¼å®é
ä¸CRISPRä¹æ以ç«ï¼è¿æå¾å¤§ä¸é¨ååå å¨äºé«æ£äº¤æ§ãé«éç¨æ§ãé«å·¥ç¨åæ½å带æ¥ç广æ³åºç¨ï¼è¯¸å¦è½¬å½åè°æ§ã人工TFççãæ ¸é
¸å®ç¹å
åæ¯ä¸ä¸ªåºç¡æ§è´¨ï¼å¸¦æ¥äºä¸è¿°æ§è´¨çå¯è½æ§ï¼éè¦ç»è¿éªè¯æè½å°±æ¯å¦å¯ä»¥ä½ä¸ºCRISPRçæ¿ä»£ææ¯æä¸ä¸ªç»è®ºã
æ¯å¦å¨ä¸åç©ç§ç»èç³»ç»ä¸çæ£äº¤æ§å¦ä½ï¼ç®åç¥éAgoå¨çæ ¸å°åæ ¸ç»èä¸å¹¿æ³åå¨åæºåºå ï¼è¿ä¸ªå¨æç« ä¸æ¯ä½ä¸ºæç§ä¼å¿æ¥è®²çï¼ä½å®é
ä¸å¯è½æ¯ä¸ä¸ªå£å¿ãå 为æ£äº¤æ§é®é¢ï¼å¨äººç±»ç»èä¸å¥½ç¨æªå¿
å¨å
¶å®ç©ç§ä¸å¥½ç¨ã大家å¯è½æ³¨æå°äºï¼æç« å¼ç¯ç¬¬äºä¸ªå®éªå°±æ¯åäºä¸ä¸ªç®åçæ£äº¤æ§æµè¯ï¼æµè¯ç»æè¿ä¸éï¼æä¸æå°
which implies that there is very limited 5 â² phosphorylated ssDNA present in human cells, suggesting that endogenous ssDNAs will not mislead NgAgo to off-target sites
è¿å½ç¶ä¸è¶³ä»¥è¯´æNgAgoå
·æé«æ£äº¤æ§ï¼ä½è
ä¹ç¨äºimpliesçæªè¾ï¼è¿æå¾
è¿ä¸æ¥è¯å®ãå¯ä»¥ä»æä¸çåºï¼ä½è
ææ
å¿çé®é¢å
¶å®ä¹æ¯ç³»ç»æ£äº¤æ§ï¼å¨æç« æåï¼ä½è
ç¹æ强è°éè¦è¿ä¸æ¥çé«ééå®éªæ¥ç 究è¿ä¸ªé®é¢ï¼å¯è§ä½è
并é没ææè¯å°ï¼èæ¯åéäºæ¶é´æå
¶å®ç§ç æ¡ä»¶ã
é¤æ¤ä¹å¤ï¼å¤ä½ç¹ç¼è¾è½åæ¯CRISPRçä¸é¡¹é大ä¼å¿ãæ们ç°å¨ç¥éNgAgoåssDNAçç»åæ¯é常稳å®çï¼å¨37âæ¡ä»¶ä¸ä¸å¯éï¼ï¼è¿å½ç¶æ¯æç§æé«ç¹å¼æ§çä¼å¿ï¼ä½ç¨³å®ä¹æ¯åååï¼æå¯è½æ为å£å¿ãæä¸æå°ï¼
similar to mammalian Ago2, which cannot exchange its bound oligos with free oligos at 37 °C
è¿æ ·çæ§è´¨æ¯å¦ä¼å½±åå°NgAgoå¨å¤ä½ç¹ç¼è¾ä¸çååºæçä¸å¾èç¥ãæçå°å¤§é¨åè¯è®ºé½æ²¡ææå°è¿ä¸ªé®é¢ã
CRISPRè¿æä¸ä¸ªäº®ç¹æ¯Cas9ç模ååæ§è´¨ãå¯ä»¥å®¹è®¸è¿è¡è¾å¤çèç½å·¥ç¨æ¹é ï¼NgAgoæ¯å¦æè¿æ ·çé«åº¦æ¨¡ååçæ§è´¨ä¹å°ç´æ¥å³å®è¿é¡¹ææ¯è½å¦é®é¼ä¼ç§DNAç¼è¾ææ¯çå°ä½ãå¦æ对èç½è´¨å·¥ç¨è®¾è®¡çå¯æ©å±æ§ä¸å¥½ï¼é£ä¹å
¶åºç¨å¾æå¯è½åéã
æåè¿æssDNAå¨åç§ç»èä¸å¦ä½ä½¿ç¨çé®é¢ï¼æ们ç°å¨ç¥éCRISPRç³»ç»å¸¸å¯¼å
¥gRNAæéè¿sgRNA transcription vectoræ¥å¶é ä»å¯¼æ ¸é
¸ï¼DNAæ¾ç¶ä¸è½ç¨è¿æï¼ç´æ¥å¯¼å
¥DNAæ¬èº«å°±éå¶äºNgAgoå¨ä¸å¤§ç±»ç©ç§å½ä¸çåºç¨æ½åãæåå¦å±æ天ç¶ç5'p-ssDNAçå å·¥æºå¶æªæ¥å¯ä»¥å©ç¨ï¼ç¶èæç« ä¸æå°äººç±»ç»èä¸5'p-ssDNA并ä¸ä¸°å¯ãè¿æå©äºç³»ç»æ£äº¤æ§ï¼å´ä¹æ示ssDNAç使ç¨æå¯è½æ为è¿é¡¹ææ¯çä¸ä¸ªç¶é¢ãæä¸ä½ç主说ssDNAçç¨éåªè¦CRISPRææ¯ä¸RNAç¨éç1/10ï¼è¿æ¾ç¶æ¯è±æä¸å¥½é æäºè¯¯è¯»ï¼æç« ä¸åææ¯ï¼
if the sgRNA is expressed from a plasmid and the normal dosage of an ssDNA guide is only ~1/10 of that of a sgRNA expression plasmid
å¯è§ï¼ç¹å¼æ§ssDNAå¨è¿é¡¹å®éªä¸æ¯å¯ä»¥éè¿ä½¿ç¨æµåº¦é¥±åæ¥å¢å¼ºNgAgoçç¹å¼æ§çï¼ä¸ç ´åæ£äº¤æ§çssDNAç«äºæ§ç»åNgAgoï¼ï¼ä½æ¯è¿ä¸ªrobustnessæ¯ä¸æåååãDNAååæ¬èº«ç稳å®æ§ãç³»ç»çé²æ£æ§å使ç¨æ¹å¼ä¼å½±åè¿ä¸ªç³»ç»çå¯æ§æ§ãä¸æ¦ç³»ç»å¯æ§æ§ä¸å¥½ï¼åºæ¬ä¸ä¼è¢«æéè¦ççç©å»çé¢åæé¤å¨å¤ã
温馨提示:答案为网友推荐,仅供参考