CRISPR/Cas£¨Clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein£©ÏµÍ³ÊÇÒ»ÖÖÔ´ÓÚϸ¾úºÍ¹Åϸ¾úµÄ¿¹²¡¶¾»ñµÃÐÔÃâÒß»úÖÆ¡£¡£¡£¡£CRISPR/CasϵͳÒÔRNAÂѰ×Öʸ´ºÏÌåµÄÐÎʽʩչÃâÒß×÷Ó㬣¬£¬ÆäÖÐЧӦÂѰװüÀ¨Cas9£¬£¬£¬Cas12a£¬£¬£¬Cas12b£¬£¬£¬CasXµÈ¡£¡£¡£¡£¾¿ÆÑ§¼Òˢн«crRNAºÍtracrRNAºÏ¶þΪһÐγɵ¥Á´Ö¸µ¼RNA£¨guide RNA£¬£¬£¬gRNA£©¡£¡£¡£¡£CasÂѰ×Ê×ÏÈʶ±ð»ùÒò×éÉϵÄPAM(Protospacer adjacement motif)ÐòÁУ¬£¬£¬È»ºógRNA ÌØÒìÐÔʶ±ð²¢Óë°Ðλµã»¥²¹Åä¶Ô£¬£¬£¬´Ó¶ø¼¤»îCasµ°º×ʩչºËËáÄÚÇÐøµÄ¹¦Ð§ÇиîDNAË«Á´£¬£¬£¬ÊµÏÖÌØ¶¨Î»µãµÄ»ùÒò±à¼¡£¡£¡£¡£×Ô¿ª·¢³ÉΪÓÐÓõĻùÒò±à¼¹¤¾ßÒÔÀ´ÔÚÖÖÖÖÉúÎïÌåÖлñµÃÁËÆÕ±éµÄÓ¦Ó㬣¬£¬À´×Ô»¯Å§Á´Çò¾ú(Streptococcus pyogenes, Sp)µÄCRISPR/Cas9ϵͳÊÇÆäÖÐÓ¦ÓÃ×îÆÕ±éµÄ»ùÒò±à¼¹¤¾ß¡£¡£¡£¡£È»¶ø£¬£¬£¬ÓÉÓÚÈËÀàÒÅ´«±äÒì¶àΪµãÍ»±ä¡¢²åÈë»òȱʧ£¬£¬£¬¶øCRISPR/Cas9ϵͳͨ¹ýÍ¬Ô´ÖØ×éµÄ·½Ê½ÊµÏÖ׼ȷµÄ¼î»ùÐÞ¸´µÄЧÂʵÍÏ£¬£¬£¬ÇÒÔÚÐÞ¸´Àú³ÌÖлᵼÖÂË«Á´ËðÉËÒý·¢ÍѰзçÏÕ£¬£¬£¬ÏÞÖÆÁËCRISPR/Cas9ϵͳÔÚ׼ȷµÄ¼î»ùÐÞ¸´ÁìÓòµÄÓ¦Óà (Science 2012; Science 2013; Cell 2017; Nature Biotechnology 2020)¡£¡£¡£¡£
ÔÚCas9 H840AÇпÚø(Cas9n-H840AÇпÚø)µÄC¶ËÈÚºÏÄæ×ªÂ¼Ã¸£¬£¬£¬²¢ÔÚÏòµ¼RNA(sgRNA)µÄ»ù´¡ÉϾÙÐÐˢУ¬£¬£¬Ê¹Æä°üÀ¨Ò»¶ÎÓÕµ¼Ä¿µÄ¼î»ùÍ»±äµÄRTÄ£°åÒÔ¼°ÒýÎïÁ¬ÏµÎ»µã(primer binding site£¬£¬£¬PBS)£¬£¬£¬´Ó¶ø¹¹½¨»ñµÃÖ¸µ¼±à¼ÊÖÒÕµÄÏòµ¼RNA(prime editing guide RNA£¬£¬£¬pegRNA£¬£¬£¬»ñµÃÄ¿µÄPrime editing£¨PE£©ÏµÍ³¡£¡£¡£¡£PEϵͳͨ¹ýpegRNA°ÐÏò»ùÒò×éÄÚÌØ¶¨Î»µã£¬£¬£¬¶ø±»Cas9n-H840AÇпÚøÇжϵķǻ¥²¹Á´ÓëpegRNAЯ´øµÄPBS½á¹¹Á¬Ïµ£¬£¬£¬ÔÚÄæ×ªÂ¼Ã¸×÷ÓÃÏ£¬£¬£¬Ê¹ÓÃÄæ×ªÂ¼Ä£°å£¨RT template£©¾ÙÐÐÐÞ¸´£¬£¬£¬´Ó¶øÔÚ°ÐλµãʵÏÖÌØ¶¨¼î»ùµÄת»»¡¢²åÈëºÍȱʧ (Nature 2019)¡£¡£¡£¡£
µ«ÓÉÓÚPE±à¼ÏµÍ³µÄÂѰױàÂëÐòÁг¤´ï6.2kb£¬£¬£¬Òò´ËÎÞ·¨¸ßЧµØ×°Ôص½µ¥¸ö×°ÔØÈÝÁ¿½öΪ4.7kbÏÙÏà¹Ø²¡¶¾£¨adeno-associated virus£¬£¬£¬AAV£©£¬£¬£¬½ø¶øÏÞÖÆÆäÔÚÌåÄÚÖÎÁÆÑо¿¡£¡£¡£¡£Òò´ËսʤAAV²¡¶¾µÄ³ÐÔØÄÜÁ¦ÏÞÖÆ£¬£¬£¬ÌåÄڳʵݸ߻îÐÔµÄÖ¸µ¼±à¼Æ÷ÊÇÏÖÔÚØ½´ý½â¾öµÄÄÑÌâ (Nature 2019; Nature Reviews Genetics 2020)¡£¡£¡£¡£
Ϊ½â¾ö¸ÃÄÑÌ⣬£¬£¬ngµç×ÓÓÎÏ·ÉúÃü¿ÆÑ§Ñ§Ôº»Æ¾ü¾Í½ÌÊÚ¿ÎÌâ×齫PE¿ÆÑ§µØ²ð·ÖÖÁÁ½¸ö²î±ðµÄAAVÉÏ£¬£¬£¬Í¨¹ýÄÚº¬ëÄ£¨intein£©Ê¹ËüÃÇÄÜÔÚϸ°ûÄÚÖØÐºϲ¢³ÉÈ«³¤¾ßÓй¦Ð§µÄPEÂѰף¬£¬£¬¹¹½¨Ë«AAV½éµ¼µÄSplit-PEϵͳ¡£¡£¡£¡£Ê×ÏÈÔÚÌåÍâÀÖ³Éɸѡ³ö¾ßÓи߻îÐÔµÄSplit-PE1024±äÌå, ²¢Í¨¹ýË«AAV8»®·Ö°ü×°split-PE1024µÄN¶ËºÍC¶Ë(»®·Ö´øÓÐpegRNAºÍgRNA)£¬£¬£¬Í¨¹ýÊÓÍøÄ¤ÏÂÇ»×¢ÉäÔÚÊÓÍøÄ¤ÇøÓòʵÏְлùÒòDnmt1 (p.P55Q, c.G164T)µÄ¼î»ùµß»»¡£¡£¡£¡£Í¨¹ýÉî¶È²âÐò¼ì²â°ÐλµãµÄ±à¼Ð§ÂÊ£¬£¬£¬Ð§¹û·¢Ã÷CMVÆô¶¯×ÓºÍEF1¦Á½¹µãÆô¶¯×ÓÇý¶¯µÄsplit-PE1024¶¼ÀÖ³ÉÔÚÊÓÍøÄ¤Dnmt1»ùÒòλµãʵÏÖ+5 G-to-TµÄ¼î»ùµß»»¡£¡£¡£¡£

ͼ1£ºË«AAV½éµ¼Split-PE1024ÓÕµ¼Ð¡ÊóÑÛµ×ÊÓÍøÄ¤µãÍ»±ä
Ñо¿Ð§¹û¡°Dual-AAV delivering split prime editor system for in vivo genome editing¡±½üÆÚ½ÒÏþÔÚMolecular TherapyÔÓÖ¾¡£¡£¡£¡£¸ÃÑо¿¹¹½¨µÄË«AAV½éµ¼µÄSplit-PEϵͳÀÖ³ÉÔÚСÊóÊÓÍøÄ¤ÊµÏÖ¾«×¼µÄÌåÄڱ༣¬£¬£¬ÎªÒÅ´«²¡µÄÌåÄÚÖÎÁÆÌṩÓÐÓõŤ¾ß¡£¡£¡£¡£
ngµç×ÓÓÎÏ·ÉúÃü¿ÆÑ§Ñ§Ôº»Æ¾ü¾Í½ÌÊÚºÍÁºÆÕƽ¸±½ÌÊÚΪ¸ÃÂÛÎĵÄÅäºÏͨѶ×÷Õߣ¬£¬£¬²©Ê¿Éú֧ʤҢºÍ³ÂêÅÙÒ²©Ê¿ÎªÂÛÎÄÅäºÏµÚÒ»×÷Õß¡£¡£¡£¡£¸ÃÑо¿»ñµÃ¹ú¼ÒÖØµãÑз¢ÍýÏë¡¢¹ú¼Ò×ÔÈ»¿ÆÑ§»ù½ðµÈÏîÄ¿×ÊÖú¡£¡£¡£¡£
ÂÛÎÄÁ´½Ó£ºhttps://doi.org/10.1016/j.ymthe.2021.07.011