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鏂囧簱璇存槑

涓銆佷骇鍝佺粍鎴

聽Edi-HTS鈩 KPP鎱㈢梾姣掗绮

鐥呮瘨婊村害锛2Xl08TU/ml锛涜鏍硷細200 碌L锛忕锛涙暟閲忥細12绠°

浜屻佷骇鍝佽鏄

浜虹被Kinase & Phosphotase & Plasma membrane鐥呮瘨鏂囧簱Edi-HTSTM KPP鏄痵gRNA杞戒綋鏂囧簱锛屽叾涓瘡涓紪杈戝熀鍥犲潎鏈6-10涓澏鐐箂gRNA锛屽彲浠ュ湪浜虹被缁嗚優鍐呮暡闄ゆ墍鏈夌殑Kinase & Phosphotase & Plasma membrane鐩稿叧缂栫爜鍩哄洜锛岀粨鍚堣嵂鐗╃瓫閫夊強浜屼唬娴嬪簭鍙互蹇熷緱鍒扮浉鍏崇殑鑰愯嵂鎴栬呭鏁忓熀鍥犮侲diGene鐨凟di-HTSTM KPP鐥呮瘨鏂囧簱鏍规嵁浜虹被鍩哄洜缁凥g38鐗堟湰浣滀负鍩虹锛屼緷鎹瓻diGene鑷富寮鍙戠殑DeepRank绠楁硶杩涜璁捐锛岄夋嫨鍦ㄤ汉绫诲熀鍥犵粍涓婃病鏈夎劚闈朵綅鐐圭殑sgRNA浣滀负鐩爣鍩哄洜鐨剆gRNA锛岃繍鐢ㄨ绠楁硶璁捐鐨剆gRNA鏁查櫎鏁堢巼宸茬粡杩囦笂鍗冧釜鍩哄洜鏁查櫎缁嗚優绯荤殑楠岃瘉銆傝繍鐢ㄨ鐥呮瘨鏂囧簱杩涜缁嗚優鏂囧簱鏋勫缓锛屽彲鍦–as9绋冲畾涓旈珮鏁堣〃杈剧殑缁嗚優绯讳腑杩涜Kinase & Phosphotase & Plasma membrane鐩稿叧鍩哄洜鐨勬暡闄わ紝浠庤屼繚璇侀珮鏁堢殑缁嗚優鏂囧簱鏁查櫎鏁堢巼銆傝鏂囧簱宸茶繘琛屽ぇ閲忔墿澧炲拰鍖呰锛屼笖缁忚繃NGS澶氭楠岃瘉鍏跺畬鏁存с

涓夈佸簲鐢ㄨ寖鍥

1.聽聽 聽绛涢夌粏鑳炲娈栫浉鍏冲熀鍥
绛涢夌粏鑳炲娈栫浉鍏冲熀鍥犳槸鍩哄洜缁勬枃搴撶殑鍩虹搴旂敤锛孍di-HTSTM KPP鐥呮瘨鏂囧簱澶ц妯℃劅鏌撻澏鍚戠粏鑳炲悗锛屽叧閿熀鍥犺鏁查櫎鐨勭粏鑳炲娈栧彉鎱㈡垨鑰呮浜★紝鑰岄潪鍏抽敭鍩哄洜鏁查櫎鐨勭粏鑳炰粛鍙甯稿垎瑁傘傞殢鐫缁嗚優鍒嗚锛屼袱绉嶇粏鑳炵殑鏁扮洰宸紓瓒婃潵瓒婂ぇ锛岄氳繃楂橀氶噺娴嬪簭姣旇緝鎰熸煋鏃╂湡鍜屾劅鏌撳悗鏈焥gRNA宸紓渚垮彲鎵惧埌涓庣粏鑳炲娈栫浉鍏崇殑鍩哄洜銆
2.聽聽 聽鑽墿闈剁偣绛涢夛細鑰愯嵂鍩哄洜鍜屽鏁忓熀鍥
Edi-HTSTM KPP鐥呮瘨鏂囧簱澶ц妯℃劅鏌撻澏鍚戠粏鑳炲悗锛岃繘琛岃嵂鐗╁鐞嗭紙ICMax锛夛紝鏁查櫎浜嗚嵂鐗╅澏鍚戝熀鍥犵殑缁嗚優鐢变簬瑙i櫎浜嗚嵂鐗╂姂鍒讹紝浠庤屽鑷寸粏鑳炲垎瑁傚彉蹇紝鑰屾暡闄や簡鑽墿闈為澏鍚戝熀鍥犵殑缁嗚優鐢熼暱浠嶈鎶戝埗銆傞殢鐫缁嗚優鍒嗚锛屼袱绉嶇粏鑳炵殑鏁扮洰宸紓瓒婃潵瓒婂ぇ锛岄氳繃楂橀氶噺娴嬪簭姣旇緝鎰熸煋鏃╂湡鍜屾劅鏌撳悗鏈焥gRNA宸紓渚垮彲鎵惧埌鑰愯嵂鍩哄洜銆
Edi-HTSTM KPP鐥呮瘨鏂囧簱澶ц妯℃劅鏌撻澏鍚戠粏鑳炲悗锛岃繘琛岃嵂鐗╁鐞嗭紙IC20锛夛紝鏁查櫎浜嗚嵂鐗╅澏鍚戝熀鍥犵殑缁嗚優鐢变簬澧炲己浜嗚嵂鐗╂晱鎰熸э紝浠庤屽鑷寸粏鑳炲垎瑁傚彉鎱㈡垨姝讳骸锛岃屾暡闄や簡鑽墿闈為澏鍚戝熀鍥犵殑缁嗚優鐢熼暱涓嶅彈褰卞搷銆傞殢鐫缁嗚優鍒嗚锛屼袱绉嶇粏鑳炵殑鏁扮洰宸紓瓒婃潵瓒婂ぇ锛岄氳繃楂橀氶噺娴嬪簭姣旇緝鎰熸煋鏃╂湡鍜屾劅鏌撳悗鏈焥gRNA宸紓渚垮彲鎵惧埌鏁忔劅鍩哄洜銆
3.聽聽 聽澶氶澏鐐瑰崗鍚屼綔鐢ㄧ爺绌
閫氳繃Edi-HTSTM KPP鐥呮瘨鏂囧簱绛涢夊緱鍒扮粏鑳炲娈栫浉鍏冲熀鍥犳垨鑰呰嵂鐗╅澏鍚戝熀鍥犱箣鍚庯紝鍙簲鐢ㄨ淇℃伅杩涜涓や袱缁勫悎锛屽啀娆¤繘琛岀瓫閫夛紝浠庤岃幏寰楀崗鍚岃嚧姝诲熀鍥犮佽泲鐧戒簰浣滃熀鍥犳垨鑰呴氳矾鍩哄洜銆

鏂囧簱璐ㄩ噺妫娴
Parameters sgRNA Library
Sequencing Depth 100脳
sgRNA Lost 0.03%
Gene Lost 0%
sgRNA distribution ( 90% : 10%) 7.31
Total sgRNA 33776
Total Gene 4255

鏂囧簱鎱㈢梾姣掑寘瑁

涓銆佸疄楠屾祦绋

EdiGene閲囩敤澶辨椿鍨嬫參鐥呮瘨鍖呰绯荤粺瀹屾垚鐥呮瘨鏂囧簱鍖呰杩囩▼銆傜梾姣掑寘瑁呭叡闇瑕佷笁涓川绮掞紝鍒嗗埆涓烘惡甯di-HTSTM KPP鏂囧簱鐨勮川绮掑拰涓や釜鐥呮瘨鍖呰杈呭姪璐ㄧ矑pHelper 1鍜宲Helper 2銆
Edi-HTSTM KPP閲囩敤涓夎川绮掑叡杞煋293T缁嗚優銆傚湪杞煋瀹屾垚鍚72 h鏀惰幏鐥呮瘨锛屽苟娴撶缉绾寲寰楀埌楂樻淮搴︾殑鎱㈢梾姣掍繚瀛樻恫锛屾祴瀹氱梾姣掓淮搴︺

浜屻丒di-HTSTM KPP鏂囧簱鐥呮瘨鍖呰

1. 鎺ョ1X107涓粏鑳炰簬缁嗚優鍩瑰吇鐡朵腑锛岀疆浜37鈩冦5% CO2缁嗚優鍩瑰吇绠变腑鍩瑰吇24 h鍚庯紝缁嗚優瀵嗗害杈緙80%锛屽彲鐢ㄤ簬杞煋锛
2. 杞煋鍓2 h鏇存崲涓烘棤琛娓呭煿鍏诲熀锛
3. 鍚戜竴鏃犺弻绂诲績绠′腑鍔犲叆璐ㄧ矑杞煋娣峰悎璇曞墏锛屽叾涓寘鍚獷di-HTSTM KPP鏂囧簱璐ㄧ矑20 ug銆 pHelper 1璐ㄧ矑20 ug浠ュ強pHelper 2璐ㄧ矑5 ug锛岃娣峰悎璇曞墏鍦ㄥ娓╀笅闈欑疆15 min鍚庯紝鍔犲叆缁嗚優鍩瑰吇鐡朵腑锛岀疆浜37鈩冦5% CO2缁嗚優鍩瑰吇绠变腑鍩瑰吇锛
4. 鍩瑰吇6 h鍚庯紝寮冨幓鍚湁杞煋娣峰悎鐗╃殑缁嗚優鍩瑰吇鍩猴紝鐢≒BS缂撳啿娑叉竻娲楀悗锛屾洿鎹负鏂伴矞閰嶅埗鐨勫叏鍩瑰吇鍩猴紝缃簬37鈩冦5% CO2缁嗚優鍩瑰吇绠变腑鍩瑰吇72 h鍚庯紝鏀堕泦缁嗚優涓婃竻娑诧紝鑾峰緱Edi-HTSTM KPP鏂囧簱鐥呮瘨锛

涓夈丒di-HTSTM KPP鏂囧簱鐥呮瘨娴撶缉涓庣函鍖

1. 灏嗙粏鑳炰笂娓呮恫浜1000 rpm銆4鈩冪蹇10 min锛岄櫎鍘荤粏鑳炵鐗囷紱
2.聽聽 聽鐢0.45 um婊ゅ櫒杩囨护涓婃竻娑蹭簬娴撶缉瓒呮护绠′腑锛屼簬4000 g銆4鈩冪蹇30 min锛岃繘琛岀梾姣掓祿缂╋紱
3. 灏嗘祿缂╁悗鐨勭梾姣掓寜瑙勬牸杩涜鍒嗚锛屽苟涓旀娴嬬梾姣掓淮搴︼紱

鍥涖佹參鐥呮瘨婊村害妫娴

1. 鎺ョ1X104涓粏鑳炰簬96瀛旀澘涓紝缃簬37鈩冦5% CO2缁嗚優鍩瑰吇绠变腑鍩瑰吇锛
2. 缁嗚優鎺ョ24 h鍚庯紝鎸夌収姊害绋閲婄殑鏂规硶灏嗙梾姣掑姞鍏ョ粏鑳炲瓟涓紝缃簬37鈩冦5% CO2缁嗚優鍩瑰吇绠变腑鍩瑰吇锛
3. 鐥呮瘨渚垫煋24 h鍚庯紝鏇存崲鏂伴矞閰嶅埗鐨勫畬鍏ㄥ煿鍏诲熀锛岀疆浜37鈩冦5% CO2缁嗚優鍩瑰吇绠变腑鍩瑰吇锛
4. 鐥呮瘨渚垫煋72 h鍚庯紝鍔犲叆1 ug/ml Puromycin杩涜鎶楁х瓫閫夛紱
5. 鎶楁х瓫閫48 h鍚庯紝鏈粡鐥呮瘨渚垫煋鐨勭粏鑳烇紙Negative Control锛夊叏閮ㄦ浜★紝妫娴嬬粏鑳炴椿鐜囥
6. 閲囩敤缁嗚優渚垫煋鏁堢巼涓10%-20%鐨勭梾姣掑姞鍏ラ噺璁$畻鐥呮瘨婊村害銆

浜斻佹參鐥呮瘨婊村害妫娴嬬粨鏋

聽缁忔娴嬶紝Edi-HTSTM KPP鐥呮瘨婊村害妫娴嬬粨鏋滀负2X108 TU/ml锛堣〃2锛夈
琛2. Edi-HTSTM KPP鐥呮瘨婊村害妫娴

鐥呮瘨閲忥紙ul锛 鐥呮瘨渚垫煋鏁堢巼锛%锛
0.02 2.98
0.04 10.65
0.08 21.83
0.16 36.17
0.31 62.25

鏂囧簱浣跨敤

涓銆佸疄楠屾祦绋

楂橀氶噺閬椾紶绛涢夋祦绋嬪寘鎷互涓嬫楠わ細鐥呮瘨鏂囧簱渚垫煋闈跺悜缁嗚優锛涚粏鑳炴枃搴撴姉鎬х瓫閫夛紱缁嗚優鏂囧簱鑽墿绛涢夛紱绛涢夊悗瀵岄泦缁嗚優鍩哄洜缁凞NA鎻愬彇锛涘瘜闆唖gRNA浜屼唬娴嬪簭锛涙祴搴忕粨鏋滃垎鏋愶紙鍥5锛夈
2

浜屻佸疄楠屾楠

1. Puromycin缁嗚優姣掓ф祴瀹
1.1 鎺ョ閫傞噺缁嗚優浜96瀛旀澘涓紝缃簬37鈩冦5% CO2缁嗚優鍩瑰吇绠变腑鍩瑰吇锛
1.2 缁嗚優鎺ョ24 h鍚庯紝鎸夌収姊害绋閲婄殑鏂规硶灏哖uromycin鍔犲叆缁嗚優瀛斾腑锛岀疆浜37鈩冦5% CO2缁嗚優鍩瑰吇绠变腑鍩瑰吇锛
1.3 鍔犲叆Puromycin澶勭悊48 h鍚庯紝妫娴嬬粏鑳炴椿鐜囷紝閫夌敤鍙潃姝荤洰鐨勭粏鑳炵殑鏈浣庢祿搴︿綔涓虹粏鑳炴枃搴撴姉鎬х瓫閫夋墍浣跨敤鐨凱uromycin娴撳害銆
2. 鏂囧簱鐥呮瘨瀵圭洰鐨勭粏鑳炴淮搴︽娴
2.1 鎺ョ閫傞噺鐩殑缁嗚優浜96瀛旀澘涓紝缃簬37鈩冦5% CO2缁嗚優鍩瑰吇绠变腑鍩瑰吇锛
2.2 鐩殑缁嗚優鎺ョ24 h鍚庯紝鎸夌収姊害绋閲婄殑鏂规硶灏嗙梾姣掑姞鍏ョ粏鑳炲瓟涓紝缃簬37鈩冦5% CO2缁嗚優鍩瑰吇绠变腑鍩瑰吇锛
2.3 鐥呮瘨渚垫煋24 h鍚庯紝鏇存崲鏂伴矞閰嶅埗鐨勫畬鍏ㄥ煿鍏诲熀锛岀疆浜37鈩冦5% CO2缁嗚優鍩瑰吇绠变腑鍩瑰吇锛
2.4 鐥呮瘨渚垫煋72 h鍚庯紝鍔犲叆棰勫厛鎽哥储鐨凱uromycin娴撳害杩涜鎶楁х瓫閫夛紱
2.5 鎶楁х瓫閫48 h鍚庯紝鏈粡鐥呮瘨渚垫煋鐨勭粏鑳烇紙Negative Control锛夊叏閮ㄦ浜★紝妫娴嬬粏鑳炴椿鐜囥
2.6 閲囩敤缁嗚優渚垫煋鏁堢巼涓10%-20%鐨勭梾姣掑姞鍏ラ噺璁$畻鐥呮瘨婊村害銆
3. 缁嗚優鏂囧簱鏋勫缓
3.1鍦ㄧ粏鑳炲煿鍏荤毧涓摵閫傞噺Cas9绋冲畾琛ㄨ揪鐨勭洰鐨勭粏鑳烇紝缁嗚優鏁版牴鎹枃搴撳ぇ灏忋佽鐩栧嶆暟銆丮OI浠ュ強鐢熺墿瀛﹂噸澶嶇殑涓暟鍐冲畾锛屽彟璁剧┖鐧藉鐓х粍銆
In Total Cell Number for Transduction=(Number of sgRNA脳1000)/(30%)脳N Replicates
3.2 寮冨幓缁嗚優娑诧紝鏇存崲鎴愬惈鏈夌浉搴斾綋绉殑鐥呮瘨杩涜渚垫煋銆傚彟璁剧┖鐧藉鐓х粍锛屼笉鍔犵梾姣掋
Volume of Virus for Each Dish=(Cell Number of transduction in each dish脳MOI)/(Titer of Virus) 锛圡OI=0.3锛
3.3鐥呮瘨渚垫煋24h涔嬪悗锛屽純鍘诲師缁嗚優鍩瑰吇鍩猴紝鏇存崲鎴愭柊椴滅殑缁嗚優鍩瑰吇鍩恒傜┖鐧藉鐓х粍杩涜鍚屾牱鐨勬搷浣溿
3.4 涓婅堪缁嗚優鎹㈡恫鍩瑰吇72h鍚庯紝鏇存崲鎴愬惈鏈夐鍏堟懜绱㈠ソ鐨凱uromycin娴撳害鐨勭粏鑳炲煿鍏诲熀杩涜绛涢夈傚緟绌虹櫧瀵圭収缁勭殑缁嗚優鍏ㄩ儴姝讳骸涔嬪悗锛堢害48h锛夛紝鏀堕泦鎵鏈夌粏鑳烇紝鍗充负缁嗚優鏂囧簱銆傚皢璇ョ粏鑳炴枃搴撶ǔ瀹氫紶浠1-2鍛ㄤ箣鍚庯紝灏嗗叾鍒嗕负3浠斤細涓浠界敤浜庡喕瀛橈紝涓浠界敤浜嶤ontrol锛屼竴浠界敤浜庤嵂鐗╃瓫閫夈
4. 鑽墿绛涢
4.1 灏嗕笂杩扮瓫閫夊緱鍒扮殑缁嗚優鏂囧簱鎸夌収涓瀹氱殑缁嗚優娴撳害鎺ョ浜庣粏鑳炲煿鍏荤毧涓紝鍒嗗埆鍒嗕负Control缁勫拰瀹為獙缁勶紝姣忕粍璁剧疆N涓猂eplicates銆
4.2 缁嗚優鎺ョ24h鍚庯紝鍦ㄥ疄楠岀粍涓姞鍏ラ鍏堟懜绱㈠ソ鐨勭瓫閫夌敤鑽墿娴撳害锛
4.3 鑽墿绛涢72h鍚庯紝鍒嗗埆鏀堕泦鍚勭粍缁嗚優锛屾彁鍙栧熀鍥犵粍锛岃繘琛孭CR鍚庯紝灏哖CR浜х墿鐢ㄤ簬娴嬪簭銆