Made-ready

CRISPR/Cas9鎶鏈槸鐩墠鏈鐑棬鐨勫熀鍥犵粍缂栬緫宸ュ叿锛屽畠閫氳繃瀵煎悜RNA寮曞Cas9铔嬬櫧璇嗗埆鐗瑰畾闈剁偣锛屽畬鎴愬鐩殑鍩哄洜鐨勬敼閫犮侰RISPR鎶鏈凡缁忓簲鐢ㄤ簬澶氱绫诲瀷鐨勭敓鐗╁鐮旂┒涓傚崥闆呰緫鍥犺嚧鍔涗簬搴旂敤CRISPR/Cas9鎶鏈紝鏋勫缓瑕嗙洊浜虹被鍏ㄥ熀鍥犵粍鐨勫熀鍥犳暡闄ょ粏鑳炵郴锛屽悓鏃舵彁渚涘熀鍥犵紪杈戠浉鍏虫湇鍔°傛墍鏈変骇鍝佸強鏈嶅姟鍧囪幏寰楃編鍥介夯鐪佺悊宸ュ闄 Broad 鐮旂┒鎵寮犲嘲瀹為獙瀹ゅ強鍝堜經澶у鐨勪笓鍒╂巿鏉冭鍙

鍩哄洜鏁查櫎缁嗚優绯诲強鍏惰鐢熺墿

鍗氶泤杈戝洜鎷ユ湁涓撲笟鐨凜RISPR/Cas9鎶鏈钩鍙帮紝鑷村姏浜庢瀯寤鸿鐩栦汉绫诲叏鍩哄洜缁勭殑鍩哄洜鏁查櫎缁嗚優搴撱傜洰鍓嶅崥闆呰緫鍥犳彁渚涜秴杩2,000绉嶇殑鍩哄洜鏁查櫎缁嗚優绯荤幇璐э紝鍙綔涓哄熀鍥犲姛鑳界爺绌舵垨淇″彿閫氳矾鐮旂┒鐨勫伐鍏枫

cell lines

鍩哄洜鏁查櫎缁嗚優绯

鎻愪緵瓒呰繃2,000绉嶅熀鍥犳暡闄ょ粏鑳炵幇璐э紝瀹屽叏鐪佺暐鍩哄洜鏁查櫎缁嗚優绯诲埗澶囪繃绋嬶紙绾3-6涓湀锛夛紝鐪佹椂鐪佸姏锛屽姞閫熷疄楠岋紒

cell lysate

鍩哄洜鏁查櫎缁嗚優瑁傝В铔嬬櫧

鎶椾綋楠岃瘉鏂版爣鍑嗭紒

搴旂敤鍩哄洜鏁查櫎缁嗚優瑁傝В铔嬬櫧锛屽畬缇庨獙璇佹偍鐨勬姉浣撶壒寮傛э紝浠庢鎷掔粷鎶椾綋鍋囬槼鎬у拰瀹為獙涓嶅彲閲嶅銆

鍩哄洜缂栬緫宸ュ叿绫讳骇鍝

CRISPR/Cas9鍩哄洜缁勭紪杈戠郴缁熶富瑕佺敱涓ら儴鍒嗙粍鎴愶細Cas9鏍搁吀閰跺拰璇嗗埆鐗瑰紓鎬ч澏鍩哄洜浣嶇偣鐨勫悜瀵糝NA锛坰gRNA锛夈傚崥闆呰緫鍥犳彁渚涢珮鏁堢巼鐨凜RISPR宸ュ叿绫讳骇鍝侊紝鍖呮嫭绋冲畾琛ㄨ揪Cas9铔嬬櫧鐨勭粏鑳炵郴鍙婄粡杩囬獙璇佸悗鐨剆gRNA銆

搴旂敤杩欎簺宸ュ叿绫讳骇鍝侊紝璁╂偍鐨凜RISPR瀹為獙鏇存祦鐣咃紒

路聽缁忛獙璇佺殑sgRNA

sisgRNA涓嶤as9铔嬬櫧缁撳悎褰㈡垚澶嶅悎鐗╋紝鐗瑰紓鎬ч澏瀹氬熀鍥犵粍鐗瑰畾浣嶇疆锛屽畬鎴愬熀鍥犵紪杈戝伐浣溿傝璁¢珮鏁堢殑锛岀壒寮傛у己鐨剆gRNA瀵逛簬绮剧‘鐨勫畬鎴愬熀鍥犵紪杈戝伐浣滆嚦鍏抽噸瑕併傚崥闆呰緫鍥犳彁渚涚殑sgRNA鍧囧凡缁忚繃楠岃瘉骞剁敓鎴愮ǔ瀹氱殑鍩哄洜鏁查櫎缁嗚優绯汇傚簲鐢ㄧ粡楠岃瘉鐨剆gRNA瀹屾垚鎮ㄧ殑鍩哄洜缂栬緫锛屽彲浠ュ畬鍏ㄧ渷鍘婚瀹為獙鐨勬椂闂淬

路聽绋冲畾琛ㄨ揪Cas9铔嬬櫧缁嗚優绯

鍒╃敤鎱㈢梾姣掔殑鏂瑰紡灏嗚〃杈綜as9铔嬬櫧鐨勭墖娈垫暣鍚堝湪缁嗚優绯诲熀鍥犵粍涓紝浣挎暣涓粏鑳炵郴鍙互鎸佺画绋冲畾鐨勮〃杈綜as9铔嬬櫧锛岀渷鍘昏浆鏌揅as9璐ㄧ矑杩涘叆缁嗚優鐨勬楠わ紝鍚屾椂鎻愰珮鍩哄洜缁勭紪杈戞晥鐜囥

ca9

CRISPR sgRNA 鏂囧簱

CRISPR sgRNA 鏁查櫎鏂囧簱鑳藉鍦ㄥ摵涔冲姩鐗╃粏鑳炲唴蹇熺敓鎴愬姛鑳界己澶辩殑绐佸彉浣撳苟涓旂瓫閫夋墍鏈熸湜鐨勮〃鍨嬨傚簲鐢–RISPR鎶鏈繘琛岄珮閫氶噺鍩哄洜鏁查櫎鏄揩閫熷彂鐜拌嵂鐗╂柊闈剁偣锛岀爺绌朵俊鍙烽氳矾鏈哄埗鐨勬柊鏂规硶銆傚崥闆呰緫鍥犳彁渚涗竴绯诲垪棰勫埗CRISPR sgRNA 鏂囧簱锛屽寘鎷汉绫诲叏鍩哄洜缁勬枃搴撳強鍩轰簬涓嶅悓鍔熻兘鍒嗙被鐨勪簹鏂囧簱锛屽彲鍚屾椂鏁查櫎涓婁竾涓熀鍥狅紝鏄珮閫氶噺閬椾紶绛涢夌殑閲嶈宸ュ叿銆

sgRNA鏂囧簱2

sgRNA鏂囧簱

鍗氶泤杈戝洜鐨剆gRNA娣峰悎鏂囧簱锛屽簲鐢ㄦ敼杩涘悗鐨勮璁$畻娉曪紝閽堝姣忎釜鍩哄洜璁捐8-10鏉′笉鍚岀殑sgRNA锛屽叿鏈夋洿楂樻晥鐨勫熀鍥犳暡闄ゆ晥鐜囷紝鍚屾椂璁捐闈為澏鍚憇gRNA浣滀负瀵圭収锛屼繚璇佹枃搴撶殑鍑嗙‘鎬с

闀块潪缂栫爜鍖篟NA鏂囧簱锛圠ncRNA Library锛

闀块潪缂栫爜RNA涓庡绉嶈偪鐦ょ殑鍙戠敓鍙戝睍瀵嗗垏鐩稿叧锛屼絾鍏跺姛鑳藉皻涓嶆槑纭傞拡瀵归潪缂栫爜鍖烘瀯寤虹殑LncRNA 鏂囧簱鍙府鍔╃瀛﹀鐮旂┒闈炵紪鐮佸尯瀵逛簬鐧岀棁鐨勫姛鑳藉強鏈哄埗銆

鐩稿叧鍙傝冩枃鐚

1 Shalem, et al. (2014). Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343, 84.

2 Wang, et al. (2014). Genetic screens in human cells using the CRISPR-Cas9 system. Science 343, 80.

3 Zhou, et al. (2014). High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells. Nature 509, 487.

4 Zhu, et al.(2016). Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR鈥揅as9 library. Nature Biotechnology