kit
Contents Specifications
Validated sgRNA plasmid 2ug, 200ng/ul
Blank vector 2ug, 200ng/ul
Cas9 vector 2ug, 200ng/ul
Donor (homozygous arm, with Loxp) 聽 聽 2ug, 200ng/ul
浜у搧鍐呭
缁勪欢 瑙勬牸
Validated sgRNA plasmid 2ug, 200ng/ul
Blank vector 2ug, 200ng/ul
Cas9 vector 2ug, 200ng/ul
Donor (homozygous arm, with Loxp) 2ug, 200ng/ul

闄勪欢1. U6-sgRNA-mCherry鍥捐氨

U6-sgRNA-mCherry鍥捐氨

闄勪欢2. Cas9铔嬬櫧璐ㄧ矑

Cas9 vector

闄勪欢1鍜岄檮浠2鐨勮浇浣撳師浠惰鏄庯細
CMV鍚姩瀛愶細鏄湡鏍哥敓鐗╀腑浣跨敤鏈骞挎硾鐨勫惎鍔ㄥ瓙涔嬩竴锛岃兘澶熷湪鍝轰钩鍔ㄧ墿缁嗚優涓珮鏁堣〃杈俱
Cas9铔嬬櫧锛氭牳閰稿唴鍒囬叾锛岃兘澶熷皢DNA鍙岄摼鎵撴柇銆
Amp鎶楁у熀鍥狅細鍦ㄥ師鏍稿厠闅嗚繃绋嬩腑锛屽彲鐢ㄤ簬绛涢夐槼鎬у厠闅嗘牚銆
mCherry锛氳崸鍏夋爣璁板熀鍥狅紝杞煋鍚庡彲鐢ㄤ簬瑙傚療杞煋鏁堢巼銆
U6鍚姩瀛愶細鏈夌簿纭殑杞綍璧峰浣嶇偣鍜岀粓姝綅鐐癸紝鑳藉鍑嗙‘鐨勮浆褰曞嚭sgRNA锛屼富瑕侀傜敤浜庡摵涔冲姩鐗╃粏鑳炪
sgRNA锛涜瘑鍒獶NA鐗瑰畾浣嶇偣銆

闄勪欢3. Donor搴忓垪

鍦―onor鎻掑叆鍒板熀鍥犵粍涓婁箣鍚庯紝鍙氳繃Cre-Loxp鐨勫伐浣滃師鐞嗗皢Puro鐨勭瓫閫夋爣璁板幓闄ゃ
Left-Homologuos Arm-Loxp-Puro-Loxp-Right-Homologus Arm

donor鐗囨

鎶鏈師鐞

CRISPR/Cas9 鎶鏈槸涓绉嶅瓨鍦ㄤ笌缁嗚弻鎴栧彜缁嗚弻涓紝鍒╃敤鑷韩闃插尽鏈哄埗鎶靛尽澶栨簮鐥呮瘨鎴朌NA鍏ヤ镜鑷韩鐨勫厤鐤郴缁熴傚湪璇ユ満鍒朵腑锛岀梾姣扗NA鍏ヤ镜缁嗚弻鍚庯紝琚蹇嗗湪缁嗚弻鐨勫熀鍥犵粍涓傚綋璇ョ梾姣扗NA鍐嶆鍏ヤ镜鏃讹紝瀛樺湪浜庣粏鑿屼腑鐨勮蹇嗙墖娈典細涓嶤as9鏍搁吀閰剁粨鍚堟垚澶嶅悎鐗╋紝璇嗗埆澶栨簮DNA骞惰繘琛屽壀鍒囷紝鏈缁堥犳垚澶栨簮DNA鍙岄摼鏂銆

CRISPR/Cas9鍒囧壊绀烘剰鍥


鍦ㄤ紶缁熺殑CRISPR/Cas9绯荤粺涓紝鏄湁crRNA銆乼racrRNA鍜孋as9铔嬬櫧3涓師浠剁粍鎴愬苟瀹屾垚DNA鐨勮瘑鍒笌鍓垏銆傝屽湪鍚庢湡浼樺寲鍚庯紝灏哻rRNA鍜宼racrRNA閾炬帴鍦ㄤ竴璧凤紝鏋勬垚浜嗗ぇ瀹跺父璇寸殑guideRNA銆傚湪涓嶉檷浣庤瘑鍒晥鐜囧拰鍒囧壊鏁堢巼鐨勫熀纭涓婏紝澧炲姞浜嗗疄楠岀殑绠渚挎с

Cas9-single RNA

鍚屾簮淇锛氶氬父鏄寜鐓х壒瀹氱殑瑕佹眰杩涜绮惧噯鐨勪慨澶嶆満鍒讹紝浣嗗悓婧愪慨澶嶇殑姣斾緥寰堜綆锛岃繖涔熸槸绮惧噯鍩哄洜缂栬緫鐨勯毦鐐广

闈炲悓婧愭湯绔繛鎺ワ細璇ヤ慨澶嶆満鍒跺湪缁嗚優涓崰寰堝ぇ姣斾緥锛岄氬父浼氬嚭鐜版彃鍏ユ垨鍒犻櫎鏁颁釜纰卞熀鐨勬柟寮忚繘琛屻傝繖绉嶄慨澶嶆ā寮忛殢鏈烘у緢澶э紝鎵浠ユ槸鐢ㄤ簬鏁查櫎鍩哄洜鐨勬湁鍔涙墜娈点

鎶鏈祦绋

CRISPR/Cas9 鍩哄洜鏁查櫎缁嗚優鎶鏈祦绋嬪浘锛

甯歌闂

鍑虹幇鐨勯棶棰

瑙e喅鏂规

鎻愬彇璐ㄧ矑娴撳害杩囦綆 鍦ㄦ憞鑿岃繃绋嬩腑瑕佹敞鎰忚瀵熻弻娑茬殑娴撳害锛岃弻娑茶佸寲鎴栨祿搴﹁繃浣庡潎浼氬嚭鐜拌川绮掓彁鍙栨祿搴﹁緝浣庣殑鐜拌薄銆
杞煋鏁堢巼浣 1. 瀵逛簬涓嶅悓鐨勭粏鑳烇紝杞煋鏉′欢闇杩涜浼樺寲锛屽彲鍥哄畾涓涓硷紝瀵瑰彟涓涓艰繘琛屾搴︾殑娴撳害绋閲婏紝浠ヨ幏寰楁渶浣崇殑杞煋鏉′欢
2. 鍙皟鏁寸粏鑳炶浆鏌撲綋绯讳腑鍚勮川绮掍笌Donor鐨勬瘮渚嬨
寮曠墿涓嶅伐浣

鍦≒CR瀹為獙涓渶鍔犲叆閲庣敓鍨嬫牱鍝佷綔涓洪槼鎬у鐓э紝濡傞槼鎬у鐓у嚭鐜版纭殑鏉″甫锛岃鏄庡紩鐗╁彲鐢紝闇閲嶆柊鏀堕泦鏁查櫎鏍峰搧锛涘闃虫у鐓ф湭鍑虹幇姝g‘鏉″甫锛岄渶閲嶆柊璁捐寮曠墿銆

娴嬪簭缁撴灉涓嶆纭 鍒囪兌杩囩▼涓夋嫨鐨勪綅缃笉鍑嗙‘銆
1. 濡傚湪閲庣敓鍨嬬殑浣嶇疆鍑虹幇鐩稿簲鐨勬潯甯︼紝鍙鍒囪兌鐨勪骇鐗╄繘琛孴A鍏嬮殕骞舵祴搴忛壌瀹氥
2. 濡傚湪闈為噹鐢熷瀷鐨勪綅缃嚭鐜版潯甯︼紝涔熷彲浠ヨ繘琛屽垏鑳舵娴嬶紝姝ょ墖娈靛涓哄ぇ鐗囨鎻掑叆銆
PCR浜х墿娴嬪簭缁撴灉涓哄宄 鐢变簬鍩哄洜鍦ㄦ煋鑹蹭綋涓婂瓨鍦ㄤ袱涓垨涓や釜浠ヤ笂绛変綅鍩哄洜锛屽悓鏃惰繕鏈夋嫹璐濇暟鐨勫瓨鍦ㄣ傚洜姝CR浜х墿鐩存帴娴嬪簭浼氬嚭鐜板宄扮殑缁撴灉銆
鍙氳繃鎷嗗嘲杞欢鍒嗘瀽鍚勭缂栬緫鐨勬儏鍐点傚啀閫氳繃鍒囪兌鍥炴敹PCR浜х墿锛岄氳繃TA鍏嬮殕鏉ラ獙璇併

浜у搧鐩綍

浠ヤ笅鍒楄〃涓殑鍩哄洜鍧囧凡閰嶅鐩稿簲CRISPR Knockout 璇曞墏鐩掞紝骞跺凡缁忚繃楠岃瘉锛屽悓鏃舵湁鐩稿簲鐨勫熀鍥犳暡闄ょ粏鑳炵郴浜у搧銆

AKT1 CTSB IRS4 MRAS PTEN
AKT2 DAPK3 ITPR1 MTMR4 RAF1
ATG12 DDIT4 KRAS MTOR RPS6KB1
ATG14 EIF2AK3 LAMP2 NRAS RPS6KB2
ATG16L1 ERN1 MAP2K1 NRBF2 RRAS
ATG2B GABARAP MAP2K2 PIK3R1 RRAS2
ATG3 GABARAPL2 MAP3K7 PIK3R2 STK11
ATG4D HIF1A MAPK1 PIK3R3 TP53INP2
BAD HMGB1 MAPK9 PRKACA VAMP8
CAMKK2 IGF1R MLST8 PRKACB WIPI1