Pool sgRNA
CRISPR sgRNA鏂囧簱鏄敤浜庨珮閫氶噺鏁查櫎浜虹被鍏ㄥ熀鍥犵粍鎴栫壒瀹氬熀鍥犵被缇ょ殑閲嶈宸ュ叿銆傞氳繃鍩哄洜鏁查櫎鍙疄鐜板熀鍥犲姛鑳界己澶辩瓫閫夛紝杩涜岃繘琛屽摵涔冲姩鐗╃郴缁熷熀鍥犲垎鏋愩佸熀鍥犲姛鑳界爺绌躲佸熀鍥犵粍绾у埆鍔熻兘鐮旂┒锛堜俊鍙烽氳矾鐮旂┒銆佺檶鐥囨満鍒剁爺绌讹級浠ュ強鍙戠幇楠岃瘉鑽墿闈剁偣绛変竴绯诲垪鐮旂┒銆

EdiGene鎻愪緵鍩哄洜鏁查櫎鏂瑰紡鐨勯珮閫氶噺閬椾紶绛涢夋墍鐢ㄧ殑CRISPR sgRNA鏂囧簱锛屽寘鎷叏浜虹被鍩哄洜缁勬暡闄ゆ枃搴撲互鍙婂熀浜庝笉鍚屽姛鑳芥х殑鍩哄洜缁勬暡闄や簹鏂囧簱銆傞氳繃鍩哄洜鍔熻兘瀹屽叏涓уけ鐨勬柟寮忕爺绌朵俊鍙烽氳矾鏈哄埗鎴栫瓫閫夎嵂鐗╃浉鍏抽澏鐐圭瓑銆

sgRNA 鏂囧簱

鍗氶泤杈戝洜鍙畾鍒跺悎鎴愪汉绫诲叏鍩哄洜缁勬枃搴撳強鍩轰簬涓嶅悓鍔熻兘鎴栧熀鍥犲畾浣嶇殑浜氭枃搴撱傚悓鏃跺彲鎻愪緵鍚庢湡鏁版嵁鍒嗘瀽鏈嶅姟锛岃鎮ㄧ殑楂橀氶噺鏈嶅姟鏇村姞绠鍗曞揩閫燂紒

璁捐鐞嗗康

鎴戜滑閽堝鍦║SCS hg38鏁版嵁搴撲腑娉ㄦ槑鐨18,513涓熀鍥犳瀯寤轰簡浜虹被鍏ㄥ熀鍥犳暡闄gRNA鏂囧簱銆 鎵鏈夎泲鐧借川缂栫爜鍩哄洜鐨勯澏鍚慍DS鍖哄煙鍧囪璁gRNA锛岄櫎鍘籊C鍚噺楂樹簬80锛呮垨浣庝簬20锛呯殑sgRNA锛 鍙繚鐣欎簡闈跺悜鍓50锛呴儴鍒咰DS鐨剆gRNA銆 鍚屾椂璁捐2000鏉¢槾鎬у鐓gRNA锛 閫氳繃John Doench绛変汉鍙戣〃鐨勭畻娉曞鍊欓塻gRNA杩涜浜嗘帓鍚嶃偮 鏈缁堢殑鏂囧簱鍖呭惈170,815涓猻gRNA锛屽苟鏍规嵁鍩哄洜浣嶇疆鍒嗕负10涓簹鏂囧簱銆

浼樺娍

閫氳繃浼樺寲鍏紑鏁版嵁鐨勭畻娉曪紝浣縮gRNA鍏锋湁鏇撮珮鐨勫熀鍥犳暡闄ゆ晥鐜囷紱

98.7%鐨勫熀鍥犲叿澶6鏉′互涓妔gRNA锛屾槸CRISPR绛涢夊己鏈夊姏鐨勫伐鍏凤紱

鎻愪緵涓嶅悓鐨勫姛鑳芥т簹鏂囧簱锛屼綋閲忔洿灏忥紝浣跨敤鏇村叿鐗瑰紓鎬э紱

鏂囧簱鏋勫缓娴佺▼

路鍙彁渚沢RNA娣峰悎鏂囧簱瀹氬埗鏈嶅姟锛屽寘鎷川绮掑拰鐥呮瘨褰㈠紡锛毬

pooled sgRNA library

璐у彿

浜у搧鍚嶇О聽

闈跺熀鍥犳暟閲

浠锋牸

HTSLV60301

Edi-HTSTM Whole Mouse Genome Lentiviral CRISPR Library

19357

璇环

HTSLV60401

Edi-HTSTM Whole Human Genome Lentiviral CRISPR Library

18823

璇环

HTSLV60402

Edi-HTSTM Human Kinase Lentiviral CRISPR Library

738

璇环

HTSLV60403

Edi-HTSTM Human Phosphatase Lentiviral CRISPR Library

265

璇环

HTSLV60404

Edi-HTSTM Human Kinase & Phosphatase Lentiviral CRISPR Library

1003

璇环

HTSLV60405

Edi-HTSTM Human Cytokine Lentiviral CRISPR Library

175

璇环

HTSLV60406

Edi-HTS鈩 Human Kinase & Phosphotase & Plasma Membrane Lentiviral聽
CRISPR Library

4255

璇环

HTSLV60407

Edi-HTSTM Human Enzyme Lentiviral CRISPR Library

2948

璇环

HTSLV60408

Edi-HTSTMHuman GPCR Lentiviral CRISPR Library

692

璇环

HTSLV60409

Edi-HTSTM Human Growth Factor Lentiviral CRISPR Library

131

璇环

HTSLV60410

Edi-HTSTM Human Ion Channel Lentiviral CRISPR Library

337

璇环

HTSLV60411

Edi-HTSTM Human Ligand-dependent Nuclear Receptor Lentiviral CRISPR Library

46

璇环

HTSLV60412

Edi-HTSTM Human Peptidase Lentiviral CRISPR Library

530

璇环

HTSLV60413

Edi-HTSTM Human Transcription Regulator Lentiviral
CRISPR Library

1515

璇环

HTSLV60414

Edi-HISTM Human Translation Regulator Lentiviral CRISPR
Library

103

璇环

HTSLV60415

HEdi-HTSTM Human Transmembrane Receptor Lentiviral
CRISPR Library

354

璇环

HTSLV60416

Edi-HTSTM Human Transporter Lentiviral CRISPR Library

1033

璇环

HTSLV60417

Edi-HTSTM Human Deubiquitin Lentiviral CRISPR Library

璇环

HTSLV60418

Edi-HTSTM Human Ubiquitin Lentiviral CRISPR Library

438

璇环

HTSLV60419

Edi-HTSTM Human Epigenetics Lentiviral CRISPR Library

209

璇环

HTSLV60420

HEdi-HTSTM Human Druggable Target Lentiviral CRISPR Library

2897

璇环

LncRNA聽鏂囧簱

鐪熸牳鍩哄洜缁勫寘鍚泲鐧借川缂栫爜鍖哄拰闈炵紪鐮佸尯銆傝浆褰曠粍鍒嗘瀽鏄剧ず锛岄珮杈90锛呯殑浜虹被鍩哄洜缁勮浆褰曚负闈炵紪鐮丷NA銆傞潪缂栫爜RNA鏍规嵁鍏跺ぇ灏忚繘涓姝ョ粏鍒嗕负灏忕殑闈炵紪鐮丷NA锛<200nt锛夊拰闀块潪缂栫爜RNA銆 lncRNA涓庝笉鍚岀殑鍒嗗瓙鍜岀粏鑳炲姛鑳界浉鍏筹紝骞朵笌鐧岀棁锛屽彂鑲插拰鐤剧梾鏈夊叧銆傜劧鑰岋紝澶у鏁拌繖浜涙柊閴村畾鐨刲ncRNA鐨勭粏鑳炲姛鑳藉皻鏈槓鏄庛傛澶栵紝lncRNA鍙兘閫氳繃涓嶅悓鐨勬満鍒讹紝鍖呮嫭鏌撹壊璐ㄤ慨楗帮紝杞綍锛岃浆褰曞悗杞綍锛屼笌RNA缁撳悎铔嬬櫧鐨勭浉浜掍綔鐢紝杞綍鍥犲瓙鐨勫叡婵娲诲拰鍚姩瀛愮殑鎶戝埗绛夊洜绱犲湪鍩哄洜璋冩帶涓捣閲嶈浣滅敤銆傚熀浜庡叾涓撴湁鐨勯厤瀵筭RNA锛坧gRNA锛夊簱璁捐绛栫暐锛孍diGene宸茬粡鏋勫缓浜嗛澏鍚戠害700涓猯ncRNAs鐨刾gRNA鏂囧簱锛岃繖灏嗕娇鐮旂┒浜哄憳鑳藉璇嗗埆鐧岀棁涓璉ncRNAs鐨勫垎瀛愬姛鑳

鐩稿叧鍙傝冩枃鐚

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

  2. Peng J, et al. (2015) High-throughput screens in mammalian cells using the CRISPR-Cas9 system. FEBS J. 282 : 2089.

  3. Zhu S, et al. (2016) Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR鈥揅as9 library. Nat. Biotechnol. 34 : 1279.

FAQs:

1. 璇烽棶浣犱滑鐢ㄧ殑鏄摢绉嶈浆鏌撶殑鏂规硶? 鍖呰鐥呮瘨鏄敤鐨勫煿鍏荤毧鏄粈涔堣鏍肩殑锛熉

聽鎴戜滑鐢ㄧ殑鏄笁璐ㄧ矑绯荤粺鐨勭梾姣掑寘瑁呮柟娉曪紝鎵鐢ㄧ殑鍖呰鍣ㄧ毧涓150T鐨勭粏鑳炲煿鍏荤摱锛

2. 璇烽棶浣犱滑鐢ㄧ殑鍖呰璐ㄧ矑锛歱Helper1鍜宲Helper2鏄笉鏄氨鏄痑ddgene鐨刾MD2.G鍜宲sPAX2锛熸垜鐪嬪埌浣犱滑鐢ㄧ殑姣斾緥鏄5:1锛20ug鍜5ug锛夛紝浣犱滑鏄笉鏄祴璇曡繃涓嶅悓鐨勬瘮渚嬶紝鍙戠幇杩欎釜鏉′欢鏈濂藉憿锛

鎴戜滑鐢ㄧ殑鍖呰璐ㄧ矑鏄痯R8.74鍜宲VSV-G锛岀梾姣掑寘瑁呮柟娉曟槸鍏徃鐥呮瘨鍖呰骞冲彴鐨勫悓浜嬪仛杩囦紭鍖栫殑锛

3. 浣犱滑鐨勬枃搴撶梾姣掔矘搴﹀緢澶э紝鏄笉鏄祿缂╄繃绋嬩腑鍩瑰吇鍩轰腑鐨勮娓呬篃琚祿缂╀簡鍛紵

姝e儚鎮ㄦ墍璇寸殑锛屾枃搴撶梾姣掔矘搴︽瘮杈冨ぇ鏄洜涓哄湪鐥呮瘨娴撶缉杩囩▼涓紝鍩瑰吇鍩轰腑鐨勬垚鍒嗕篃琚祿缂╀簡锛

4. 杞煋璇曞墏鐢ㄧ殑鏄粈涔堬紵

杞煋璇曞墏锛歅EI锛

5. PCR娴嬪簭鏈変粈涔堥渶瑕佹敞鎰忕殑鍦版柟鍚楋紵

PCR娌′粈涔堣娉ㄦ剰鐨勶紝鍙淇濊瘉鏀堕泦鐨勭粏鑳炴暟鑳藉瑕嗙洊鏁翠釜鏂囧簱灏卞彲浠ワ紝 鐒跺悗杩欎簺缁嗚優鎻愬嚭鏉ョ殑鍩哄洜缁勫叏閮ㄧ敤鏉ユ墿澧濸CR锛 寰幆鏁颁笉鑳藉お澶 20-25涓惊鐜紱

6. 鏂囧簱鐥呮瘨鐨勬淮搴﹀ソ鍍忔瘮璇存槑涔﹂噷闈㈡彁渚涚殑婊村害瑕侀珮寰堝锛屾垜鏄敤鐨293T缁嗚優锛屽ぇ鑷存祴涓涓嬫劅鏌撶殑鏁堢巼锛屽彂鐜1:10000绋閲婁箣鍚庣敤200ul鍩瑰吇鍩猴紝鎰熸煋涔嬪悗锛宲uro鍩烘湰鏉涓嶆浠讳綍缁嗚優锛屽鐓ф湭鎰熸煋鐨勭粏鑳炲叏閮ㄦ潃姝伙紝鐥呮瘨鐨勬淮搴﹁繙姣斾綘浠彁渚涚殑鏁板奸珮锛

铏界劧缁嗚優鏍竴鏍凤紝浣嗘槸缁嗚優鐘舵佷笉涓鏍风殑璇濓紝婊村害涔熻繕鏄細鏈夊樊鍒殑锛涙墍浠ユ垜浠細寤鸿鍦ㄦ嬁鍒扮梾姣掍箣鍚庯紝鍦ㄩ澏缁嗚優涓婇噸鏂版祴瀹氱梾姣掓淮搴︼紝鍐冲畾鐥呮瘨鐢ㄩ噺

7. 绛涢夊嚭闃虫х殑缁嗚優涔嬪悗锛岃鎻愬熀鍥犵粍杩涜PCR锛屼綘浠湭鎻愪緵PCR寮曠墿鍝

Library-Seq-F1: N25TATCTTGTGGAAAGGACGAAACACC
Library-Seq-R锛歂25AATACGGTTATCCACGCGGC