铔嬬櫧缂栫爜鍩哄洜浠呭崰3%涓嶅埌鐨勫熀鍥犵粍锛岀爺绌惰呭彂鐜拌秺鏉ヨ秺澶氱殑闈炵紪鐮佸厓浠跺湪鐢熷懡娲诲姩涓彂鎸ユ瀬鍏堕噸瑕佺殑浣滅敤锛屾瘮濡傞潪缂栫爜RNA銆佺壒鍒槸闀块潪缂栫爜RNA锛圠ncRNA锛夌殑寮傚父涓庣檶鐥囩瓑寰堝鐤剧梾鐨勫彂鐢熷彂灞曠浉鍏炽

瀵圭紪鐮佽泲鐧藉熀鍥犵殑鏁查櫎鍙互閫氳繃鍩哄洜缁勭紪杈戞妧鏈鐩爣鍖哄煙瀹炶鍗曠偣鍒囧壊銆佸紩鍏ュ皬鐗囨鎻掑叆鎴栫己澶憋紙indels锛変互閫犳垚鍩哄洜缈昏瘧璇荤爜妗嗙牬鍧忥紝鐒惰岃繖涓鏂规硶瀵逛簬涓嶄緷璧栬鐮佹鐨勯潪缂栫爜鍏冧欢浣滅敤鏈夐檺銆

鍗氶泤杈戝洜鍏徃涓烘寤虹珛浜唒aired-guide RNA (pgRNA) 鏂囧簱鐨勬瀯寤烘柟娉曪紝閫氳繃pgRNA寮曞叆鐨勫熀鍥犵粍澶х墖娈靛垹闄ゆ潵鐮村潖LncRNA琛ㄨ揪鍙婂姛鑳斤紝骞剁敱鎱㈢梾姣掍粙瀵煎湪澶氫釜鐧岀粏鑳炵郴涓疄鐜颁簡鍔熻兘绛涢夈

宸ヤ綔娴佺▼

鍗氶泤杈戝洜浣跨敤鍙宻gRNA锛坧gRNAs锛夌殑绛涢夌瓥鐣ワ紝鎴戜滑鏍规嵁瀹㈡埛鐨勯渶姹傝璁ncRNA澶х墖娈靛垹闄ょ殑sgRNA鏂囧簱銆傛暣涓瓫閫夌敱浣跨敤鍖呭惈pgRNA鏂囧簱鐨勬參鐥呮瘨渚垫煋缁嗚優寮濮嬶紝鍦ㄥ悎鐞嗘帶鍒朵镜鏌撳鏁帮紙multiplicity of infection锛孧OI锛夌殑鍩虹涓婏紝姣忎釜琚镜鏌撶殑缁嗚優锛屼粎鏈夊崟涓鐨刾gRNA琚暣鍚堣繘缁嗚優鐨勫熀鍥犵粍銆傚湪渚垫煋瀹屾垚鍚庯紝浣跨敤娴佸紡杩涜鍒嗛夋垨鍏朵粬瀵岄泦鏂瑰紡锛岄氳繃瀵圭粏鑳炶〃鍨嬬殑瑙傚療瀵圭粏鑳炶繘琛岃仛绫伙紝鍚庣画鍒欏埄鐢ㄦ繁搴︽祴搴忚瀵熶笉鍚岀被鍒粏鑳炵兢鍐卲gRNA涓板害鐨勫彉鍖栵紝鏉ュ垽鏂摢浜汱ncRNA鍙備笌鍦ㄦ垜浠爺绌剁殑鐢熺墿闂涓

3

CRISPR楂橀氶噺LncRNA绛涢夐傜敤浜

  • 鐮旂┒褰卞搷缁嗚優澧炴畺鐨勯潪缂栫爜鍏冧欢锛
  • 绛涢夊彂鎸ュ叾浠栭噸瑕佷綔鐢ㄧ殑闈炵紪鐮佸厓浠讹紱
  • 鐮旂┒鍩哄洜缁勪腑鐨勫姛鑳芥湭娉ㄩ噴鍖哄煙锛
  • 瀵绘壘鐧岀棁鐨勮嵂鐗╅澏鐐癸紱
  • 鍙戠幇娼滃湪鑱斿悎娌荤枟鐨勯澏鐐规垨淇″彿閫氳矾锛

妗堜緥灞曠ず锛歭ncRNA绛涢夐壌瀹氬嚭姝e悜鍙婅礋鍚戣皟鎺х檶缁嗚優澧炴畺鐨刲ncRNA

鐩殑锛閫氳繃鏋勫缓12,000 pgRNAs 鐨凜RISPR鏂囧簱锛屼粠涓壌瀹氬嚭姝e悜鍙婅礋鍚戣皟鎺х檶缁嗚優澧炴畺鐨刲ncRNA銆傚啀閫氳繃澶氱閬椾紶瀛︽墜娈甸獙璇佸欓塴ncRNA鐨勫姛鑳斤紝骞朵娇鐢ㄧ敓鐗╀俊鎭垎鏋愬拰琛ㄨ揪璋辨祴搴忔帰绌朵簡鍏跺彂鎸ヤ綔鐢ㄧ殑鏈哄埗銆


鏂规硶锛

  • 浣跨敤鍗氶泤杈戝洜鐙壒鐨勬瀯寤烘柟娉曞缓绔嬭妯″彲鎺х殑pgRNA鏂囧簱聽;
  • 鍒╃敤pgRNA鏂囧簱鏂囧簱鏋勫缓Huh7.5缁嗚優鏂囧簱;
  • 閫氳繃鐢熼暱閫熺巼鐨勪笉鍚屾潵绛涢夊嚭涓嶅悓鐨勭粏鑳;
  • NGS娴嬪簭barcode浣嶇偣鏉ラ壌鍒叧杩沴ncRNA;
  • 鍚庣画瀹為獙杩涗竴姝ラ獙璇佸叧閿甃ncRNA鐨勪綔鐢ㄣ

5

4

鍥2 姝e悜鍜岃礋鍚戠瓫閫夎幏寰楃殑鍊欓塋ncRNA

聽 聽

CRISPR楂橀氶噺缂栫爜鍩哄洜绛涢


Reference

Zhu S, Li W, Liu J, Chen C-H, Liao Q, Xu P, Xu H, Xiao T,Cao Z, Peng J, Yuan P, Brown M, Liu X* & Wei W*. (2016) CRISPR/Cas9-mediated genomic deletion screening for long non-coding RNAs using paired-gRNAs. Nat Biotechnol, 34(12):1279..