完美基因编辑：新方法可消除CRISPR-Cas的脱靶效应（附原文）

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DNA targeting specificity of RNA-guided Cas9 nucleases
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Patrick D Hsu,         David A Scott,         Joshua A Weinstein,         F Ann Ran,         Silvana Konermann,         Vineeta Agarwala,         Yinqing Li,         Eli J Fine,         Xuebing Wu,         Ophir Shalem,         Thomas J Cradick,         Luciano A Marraffini,         Gang Bao         & Feng Zhang
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2 h& N2 h. W* @" a0 n" {* rNature Biotechnology (2013) doi:10.1038/nbt.2647
0 y* y; g' ~+ tReceived 30 March 2013 Accepted 30 June 2013 Published online 21 July 2013  O0 n/ }' L2 v8 f$ a) n$ I
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Abstract
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Here we use the clustered, regularly interspaced, short palindromic repeats (CRISPR)–associated Cas9 endonuclease complexed with dual-RNAs to introduce precise mutations in the genomes of Streptococcus pneumoniae and Escherichia coli. The approach relies on dual-RNA:Cas9-directed cleavage at the targeted genomic site to kill unmutated cells and circumvents the need for selectable markers or counter-selection systems. We reprogram dual-RNA:Cas9 specificity by changing the sequence of short CRISPR RNA (crRNA) to make single- and multinucleotide changes carried on editing templates. Simultaneous use of two crRNAs enables multiplex mutagenesis. In S. pneumoniae, nearly 100% of cells that were recovered using our approach contained the desired mutation, and in E. coli, 65% that were recovered contained the mutation, when the approach was used in combination with recombineering. We exhaustively analyze dual-RNA:Cas9 target requirements to define the range of targetable sequences and show strategies for editing sites that do not meet these requirements, suggesting the versatility of this technique for bacterial genome engineering.