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10.1038/nprot.2013.143

http://scihub22266oqcxt.onion/10.1038/nprot.2013.143
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24157548!3969860!24157548
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suck abstract from ncbi

pmid24157548      Nat+Protoc 2013 ; 8 (11): 2281-2308
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  • Genome engineering using the CRISPR-Cas9 system #MMPMID24157548
  • Ran FA; Hsu PD; Wright J; Agarwala V; Scott DA; Zhang F
  • Nat Protoc 2013[Nov]; 8 (11): 2281-2308 PMID24157548show ga
  • Targeted nucleases are powerful tools for mediating genome alteration with high precision. The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA. Here we describe a set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies. To minimize off-target cleavage, we further describe a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs. This protocol provides experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. Beginning with target design, gene modifications can be achieved within as little as 1-2 weeks, and modified clonal cell lines can be derived within 2-3 weeks.
  • |*Clustered Regularly Interspaced Short Palindromic Repeats[MESH]
  • |*Genome[MESH]
  • |Base Sequence[MESH]
  • |Cell Culture Techniques[MESH]
  • |Cell Line[MESH]
  • |DNA End-Joining Repair[MESH]
  • |DNA Mutational Analysis[MESH]
  • |DNA Repair[MESH]
  • |Deoxyribonucleases/chemistry/genetics[MESH]
  • |Genetic Engineering/*methods[MESH]
  • |Genotyping Techniques[MESH]
  • |HEK293 Cells[MESH]
  • |Humans[MESH]
  • |Molecular Sequence Data[MESH]
  • |Mutagenesis[MESH]


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