cas9 质粒转染protocal.pdf

protocol Genome engineering using the CRISPR-Cas9 system 1–5,8 1–5,8 1 1,6,7 1–4 1–4 F Ann Ran , Patrick D Hsu , Jason Wright , Vineeta Agarwala , David A Scott Feng Zhang 1Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard, Cambridge, Massachusetts, USA. 2McGovern Institute for Brain Research, Cambridge, Massachusetts, USA. 3Department of Brain and Cognitive Sciences, MIT, Cambridge, Massachusetts, USA. 4Department of Biological Engineering, MIT, Cambridge, Massachusetts, USA. 5Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA. 6Program in Biophysics, Harvard University, MIT, Cambridge, Massachusetts, USA. 7Harvard-MIT Division of Health Sciences and Technology, MIT, Cambridge, Massachusetts, USA. 8These authors contributed equally to this work. Correspondence should be addressed to F.Z. (zhang@). Published online 24 October 2013; doi:10.1038/nprot.2013.143 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 cleavag