Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.

Multiplex Genome Engineering Using CRISPR/Cas Systems

Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02142, USA, and McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences, Department of Biological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA.

Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, MA 02115, USA.

Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.

Harvard-MIT Health Sciences and Technology, Harvard Medical School, Boston, MA 02115, USA.

School of Life Sciences, Tsinghua University, Beijing 100084, China.

Department of Biochemistry and Molecular Biophysics, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA.

Computational and Systems Biology Graduate Program and Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.