This study introduces a method for precise genome engineering using CRISPR/Cas systems. The researchers engineered two different type II CRISPR/Cas systems and demonstrated 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. Multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating the easy programmability and wide applicability of the RNA-guided nuclease technology. The study shows that the S. pyogenes CRISPR system can be heterologously reconstituted in mammalian cells to facilitate efficient genome editing. The researchers tested whether heterologous expression of the CRISPR system could achieve targeted cleavage of mammalian chromosomes and found that it could. They also demonstrated the broad applicability of the CRISPR/Cas system in modifying different loci across multiple organisms. The study also shows that the CRISPR/Cas system can be used for targeted genomic insertions and that the use of a nickase may reduce off-target mutations. The natural architecture of CRISPR loci with arrayed spacers suggests the possibility of multiplexed genome engineering. The researchers used a single CRISPR array encoding a pair of EMX1- and PVALB-targeting spacers to detect efficient cleavage at both loci. They further tested targeted deletion of larger genomic regions through concurrent DSBs and observed a 1.6% deletion efficacy, demonstrating the CRISPR/Cas system can mediate multiplexed editing within a single genome. The ability to use RNA to program sequence-specific DNA cleavage defines a new class of genome engineering tools.This study introduces a method for precise genome engineering using CRISPR/Cas systems. The researchers engineered two different type II CRISPR/Cas systems and demonstrated 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. Multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating the easy programmability and wide applicability of the RNA-guided nuclease technology. The study shows that the S. pyogenes CRISPR system can be heterologously reconstituted in mammalian cells to facilitate efficient genome editing. The researchers tested whether heterologous expression of the CRISPR system could achieve targeted cleavage of mammalian chromosomes and found that it could. They also demonstrated the broad applicability of the CRISPR/Cas system in modifying different loci across multiple organisms. The study also shows that the CRISPR/Cas system can be used for targeted genomic insertions and that the use of a nickase may reduce off-target mutations. The natural architecture of CRISPR loci with arrayed spacers suggests the possibility of multiplexed genome engineering. The researchers used a single CRISPR array encoding a pair of EMX1- and PVALB-targeting spacers to detect efficient cleavage at both loci. They further tested targeted deletion of larger genomic regions through concurrent DSBs and observed a 1.6% deletion efficacy, demonstrating the CRISPR/Cas system can mediate multiplexed editing within a single genome. The ability to use RNA to program sequence-specific DNA cleavage defines a new class of genome engineering tools.