Genome engineering

A method of multiplex altering of a plurality of target DNA at multiple genomic loci in a eukaryotic cell in vitro wherein the cell genome is genetically modified to include a nucleic acid encoding a Cas 9 enzyme and wherein the Cas 9 enzyme is expressed, comprising

• (a) introducing into the cell a DNA donor nucleic acid sequence,
• (b) introducing into the cell guide RNA complementary to the target DNA, wherein the guide RNA and the Cas 9 enzyme are members of a co-localization complex for the target DNA, wherein the guide RNA is modified from native form to include a 5′ Cap structure,
• wherein the guide RNA and the Cas 9 enzyme co-localize to the target DNA, the Cas 9 enzyme cleaves the target DNA and the donor nucleic acid is inserted into the target DNA to produce altered DNA in the cell, and
• repeating steps (a) and (b) to result in multiple exogenous nucleic acid insertions in the cell wherein steps (a) and (b) are carried out without further introducing a nucleic acid encoding a Cas 9 enzyme.

A method of multiplex altering of a plurality of target DNA at multiple genomic loci in a eukaryotic cell in vitro wherein the cell genome is genetically modified to include a nucleic acid encoding a Cas 9 enzyme and wherein the Cas 9 enzyme is expressed, comprising

• (a) introducing into the cell a DNA donor nucleic acid sequence,
• (b) introducing into the cell guide RNA complementary to the target DNA, wherein the guide RNA and the Cas 9 enzyme are members of a co-localization complex for the target DNA, wherein the guide RNA is modified from native form by treating with phosphatase,
• wherein the guide RNA and the Cas 9 enzyme co-localize to the target DNA, the Cas 9 enzyme cleaves the target DNA and the donor nucleic acid is inserted into the target DNA to produce altered DNA in the cell, and
• repeating steps (a) and (b) to result in multiple exogenous nucleic acid insertions in the cell wherein steps (a) and (b) are carried out without further introducing a nucleic acid encoding a Cas 9 enzyme.