Producing Engineered Cells by Introducing Cas9/sgRNA Complexes

A method of producing an engineered human cell, comprising:

• providing a composition for inducing a modification of a target endogenous nucleic acid sequence in a nucleus of a human cell,
• wherein the composition comprises an amount of a recombinant Cas9 protein and an amount of a single-guide RNA (sgRNA) in an in vitro environment free of the human cell,
• wherein the recombinant Cas9 protein comprises a nuclear localization signal (NLS);
• wherein the sgRNA comprises a crRNA and a tracrRNA;
• wherein the amount of sgRNA and the amount of Cas9 protein are present in the composition at a weight ratio of 4:9 to 4:3;
• and
• wherein a Cas9/sgRNA complex forms in the in vitro environment prior to being introduced into the human cell;
• and
• introducing the Cas9/sgRNA complex into the human cell via electroporation, wherein the Cas9/sgRNA complex induces the modification of the target endogenous nucleic acid sequence in the nucleus of the human cell to provide for the engineered human cell.