Methods and compositions for producing double allele knock outs

A method of producing a double allele knock-out of a target gene in a eukaryotic cell, comprising the steps of:

• providing the cells with a CRISPR system comprising a wild-type Cas9 nuclease, wherein the Cas9 nuclease has two functional nuclease domains that produce a double-stranded break, and three to seven targeting RNAs located in the same gene, wherein each targeting RNA is comprised of a crRNA and a tracrRNA, wherein each crRNA has a different sequence, and expressing the CRISPR nuclease and the targeting RNAs whereby the target gene is knocked out in both alleles of the cell.