the tal effector nuclease heterodimer talen are underlined as shown in fig1 c bar graph depict hr efficiencies induced by t1 t2 and talen mediated nuclease activity at the target locus as measured by facs representative facs plots and microscopy images of the targeted cells are depicted below scale bar is 100 microns data is mean u2212 sem n 3 according to one aspect to direct cas9 to cleave sequences of interest crrna tracrrna fusion transcripts are expressed hereafter referred to as guide rnas grnas from the human u6 polymerase iii promoter according to one aspect grnas are directly transcribed by the cell this aspect advantageously avoids reconstituting the rna processing machinery employed by bacterial crispr systems fig1 a and fig3 b see references 4 7 9 according to one aspect a method is provided for altering genomic dna using a u6 transcription initiating with g and a pam protospacer adjacent motif sequence ngg following the 20 bp crrna target according to this aspect the target genomic site is in the form of gn 20 gg see fig3 c according to one aspect a gfp reporter assay fig1 b in 293t cells was developed similar to one previously described see reference 10 to test the functionality of the genome engineering methods described herein according to one aspect a stable cell line was established bearing a genomically integrated gfp coding sequence disrupted by the insertion of a stop codon and a 68 bp genomic fragment from the aavs1 locus that renders the expressed protein fragment non fluorescent homologous recombination hr using an appropriate repair donor can restore the normal gfp sequence which allows.