DELIVERY， ENGINEERING AND OPTIMIZATION OF TANDEM GUIDE SYSTEMS， METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION

PROBLEM TO BE SOLVED: To provide methods for modifying genomic locus of interest in a cell. SOLUTION: Provided is a method comprising delivering a non-naturally occurring or engineered composition comprising a tandem guide RNA construct comprising: I. a CRISPR-Cas system chimeric RNA polynucleotide sequence comprising (a) a first guide sequence capable of hybridizing to the first target sequence， (b) a first tracr mate sequence， (c) a first tracr sequence， (d) a second guide sequence capable of hybridizing to the second target sequence， (e) a second tracr mate sequence， and (f) a second tracr sequence， as well as II. a polynucleotide sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences， the first guide sequence directing the cleavage of one strand of a DNA duplex in the vicinity of the first target sequence， and thereby the genomic locus being modified. SELECTED DRAWING: None