CRISPR is a revolutionary new system that can be used to efficiently and easily modify the genome.  By introducing genome-wide gene knockouts, or transcriptional activation or repression, both applications that have been demonstrated with CRISPR libraries, the physiological relevance of drug targets can be better understood. Agilent’s parallel DNA synthesis capability is an ideal platform for generating these libraries for custom and standard applications. The ability to design and synthesize any DNA sequence is at the core of genome engineering efforts.  Agilent’s capability to synthesize up to 2.44 x 10⁵ high quality DNA oligomers of 230 nt in length on a single chip enables a variety of experimental approaches otherwise not accessible. This poster describes the synthesis process that enable higher fidelity and representation in the construction of libraries of DNA encoding short guide RNAs for CAS9 to enable genome-wide gene knock out screening or CAS mediated transcriptional activation or repression (CRISPR-A/CRISPR-I).  In addition, a novel, overlap-based cloning method is described that enables a consistent workflow for generating ultra-high quality, low-bias CRISPR plasmid libraries.  These improvements allow fast and efficient construction of libraries far exceeding the quality of competing platforms.