CRISPR/Cas systems have revolutionized biomedical research by providing easily programmable nucleic acid-targeting proteins. However, the discovery of different CRISPR systems in various organisms and their rapid proliferation in research settings have crowded the field and ultimately complicated the use of CRISPR tools. During my research, I investigated how different CRISPR/Cas9 design strategies impact the efficiency of these tools. Additionally, I quantified and compared the efficacy of CRISPR/Cas systems from a variety of different species for genome editing and transcriptional control. Finally, I leveraged these optimized design parameters to create a new synthetic system for controlling the generation of new mitochondria within living human cells.