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Nme2Cas9 has been established as a genome editing platform with compact size, high accuracy, and broad targeting range, including single-AAV-deliverable adenine base editors. Here, we have engineered Nme2Cas9 to further increase the activity and targeting scope of compact Nme2Cas9 base editors. We first used domain insertion to position the deaminase domain nearer the displaced DNA strand in the target-bound complex. These domain-inlaid Nme2Cas9 variants exhibited shifted editing windows and increased activity in comparison to the N-terminally fused Nme2-ABE. We next expanded the editing scope by swapping the Nme2Cas9 PAM-interacting domain with that of SmuCas9, which we had previously defined as recognizing a single-cytidine PAM. We used these enhancements to correct two commonMECP2mutations associated with Rett syndrome with little or no bystander editing. Finally, we validated domain-inlaid Nme2-ABEs for single-AAV deliveryin vivo.

Guide RNAs offer programmability for CRISPR-Cas9 genome editing but also add challenges for delivery. Chemical modification, which has been key to the success of oligonucleotide therapeutics, can enhance the stability, distribution, cellular uptake, and safety of nucleic acids. Previously, we engineered heavily and fully modified SpyCas9 crRNA and tracrRNA, which showed enhanced stability and retained activity when delivered to cultured cells in the form of the ribonucleoprotein complex. In this study, we report that a short, fully stabilized oligonucleotide (a “protecting oligo”), which can be displaced by tracrRNA annealing, can significantly enhance the potency and stability of a heavily modified crRNA. Furthermore, protecting oligos allow various bioconjugates to be appended, thereby improving cellular uptake and biodistribution of crRNAin vivo. Finally, we achievedin vivogenome editing in adult mouse liver and central nervous system via co-delivery of unformulated, chemically modified crRNAs with protecting oligos and AAV vectors that express tracrRNA and either SpyCas9 or a base editor derivative. Our proof-of-concept establishment of AAV/crRNA co-delivery offers a route towards transient editing activity, target multiplexing, guide redosing, and vector inactivation.

For our Junior Project course we worked with John Anderson, apart of the Mechanical and Manufacturing Engineering and Technology department at Oregon Institute of Technology, to build a Robotic Control System. This system would allow a more cost effective way for teachers and students to create scripts for running a robot in an industry style assembly line. The basic functionality of such a program is to allow the user to create, manage, debug, run and save scripts that will directly manipulate a connected robot. The long term goal was to have a system that will allow any kind of robot to be implemented and have our front end connect to it and work exactly the same. We accomplished this using careful planning to give it the most agile feel possible. We are also attempting to utilize a combination of Microsoft Robotics Studio and direct serial port communication to interact with a Lynxmotion Lynx6 robot.Copyright © 2007 by ASME