AAV mediated gene delivery has unlocked many new possibilities from enabling researchers in basic science to providing hope for the treatment of genetic diseases. Whether it is probing at molecular mechanistic questions in complex organisms or tackling rare genetic diseases, current AAV capsid technology falls short from being a perfect tool. AAV mediated gene therapies are among the most expensive treatment options available, in part, due to the costly inefficient yields with current AAV production methods. Second, the current capsid technology has unfavorable biodistribution yielding cautionary safety profiles. All these limitations can be tackled with AAV capsid engineering. We have previously published how PacBio’s SMRT sequencing has enabled the high throughput capabilities of our whole capsid engineering strategy using capsid shuffled libraries. Herein, we expand on the use of long read technology with a novel AAV9 controlled randomization library. We have identified novel AAV9 based variants yielding enhanced production yields and enhanced neuronal and muscle tissue transduction in mice and nonhuman primates via intrathecal delivery. We show that two amino acid changes spanning two distinct variable regions are sufficient to improve both properties. Using multimodal approaches we demonstrate the effects of these two amino acid changes on atomic resolution structure and how ultimately these result in significant functional differences. This lays the foundation for future generations of AAV technology using whole capsid engineering strategies.