While the genetic and pathogenic basis of human diseases continues to grow, translation is currently bottlenecked by lack of tools and technologies to administer and evaluate corresponding gene-based therapeutics. Consequently, development of safe and efficient in vivo gene transfer platforms, coupled with emerging genome and epigenome engineering tools, will transform our ability to target a range of human diseases. In this regard, the holy grail of in vivo genome engineering is the ability to achieve the trifecta of: 1) efficient and safe delivery; 2) temporally regulatable and tunable payload delivery; and 3) immune stealth to minimize dosage & enable re-administration of nucleic acid or protein therapeutics. Towards this, the objective of this dissertation was to develop a platform to enable efficacious in vivo genome and epigenome engineering with a focus on enabling in situ therapeutic efficacy. The studies in this dissertation are independent bodies of work that explore the optimization and engineering of CRISPR-Cas9 systems to bring these one step closer to their eventual translation into the clinic. Towards these, I first developed a robust and generalizable platform for in situ genome editing and regulation via AAV CRISPR-Cas9. Towards this, I utilized split-Cas9 systems to develop a modular adeno-associated viral (AAV) vector platform for CRISPR-Cas9 delivery to enable the full spectrum of targeted in situ gene regulation functionalities, demonstrating robust transcriptional repression (up to 80%) and activation (up to 6-fold) of target genes in cell culture and mice. We also applied our platform for targeted in vivo gene-repression-mediated gene therapy for retinitis pigmentosa. Specifically, we engineered targeted repression of Nrl, a master regulator of rod photoreceptor determination, and demonstrated Nrl knockdown mediates in situ reprogramming of rod cells into cone-like cells that are resistant to retinitis pigmentosa-specific mutations, with concomitant prevention of secondary cone loss. Furthermore, we benchmarked our results from Nrl knockdown with those from in vivo Nrl knockout via gene editing. Taken together, our AAV-CRISPR-Cas9 platform for in vivo epigenome engineering enables a robust approach to target disease in a genomically scarless and potentially reversible manner. Additionally, this is the first time that the utility of AAV-KRAB-dCas9 mediated in situ gene repression in the context of gene therapy was demonstrated (Moreno et al., WIREs Systems Biology and Medicine, 2017; Moreno et al., Molecular Therapy, 2018). Next, I focused on addressing, arguably the most important hurdle for CRISPR-Cas based gene therapies, which is the interaction of these non-host derived systems with the adaptive immune system which can lead to neutralization by circulating antibodies and clearance of treated cells by cytotoxic T-lymphocytes. To address this issue, I proposed a new approach: sequential use of orthologous proteins that are orthogonal in immune space. This would, in principle, allow for repeated treatments by thus chosen orthologs without reduced efficacy due to lack of immune cross-reactivity among the proteins. To explore and validate this concept we chose 284 DNA targeting and 84 RNA targeting CRISPR effectors (including Cas9, Cpf1/Cas12a, and Cas13a, b, and c), and 167 Adeno-associated virus (AAV) capsid protein orthologs and developed a pipeline to compare total sequence similarity as well as predicted binding to class I and class II Major Histocompatibility Complex (MHC) proteins. Our MHC binding predictions revealed wide diversity among the set of DNA-targeting Cas orthologs, with 79% of pairs predicted not to elicit cross-reacting immune responses, while no global immune orthogonality among AAV serotypes was observed. We validated the computationally predicted immune orthogonality among three important Cas9 orthologs, from S. pyogenes, S. aureus, and C. jejuni observing cross-reacting antibodies against AAV but not Cas9 orthologs in sera from immunized mice. Finally, to demonstrate the efficacy of multiple dosing with immune orthogonal orthologs, we delivered AAV-Cas9 targeting PCSK9 into BALB/c mice previously immunized against the AAV vector and/or the Cas9 payload, demonstrating that editing efficiency is compromised by immune recognition of either the AAV or Cas9, but, importantly, this effect is abrogated when using immune orthogonal orthologs. Moving forward, we anticipate this framework can be applied to prescribe sequential transient regimens of immune orthogonal protein therapeutics to circumvent pre-existing or induced immunity, and eventually, to rationally engineer immune orthogonality among protein orthologs. (Moreno, Palmer et al., Nature Biomedical Engineering, in press, 2019). Lastly, I then proceeded to integrate the advances accomplished in the previous chapters to enable pain management via in situ genome repression. In the US and worldwide, pain is a leading cause of disability, which leads to a diminished quality of life. Patients have come to routinely expect pharmacological management, with the prevalent aggressive approach for managing pain states being based on opiates. While the utility of opiates has made them a mainstay of pain management, there are at least four key reasons supporting the need for new and alternative pain therapeutics: limited efficacy, abuse potential, tolerance after continued exposure, and an enhancement of post-wound pain states. Despite decades of research, broad-acting, longer-lived, non-addictive, and effective drugs for chronic pain remain elusive. Notably, genetic studies have correlated a hereditary loss-of-function mutation in a human Na+ channel isoform--NaV1.7--with a rare genetic disorder, Congenital Insensitivity to Pain (CIP), which leads to insensitivity to pain without other neurodevelopmental alterations. While an excellent target, the creation of blockers for this site has not led yet to an efficient and safe drug, due to their lack of specificity, leading to unwanted side-effects. Taking advantage of this druggable target in the human genome, the aim of this work was to develop a novel therapeutic regiment via in situ NaV1.7 repression to regulate the development and maintenance of impending chronic pain states. In this regard, I demonstrated robust in vitro repression using two epigenome engineering tools (KRAB-CRISPR-Cas9 and KRAB-Zinc-Fingers) with ~71% and ~88% repression respectively. To enable pain management, I injected mice intrathecally using the constructs with the highest in vitro efficacy and demonstrated robust NaV1.7 repression with a significant improvement in pain response in a carrageenan inflammatory pain model. I demonstrated a 133% improvement in paw withdrawal latency as compared to a negative control (mCherry) and 62% improvement over the positive control (Gabapentin, 100 mg/kg). Taken together, the use of these in situ engineering approaches could thus represent a viable replacement for opioids and a potential therapeutic approach that is tunable and reversible (Moreno et al., in preparation). Together, the advances in these bodies of work, which demonstrate efficacious in vivo delivery and gene editing/regulation is a significant step toward their implementation for gene therapeutic applications.