P21-activated kinase-1 signaling is required to preserve adipose tissue homeostasis and cardiac function.

While P21-activated kinase-1 (PAK1) has been extensively studied in relation to cardiovascular health and glucose metabolism, its roles within adipose tissue and cardiometabolic diseases are less understood. In this study, we explored the effects of PAK1 deletion on energy balance, adipose tissue homeostasis, and cardiac function utilizing a whole-body PAK1 knockout (PAK1-/-) mouse model. Our findings revealed that body weight differences between PAK1-/- and WT mice emerged at 9 weeks of age, with further increases observed at 12 weeks. Furthermore, PAK1-/- mice displayed increased fat mass and decreased lean mass at 12 weeks, indicating a shift towards adiposity. In conjunction with the increased body weight, PAK1-/- mice had increased food intake and reduced energy expenditure. At a mechanistic level, PAK1 deletion boosted the expression of lipogenic markers while diminishing thermogenic markers expression in adipose tissues, contributing to reduced energy expenditure and the overall obesogenic phenotype. Moreover, our findings highlighted a significant impact on cardiac function following PAK1 deletion, including alterations in calcium kinetics and compromised systolic and lusitropy functions. In summary, our study emphasizes the significant role of PAK1 in weight regulation and cardiac function, enriching our comprehension of heart health and metabolism. These findings could potentially facilitate the identification of novel therapeutic targets in cardiometabolic diseases.

Analysis of NIH K99/R00 awards and the career progression of awardees.

Many postdoctoral fellows and scholars who hope to secure tenure-track faculty positions in the United States apply to the National Institutes of Health (NIH) for a Pathway to Independence Award. This award has two phases (K99 and R00) and provides funding for up to 5 years. Using NIH data for the period 2006-2022, we report that ~230 K99 awards were made every year, representing up to ~$250 million annual investment. About 40% of K99 awardees were women and ~89% of K99 awardees went on to receive an R00 award annually. Institutions with the most NIH funding produced the most recipients of K99 awards and recruited the most recipients of R00 awards. The time between a researcher starting an R00 award and receiving a major NIH award (such as an R01) ranged between 4.6 and 7.4 years, and was significantly longer for women, for those who remained at their home institution, and for those hired by an institution that was not one of the 25 institutions with the most NIH funding. Shockingly, there has yet to be a K99 awardee at a historically Black college or university. We go on to show how K99 awardees flow to faculty positions, and to identify various factors that influence the future success of individual researchers and, therefore, also influence the composition of biomedical faculty at universities in the United States.

Corticotropin-releasing hormone signaling from prefrontal cortex to lateral septum suppresses interaction with familiar mice.

Social preference, the decision to interact with one member of the same species over another, is critical to optimize social interactions. Thus, adult rodents favor interacting with novel conspecifics over familiar ones, but whether this social preference stems from neural circuits facilitating interactions with novel individuals or suppressing interactions with familiar ones remains unknown. Here, we identify neurons in the infra-limbic area (ILA) of the mouse prefrontal cortex that express the neuropeptide corticotropin-releasing hormone (CRH) and project to the dorsal region of the rostral lateral septum (rLS). We show how release of CRH during familiar encounters disinhibits rLS neurons, thereby suppressing social interactions with familiar mice and contributing to social novelty preference. We further demonstrate how the maturation of CRH expression in ILA during the first 2 post-natal weeks enables the developmental shift from a preference for littermates in juveniles to a preference for novel mice in adults.

Changes in the levels of free sialic acid during ex vivo lung perfusion do not correlate with pulmonary function. Experimental model.

BACKGROUND: Ex vivo lung perfusion (EVLP) constitutes a tool with great research potential due to its advantages over in vivo and in vitro models. Despite its important contribution to lung reconditioning, this technique has the disadvantage of incurring high costs and can induce pulmonary endothelial injury through perfusion and ventilation. The pulmonary endothelium is made up of endothelial glycocalyx (EG), a coating of proteoglycans (PG) on the luminal surface. PGs are glycoproteins linked to terminal sialic acids (Sia) that can affect homeostasis with responses leading to edema formation. This study evaluated the effect of two ex vivo perfusion solutions on lung function and endothelial injury. METHODS: We divided ten landrace swine into two groups and subjected them to EVLP for 120 min: Group I (n = 5) was perfused with Steen® solution, and Group II (n = 5) was perfused with low-potassium dextran-albumin solution. Ventilatory mechanics, histology, gravimetry, and sialic acid concentrations were evaluated. RESULTS: Both groups showed changes in pulmonary vascular resistance and ventilatory mechanics (p < 0.05, Student's t-test). In addition, the lung injury severity score was better in Group I than in Group II (p < 0.05, Mann-Whitney U); and both groups exhibited a significant increase in Sia concentrations in the perfusate (p < 0.05 t-Student) and Sia immunohistochemical expression. CONCLUSIONS: Sia, as a product of EG disruption during EVLP, was found in all samples obtained in the system; however, the changes in its concentration showed no apparent correlation with lung function.

Drug screening in human physiologic medium identifies uric acid as an inhibitor of rigosertib efficacy.

The non-physiological nutrient levels found in traditional culture media have been shown to affect numerous aspects of cancer cell physiology, including how cells respond to certain therapeutic agents. Here, we comprehensively evaluated how physiological nutrient levels impact therapeutic response by performing drug screening in human plasma-like medium (HPLM). We observed dramatic nutrient-dependent changes in sensitivity to a variety of FDA-approved and clinically trialed compounds, including rigosertib, an experimental cancer therapeutic that has recently failed in phase 3 clinical trials. Mechanistically, we found that the ability of rigosertib to destabilize microtubules is strongly inhibited by the purine metabolism waste product uric acid, which is uniquely abundant in humans relative to traditional in vitro and in vivo cancer models. Structural modelling studies suggest that uric acid interacts with the tubulin-rigosertib complex and may act as an uncompetitive inhibitor of rigosertib. These results offer a possible explanation for the failure of rigosertib in clinical trials and demonstrate the utility of physiological media to achieve in vitro results that better represent human therapeutic responses.

Cardiomyocyte external mechanical unloading activates modifications of α-actinin differently from sarcomere-originated unloading.

Loss of myocardial mass in a neonatal rat cardiomyocyte culture is studied to determine whether there is a distinguishable cellular response based on the origin of mechano-signals. The approach herein compares the sarcomeric assembly and disassembly processes in heart cells by imposing mechano-signals at the interface with the extracellular matrix (extrinsic) and at the level of the myofilaments (intrinsic). Experiments compared the effects of imposed internal (inside/out) and external (outside/in) loading and unloading on modifications in neonatal rat cardiomyocytes. Unloading of the cellular substrate by myosin inhibition (1 µm mavacamten), or cessation of cyclic strain (1 Hz, 10% strain) after preconditioning, led to significant disassembly of sarcomeric α-actinin by 6 h. In myosin inhibition, this was accompanied by redistribution of intracellular poly-ubiquitin K48 to the cellular periphery relative to the poly-ubiquitin K48 reservoir at the I-band. Moreover, loading and unloading of the cellular substrate led to a three-fold increase in post-translational modifications (PTMs) when compared to the myosin-specific activation or inhibition. Specifically, phosphorylation increased with loading while ubiquitination increased with unloading, which may involve extracellular signal-regulated kinase 1/2 and focal adhesion kinase activation. The identified PTMs, including ubiquitination, acetylation, and phosphorylation, are proposed to modify internal domains in α-actinin to increase its propensity to bind F-actin. These results demonstrate a link between mechanical feedback and sarcomere protein homeostasis via PTMs of α-actinin that exemplify how cardiomyocytes exhibit differential responses to the origin of force. The implications of sarcomere regulation governed by PTMs of α-actinin are discussed with respect to cardiac atrophy and heart failure.

Analysis of NIH K99/R00 Awards and the Career Progression of Awardees.

Many postdoctoral fellows and scholars who hope to secure tenure-track faculty positions in the United States apply to the National Institutes of Health (NIH) for a Pathway to Independence Award. This award has two phases (K99 and R00) and provides funding for up to five years. Using NIH data for the period 2006-2022, we report that ~230 K99 awards were made every year, representing up to ~$250 million annual investment. About 40% of K99 awardees were women and ~89% of K99 awardees went on to receive an R00 award annually. Institutions with the most NIH funding produced the most recipients of K99 awards and recruited the most recipients of R00 awards. The time between a researcher starting an R00 award and receiving a major NIH award (such as an R01) ranged between 4.6 and 7.4 years, and was significantly longer for women, for those who remained at their home institution, and for those hired by an institution that was not one of the 25 institutions with the most NIH funding. Shockingly, there has yet to be a K99 awardee at a historically Black college or university. We go on to show how K99 awardees flow to faculty positions, and to identify various factors that influence the future success of individual researchers and, therefore, also influence the composition of biomedical faculty at universities in the US.

CPEB3 low-complexity motif regulates local protein synthesis via protein-protein interactions in neuronal ribonucleoprotein granules.

Biomolecular condensates, membraneless organelles found throughout the cell, play critical roles in many aspects of cellular function. Ribonucleoprotein granules (RNPs) are a type of biomolecular condensate necessary for local protein synthesis and are involved in synaptic plasticity and long-term memory. Most of the proteins in RNPs possess low-complexity motifs (LCM), allowing for increased promiscuity of protein-protein interactions. Here, we describe the importance of protein-protein interactions mediated by the LCM of RNA-binding protein cytoplasmic polyadenylation element binding protein 3 (CPEB3). CPEB3 is necessary for long-term synaptic plasticity and memory persistence, but the mechanisms involved are still not completely elucidated. We now present key mechanisms involved in its regulation of synaptic plasticity. We find that CPEB3-LCM plays a role in appropriate local protein synthesis of messenger ribonucleic acid (mRNA) targets, through crucial protein-protein interactions that drive localization to neuronal Decapping protein 1 (DCP1)-bodies. Translation-promoting CPEB3 and translation-inhibiting CPEB1 are packaged into neuronal RNP granules immediately after chemical long-term potentiation is induced, but only translation-promoting CPEB3 is repackaged to these organelles at later time points. This localization to neuronal RNP granules is critical for functional influence on translation as well as overall local protein synthesis (measured as α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) insertion into the membrane and localization to the synapse). We therefore conclude that protein-protein interaction between the LCM of CPEB3 plays a critical role in local protein synthesis by utilizing neuronal RNP granules.

Mechano-growth factor E-domain modulates cardiac contractile function through 14-3-3 protein interactomes.

In the heart, alternative splicing of the igf-I gene produces two isoforms: IGF-IEa and IGF-IEc, (Mechano-growth factor, MGF). The sequence divergence between their E-domain regions suggests differential isoform function. To define the biological actions of MGF's E-domain, we performed in silico analysis of the unique C-terminal sequence and identified a phosphorylation consensus site residing within a putative 14-3-3 binding motif. To test the functional significance of Ser 18 phosphorylation, phospho-mimetic (S/E18) and phospho-null (S/A18) peptides were delivered to mice at different doses for 2 weeks. Cardiovascular function was measured using echocardiography and a pressure-volume catheter. At the lowest (2.25 mg/kg/day) and highest (9 mg/kg/day) doses, the peptides produced a depression in systolic and diastolic parameters. However, at 4.5 mg/kg/day the peptides produced opposing effects on cardiac function. Fractional shortening analysis also showed a similar trend, but with no significant change in cardiac geometry. Microarray analysis discovered 21 genes (FDR p < 0.01), that were expressed accordant with the opposing effects on contractile function at 4.5 mg/kg/day, with the nuclear receptor subfamily 4 group A member 2 (Nr4a2) identified as a potential target of peptide regulation. Testing the regulation of the Nr4a family, showed the E-domain peptides modulate Nr4a gene expression following membrane depolarization with KCl in vitro. To determine the potential role of 14-3-3 proteins, we examined 14-3-3 isoform expression and distribution. 14-3-3γ localized to the myofilaments in neonatal cardiac myocytes, the cardiac myocytes and myofilament extracts from the adult heart. Thermal shift analysis of recombinant 14-3-3γ protein showed the S/A18 peptide destabilized 14-3-3γ folding. Also, the S/A18 peptide significantly inhibited 14-3-3γ's ability to interact with myosin binding protein C (MYPC3) and phospholamban (PLN) in heart lysates from dobutamine injected mice. Conversely, the S/E18 peptide showed no effect on 14-3-3γ stability, did not inhibit 14-3-3γ's interaction with PLN but did inhibit the interaction with MYPC3. Replacing the glutamic acid with a phosphate group on Ser 18 (pSer18), significantly increased 14-3-3γ protein stability. We conclude that the state of Ser 18 phosphorylation within the 14-3-3 binding motif of MGF's E-domain, modulates protein-protein interactions within the 14-3-3γ interactome, which includes proteins involved in the regulation of contractile function.

Editorial for 'Issue focus on 2nd Costa Rica biophysics symposium - March 11th-12th, 2021'.

This Editorial describes both the motivation for, and the five articles appearing in, the Issue Focus dedicated to the 2nd Costa Rica Biophysics Symposium which was held in March 2021. Some recent history about both the symposium and developments in science occurring within Costa Rica is described.

Transthyretin deposition alters cardiomyocyte sarcomeric architecture, calcium transients, and contractile force.

Age-related wild-type transthyretin amyloidosis (wtATTR) is characterized by systemic deposition of amyloidogenic fibrils of misfolded transthyretin (TTR) in the connective tissue of many organs. In the heart, this leads to age-related heart failure with preserved ejection fraction (HFpEF). The hypothesis tested is that TTR deposited in vitro disrupts cardiac myocyte cell-to-cell and cell-to-matrix adhesion complexes, resulting in altered calcium handling, force generation, and sarcomeric disorganization. Human iPSC-derived cardiomyocytes and neonatal rat ventricular myocytes (NRVMs), when grown on TTR-coated polymeric substrata mimicking the stiffness of the healthy human myocardium (10 kPa), had decreased contraction and relaxation velocities as well as decreased force production measured using traction force microscopy. Both NRVMs and adult mouse atrial cardiomyocytes had altered calcium kinetics with prolonged transients when cultured on TTR fibril-coated substrates. Furthermore, NRVMs grown on stiff (~GPa), flat or microgrooved substrates coated with TTR fibrils exhibited significantly decreased intercellular electrical coupling as shown by FRAP dynamics of cells loaded with the gap junction-permeable dye calcein-AM, along with decreased gap junction content as determined by quantitative connexin 43 staining. Significant sarcomeric disorganization and loss of sarcomere content, with increased ubiquitin localization to the sarcomere, were seen in NRVMs on various TTR fibril-coated substrata. TTR presence decreased intercellular mechanical junctions as evidenced by quantitative immunofluorescence staining of N-cadherin and vinculin. Current therapies for wtATTR are cost-prohibitive and only slow the disease progression; therefore, better understanding of cardiomyocyte maladaptation induced by TTR amyloid may identify novel therapeutic targets.

A direct lateral entorhinal cortex to hippocampal CA2 circuit conveys social information required for social memory.

The hippocampus is essential for different forms of declarative memory, including social memory, the ability to recognize and remember a conspecific. Although recent studies identify the importance of the dorsal CA2 region of the hippocampus in social memory storage, little is known about its sources of social information. Because CA2, like other hippocampal regions, receives its major source of spatial and non-spatial information from the medial and lateral subdivisions of entorhinal cortex (MEC and LEC), respectively, we investigated the importance of these inputs for social memory. Whereas MEC inputs to CA2 are dispensable, the direct inputs to CA2 from LEC are both selectively activated during social exploration and required for social memory. This selective behavioral role of LEC is reflected in the stronger excitatory drive it provides to CA2 compared with MEC. Thus, a direct LEC → CA2 circuit is tuned to convey social information that is critical for social memory.

Enkephalin release from VIP interneurons in the hippocampal CA2/3a region mediates heterosynaptic plasticity and social memory.

The hippocampus contains a diverse array of inhibitory interneurons that gate information flow through local cortico-hippocampal circuits to regulate memory storage. Although most studies of interneurons have focused on their role in fast synaptic inhibition mediated by GABA release, different classes of interneurons express unique sets of neuropeptides, many of which have been shown to exert powerful effects on neuronal function and memory when applied pharmacologically. However, relatively little is known about whether and how release of endogenous neuropeptides from inhibitory cells contributes to their behavioral role in regulating memory formation. Here we report that vasoactive intestinal peptide (VIP)-expressing interneurons participate in social memory storage by enhancing information transfer from hippocampal CA3 pyramidal neurons to CA2 pyramidal neurons. Notably, this action depends on release of the neuropeptide enkephalin from VIP neurons, causing long-term depression of feedforward inhibition onto CA2 pyramidal cells. Moreover, VIP neuron activity in the CA2 region is increased selectively during exploration of a novel conspecific. Our findings, thus, enhance our appreciation of how GABAergic neurons can regulate synaptic plasticity and mnemonic behavior by demonstrating that such actions can be mediated by release of a specific neuropeptide, rather than through classic fast inhibitory transmission.

Striated muscle proteins are regulated both by mechanical deformation and by chemical post-translational modification.

All cells sense force and build their cytoskeleton to optimize function. How is this achieved? Two major systems are involved. The first is that load deforms specific protein structures in a proportional and orientation-dependent manner. The second is post-translational modification of proteins as a consequence of signaling pathway activation. These two processes work together in a complex way so that local subcellular assembly as well as overall cell function are controlled. This review discusses many cell types but focuses on striated muscle. Detailed information is provided on how load deforms the structure of proteins in the focal adhesions and filaments, using α-actinin, vinculin, talin, focal adhesion kinase, LIM domain-containing proteins, filamin, myosin, titin, and telethonin as examples. Second messenger signals arising from external triggers are distributed throughout the cell causing post-translational or chemical modifications of protein structures, with the actin capping protein CapZ and troponin as examples. There are numerous unanswered questions of how mechanical and chemical signals are integrated by muscle proteins to regulate sarcomere structure and function yet to be studied. Therefore, more research is needed to see how external triggers are integrated with local tension generated within the cell. Nonetheless, maintenance of tension in the sarcomere is the essential and dominant mechanism, leading to the well-known phrase in exercise physiology: "use it or lose it."

Announcing the call for the Issue Focus on the 2nd Costa Rican Biophysics Symposium-virtual meeting, March 2021.

This Commentary is a call for submissions for the upcoming Issue Focus that will highlight some of the scientific topics discussed during the 2nd Costa Rica Biophysics Symposium.

Novel insights into sarcomere regulatory systems control of cardiac thin filament activation.

Our review focuses on sarcomere regulatory mechanisms with a discussion of cardiac-specific modifications to the three-state model of thin filament activation from a blocked to closed to open state. We discuss modulation of these thin filament transitions by Ca2+, by crossbridge interactions, and by thick filament-associated proteins, cardiac myosin-binding protein C (cMyBP-C), cardiac regulatory light chain (cRLC), and titin. Emerging evidence supports the idea that the cooperative activation of the thin filaments despite a single Ca2+ triggering regulatory site on troponin C (cTnC) cannot be considered in isolation of other functional domains of the sarcomere. We discuss long- and short-range interactions among these domains with the regulatory units of thin filaments, including proteins at the barbed end at the Z-disc and the pointed end near the M-band. Important to these discussions is the ever-increasing understanding of the role of cMyBP-C, cRLC, and titin filaments. Detailed knowledge of these control processes is critical to the understanding of mechanisms sustaining physiological cardiac state with varying hemodynamic load, to better defining genetic and acquired cardiac disorders, and to developing targets for therapies at the level of the sarcomeres.

Mechanosignaling pathways alter muscle structure and function by post-translational modification of existing sarcomeric proteins to optimize energy usage.

A transduced mechanical signal arriving at its destination in muscle alters sarcomeric structure and function. A major question addressed is how muscle mass and tension generation are optimized to match actual performance demands so that little energy is wasted. Three cases for improved energy efficiency are examined: the troponin complex for tuning force production, control of the myosin heads in a resting state, and the Z-disc proteins for sarcomere assembly. On arrival, the regulation of protein complexes is often controlled by post-translational modification (PTM), of which the most common are phosphorylation by kinases, deacetylation by histone deacetylases and ubiquitination by E3 ligases. Another branch of signals acts not through peptide covalent bonding but via ligand interactions (e.g. Ca2+ and phosphoinositide binding). The myosin head and the regulation of its binding to actin by the troponin complex is the best and earliest example of signal destinations that modify myofibrillar contractility. PTMs in the troponin complex regulate both the efficiency of the contractile function to match physiologic demand for work, and muscle mass via protein degradation. The regulation of sarcomere assembly by integration of incoming signaling pathways causing the same PTMs or ligand binding are discussed in response to mechanical loading and unloading by the Z-disc proteins CapZ, α-actinin, telethonin, titin N-termini, and others. Many human mutations that lead to cardiomyopathy and heart disease occur in the proteins discussed above, which often occur at their PTM or ligand binding sites.

Cardiac troponin and tropomyosin bind to F-actin cooperatively, as revealed by fluorescence microscopy.

In cardiac muscle, binding of troponin (Tn) and tropomyosin (Tpm) to filamentous (F)-actin forms thin filaments capable of Ca2+ -dependent regulation of contraction. Tpm binds to F-actin in a head-to-tail fashion, while Tn stabilizes these linkages. Valuable structural and functional information has come from biochemical, X-ray, and electron microscopy data. However, the use of fluorescence microscopy to study thin filament assembly remains relatively underdeveloped. Here, triple fluorescent labeling of Tn, Tpm, and F-actin allowed us to track thin filament assembly by fluorescence microscopy. It is shown here that Tn and Tpm molecules self-organize on actin filaments and give rise to decorated and undecorated regions. Binding curves based on colocalization of Tn and Tpm on F-actin exhibit cooperative binding with a dissociation constant Kd of ~ 0.5 µm that is independent of the Ca2+ concentration. Binding isotherms based on the intensity profile of fluorescently labeled Tn and Tpm on F-actin show that binding of Tn is less cooperative relative to Tpm. Computational modeling of Tn-Tpm binding to F-actin suggests two equilibrium steps involving the binding of an initial Tn-Tpm unit (nucleation) and subsequent recruitment of adjacent Tn-Tpm units (elongation) that stabilize the assembly. The results presented here highlight the utility of employing fluorescence microscopy to study supramolecular protein assemblies.

CapZ integrates several signaling pathways in response to mechanical stiffness.

Muscle adaptation is a response to physiological demand elicited by changes in mechanical load, hormones, or metabolic stress. Cytoskeletal remodeling processes in many cell types are thought to be primarily regulated by thin filament formation due to actin-binding accessory proteins, such as the actin-capping protein. Here, we hypothesize that in muscle, the actin-capping protein (named CapZ) integrates signaling by a variety of pathways, including phosphorylation and phosphatidylinositol 4,5-bisphosphate (PIP2) binding, to regulate muscle fiber growth in response to mechanical load. To test this hypothesis, we assess mechanotransduction signaling that regulates muscle growth using neonatal rat ventricular myocytes cultured on substrates with the stiffness of the healthy myocardium (10 kPa), fibrotic myocardium (100 kPa), or glass. We investigate how PIP2 signaling affects CapZ using the PIP2 sequestering agent neomycin and the effect of PKC-mediated CapZ phosphorylation using the PKC-activating drug phorbol 12-myristate 13-acetate (PMA). Molecular simulations suggest that close interactions between PIP2 and the ß-tentacle of CapZ are modified by phosphorylation at T267. Fluorescence recovery after photobleaching (FRAP) demonstrates that the kinetic binding constant of CapZ to sarcomeric thin filaments in living muscle cells increases with stiffness or PMA treatment but is diminished by PIP2 reduction. Furthermore, CapZ with a deletion of the ß-tentacle that lacks the phosphorylation site T267 shows increased FRAP kinetics with lack of sensitivity to PMA treatment or PIP2 reduction. Förster resonance energy transfer (FRET) probes the molecular interactions between PIP2 and CapZ, which are decreased by PIP2 availability or by the ß-tentacle truncation. These data suggest that CapZ is bound to actin tightly in the idle, locked state, with little phosphorylation or PIP2 binding. However, this tight binding is loosened in growth states triggered by mechanical stimuli such as substrate stiffness, which may have relevance to fibrotic heart disease.

The Development of an AAV-Based CRISPR SaCas9 Genome Editing System That Can Be Delivered to Neurons in vivo and Regulated via Doxycycline and Cre-Recombinase.

The RNA-guided Cas9 nuclease, from the type II prokaryotic clustered regularly interspersed short palindromic repeats (CRISPR) adaptive immune system, has been adapted by scientists to enable site specific genome editing of eukaryotic cells both in vitro and in vivo. Previously, we reported the development of an adeno-associated virus (AAV)-mediated CRISPR Streptococcus pyogenes (Sp) Cas9 system, in which the genome editing function can be regulated by controlling the expression of the guide RNA (sgRNA) in a doxycycline (Dox)-dependent manner. Here, we report the development of an AAV vector tool kit utilizing the Cas9 from Staphylococcus aureus (SaCas9). We demonstrate in vitro genome editing in human derived 293FT cells and mouse derived Neuro2A (N2A) cells and in vivo in neurons of the mouse brain. We also demonstrate the ability to regulate the induction of genome editing temporally with Dox and spatially with Cre-recombinase. The combination of these systems enables AAV-mediated CRISPR/Cas9 genome editing to be regulated both spatially and temporally.