Neuronal DNA repair reveals strategies to influence CRISPR editing outcomes.

Genome editing is poised to revolutionize treatment of genetic diseases, but poor understanding and control of DNA repair outcomes hinders its therapeutic potential. DNA repair is especially understudied in nondividing cells like neurons, which must withstand decades of DNA damage without replicating. This lack of knowledge limits the efficiency and precision of genome editing in clinically relevant cells. To address this, we used induced pluripotent stem cells (iPSCs) and iPSC-derived neurons to examine how postmitotic human neurons repair Cas9-induced DNA damage. We discovered that neurons can take weeks to fully resolve this damage, compared to just days in isogenic iPSCs. Furthermore, Cas9-treated neurons upregulated unexpected DNA repair genes, including factors canonically associated with replication. Manipulating this response with chemical or genetic perturbations allowed us to direct neuronal repair toward desired editing outcomes. By studying DNA repair in postmitotic human cells, we uncovered unforeseen challenges and opportunities for precise therapeutic editing.

Protocol for Delivery of CRISPR/dCas9 Systems for Epigenetic Editing into Solid Tumors Using Lipid Nanoparticles Encapsulating RNA.

Genome editing tools, particularly the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems (e.g., CRISPR/Cas9), and their repurposing into epigenetic editing platforms, offer enormous potential as safe and customizable therapies for cancer. Specifically, various transcriptional abnormalities in human malignancies, such as silencing of tumor suppressors and ectopic re-expression of oncogenes, have been successfully targeted with virtually no off-target effects using CRISPR activation and repression systems. In these systems, the nuclease-deactivated Cas9 protein (dCas9) is fused to one or more domains inducing selective activation or repression of the targeted genes. Despite these advances, the efficient in vivo delivery of these molecules into the target cancer cells represents a critical barrier to accomplishing translation into a clinical therapy setting for cancer. Major obstacles include the large size of dCas9 fusion proteins, the necessity of multimodal delivery of protein and gRNAs, and the potential of these formulations to elicit detrimental immune responses.In this context, viral methods for delivering CRISPR face several limitations, such as the packaging capacity of the viral genome, the potential for integration of the nucleic acids into the host cells genome, and immunogenicity of viral proteins, posing serious safety concerns. The rapid development of mRNA vaccines in response to the COVID-19 pandemic has rekindled interest in mRNA-based approaches for CRISPR/dCas9 delivery. Simultaneously, due to their high loading capacity, scalability, customizable surface modification for cell targeting, and low immunogenicity, lipid nanoparticles (LNPs) have been widely explored as nonviral vectors. In this chapter, we first describe the design of optimized dCas9-effector mRNAs and gRNAs for epigenetic editing. We outline formulations of LNPs suitable for dCas9 mRNA delivery. Additionally, we provide a protocol for the co-encapsulation of the dCas9-effector mRNAs and gRNA into these LNPs, along with detailed methods for delivering these formulations to both cell lines (in vitro) and mouse models of breast cancer (in vivo).

Isothermal titration calorimetry analysis of the binding between the maltodextrin binding protein malE of Staphylococcus aureus with maltodextrins of various lengths.

Staphylococcus aureus (S. aureus), a Gram-positive bacterium, causes a wide range of infections, and diagnosis at an early stage is challenging. Targeting the maltodextrin transporter has emerged as a promising strategy for imaging bacteria and has been able to image a wide range of bacteria including S. aureus. However, little is known about the maltodextrin transporter in S. aureus, and this prevents new S. aureus specific ligands for the maltodextrin transporter from being developed. In Gram-positive bacteria, including S. aureus, the first step of maltodextrin transport is the binding of the maltodextrin-binding protein malE to maltodextrins. Thus, understanding the binding affinity and characteristics of malE from S. aureus is important to developing efficient maltodextrin-based imaging probes. We evaluated the affinity of malE of S. aureus to maltodextrins of various lengths. MalE of S. aureus (SAmalE) was expressed in E. coli BL21(DE3) and purified by Ni-NTA resin. The affinities of SAmalE to maltodextrins were evaluated with isothermal titration calorimetry. SAmalE has low affinity to maltose but binds to maltotriose and longer maltodextrins up to maltoheptaose with affinities up to Ka = 9.02 ± 0.49 × 105 M-1. SAmalE binding to maltotriose-maltoheptaose was exothermic and fit a single-binding site model. The van't Hoff enthalpy in the binding reaction of SAmalE with maltotriose was 9.9 ± 1.3 kcal/mol, and the highest affinity of SAmalE was observed with maltotetraose with Ka = 9.02 ± 0.49 × 105 M-1. In the plot of ΔH-T*ΔS, the of Enthalpy-Entropy Compensation effect was observed in binding reaction of SAmalE to maltodextrins. Acarbose and maltotetraiol bind with SAmalE indicating that SAmalE is tolerant of modifications on both the reducing and non-reducing ends of maltodextrins. Our results show that unlike ECmalE and similar to the maltodextrin binding protein of Streptococci, SAmalE primarily binds to maltodextrins via hydrogen bonds. This is distinct from the maltodextrin binding protein of Streptococci, SAmalE that binds to maltotetraiol with high affinity. Understanding the binding characteristics and tolerance to maltodextrins modifications by maltodextrin binding proteins will hopefully provide the basis for developing bacterial species-specific maltodextrin-based imaging probes.