Engineering self-deliverable ribonucleoproteins for genome editing in the brain.

The delivery of CRISPR ribonucleoproteins (RNPs) for genome editing in vitro and in vivo has important advantages over other delivery methods, including reduced off-target and immunogenic effects. However, effective delivery of RNPs remains challenging in certain cell types due to low efficiency and cell toxicity. To address these issues, we engineer self-deliverable RNPs that can promote efficient cellular uptake and carry out robust genome editing without the need for helper materials or biomolecules.

Engineering self-deliverable ribonucleoproteins for genome editing in the brain.

The delivery of CRISPR ribonucleoproteins (RNPs) for genome editing and has important advantages over other delivery methods, including reduced off-target and immunogenic effects . However, effective delivery of RNPs remains challenging in certain cell types due to low efficiency and cell toxicity. To address these issues, we engineered self-deliverable RNPs that can promote efficient cellular uptake and carry out robust genome editing without the need for helper materials or biomolecules.

Genome editing in the mouse brain with minimally immunogenic Cas9 RNPs.

Transient delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) into the central nervous system (CNS) for therapeutic genome editing could avoid limitations of viral vector-based delivery including cargo capacity, immunogenicity, and cost. Here, we tested the ability of cell-penetrant Cas9 RNPs to edit the mouse striatum when introduced using a convection-enhanced delivery system. These transient Cas9 RNPs showed comparable editing of neurons and reduced adaptive immune responses relative to one formulation of Cas9 delivered using AAV serotype 9.

Improved genome editing by an engineered CRISPR-Cas12a.

CRISPR-Cas12a is an RNA-guided, programmable genome editing enzyme found within bacterial adaptive immune pathways. Unlike CRISPR-Cas9, Cas12a uses only a single catalytic site to both cleave target double-stranded DNA (dsDNA) (cis-activity) and indiscriminately degrade single-stranded DNA (ssDNA) (trans-activity). To investigate how the relative potency of cis- versus trans-DNase activity affects Cas12a-mediated genome editing, we first used structure-guided engineering to generate variants of Lachnospiraceae bacterium Cas12a that selectively disrupt trans-activity.

CRISPR-Cas9-mediated nuclear transport and genomic integration of nanostructured genes in human primary cells.

DNA nanostructures are a promising tool to deliver molecular payloads to cells. DNA origami structures, where long single-stranded DNA is folded into a compact nanostructure, present an attractive approach to package genes; however, effective delivery of genetic material into cell nuclei has remained a critical challenge. Here, we describe the use of DNA nanostructures encoding an intact human gene and a fluorescent protein encoding gene as compact templates for gene integration by CRISPR-mediated homology-directed repair (HDR).

LuNER: Multiplexed SARS-CoV-2 detection in clinical swab and wastewater samples.

Clinical and surveillance testing for the SARS-CoV-2 virus relies overwhelmingly on RT-qPCR-based diagnostics, yet several popular assays require 2-3 separate reactions or rely on detection of a single viral target, which adds significant time, cost, and risk of false-negative results. Furthermore, multiplexed RT-qPCR tests that detect at least two SARS-CoV-2 genes in a single reaction are typically not affordable for large scale clinical surveillance or adaptable to multiple PCR machines and plate layouts. We developed a RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (LuNER) to address these shortcomings and meet the testing demands of a university campus and the local community.

Robotic RNA extraction for SARS-CoV-2 surveillance using saliva samples.

Saliva is an attractive specimen type for asymptomatic surveillance of COVID-19 in large populations due to its ease of collection and its demonstrated utility for detecting RNA from SARS-CoV-2. Multiple saliva-based viral detection protocols use a direct-to-RT-qPCR approach that eliminates nucleic acid extraction but can reduce viral RNA detection sensitivity. To improve test sensitivity while maintaining speed, we developed a robotic nucleic acid extraction method for detecting SARS-CoV-2 RNA in saliva samples with high throughput.

Launching a saliva-based SARS-CoV-2 surveillance testing program on a university campus.

Regular surveillance testing of asymptomatic individuals for SARS-CoV-2 has been center to SARS-CoV-2 outbreak prevention on college and university campuses. Here we describe the voluntary saliva testing program instituted at the University of California, Berkeley during an early period of the SARS-CoV-2 pandemic in 2020. The program was administered as a research study ahead of clinical implementation, enabling us to launch surveillance testing while continuing to optimize the assay.

Robotic RNA extraction for SARS-CoV-2 surveillance using saliva samples.

Saliva is an attractive specimen type for asymptomatic surveillance of COVID-19 in large populations due to its ease of collection and its demonstrated utility for detecting RNA from SARS-CoV-2. Multiple saliva-based viral detection protocols use a direct-to-RT-qPCR approach that eliminates nucleic acid extraction but can reduce viral RNA detection sensitivity. To improve test sensitivity while maintaining speed, we developed a robotic nucleic acid extraction method for detecting SARS-CoV-2 RNA in saliva samples with high throughput.

IGI-LuNER: single-well multiplexed RT-qPCR test for SARS-CoV-2.

Commonly used RT-qPCR-based SARS-CoV-2 diagnostics require 2-3 separate reactions or rely on detection of a single viral target, adding time and cost or risk of false-negative results. Currently, no test combines detection of widely used SARS-CoV-2 E- and N-gene targets and a sample control in a single, multiplexed reaction. We developed the IGI-LuNER RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (NER).

An Exploratory Study into the Implantation of Arytenoid Cartilage Scaffold in the Horse.

Respiratory function in the horse can be severely compromised by arytenoid chondritis, or arytenoid chondropathy, a pathologic condition leading to deformity and dysfunction of the affected cartilage. Current treatment in cases unresponsive to medical management is removal of the cartilage, which can improve the airway obstruction, but predisposes the patient to other complications like tracheal penetration of oropharyngeal content and dynamic collapse of the now unsupported soft tissue lateral to the cartilage. A tissue engineering approach to reconstructing the arytenoid cartilage would represent a significant advantage in the management of arytenoid chondritis.

Macrophages in the Aging Liver and Age-Related Liver Disease.

The number of individuals aged 65 or older is projected to increase globally from 524 million in 2010 to nearly 1. 5 billion in 2050. Aged individuals are particularly at risk for developing chronic illness, while being less able to regenerate healthy tissue and tolerate whole organ transplantation procedures.

Evaluation of the host immune response to decellularized lung scaffolds derived from α-Gal knockout pigs in a non-human primate model.

Whole organ tissue engineering is a promising approach to address organ shortages in many applications, including lung transplantation for patients with chronic pulmonary disease. Engineered lungs may be derived from animal sources after removing cellular content, exposing the extracellular matrix to serve as a scaffold for recellularization with human cells. However, the use of xenogeneic tissue sources in human transplantation raises concerns due to the presence of the antigenic Gal epitope.

The Polyploid State Restricts Hepatocyte Proliferation and Liver Regeneration in Mice.

The liver contains a mixture of hepatocytes with diploid or polyploid (tetraploid, octaploid, etc.) nuclear content. Polyploid hepatocytes are commonly found in adult mammals, representing ~90% of the entire hepatic pool in rodents.

Effects of age-related shifts in cellular function and local microenvironment upon the innate immune response to implants.

The host macrophage response is now well recognized as a predictor of the success or failure of biomaterial implants following placement. More specifically, shifts from an "M1" pro-inflammatory towards a more "M2-like" anti-inflammatory macrophage polarization profile have been shown to result in enhanced material integration and/or tissue regeneration downstream. As a result, a number of biomaterials-based approaches to controlling macrophage polarization have been developed.

Tumor-Derived α-Fetoprotein Directly Drives Human Natural Killer-Cell Activation and Subsequent Cell Death.

Hepatocellular carcinoma (HCC) patients with reduced natural killer (NK)-cell numbers and function have been shown to have a poor disease outcome. Mechanisms underlying NK-cell deficiency and dysfunction in HCC patients remain largely unresolved. α-Fetoprotein (AFP) is an oncofetal antigen produced by HCC.

Effects of aging upon the host response to implants.

Macrophage polarization during the host response is now a well-accepted predictor of outcomes following material implantation. Immunosenescence, dysregulation of macrophage function, and delayed resolution of immune responses in aged individuals have all been demonstrated, suggesting that host responses to materials in aged individuals should differ from those in younger individuals. However, few studies examining the effects of aging upon the host response have been performed.

Cell Therapy Strategies to Combat Immunosenescence.

Declining function of the immune system, termed "immunosenescence," leads to a higher incidence of infection, cancer, and autoimmune disease related mortalities in the elderly population. (1) Increasing interest in the field of immunosenescence is well-timed, as 20% of the United States population is expected to surpass the age of 65 by the year 2030. (2) Our current understanding of immunosenescence involves a shift in function of both adaptive and innate immune cells, leading to a reduced capacity to recognize new antigens and widespread chronic inflammation.