High flux (e.g., greater than 1012 n/s/cm2) neutrons with energies between 8 and 30 MeV are needed for a number of applications including radioisotope production. However, none of the existing neutron sources available can fulfill these requirements. Neutron flux intensities from typical neutron sources using Deuterium-Tritium (DT) fusion are typically more than 2 orders of magnitude lower in intensity than what is needed for making production practical. Deuterium-Deuterium (DD) fusion sources provide a spectrum which is too low in energy to perform the nuclear reactions needed for isotope production. High-energy proton accelerator-driven spallation sources produce isotopes with significant co-production of unwanted radioisotopes, due to a neutron spectrum which is far higher in energy than required. While accelerator-driven neutron sources using deuteron breakup have been shown to be a viable pathway for producing a range of isotopes including actinium-225 1, a limited number of machines capable of producing ~30 MeV deuteron beams exist commercially. To address this problem, researchers at UC Berkeley have developed systems and methods for producing radionuclides using accelerator-driven fast neutron sources, and more specifically for producing actinium-225, an inherently-safe, fast neutron source based on low energy proton accelerators used throughout the world to support positron emission tomography.

UC Berkeley researchers have developed systems and methods of using a split guide RNA (gRNA) to extend the lower size range of RNA detectable by Cas13a. When Cas13a is in complex with a split gRNA and capture RNA (capRNA), it can directly detect single-stranded RNA ranging from 8-24 nucleotides. The Cas13a split gRNA system is sensitive, enabling detection of femtomolar levels of RNA, and specific to sequence mismatches and gaps. We show that the split Cas13a RNP can detect miRNAs from extracted cell RNA. To detect a new RNA target, only the sequence of the capRNA needs to be modified; the same Cas13a RNP can be used for all targets. The capRNA can be tuned to maximize sensitivity of specificity, depending on the desired application. The split gRNA system expands the current use of Cas13a in molecular diagnostics and opens the door for its use in miRNA discovery.

Understanding the function of RNAs requires visualizing their location and dynamics in live cells. However, direct labeling and imaging individual endogenous RNAs in living cells is still needed. UC Berkeley researchers have developed a method to directly resolve individual endogenous RNA transcripts in living cells using programmable RNA-guided and RNA-targeting CRISPR-Csm complexes coupled with a variety of crRNAs that collectively span along the transcripts of interest.  The researchers demonstrated robust labeling of MAP1B and NOTCH2 mRNAs in several cell lines. We tracked NOTCH2 and MAP1B transcript transient dynamics in living cells, captured distinct mobilities of individual transcripts in different subcellular compartments, and detected translation dependent and independent RNA motions.  

Rapid virus evolution generates proteins essential to infectivity and replication but with unknown function due to extreme sequence divergence. Using a database of 67,715 newly predicted protein structures from 4,463 eukaryotic viral species, it was found that 62% of viral proteins are structurally distinct and lack homologs in the Alphafold database. Structural comparisons suggested putative functions for >25% of unannotated viral proteins.  UC Berkeley researcher have created new single stranded DNA (ssDNA) bindingproteins and double stranded (dsDNA) binding proteins, and methods and compositions for using them, such as binding to target DNA.   

UC Berkeley researchers have indentified and characterized a novel CRISPR Cas13 subtype that exhibits unique and advantageous features for transcriptome editing applications. At approximately half the size of the smallest known Cas13 subtype, this novel subtype is the smallest CRISPR Cas effector identified to date. The compactness of this novel Cas13 subtype facilitates its delivery into a wide array of cell types using various delivery mechanisms, significantly enhancing its utility in genomic research and therapeutic applications. The novel Cas13 subtype retains the hallmark programmable RNA-targeting capability of the Cas13 family, enabling precise and efficient editing of RNA sequences. This feature is particularly valuable in the context of transcriptome engineering, where specific alterations to RNA molecules can modulate gene expression, correct genetic errors, or modulate the function of non-coding RNAs. The discovery of this compact Cas13 subtype opens new avenues for transcriptome editing, offering potential applications in functional genomics, gene therapy, and the development of novel therapeutic strategies targeting RNA. Its ease of delivery and potent RNA-editing capabilities position this novel Cas13 subtype as a valuable tool for both basic research and clinical applications in the field of genetic engineering and precision medicine.

Class 2 CRISPR-Cas are streamlined versions in which a single Cas protein bound to RNA is responsible for binding to and cleavage of a targeted sequence. Theprogrammable nature of these minimal systems has facilitated their use as a versatile technology for genome editing.  CRISPR-Cas enzymes with reduced requirements for a protospacer-adjacent motif (PAM) sequence adjacent to the target site could improve the breadth of target sites available for genome editing.  UC Berkeley researchers have developed a novel PAM-loose 12a variants, nucleic acids encoding the variant Cas12a proteins and systems using these variants that make the Cas12a-based CRISPR technology much easier to design a DNA target for carrying out genome editing in human cells. 

mRNA-based cancer therapies include vaccination via mRNA delivery of tumor neoantigens, delivery of mRNA encoding for immune checkpoint and other protein therapeutics, and induced expression of anticancer surface proteins such as CAR expression in T cells. Success requires transfection of a critical number of immune cells together with appropriate immune-stimulation to effectively drive anti-tumor responses. UC Berkeley researchers have developed an adjuvant-assisted mRNA LNP delivery method that uses mRNA LNP and adjuvant to enhance delivery of nucleic acids to immune cells in vivo and stimulate immune cells. They demonstrated the use of this system to reduce mRNA reporter protein expression in the liver and enhance protein expression in the spleen in mice and also demonstrated this system can be used to genetically engineer T cells by delivering a Cre-recombinase mRNA construct- transfection and editing of approximately 4% of T cells is achieved in vivo. The immune response is superior in our system compared to current, commercial lipid nanoparticle delivery technologies.

Antibodies against poly(ethylene glycol) (PEG) have the potential to cripple the development of lipid nanoparticle (LNP) based therapeutics and new polymers that can stabilize LNPs are greatly needed. Developing alternatives to the PEG-lipid has been challenging partially because of the synthetic challenges associated with making PEG-lipids.  UC Berkeley researcher have created methods for synthesizing PEG replacement polymers based on RAFT block copolymerization and have shown that the block co-polymers synthesized via RAFT polymerization can replace the PEG on LNPs and can generate LNPs that are more efficient at delivering mRNA than PEG-LNPs and are less immunogenic. Block copolymers synthesized via RAFT have great potential for improving the performance of LNPs given their ability to be rapidly synthesized, the commercial availability of numerous methacrylate monomers and the large functional group tolerance of RAFT.