Delivery technologies such as lipid nanoparticles (LNP) offer significant advantages over the delivery of free RNA for various RNA therapeutic, vaccine, and basic science applications. UC Berkeley researchers developed a new class of lipid nanoparticle (LNP) which is effective in delivering various types of nuclei acids in different tissues.  The LNP was successfully tested in in-vivo mouse models and therefore poses a significant promise in the gene editing field. The lipid formulation was packaged together with CRISPR Cas9 and a gRNA targeting the endogenous Tau locus. Tau dysrregulation is a pathological feature of Alzheimers disease, thus the invention provides a means to intervene in the development of pathological states associated with Tau aggregate formation. 

Cellulose, the major component of plant biomass, is considered the most abundant biopolymer. Certain microorganisms are able to convert the monomer of cellulose, glucose, into various products useful in the production of biofuels and other methods. Cellulose is highly stable, has a high storage potential, low cost, and plentiful supply. Based on these and other properties, cellulose and enzymes capable of degrading and hydrolyzing it are useful in the sequestration, storage, and production of bioenergy.  Crystalline cellulose is composed of linear polymers of β1-4 linked glucose, held in a tightly crosslinked crystalline lattice by a high degree of intermolecular hydrogen bonding. This structure confers stability but also hinders efficient deconstruction of cellulose. Strategies for commercial depolymerization of cellulose typically combine pretreatment to disrupt the crystalline structure, followed by enzymatic hydrolysis. Disruption of the crystalline structure and chemical hydrolysis typically requires high temperatures and low pH. Enzymatic hydrolysis generally occurs under milder conditions. The degree of pretreatment required and the expense of subsequent cleanup steps are affected by properties of the enzymes used. Bacteria capable of degrading cellulose include those belonging to the genera Aquifex, Rhodothermus, Thermobifida, Anaerocellum, and Caldicellulosiruptor. A recombinant thermostable endoglucanase of Aquifex aeolicus produced in E. coli showed maximal activity at 80° C. and pH 7.0 with a half-life of 2 h at 100° C.  UC Berkeley investigators have engineered a polypeptide having cellulase activity for hydrolysis and degradation of cellulose-containing biomass.

Researchers at UC Berkeley have developed methods to deliver biomolecules to plant cells using new plant-derived cell penetrating peptides (CPPs). Despite the revolution in DNA editing that the last decade has brought, plant genetic engineering has not been able to benefit to the same extent. This is due to certain challenges in plant physiology that limit the delivery of exogenous protein cargos, as required in the CRISPR-Cas9 system, primarily due to the plant cell wall. In mammalian cells, for instance, cargo delivery can be accomplished using cell-penetrating peptides (CPPs) which are short peptides that facilitate the transport of cargo molecules through the plasma membrane to the cytosol. While this technology has been optimized in mammalian cells, few have studied the delivery of CPPs in plants to verify whether the cell wall is permissible to these materials. Another barrier to the use of nanotechnologies for plant biomolecule delivery is the lack of quantitative validation of successful intracellular protein delivery. The near universal dependence on confocal microscopy to validate delivery of fluorescent proxy cargoes can be inappropriate for use in plants due to various physiological plant properties, for example intrinsic autofluorescence of plant tissues. Therefore, there exists an unmet need for new materials and methods to deliver biomolecules to plant cells and to confirm the delivery of proteins of varying sizes into walled plant tissues. Stage of Research The inventors have developed methods to deliver proteins into plant cells using cell penetrating peptides which are appropriate for use with CRISPR-Cas9 technology, siRNAs, zinc-finger nucleases, TALENs, and other DNA editing methods. They have also developed a biomolecule fluorophore-based assay to accurately quantitate protein delivery to plants cells.Stage of DevelopmentResearch - in vitro 

Within the plant kingdom, a wide variety of species possess an extraordinary ability to regenerate whole organs and tissues naturally. Invasive weeds such as Japanese knotweed can regenerate from tiny root fragments in the soil, and many gardeners’ favorites can be propagated by taking cuttings from fully-grown plants. However, this flexible ability to regenerate organs is missing from most economically important crop species, and is currently the single biggest bottleneck for plant biotechnology.  While there is an increasingly impressive array of tools to edit the genes of a plant cell, regenerating whole organs and body plans from edited cells via labor-intensive tissue culture remains a painstaking process – often requiring a year or more – and resulting in undesirable mutations and chromosome instability.  UCB researchers have discovered that complete genetic knockout of the DNA demethylation pathway in the model plant Arabidopsis dramatically enhances the ability of plant organs to regenerate after wounding. In many plants, including Arabidopsis, regeneration after wounding does not occur naturally and requires intensive tissue culture. By contrast, quadruple homozygous mutant plants harboring loss of function mutations to all four DNA demethylase enzymes capably regenerate all organs and complete body plans after cutting, even in the absence of exogenous plant hormones and tissue culture. 

Rhamnolipids (RLs) are a class of bacterially produced biosurfactants that possess antimicrobial as well as surface-active properties. While RLs have broad utility in industry as antimicrobial biosurfactants, their anionic nature limits the efficacy of these molecules in certain applications. Alternatively, rhamnolipid esters (RLEs) exhibit improved properties as nonionic surfactants. However, a major challenge in RLE application in the commercial arena is that, to date, they are only reliably accessed via chemical synthesis, a costly and unsustainable approach.To address this problem, UC Berkeley researchers have developed a novel, reliable microbial source for biosynthesized RLEs enabling their production in an efficient, sustainable, and renewable manner. Additionally, three novel rhamnolipid methyl ester (RLME) congeners have been produced and a new enzyme for RLE production identified. The produced RLEs are expected to be more effective than RLs in many ways, including antifungal activity and hydrocarbon solubilization.

Cardiovascular disease is the leading cause of death both worldwide and in the United States, with associated costs in the U.S. reaching approximately $229 billion, each, in 2017 and 2018. Early detection, which can drastically reduce both rates of death and treatment costs, requires access to facilities and highly-trained physicians that can be difficult to access in rural areas and developing countries—despite their prevalence of cardiovascular disease. Computer-based models that use, e.g., PCG (phonocardiogram), EKG (electrocardiogram), or other cardiac data, are a promising route to bridge the gap in standard-of-care for these underserved areas. However, current algorithms are unable to account for demographic features, such as race, sex, or other characteristics, which are known to affect both the structure of the heart and presentation of heart disease. To address this problem, UC Berkeley researchers have developed a new, cloud-based system for collecting a patient's continuous cardiovascular data, monitoring for and detecting disease, and keeping a doctor informed about the cardiac health of the patient. The system sends an alarm when disease or heart attack are detected. To generate the most accurate diagnoses by taking into account demographic information, the system includes private and ethical dataset collection and model-training techniques.

Plasma medicine has emerged as a promising approach for treatment of biofilm-related and virus infections, assistance in cancer treatment, and treatment of wounds and skin diseases. However, an important challenge arises with the need to adapt control policies, often only determined after each treatment and using limited observations of therapeutic effects. Control policy adaptation that accounts for the variable characteristics of plasma and of target surfaces across different subjects and treatment scenarios is needed. Personalized, point-of-care plasma medicine can only advance efficaciously with new control policy strategies.To address this opportunity, UC Berkeley researchers have developed a novel control scheme for tailored and personalized plasma treatment of surfaces. The approach draws from concepts in deep learning, Bayesian optimization and embedded control. The approach has been demonstrated in experiments on a cold atmospheric plasma jet, with prototypical applications in plasma medicine.

RNA-mediated adaptive immune systems in bacteria and archaea rely on Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) genomic loci and CRISPR associated (Cas) proteins that function together to provide protection from invading viruses and plasmids. Genome editing can be carried out using a CRISPR-Cas system comprising a CRISPR-Cas effector polypeptide and a guide nucleic acid, such as a guide RNA. However, unintended chromosomal abnormalities following on-target genome editing, such as chromosome loss, are potential concerns for genome editing. UC Berkeley researchers and others have developed a method to modulate the expression levels of the DNA damage response factor p53 in order to mitigate chromosomal abnormalities that occur after genome editing by nucleases like Cas9. The invention provides treatment methods by generating a modified cell and then administering the modified cell to an individual in need thereof and compositions having a CRISPR-Cas effector polypeptide, a guide nucleic acid, and an agent that increases the level of a p53 polypeptide in a mammalian cell.

Plastics are essential for the modern world but are also non-sustainable products of the petrochemical industry that negatively impact our health, environment, and food chain. Natural biogenic plastics, such as polyhydroxyalkanoates (PHA), are readily biodegradable, can be produced more sustainably, and offer an attractive alternative. The global demand for bioplastics is increasing with the 2019 market value of $8.3B expected to reach a compound annual growth rate of 16.1% from 2020-2027 (https://www.grandviewresearch.com/industry-analysis/bioplastics-industry). However, current PHA production is constrained by the underlying physiology of the microorganisms which produce them, meaning bioplastic production is currently limited to inefficient, batch fermentation processes that are difficult to scale.To address this problem, UC Berkeley researchers have developed a new system for PHA production wherein the PHA are generated continuously throughout microorganism growth lifecycles. The invention allows these sustainable bioplastics to be produced via precision continuous fermentation technology, a scalable and efficient approach.

Biological fluids are complex, with compositions that vary constantly and evade molecular definition. Nevertheless, within these fluids proteins fluctuate, fold, function, and evolve as programmed. Synthetic heteropolymers capable of emulating such interactions would replicate how proteins behave in biological fluids, individually and collectively, leading the way toward synthetic biological fluids. However, while there exist known monomeric sequence requirements, the chemical and sequence characteristics of proteins at the segmental level, rather than the monomeric level, may be the key factor governing how proteins transiently interact with neighboring molecules (and how biological fluids collectively behave). To address this opportunity, UC Berkeley researchers have developed a new process of heteropolymer design for protein stabilization and synthetic mimics of biological fluids. The process leverages chemical characteristics and sequential arrangements along protein chains at the segmental level to design heteropolymer ensembles as mixtures of disordered, partially folded, and folded proteins. In studies, for each heteropolymer ensemble, the level of segmental similarity to that of natural proteins determines its ability to replicate many functions of biological fluids, including: assisting protein folding during translation; preserving the viability of fetal bovine serum without refrigeration; enhancing the thermal stability of proteins; and, behaving like synthetic cytosol under biologically relevant conditions. Molecular studies further translated protein sequence information at the segmental level into intermolecular interactions with a defined range, degree of diversity and temporal and spatial availability.

Single-cell analyses are revolutionizing biomedicine and biology, with genomics (DNA) and transcriptomics (RNA) tools leading the way. At the protein-level, single-cell analyses are limited to mass spectrometry and immunoassays. Neither assay provides comprehensive coverage of proteome for single cells, missing key protein forms (called isoforms).  UC Berkeley researchers have developed a hybrid droplet-electrophoresis device, termed “DropBlot”, to detect proteins from patient-derived tissue biospecimens relevant to clinical medicine and pathology. The DropBlot takes advantage of water-in-oil (W/O) droplets to encapsulate single cells derived from chemically fixed tissues, thus providing a picoliter-volume reaction chamber in which said cells are lysed and subjected to harsh lysis conditions (100ºC, 2 hours), as needed for fixed cells. We report an all-in-one microdevice to facilitate cell-laden droplet loading with >98% microwell occupancy. Droplets remain intact under the electric field and protein isoforms are shown to electromigrate out of the droplet and into a microfluidic separation channel where protein sizing takes place via the action of electrophoresis in a photoactive polyacrylamide (PA) gel. DropBlot has been successfully applied to live and fixed cancer cell lines and resolved proteins with high sensitivity.

Existing screening tools for respiratory pathogens, including PCR-based methods and antibody-based methods, are generally time-consuming to perform and analyze, difficult to manufacture at scale, and reliant on a detailed understanding of the targeted pathogen. Additionally, these traditional methods give little insight into the extent to which an individual is capable of spreading the disease. All of these features hamstring early responses to emerging pathogens and early-stage epidemics, as can be seen from the ongoing SARS-COV-2 pandemic. To address these problems, researchers at UC Berkeley have developed a device which ionizes large biomolecules from aerosol droplets and routes them to the inlet of a mass spectrometer or ion mobility spectrometer for identification based on size and/or mass. This can serve as the basis for a screening tool which measures the concentration of pathogenic particles, including common respiratory viruses and bacteria, in the breath. Results from this test could be read out in a matter of seconds, and it does not depend on detailed knowledge of the pathogen in question. Researchers have demonstrated the efficacy of such a device in detecting both large human proteins and virus-sized styrofoam particles.

Cardiovascular disease is among the leading causes of death for citizens in affluent nations, and the most significant cause of morbidity in those with cardiovascular disease is hypertension. Often called the “silent killer” because it has few clinical signs in its early stages, elevated blood pressure is often in an advanced stage before it is treated, leading to a substantially worse prognosis than if it had been detected earlier.In order to address this problem, researchers at UC Berkeley have developed a wearable device which continuously monitors diastolic blood pressure, transmitting data to a portable device such as a cell phone, where it can be stored and analyzed. The device utilizes piezoelectric transducers to perform the measurement, which allows the wearable device to remain small while containing a large number of sensors in order to reduce noise.

The invention are mice lacking functional expression of the Nirc4 gene that were generated using CRISPR-Cas9.  

The CRISPR-Cas system is now understood to confer bacteria and archaea with acquired immunity against phage and viruses. CRISPR-Cas systems consist of Cas proteins, which are involved in acquisition, targeting and cleavage of foreign DNA or RNA, and a CRISPR array, which includes direct repeats flanking short spacer sequences that guide Cas proteins to their targets. The programmable nature of these systems has facilitated their use as a versatile technology that is revolutionizing the field of genome manipulation. There is a need in the art for additional CRISPR-Cas systems with improved cleavage and manipulation under a variety of conditions and ones that are particularly thermostable under those conditions. UCB researchers created a set of efficient CRISPR-Cas9 proteins from a thermostable Cas9 from the thermophilic bacterium Geobacillus stearothermophilus (GeoCas9) through directed evolution. The gene editing activity of the evolved mutant proteins was improved by up to four orders of magnitude compared to the wild-type GeoCas9. The researchers showed that the gene editors based on the evolved GeoCas9 can be effectively assembled into lipid nanoparticles (LNP) for the rapid delivery to different cell lines in vitro as well as different organs or tissues in vivo. The LNP-based delivery strategy could also be extended to other gene editors.  

There are no approved disease-modifying therapies for Huntington’s disease (HD), a fatal neurodegenerative condition caused by a heterozygous expansion of a CAG array in exon 1 of Huntingtin (Htt). Typically, HD patients are heterozygous for the toxic gain of function disease allele, yet expression of the wildtype version of the gene is essential. The inventors have developed methods and compositions to selectively silence expression from the disease-associated allele while leaving the wildtype version intact. The invention relies on the introduction of a 'poison' exon into the diseased allele wherein introduction of the poison exon may be accomplished by standard methods in the art, such as introduction of the exon sequences through homology-directed repair following targeted nuclease cleavage, transposon-associated targeted sequence introduction, base editing, and prime editing. Following the introduction of the poison exon, post-transcriptional splicing results in an RNA that is susceptible to nonsense mediated decay due to the introduction of a stop codon in the introduced exon. RNAs comprising the poison exon are subsequently degraded in the cell, effectively silencing expression of the mutant disease-associated allele.

Methyl-CpG-binding-protein 2 (MeCP2) is a nuclear protein expressed in all cell types, especially neurons. Mutations in the MECP2 gene cause Rett syndrome (RTT), an incurable neurological disorder that disproportionately affects young girls. Strategies to restore MeCP2 expression phenotypically reverse RTT-like symptoms in male and female MeCP2-deficient mice, suggesting that direct nuclear delivery of functional MeCP2 could restore MeCP2 activity.The inventors have discovered that ZF-tMeCP2, a conjugate of MeCP2(aa13-71, 313-484) and the cell-permeant mini-protein ZF5.3, binds DNA in a methylation-dependent manner and reaches the nucleus of model cell lines intact at concentrations above 700 nM. When delivered to live cells, ZF-tMeCP2 engages the NCoR/SMRT co-repressor complex and selectively represses transcription from methylated promoters. Efficient nuclear delivery of ZF-tMeCP2 relies on a unique endosomal escape portal provided by HOPS-dependent endosomal fusion.In a comparative evaluation, the inventors observed the Tat conjugate of MeCP2 (Tat-tMeCP2) (1) degrades within the nucleus, (2) is not selective for methylated promoters, and (3) traffics in a HOPS-independent manner. These results support the feasibility of a HOPS-dependent portal for delivering functional macromolecules to the cell interior using the cell-penetrant mini-protein ZF5.3. Such a strategy could broaden the impact of multiple families of protein-derived therapeutics.

Type III CRISPR-Cas systems recognize and degrade RNA molecules using an RNA-guided mechanism that occurs widely in microbes for adaptive immunity against viruses. The inventors have demonstrated that this multi-protein system can be leveraged for programmable RNA knockdown of both nuclear and cytoplasmic transcripts in mammalian cells. Using single-vector delivery of the S. thermophilus Csm complex, RNA knockdown was achieved with high efficiency (90-99%) and minimal off-targets, outperforming existing technologies of shRNA- and Cas13-mediated knockdown. Furthermore, unlike Cas13, Csm is devoid of trans-cleavage activity and thus does not induce non-specific transcriptome-wide degradation and cytotoxicity. Catalytically inactivated Csm can also be used for programmable RNA-binding, which the inventors exploit for live-cell RNA imaging. This work demonstrates the feasibility and efficacy of multi-subunit CRISPR-Cas effector complexes as RNA-targeting tools in eukaryotes.

Injectable hydrogels are attracting increasing interest for the therapeutic delivery of cells to tissue. However, these hydrogel formulations can suffer from engraftment efficiencies of less than 5% when delivered to native tissue. These poor engraftment efficiency rates are often attributed to high shear stresses during delivery and inability to provide a stable three-dimensional niche at the delivery site. The inventors have developed a technique for encapsulating cells in the pore space between microscopic hydrogel particles by employing the yield stress fluid properties of packs of microgels. The technology protects the cells from mechanical stress during delivery and facilitates integration to the native tissue. During delivery, the packs of microgels undergo plug flow in which the pressure drop across the length of the pipe is compensated solely by frictional forces at the interface between the pipe wall and microgels. At the delivery site, the pack of microgels behave as an elastic solid across the range of physiological frequencies and provide a stable 3D culture paradigm to support engraftment.Furthermore, the inventors address the challenges associated with cryopreserving, transporting, and delivering this injectable formulation from benchtop-to-bedside with a concept for a perfusable delivery device. The device encapsulates cells in the pore space of the microgels and confines the formulation to a fixed volume where researchers can perfuse liquid freeze/thaw or maintenance media, differentiation factors, and anti-inflammatory agents at virtually any time prior to delivery to the tissue. The porous microgel network facilitates this process and makes the formulation amenable to transport and storage which would otherwise be unattainable in hydrogel formulations.

The inventors have developed an engineering approach to identify novel and nonobvious membrane-associated accessory protein (MAAP) sequence variants that confer increased Adeno-associated virus (AAV) secretion during packaging. The technique is based upon the iterative process of sequence diversification and selection of functional gene variants known as directed evolution. First, the inventors generated a library of more than 1E6 MAAP variants. The variants were subjected to five rounds of packaging into an AAV2 capsid for which MAAP expression was inactivated without altering the viral protein VP1 open reading frame (ORF) (AAV2-MAAP-null). Among each iterative packaging round, the inventors observed a progressive increase in both the overall titer and ratio of secreted vector genomes conferred by the bulk selected MAAP library population. Next-generation sequencing uncovered common mutational features that were enriched up to over 10,000-fold on the amino acid level. Individual MAAP variants were isolated and systematically tested for effect on recombinant AAV2-MAAP-null packaging in HEK293 cells. The inventors predict that this work may be applicable to increasing per-cell AAV output in industrial settings, potentially reducing global costs and increasing functional vector recovery in downstream manufacturing processes.BACKGROUNDParvoviruses are small, single-stranded DNA viruses that are ubiquitously found in many animal species. AAV is a prototypic dependoparvovirus whose replication cycle requires the function of helper genes from larger co-infected viruses such as Adenoviruses or Herpesviruses. The natural genome of AAV contains ~4.7 kb of ssDNA that encodes up to ten known proteins in a highly overlapped fashion. The rep gene encodes four protein products named based on their molecular weight: Rep72 and Rep68 facilitate genomic replication, whereas Rep52, and Rep40 play essential roles in loading nascent ssDNA genomes into assembled capsids. Downstream of rep lies the cap gene, which encodes three known protein products off of overlapping reading frames: VP1, VP2, and VP3 are structural proteins that assemble to form the capsid, the assembly activating protein (AAP) targets VP proteins to the nucleus and is involved in capsid assembly. The most recently discovered AAV-encoded gene is the membrane-associated accessory protein (MAAP). MAAP is encoded by an alternative ORF in the AAV cap gene that is found in all presently reported natural serotypes. Gene delivery by recombinant AAV (rAAV) have shown significant success in both research and clinical gene therapy applications. In the rAAV system, Rep and Cap are removed from between AAV’s 5’ and 3’ inverted terminal repeats (ITRs) and provided in trans. Instead, a transgene of interest is inserted between the ITRs and subsequently packaged into the nascent AAV capsids. However, manufacturing quantities of good manufacturing practice (GMP)-grade rAAVs necessary to achieve current and projected dosing requirements–particularly in a clinical context–presents a significant hurdle to expanding rAAV-based gene therapies. Recently, evidence has emerged supporting a functional role of MAAP in AAV egress. This led to the hypothesis that MAAP could be engineered to facilitate increased levels of secreted AAV produced from HEK293 cells. 

The inventors have developed a method of mutagenizing user-defined regions of cytoplasmic DNA using a single guide RNA (sgRNA) or combinations of sgRNAs and a highly engineered fusion polypeptide comprising: a nuclear export sequence (NES)-containing, engineered nuclear localization sequence (NLS)-lacking, enzymatically active, RNA-guided endonuclease that introduces a single-stranded break in cytoplasmic DNA, and an error-prone DNA polymerase. This novel technology encompasses and provides evidence for the use of RNA-guided nucleases with relaxed PAM requirements, which are particularly useful for AT-rich targets such as the vaccinia virus genome. The inventors show that the truncation of up to several base pairs from the PAM-distal template binding region of the sgRNAs significantly increases the functional activity and specificity of the targeted mutagenesis complex. Moreover, the invention describes specific methods for the use of this technology to edit cytoplasmically replicating viruses with large DNA genomes, using poxviruses as a model system. The novel editing platform and methods selectively and continuously accelerate diversification of user-defined sites in the vaccinia genome during infection, while retaining most library members, due to significantly lowering deleterious off-target mutations. BACKGROUND Nucleocytoplasmic large DNA viruses (NCLDVs) are a group of viruses that harbor large (150 kbp - 1.2 mbp) double-stranded DNA genomes and replicate in the cytoplasm of eukaryotic cells. An example of an NCLDV that has historically been among the most prominent tools in human health is vaccinia, a poxvirus. Hundreds of millions of humans have been intentionally inoculated with vaccinia as part of a global effort to eliminate smallpox, which was declared eradicated in 1980.Vaccinia and some other poxviruses remain highly scientifically relevant in the post-eradication world. They are useful as vaccines against deadly poxvirus outbreaks that could potentially arise from natural spillover, bioterrorism, or biowarfare, as well as due to their therapeutic promise as oncolytic agents to selectively deliver anti-cancer transgenes and recruit adaptive immunity while leaving healthy cells unharmed. Directed evolution is a powerful engineering technique for evolving new phenotypes that are beneficial for biotechnological applications but for which there may have never been a selective pressure to evolve in nature. Both natural and directed evolution depend upon generation of genetic diversity, followed by a selective pressure. While natural evolution generates genetic diversity randomly and throughout the entirety of the genome, directed evolution ideally focuses mutations within specific genomic windows connected to the phenotype that one wishes to engineer. However, there is a need in the art for compositions and methods for mutagenizing a target DNA in the cytoplasm of mammalian cells. NCLDVs, which either partially or entirely express their own replicative and translational machinery independent of the nucleus, are difficult, and in many cases impossible, to produce from plasmid DNA in cells. Thus, NCLDVs are not amenable to standard in vitro molecular diversification strategies.  

The inventors have discovered PylRS enzymes that accept -thio acids, N-formyl-L-amino acids, and diverse -carboxyl acid monomers (malonic acids) that are formally precursors to polyketide natural products. These monomers are all accommodated and accepted by the translation apparatus in vitro. High-resolution structural analysis of the complex between one such PylRS enzyme and a meta-substituted 2-benzylmalonate derivative reveals an active site that discriminates pro-chiral carboxylates and accommodates the large size and distinct electrostatics of an -carboxyl acid substituent.This discovery emphasizes the potential of PylRS for evolving new enzymes capable of encoding diverse non-L-amino acids in synergy with natural or evolved ribosomes. The absence of orthogonal aminoacyl-tRNA synthetase enzymes that accept non-L-amino acids is the primary bottleneck hindering the in vivo translation of sequence-defined hetero-oligomers. 

Plants rely on systemic signaling mechanisms to establish whole-plant defense in response to insect and nematode attack. The Glutamate receptor-like (GLR) genes have been implicated in long-distance propagation of wound signals to initiate accumulation of defense hormone jasmonate (JA) at undamaged distal sites.UCB researchers have shown the ability to desensitize GLR channels, providing a potential target for engineering anti-herbivore defense in crops.

The inventors have developed a genome editing therapy for bone marrow failure (BMF) in people living with dyskeratosis (DC). This technology includes two novel endogenous, isogenic models to study TINF2-DC mutations.Human embryonic stem cells (hESCs) engineered to carry the TIN2-DC T284R mutation recapitulated the short telomere phenotype observed in DC patients. Yet, telomeres in TINF2-DC hESCs did not trigger DNA damage responses at telomeres or show exacerbated telomere shortening when differentiated into telomerase-negative cells. Disruption of the mutant TINF2 allele by introducing a frameshift mutation in exon 2 restored telomere length in stem cells and the replicative potential of differentiated cells. The inventors also established in vitro and in vivo human hematopoietic stem cell (hHSC) models to assess the changes in telomere length and proliferative capacity upon the introduction of TERT and TINF2 editing. In addition, the inventors demonstrated that editing at exon 2 of TINF2 that restored telomere length in hESCs could be generated in TINF2-DC patient HSCs. These experiments nominate TINF2 as a target for: CRISPR/CAS9 to elongate telomeres in patient with TINF2 mutations,CRISPR/CAS9 to elongate telomeres with other mutations causing TBD, and chemical interventions to elongate telomeres in general.BACKGROUNDBMF is a major cause of morbidity and mortality in DC and other telomere biology disorders (TBDs). Mutations in the TINF2 gene, encoding the shelterin protein TIN2, cause telomere shortening and the inherited bone marrow failure syndrome dyskeratosis congenita (DC). A lack of suitable model systems limits the mechanistic understanding of telomere shortening in the stem cells and thus hinders the development of treatment options for bone marrow failure.   

The inventors engineered a set of LbCas12a mutants through rational design and directed evolution. The engineered mutants can function as efficient genome editors with minimized trans-activity.