Deoxyribonucleic acid- (DNA-) based identification in forensics is typically accomplished via genotyping allele length at a defined set of short tandem repeat (STR) loci via polymerase chain reaction (PCR). These PCR assays are robust, reliable, and inexpensive. Given the multiallelic nature of each of these loci, a small panel of STR markers can provide suitable discriminatory power for personal identification. Massively parallel sequencing (MPS) technologies and genotype array technologies invite new approaches for DNA-based identification. Application of these technologies has provided catalogs of global human genetic variation at single-nucleotide polymorphic (SNP) sites and short insertion-deletion (INDEL) sites. For example, from the 1000 Genomes Project, there is now a catalog of nearly all human SNP and INDEL variation down to 1% worldwide frequency. Genotype files, generated via MPS or genotype array, can be compared between individuals to find regions that are co-inherited or identical-by-descent (IBD). These comparisons are the basis of the relative finder functions in many direct-to-consumer genetic testing products. A special case of relative-finding is self-identification. This is a trivial comparison of genotype files as self-comparisons will be identical across all sites, minus the error rate of the assay. For many forensic samples, however, the available DNA may not be suitable for PCR-based STR amplification, genotype array analysis, or MPS to the depth required for comprehensive, accurate genotype calling. In the case of PCR, one of the most common failure modes occurs when DNA is too fragmented for amplification. For these samples, it may be possible to directly observe the degree of DNA fragmentation from the decreased amplification efficiency of larger STR amplicons from a multiplex STR amplification. In the case of severely fragmented samples, where all DNA fragments are shorter than the shortest STR amplicon length, PCR simply fails with no product.

Aberrant splicing contributes to the etiology of many inherited diseases. Pathogenic variants impact pre-mRNA splicing through a variety of mechanisms. Most notably, variants remodel the cis-regulatory landscape of pre-mRNAs by ablation or creation of splice sites, and auxiliary splicing regulatory sequences such as exonic or intronic splicing enhancers (ESE and ISE, respectively) and splicing silencers (ESS and ISS, respectively). Splicing-sensitive variants cripple the integrity of the gene, resulting in the production of a faulty message that is either unstable or encodes an internally deleted protein. Antisense oligonucleotides (ASOs) are a promising therapeutic modality for rescuing pathogenic aberrant splicing patterns as their direct base pairing abilities make them highly customizable and specific to targets. Although challenges such as toxicity, delivery and stability represent barriers to the clinical translation of ASOs, solutions to these challenges exist, as exemplified by the recent FDA approval of multiple ASO drugs.Generally, ASO's that target splicing mutations are limited to mutations in and around splicing enhancers and exonic mutations are commonly not targeted because of the idea that the mutation causes a significant change in protein function. 

The strategy employed by the invention is inspired by splicing factors, a category of RNA-binding protein that influence alternative splicing outcomes. These splicing factors are trans-acting, and act to enhance or silence exon inclusion by binding near or on the target exon and promoting or repressing the activity of splicing machinery. Scientifically, a highly programmable, minimally disruptive system to increase exon inclusion could allow for higher-throughput identification of functional roles of specific exons than have been previously shown.

A patent-pending platform technology designed to work in any gravity, which includes in microgravity environments, able to execute advanced molecular biology workflows; representing a paradigm shift in automation for molecular biology.

Researchers from UC San Diego developed an invention that enables protein expression to be upregulated using specific proteins and/or peptide sequences derived from SARS-CoV-2 proteins that are engineered to recognize specific mRNA transcripts by fusion to RNA-targeting modules such as CRISPR/Cas systems. They anticipate that these proteins can be fused or tethered to any engineered RNA-targeting moiety/module such as PUF/Pum, and pentatricopeptide proteins.

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. 

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 

Long read nanopore sequencing can directly sequence RNA molecules, including rRNA, and result in full-length RNA sequences. rRNA sequencing is particularly useful for identifying microbes and full-length rRNA sequencing can identify microbes with post transcriptional modifications that confer antibiotic resistance. Such post transcriptional modifications are invisible to amplification based sequencing or other sequencing techniques that require reverse transcription.Before this technology was developed, there were few if any efficient methods for preparing rRNA libraries for direct RNA sequencing, particularly for microbial identification in either a clinical or an environmental setting.   

In cells, DNA is organized by wrapping DNA strands around histone proteins, creating protein-DNA complexes called nucleosomes which comprise the basic unit of chromatin. Chromatin is associated with regions of low gene expression, as compacted DNA is inaccessible to proteins that would promote transcription. Conversely, regions in the DNA not bound by histones experience higher gene expression, as this DNA is readily available to be transcribed.  Nucleosomes are not uniformly positioned on a DNA molecule, and they change based on factors like which genes are expressed during different cellular processes. It is beneficial to understand where nucleosomes are positioned, as this can provide insight into how genes are regulated, or how factors like epigenetic modifications or chromatin structure affect this accessibility and can additionally illuminate gene expression patterns in disease for designing therapies. Nucleosome profiling is a technique used to study the positions of nucleosomes along a DNA molecule. Typically, histones are crosslinked to DNA, then the DNA is fragmented and digested leaving only regions protected by nucleosomes left for short-read sequencing. However, this fragmentation only reveals nucleosome positioning at the resolution of a few hundred base pairs, leaving the larger genomic context of these nucleosome positions to be desired. To address this, researchers at UC Santa Cruz developed Add-SEQ, a pipeline using long-read nanopore sequencing to map nucleosomes across long stretches > 10 kb of single DNA molecules.

An abasic site (i.e., an apurinic or apyrimidinic site) in a DNA or RNA strand is one in which the base is not present, but the sugar phosphate backbone remains intact. UC Santa Cruz researchers discovered that nanopore sequencers can readily detect the positions of abasic sites within a DNA strand during sequencing. This invention capitalizes on this discovery by using enzymes to generate abasic sites at places on a DNA strand that contain modified bases. The DNA strand can then be sequenced using nanopore sequencing, thereby providing a way of detecting modified bases.

Many genomes encode Restriction-Modification systems (RMs) that act to protect the host cell from invading DNA by cutting at specific sites (frequently short 4-6 base reverse complement palindromes). RMs also protect host DNA from unfavorably being cut by modifying sites within the host DNA that could be targets by the host’s own surveillance enzymes. It is also not unusual to find that these enzymes are adjacent to each other in the host genome. Traditional approaches to understanding these sites involve finding a methylase that is typically adjacent to a restriction enzyme, and then extracting DNA, expressing protein and then testing DNA sequence for evidence of cutting. In certain laboratory research (e.g., programs that involve transforming DNA/RNA) it may be desirable to more comprehensively understand the sequences being surveilled by the host. Moreover, it may be desirable in certain laboratory research to know/predict which surveillance enzymes are present in a genome in order to affect cell transformation efficiency through evasion of those sequences.

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. 

Barcodes are identifiable nucleic acid sequences that can be coupled to a target nucleic acid, either directly or indirectly. Doing so assists in analyzing the nucleic acids of interest. There are currently methods for introducing barcodes to long DNA molecules. However, long DNA molecules can be difficult to isolate. For example, long DNA molecules cannot be recovered from formalin-fixed-paraffin-embedded (FFPE) samples, but such samples are the major source of patient tumor DNA. There is a need for more efficient methods of using barcodes in haplotype phasing and other applications.   Barcoded nucleic acids on solid supports are used in a number of applications such as genome scaffolding, haplotype phasing, and single cell transcriptomics. Current methods involve the use of DNA amplification or chemical synthesis. These techniques are error prone and cost prohibitive. 

When this invention was originally conceived, there were no systems available for sequencing the entire length of an RNA transcript. Next Generation Sequencing (exemplified by the Illumina® sequencing platform) only allows sequencing of short (70-150 base reads) and only cDNA templates.  Direct, long read RNA sequencing, (exemplified by nanopore sequences produced by Oxford Nanopore Technologies) can sequence an entire polyadenylated transcript (when a poly-T adapter is included), but not a non-polyadenylated transcript - it misses the last nucleic acids. It is also unable to accurately determine the length of the poly-A region of the polyadenylated transcript. Researchers at UC Santa Cruz developed a system that solves this problem. The entire length of a natural RNA can now be sequenced, unlocking new insights into RNA expression and variability.   

High-throughput next-generation sequencing (NGS) systems have allowed for large scale collection of transcriptomic data with single cell resolution. Within this data lies variability allowing researchers to characterize and/or infer certain morphological aspects of interest, such as single cell type, cell state, cell growth trajectories, and inter-cellular gene regulatory networks. All of these qualities are important parts of understanding how cells interact with one another, both for building better cellular models in vitro and for understanding biological processes in vivo. While the size of single cell data has increased massively, NGS techniques for key pieces of analysis have not kept pace, using slow, manual pipelines of domain experts for initial clustering. Attempts to improve NGS classification performance have fallen short as the numbers of cell types (often asymmetric) and cell subtypes have increased while the number of samples per label has become small. The technical variability between NGS experiments can make robust classification between multiple tissue samples difficult. Moreover, the high-dimensional nature of NGS transcriptomic data makes this type of analysis statistically and computationally intractable.

Long read sequencing technologies such as nanopore sequencing allow better visualization of whole polynucleotides than other methods. One challenge of long read sequencing, though, is that the presence of shorter nucleic acid strands reduces the efficiency of long read sequencing. There is a need for inexpensive, simpler, and rapid ways to remove unwanted shorter strands in sequencing libraries.

Long read sequencing (e.g. nanopore sequencing) involves a tradeoff between the length of the DNA fragment sequenced, which allows for greater ease of data assembly relative to massively parallel sequencing technologies (e.g. Illumina (R) sequencing) and accuracy of individual base calls.This technology takes advantage of the long read capabilities of nanopore sequencing to improve the accuracy of reads of highly variable nucleic acid species, including cDNAs, and which can be highly variable due to alternative RNA splicing. 

Researchers at the University of California, Davis have developed a method to stop bacterial growth while maintaining desirable metabolic functions for therapeutic and biotechnological applications.

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.  

Alternative splicing accounts for a considerable portion of transcriptomic diversity, as most protein-coding genes are spliced into multiple mRNA isoforms. However, errors in splicing patterns can give rise to mis-splicing with pathological consequences, such as the congenital diseases familial dysautonomia, Duchenne muscular dystrophy, and spinal muscular atrophy. Small nuclear RNA (snRNA) components of the U snRNP family have been proposed as a therapeutic modality for the treatment of mis-splicing. U1 snRNAs offer great promise, with prior studies demonstrating in vivo efficacy, suggesting additional preclinical development is merited. Improvements in enabling technologies, including screening methodologies, gene delivery vectors, and relevant considerations from gene editing approaches justify further advancement of U1 snRNA as a therapeutic and research tool.

Nanopore sequencing requires a processive motor or other element to control the rate of RNA movement in single nucleotide steps through the nanopore sensor. The control element is typically situated several nucleotides from the sensor, therefore it necessarily releases before the end of the native RNA strand reaches the sensor. Thus, the bases along that terminal interval cannot be sequenced using conventional nanopore strategies. Furthermore the nucleotide sequence near that end in many eukaryotic RNAs is not typical. An important example is polyadenylated (poly A) RNA which often bears a 7 methylguanosine cap at the 5 prime end. The linkage between this modied cap and its neighbor is inverted, i.e. the two nucleotides are connected via the 5 prime carbons of their ribose sugars through triphosphate linker, rather than by a typical 5 prime to 3 prime linkage via a phosphodiester bond. 

The gene expression profile of a cell can indicate the current status of the cell, such as its cell type, proliferation status, and degree of maturation or differentiation. The health of a cell in tissues is always in transition, such as diseased state (e.g., tumor cells), healthy state, and states in between. To fully understand and leverage the nature and pathways of cell states towards better diagnosis, treatment, and medical outcomes, it may be beneficial to forecast cell health as a function of certain gene-related configuration. Traditionally it has been difficult to predict ad hoc whole transcriptome alterations caused by gene-related perturbations.

Researchers at the University of California, Davis have developed a targeted epigenetic approach for the prevention and treatment CDKL5 deficiency disorder.

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.