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MODULATORS OF TYPE VI-D CRISPR-CAS EFFECTOR POLYPEPTIDES AND METHODS OF USE THEREOF

UC researchers have discovered anti-CRISPR (Acr) polypeptides that inhibit activity of a CRISPR-Cas effector polypeptide, for example, Type VI-D CRISPR-Cas effector polypeptides, nucleic acids encoding the Acr polypeptides, and systems and kits comprising the polypeptides and/or nucleic acids encoding the Acr polypeptides. The inhibitor is a small protein from a phage and is capable of strongly inhibiting gene editing in human cells.

Gasdermin-D Deficient Mice

UC researchers created a Gasdermin-D deficient mice that carry a CRISPR/Cas9-derived knock-out allele of the mouse Gsdmd gene that involves a 19 bp and a 1 bp (20 bp total) insertion in exon 2. 

Engineered/Variant Hyperactive CRISPR CasPhi Enzymes And Methods Of Use Thereof

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.  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. The programmable nature of these minimal 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 Class 2 CRISPR/Cas systems (e.g., Cas protein plus guide RNA combinations).     UC Berkeley researchers discovered a new type of CasPhi/12j protein.  Site-specific binding and/or cleavage of a target nucleic acid (e.g., genomic DNA, ds DNA, RNA, etc.) can occur at locations (e.g., target sequence of a target locus) determined by base-pairing complementarity between the Cas12 guide RNA (the guide sequence of the Cas12 guide RNA) and the target nucleic acid.  Similar to CRISPR Cas9, the compact Cas12 enzymes are expected to have a wide variety of applications in genome editing and nucleic acid manipulation.  

Optimized Virus-like Particles for Cas9 RNPs & Transgene/HDR Template Delivery

The inventors have developed optimized methods for using virus-like particles for the co-delivery of Cas9 ribonucleoprotein complexes and: a lentiviral genome that encodes a large transgene, such as a chimeric angtigen receptor (CAR) transgene a lentiviral genome that does not encode a sgRNA expression cassette a method for nucleofecting VLPs + homology directed repair (HDR) donor template together to enhance HDR in treated cells  

At-Home Health Diagnostics Using Face Mask

The inventors propose to collect and detect exhaled breath condensate (EBC) on nanostructured face masks to detect viral infections. Nanofibers can provide more than 100 times the surface area of a conventional mask. With this extensive surface area, nanostructured face masks can collect and accumulate virus with high concentrations without the time/equipment-intense amplification step in RT-PCR or RT-LAMP. By wearing the mask for 6 hours, a person may accumulate 5,400 times more virus than the amount collected in a throat swab specimen. Together, 540,000 times higher virus concentration could be achieved, which is equivalent to 19 cycles in a typical PCR amplification process - the same level as current state-of-art detection schemes for COVID-19 virus.COVID-19 is used here as an example for the possible application of the invention. Over 50% of COVID-19 patients have delayed or no symptoms while they are spreading the virus. Both healthy and symptomless people could easily conduct self-tests at home if there are at-home detection kits. Face masks have become mandatory personal protection equipment (PPE) around the world, and are widely accepted to stop the spread of COVID-19.   

Facile, Excitation-Based Spectral Microscopy For Fast Multicolor Imaging And Quantitative Biosensing

The number of color channels that can be concurrently probed in fluorescence microscopy is severely limited by the broad fluorescence spectral width. Spectral imaging offers potential solutions, yet typical approaches to disperse the local emission spectra notably impede the attainable throughput.    UC Berkeley researchers have discovered methods and systems for simultaneously imaging up to 6 subcellular targets, labeled by common fluorophores of substantial spectral overlap, in live cells at low (~1%) crosstalks and high temporal resolutions (down to ~10 ms), using a single, fixed fluorescence emission detection band. 

Improved guide RNA and Protein Design for CasX-based Gene Editing Platform

The inventors have developed two new CasX gene-editing platforms (DpbCasXv2 and PlmCasXv2) through rationale structural engineering of the CasX protein and gRNA, which yield improved in vitro and in vivo behaviors. These platforms dramatically increase DNA cleavage activity and can be used as the basis for further improving CasX tools.The RNA-guided CRISPR-associated (Cas) protein CasX has been reported as a fundamentally distinct, RNA-guided platform compared to Cas9 and Cpf1. Structural studies revealed structural differences within the nucleotide-binding loops of CasX, with a compact protein size less than 1,000 amino acids, and guide RNA (gRNA) scaffold stem. These structural differences affect the active ternary complex assembly, leading to different in vivo and in vitro behaviors of these two enzymes.

Compression of Genetic Information in Multiple Reading Frames

Techniques such as genome editing, gene therapy, and CRISPR-based gene expression require robust methods of delivering genetic information. The effectiveness of delivery depends on the amount of DNA or RNA that can be delivered.  In some cases there is a strict upper-limit on the amount of DNA or RNA that can be delivered.  For example, AAV vectors for mammalian gene delivery are limited to genetic cargos of < 5 kb.  In general, and irrespective of the delivery vector, larger DNA constructs are delivered less efficiently and so it is advantageous to use smaller constructs where possible. It is therefore advantageous to compress constructs. Methods of compression that do not require removal of genetic elements (“lossless compression”) are very desirable since size requirements can be met without compromising functionality.     In order to reduce the number of bases (DNA or RNA) required to encode larger constructs, UC Berkeley researchers have developed a method for compressing genetic information.   The method can be applied to two elements which be encoded in the same or different reading and can also be applied to a single genetic elements.