Methods and compositions for modifying the 3′ untranslated region or coding sequence of endogenous genes using rare-cutting endonucleases and donor molecules. The methods and compositions described herein can be used to modify the coding sequence of endogenous genes or to facilitate early termination of transcripts.

Embodiments of the disclosure includes methods of producing viral-specific therapy (VST) cells specific for the SARS-CoV-2 virus and uses of the cells. The methods may utilized peptide mixtures and stimulation of mononuclear cells using particular cytokine cocktails. The cells may also be genetically modified to lack expression of one or more endogenous genes, including one or more genes that renders the cells more effective and/or able to withstand deleterious conditions, such as the presence of glucocorticoids.

Provided is a genome editing system and method for gene editing at least one target sequence in a cell genome. The genome editing system comprises: (1) an expression construct comprising a Cas12f nuclease; and (2) an expression DNA sequence comprising a guide RNA corresponding to the Cas12f nuclease, and an expression construct of the targeting sequence of the target sequence. The gene editing system or method can accurately knock out a target gene from within a cell; in addition, in an in vitro cutting experiment, the target gene can be accurately cut.

Provided herein are gene editing systems and/or compositions comprising RNA guides targeting LDHA for use in genetic editing of the LDHA gene. Also provide herein are methods of using the gene editing system for introducing edits to the LDHA gene and/or for treatment of primary hyperoxaluria (PH), and processes for characterizing the gene editing system.

Provided herein are gene editing systems and/or compositions comprising RNA guides targeting HAO1 for use in genetic editing of the HAO1 gene. Also provide herein are methods of using the gene editing system for introducing edits to the HAO1 gene and/or for treatment of primary hyperoxaluria (PH), and processes for characterizing the gene editing system.

A method for gene editing in a vertebrate brain comprising: intravascular administration of a brain penetrable viral vector including a target sequence in a genomic locus of interest and a CRISPR enzyme; genomic integration via Non-Homologues End Joining (NHEJ) in post-mitotic neurons; and editing a monopartite cell-type specific gene via NHEJ knock-in a sgRNA flanked by self-cleaving ribozymes into 3′UTR to use an endogenous promoter for sgRNA expression.

The present invention relates in part to nucleic acids encoding proteins, nucleic acids containing non-canonical nucleotides, therapeutics comprising nucleic acids, methods, kits, and devices for inducing cells to express proteins, methods, kits, and devices for transfecting, gene editing, and reprogramming cells, and cells, organisms, and therapeutics produced using these methods, kits, and devices. Methods for inducing cells to express proteins and for reprogramming and gene-editing cells using RNA are disclosed. Methods for producing cells from patient samples, cells produced using these methods, and therapeutics comprising cells produced using these methods are also disclosed.

Provided herein are methods and compositions for depleting targeted nucleic acid sequences from a sample, enriching for sequences of interest from a sample, and/or partitioning of sequences from a sample. The methods and compositions are applicable to biological, clinical, forensic, and environmental samples.

Biosensors for small molecules can be used in applications that range from metabolic engineering to orthogonal control of transcription. Biosensors are produced based on a ligand-binding domain (LBD) using a method that, in principle, can be applied for any target molecule. The LBD is fused to either a fluorescent protein or a transcriptional activator and is destabilized by mutation such that the fusion accumulates only in cells containing the target ligand. The power of this method is illustrated by developing biosensors for digoxin and progesterone. Addition of ligand to cells expressing a biosensor activates transcription in yeast, mammalian cells and plants, with a dynamic range of up to about 100-fold or more. The biosensors are used to improve the biotransformation of pregnenolone to progesterone in yeast and to regulate CRISPR activity in mammalian cells. This work provides a general methodology to develop biosensors for a broad range of molecules.

Provided herein are systems, methods, and compositions for the modification of target DNA sequences. More particularly, systems, methods, and compositions for cleaving a target DNA in eukaryotic cells with a guide RNA capable of hybridizing with a target sequence and an RNA-guided DNA nuclease are provided. Also provided are vectors and vector systems which encode one or more components of a CRISPR complex, as well as methods for the design and use of such vectors. Also provided are methods for identifying and validating novel CRISPR systems.