The present disclosure provides methods of promoting a persistent effector function of immune cells, comprising modifying the cells to overexpress c-Jun and reduced levels of a NR4A gene and/or protein. Also provided are modified cells, e.g., immune cell, which have been modified to overexpress c-Jun and express reduced levels of NR4A gene and/or protein. Overexpressing c-Jun and simultaneously reducing expression levels of a NR4A gene and/or protein leads to exhaustion/dysfunction resistant cells, which are apoptosis resistant and also immune checkpoint resistant, and also to the maintenance of anti-tumor function in tumor microenvironments.

Methods for manufacturing genetically engineered T cells expressing a chimeric antigen receptor (CAR), such as a CAR that binds human CD19, BCMA, or CD70, and having multiple additional gene edits, for example, a disrupted Regnase-1 gene, a disrupted TGFBRII gene, a disrupted TRAC gene, a disrupted β2M gene, or a combination thereof, using CRISPR/Cas gene editing systems.

The present disclosure relates to systems, compositions and methods for modifying target nucleic acid sequences.

Described is an assay termed Programmable Enzyme-Assisted Selective Exponential Amplification (PASEA) that concurrently amplifies both wild type and mutant alleles while selectively cleaving the former. With time, the rare mutant alleles dominate, and are readily detectable by direct detection, Sanger sequencing, and other readily available methods. Also described are point-of-care assays and microfluidic devices for performing PASEA.

Aspects of this invention inter alia relate to novel systems for targeting, editing or manipulating DNA in a cell, comprising one or more heterologous vector(s) encoding a SluCas9 nuclease from Staphylococcus lugdunensis or variants thereof, and one or more guide RNAs (gRNAs), or a SluCas9 nuclease or variant thereof and one or more gRNAs.

The present disclosure provides RNA-guided CRISPR-Cas effector proteins, nucleic acids encoding same, and compositions comprising same. The present disclosure provides ribonucleoprotein complexes comprising: an RNA-guided CRISPR-Cas effector protein of the present disclosure; and a guide RNA. The present disclosure provides methods of modifying a target nucleic acid, using an RNA-guided CRISPR-Cas effector protein of the present disclosure and a guide RNA. The present disclosure provides methods of modulating transcription of a target nucleic acid.

The invention provides for systems, methods, and compositions for altering expression of target gene sequences and related gene products. Provided are structural information on the Cas protein of the CRISPR-Cas system, use of this information in generating modified components of the CRISPR complex, vectors and vector systems which encode one or more components or modified components of a CRISPR complex, as well as methods for the design and use of such vectors and components. Also provided are methods of directing CRISPR complex formation in eukaryotic cells and methods for utilizing the CRISPR-Cas system. In particular the present invention comprehends optimized functional CRISPR-Cas enzyme systems. In particular the present invention comprehends engineered new guide architectures and enzymes to be used in optimized Staphylococcus aureus CRISPR-Cas enzyme systems.

This disclosure provides an engineered system comprising: a first nucleic acid molecule encoding a CasX nuclease, and a guide RNA (gRNA) or a second nucleic acid molecule encoding the gRNA, where the first nucleic acid molecule is codon optimized for a eukaryotic cell, and where the gRNA is designed to hybridize with a target site in the eukaryotic cell. Further, this disclosure provides a method of modifying at least one target site in a eukaryotic genome comprising: providing a eukaryotic cell with a CasX nuclease or a first nucleic acid molecule encoding the CasX nuclease, and providing the eukaryotic cell with a guide RNA (gRNA) or a second nucleic acid molecule encoding the gRNA, where the gRNA and the CasX nuclease form a complex, where the gRNA hybridizes to the target site, and where the complex generates a modification at the target site.

In some aspects, fusosome compositions and methods are described herein that comprise membrane enclosed preparations, comprising a fusogen. In some embodiments, the fusosome can the target cell, thereby delivering complex biologic agents to the target cell cytoplasm.

Systems and methods to genetically modify B cells to express selected antibodies are described. The systems and methods can be used to: obviate the need for classical vaccinations; provide protection against infectious agents for which no vaccinations are currently available; provide protection against infectious agents when patients are otherwise immune-suppressed; and/or provide a benefit provided by a therapeutic antibody, such as in the treatment of autoimmune disorders.