The present disclosure provides instruments, modules and methods for improved detection of edited cells following nucleic acid-guided nuclease genome editing. The disclosure provides improved automated instruments that perform methods—including high throughput methods—for screening cells that have been subjected to editing and identifying cells that have been properly edited.

The application provides a gRNA targeting miR-29b, an AAV8-CRISPR/Cas9 system and use thereof, belonging to the technical field of genetic engineering. The application provides a gRNA targeting miR-29b, which has a nucleotide sequence set forth in SEQ ID No.1. The gRNA shows strong specific editing ability to miR-29b at the cellular level, only has targeted editing effect on miR-29b without influence on miR-29a and miR-29c of the same family, and also shows higher editing efficiency, thus effectively inhibiting the expression of miR-29b. An AAV8-CRISPR/Cas9 system containing said gRNA targeting miR-29b. The use of gRNA targeting miR-29b or AAV8-CRISPR/Cas9 system in preparing drugs for treating muscle atrophy.

Provided herein are genetically engineered gas vesicle expression systems (GVES) that are configured to express gas vesicles (GVs) in a mammalian cell, related gas vesicle polynucleotide constructs, gas vesicle reporting genetic circuits, vectors, genetically engineered mammalian cells, non-human mammalian hosts, compositions, methods and systems, which in several embodiments can be used together with contrast-enhanced imaging techniques to detect and report biological events in an imaging target site comprising a mammalian cell and/or organism.

Provided herein are chimeric transactivator minigenes, where the alternative splicing of the minigene determines whether a transactivator is expressed. Expression of the transactivator results in the transcription of a target gene that is under the control of a designer promoter sequence. Alternatively, provided herein are chimeric target gene minigenes, wherein the alternative splicing of the minigene directly determines whether the target gene is expressed. The target gene may encode an inhibitory RNA, a CRISPR-Cas9 protein, or a therapeutic protein.

The present invention provides methods and compositions for labeling and/or targeting cells with increased calcium by providing a CREB reporter system. In addition, methods of treating disorders with activated cells such as brain disorders or cancer are also provided herein.

The invention relates to genetic constructs comprising at least one microRNA target site, and vectors comprising such constructs. The genetic constructs and vectors can be used in diagnosis and therapy of a range of disorders, including cancers, for example T-cell acute lymphoblastic leukaemia (T-ALL).

Provided is a new method for activating transcription of a gamma-globin gene. The method uses a single-stranded oligonucleotide (ssODN) containing GATA or an antisense complementary sequence TATC thereof as guidance information, and performs gene editing in a gamma-globin gene promoter region to form a GATA-containing enhancer element, which can promote the expression of the gamma-globin gene in mature red blood cells. Hematopoietic stem cells genetically edited by the method have normal functions, can significantly improve the expression of fetal hemoglobin after being differentiated into red blood cells, and therefore can be used in clinical treatment of beta-thalassemia and sickle cell anemia.

The invention provides a T cell wherein one or more therapeutic transgenes is integrated at a within the genome of the cell such that expression of the transgene is under control of an endogenous promoter of the T cell. The invention additional provides methods of making and using such cells to treat a subject with T cell therapy. The invention also provides a T cell wherein a recombinant nucleic acid sequence encoding a chimeric antigen receptor (CAR) is integrated at a first site within the genome of the cell such that the CAR is expressed by the cell at the surface of the cell, and wherein integration of the nucleic acid encoding the CAR at the first site reduces or prevents expression of a functional T cell receptor (TCR) complex at the surface of the cell. The invention additional provides methods of making and using such cells to treat a subject with CAR therapy.

Provided herein are genetically engineered gas vesicle expression systems (GVES) that are configured to express gas vesicles (GVs) in a mammalian cell, related gas vesicle polynucleotide constructs, gas vesicle reporting genetic circuits, vectors, genetically engineered mammalian cells, non-human mammalian hosts, compositions, methods and systems, which in several embodiments can be used together with contrast-enhanced imaging techniques to detect and report biological events in an imaging target site comprising a mammalian cell and/or organism.

Aspects of this disclosure provide nucleic acid constructs A nucleic acid construct, comprising a first expression cassette comprising a nucleic acid encoding a tetracycline-responsive transactivator under the control of a cell type-specific promoter; and a second expression cassette comprising a nucleic acid encoding a transgene, for example, a chimeric antigen receptor (CAR), under the control of a tetracycline-responsive promoter.