Provided by the present disclosure are fluorescent proteins that can detect kinase activity. The fluorescent proteins of the present disclosure have us in, for example, detecting kinase activity in any eukaryotic cell.

Fusosome compositions and methods are described herein.

Methods and composition for modulating a target genome and stable integration of a transgene of interest into the genome of a cell are disclosed.

The present disclosure provides methods and compositions of modulating expression of a target nucleic acid in a eukaryotic cell. The methods include providing to the cell a guide RNA complementary to the target nucleic acid sequence, providing to the cell a fusion protein, wherein the fusion protein comprises a nuclease null Cas9 protein and a transcriptional effector domain, wherein the nuclease null Cas9 protein interacts with the guide RNA and binds to the target nucleic acid sequence in a site specific manner and wherein the transcriptional effector domain modulates expression of the target nucleic acid.

The present description relates to methods for delivering polypeptide cargos from an extracellular space to the cytosol and/or nucleus of a target eukaryotic cell. The methods involve contacting the cell with the polypeptide cargo in the presence of a peptide shuttle agent at a concentration sufficient to increase the polypeptide cargo's transduction efficiency. Also described here are parameters that may be used in the rational design of such synthetic peptide shuttle agents, peptide shuttle agents that satisfy one or more of these design parameters, as well as methods and compositions relating to the use of the synthetic peptide shuttle agents for delivery of a variety of polypeptide cargos (such as transcription factors, antibodies, CRISPR-associated nucleases and functional genome editing complexes) from an extracellular space to the cytosol and/or nucleus of target eukaryotic cells. Applications and targets for genome-editing NK cells for improved immunotherapy are also described.

Some aspects of this disclosure provide a fusion protein comprising a guide nucleotide sequence-programmable DNA binding protein domain (e.g., a nuclease-inactive variant of Cas9 such as dCas9), an optional linker, and a recombinase catalytic domain (e.g., a tyrosine recombinase catalytic domain or a serine recombinase catalytic domain such as a Gin recombinase catalytic domain). This fusion protein can recombine DNA sites containing a minimal recombinase core site flanked by guide RNA-specified sequences. The instant disclosure represents a step toward programmable, scarless genome editing in unmodified cells that is independent of endogenous cellular machinery or cell state.

In certain embodiments methods of treating X-Linked Hyper-IgM Syndrome (XHIM) in a mammal am provided where the methods comprise: i) providing differentiated T cells and/or stem/progenitor cells from the mammal; ii) performing a targeted insertion of a corrective CD40L cDNA at the CD40LG gene locus in said cells to provide a corrected CD40LG gene wherein said targeted insertion places said corrective CD40L cDNA downstream and operably linked to the endogenous CD40LG enhancer/promoter, and iii) introducing said cells into said mammal where said corrected CD40LG gene is expressed in a physiologically regulated manner.

The technology described herein is directed to regulated synthetic gene expression systems. In one aspect described herein are synthetic transcription factors (synTFs) comprising a DNA binding domain, a transcriptional effector domain, and a regulator protein. In other aspects described herein are gene expression systems comprising said synTFs and methods of treating diseases and disorders using said synTFs.

The present disclosure relates to CRISPR-Cas systems that utilize Cas 12J for editing nucleic acids in plants. Methods and compositions for using these systems for editing nucleic acids in plants are provided herein.

According to the present disclosure, a technology for efficiently introducing a desired foreign substance from the outside of eukaryotic cells into at least the cytoplasm (and the nucleolus) of the cells is provided. A method disclosed here includes a step of preparing a construct for introducing a foreign substance including a carrier peptide fragment composed of any of amino acid sequences: KKRTLRKKKRKKR (SEQ ID NO: 1), KKRTLRKRRRKKR (SEQ ID NO: 2), KKRTLRKRKRKKR (SEQ ID NO: 3) and KKRTLRKKRRKKR (SEQ ID NO: 4), and a foreign substance that is bound to an N-terminal side and/or C-terminal side of the carrier peptide fragment, a step of supplying the construct for introducing a foreign substance to a sample containing desired eukaryotic cells, and a step of incubating the sample to which the construct for introducing a foreign substance is supplied and introducing the construct into eukaryotic cells in the sample.