Systems and methods for targeted gene modification, targeted insertion, perturbation of gene transcripts, and nucleic acid editing. Novel nucleic acid targeting systems comprise components of CRISPR systems, reverse transcriptase, pegRNAs, paired pegRNAs or modified pegRNAs, DNA processing proteins, recombinases, proteins for inhibiting nucleases, and proteins for promoting ssDNA annealing.

The present disclosure generally relates to therapies involving immune effector cells such as T cells engineered to express antigen receptors such as T cell receptors (TCRs) or chimeric antigen receptors (CARs). It is demonstrated herein that such antigen receptor-engineered immune effector cells may be generated in vitro/ex vivo as well as in vitro by delivering nucleic acid encoding an antigen receptor for genetic modification to cells using particles comprising the nucleic acid and a targeting molecule for targeting the immune effector cells, wherein the targeting molecule is a designed ankyrin repeat protein (DARPin). In particular, DARPins are described herein which are high-affinity binders for CDS binding to the CDS receptor on human and non-human primate (NHP) cells. Nanoparticles functionalized with CD8− targeting DARPins (CDS-DARPin) can deliver genes exclusively and specifically to human CD8.sup.+ T cells in vitro and in vivo.

The present disclosure generally relates to therapies involving immune effector cells such as T cells engineered to express antigen receptors such as T cell receptors (TCRs) or chimeric antigen receptors (CARs). It is demonstrated herein that such antigen receptor-engineered immune effector cells may be generated in vitro/ex vivo as well as in vitro by delivering nucleic acid encoding an antigen receptor for genetic modification to cells using particles comprising the nucleic acid and a targeting molecule for targeting the immune effector cells, wherein the targeting molecule is a designed ankyrin repeat protein (DARPin). In particular, DARPins are described herein which are high-affinity binders for CDS binding to the CDS receptor on human and non-human primate (NHP) cells. Nanoparticles functionalized with CD8− targeting DARPins (CDS-DARPin) can deliver genes exclusively and specifically to human CD8.sup.+ T cells in vitro and in vivo.

Provided herein are regulatable expression systems and methods of using said regulatable expression systems to express proteins of interest. The regulatable expression systems comprise a unidirectional regulatable promoter operably linked to a single transcription unit encoding a protein of interest, a ribosome skip, and a transactivator protein.

Provided herein are regulatable expression systems and methods of using said regulatable expression systems to express proteins of interest. The regulatable expression systems comprise a unidirectional regulatable promoter operably linked to a single transcription unit encoding a protein of interest, a ribosome skip, and a transactivator protein.

Disclosed are cells that have stably integrated into their genomes exogenous nucleic acid sequences, such as transgenes, within or proximal to the integration site of a sequence comprising at least part of an endogenous retrovirus (ERV) or a LTR-retrotransposon (LTR-RT), or instead of a sequence encompassing an ERV or a LTR-RT that is part or was part of the genome of the cell, as well as method of producing and using such cells. Advantageously, a high level and/or stable production of the transgene expression product(s) can be achieved. Transgene integration and expression may be furthered by modulating the DNA repair pathways of the cell, e.g., by transiently expressing a gene encoding a protein that forms part of a DNA repair pathway during transgene integration.

Disclosed are cells that have stably integrated into their genomes exogenous nucleic acid sequences, such as transgenes, within or proximal to the integration site of a sequence comprising at least part of an endogenous retrovirus (ERV) or a LTR-retrotransposon (LTR-RT), or instead of a sequence encompassing an ERV or a LTR-RT that is part or was part of the genome of the cell, as well as method of producing and using such cells. Advantageously, a high level and/or stable production of the transgene expression product(s) can be achieved. Transgene integration and expression may be furthered by modulating the DNA repair pathways of the cell, e.g., by transiently expressing a gene encoding a protein that forms part of a DNA repair pathway during transgene integration.

Embodiments of the present disclosure are directed to a method that includes contacting a population of plant cells with a messenger ribonucleic acid (mRNA) construct including a sequence encoding a rare-cutting endonuclease and a detectable label, wherein the rare-cutting endonuclease is configured to induce a mutation at a target genomic locus. The method further includes screening the population of plant cells for the detectable label to identify target plant cells that are genetically transformed with the mRNA construct.

Embodiments of the present disclosure are directed to a method that includes contacting a population of plant cells with a messenger ribonucleic acid (mRNA) construct including a sequence encoding a rare-cutting endonuclease and a detectable label, wherein the rare-cutting endonuclease is configured to induce a mutation at a target genomic locus. The method further includes screening the population of plant cells for the detectable label to identify target plant cells that are genetically transformed with the mRNA construct.

The invention relates to logic-gate-based Type II or Type V CRISPR-Cas constructs and methods for modifying gene expression using the CRISPR-Cas constructs and CRISPR-Cas effector proteins.