The invention provides for delivery, engineering and optimization of systems, methods, and compositions for manipulation of sequences and/or activities of target sequences. Provided are delivery systems and tissues or organ which are targeted as sites for delivery. Also provided are vectors and vector systems some of 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 of directing CRISPR complex formation in eukaryotic cells to ensure enhanced specificity for target recognition and avoidance of toxicity and to edit or modify a target site in a genomic locus of interest to alter or improve the status of a disease or a condition.

This disclosure provides, among other things, strategies for minimizing antibody diversification in a transgenic animal that uses gene conversion for antibody diversification. In some embodiments, the animal may comprise a genome comprising an endogenous immunoglobulin light chain locus comprising: (a) a functional immunoglobulin light chain gene comprising a nucleic acid encoding a light chain variable region; and (b) a plurality of pseudogenes that are operably linked to the functional immunoglobulin light chain gene and that donate, by gene conversion, nucleotide sequence to the nucleic acid encoding a light chain variable region, wherein the pseudogenes are upstream or downstream of the functional immunoglobulin light chain gene and encode the same amino acid sequence as the light chain variable region of the functional immunoglobulin light chain gene of (a). In other embodiments, the locus may have a tandem array of coding sequences for the light chain.

Non-human animals, cells, methods and compositions for making and using the same are provided, wherein the non-human animals and cells comprise a humanized B-cell activating factor gene. Non-human animals and cells that express a human or humanized B-cell activating factor protein from an endogenous B-cell activating factor locus are described.

Non-human animals, cells, methods and compositions for making and using the same are provided, wherein the non-human animals and cells comprise a humanized a proliferation-inducing ligand gene. Non-human animals and cells that express a human or humanized a proliferation-inducing ligand protein from an endogenous a proliferation-inducing ligand locus are described.

The invention provides a composition comprising an extraembryonic endodermal (XEN) call and/or an embryonic fibroblast (EF) cell. The invention also provides a method of establishing a XEN cell line or a primary embryonic fibroblast (EF) cell line in vitro, the method comprising culturing a zygote or parthenote from a mammal for a time sufficient to produce one or more blastocysts; and culturing the one or more blastocysts on feeder cells in culture medium for a time sufficient to produce one or a plurality of XEN cells and/or one or a plurality of EF cells.

The invention provides methods for improving cloning efficiency and modulating an imprinting control region. In particular embodiments, the invention provides methods for activating a repressed allele within an imprinting control region, thereby treating an imprinting associated disorder. In other embodiments, the invention provides methods for improving somatic cell nuclear transfer efficiency that involve Kdm4d overexpression is a Xist knockout donor cell.

Methods and compositions are provided for assessing CRISPR/Cas-mediated non-homologous end joining (NHEJ) activity and/or CRISPR/Cas-induced recombination of a target genomic locus with an exogenous donor nucleic acid in vivo or ex vivo. The methods and compositions employ non-human animals comprising a CRISPR reporter such as a genomically integrated CRISPR reporter for detecting and measuring targeted excision of a sequence between two CRISPR/Cas nuclease cleavage sites or disruption of a sequence near a CRISPR/Cas nuclease cleavage site and/or measuring CRISPR/Cas-induced recombination of the CRISPR reporter with an exogenous donor nucleic acid to convert the coding sequence for a first reporter protein to the coding sequence for a different second reporter protein. Methods and compositions are also provided for making and using these non-human animals.

This disclosure provides, among other things, strategies for minimizing antibody diversification in a transgenic animal that uses gene conversion for antibody diversification. In some embodiments, the animal may comprise a genome comprising an endogenous immunoglobulin light chain locus comprising: (a) a functional immunoglobulin light chain gene comprising a nucleic acid encoding a light chain variable region; and (b) a plurality of pseudogenes that are operably linked to the functional immunoglobulin light chain gene and that donate, by gene conversion, nucleotide sequence to the nucleic acid encoding a light chain variable region, wherein the pseudogenes are upstream or downstream of the functional immunoglobulin light chain gene and encode the same amino acid sequence as the light chain variable region of the functional immunoglobulin light chain gene of (a). In other embodiments, the locus may have a tandem array of coding sequences for the light chain.

The invention relates to gene drives, and in particular to genetic sequences and constructs for use in a gene drive. The invention is especially concerned with ultra-conserved and ultra-constrained sequences for use as a gene drive target with the aim of overcoming the development of resistance to the drive. The invention is also concerned with methods of suppressing wild type arthropod populations by use of the gene drive construct described herein.

The invention provides methods for rearing and genetic manipulation of the genome of sap-feeding insects (e.g., white-flies and others) to identify genetic targets for pest control, insecticides for pest control, and approaches to the genetic control of these pests.