Abstract: Provided are compositions and methods for using the compositions to modify eukaryotic chromosomes. The methods involve iteratively inserting DNA payloads into a chromosomal locus, or into multiple chromosomal loci. The methods utilize positive and negative selection approaches in combination with one or more recombinases to select cells that contain a payload, eliminate cells that do not contain a payload, and sequentially replace contiguous segments of the chromosome with subsequent payload insertions. Modified cells, and modified mammals containing modified cells, are included.

Abstract: In response to a folder event received for a first folder, a first work item is dequeued from an ID queue and metadata of the first folder, and immediate children of the first folder, is fetched and enqueued as work items in a metadata queue. If further first folder children remain to be scanned, the first work item is updated with child IDs for each immediate child of the first folder that is a folder, and it is inserted into the ID queue. In a second pass, a child ID is dequeued and metadata of immediate children of the folder associated with the child ID is fetched and enqueued as work items in the metadata queue. The second pass is repeated for all child IDs in the updated work item. This process is repeated for each generation of children of the first folder or until a specified limit is met.

Abstract: In response to a folder event received for a first folder, a first work item is dequeued from an ID queue and metadata of the first folder, and immediate children of the first folder, is fetched and enqueued as work items in a metadata queue. If further first folder children remain to be scanned, the first work item is updated with child IDs for each immediate child of the first folder that is a folder, and it is inserted into the ID queue. In a second pass, a child ID is dequeued and metadata of immediate children of the folder associated with the child ID is fetched and enqueued as work items in the metadata queue. The second pass is repeated for all child IDs in the updated work item. This process is repeated for each generation of children of the first folder or until a specified limit is met.

Abstract: In response to a folder event received for a first folder, a first work item is dequeued from an ID queue and metadata of the first folder, and immediate children of the first folder, is fetched and enqueued as work items in a metadata queue. If further first folder children remain to be scanned, the first work item is updated with child IDs for each immediate child of the first folder that is a folder, and it is inserted into the ID queue. In a second pass, a child ID is dequeued and metadata of immediate children of the folder associated with the child ID is fetched and enqueued as work items in the metadata queue. The second pass is repeated for all child IDs in the updated work item. This process is repeated for each generation of children of the first folder or until a specified limit is met.

Abstract: The present invention relates to a system for F-box hormone receptor regulated protein expression in mammalian cells. The system includes a silencing nucleic acid molecule comprising a first promoter and an shRNA operably linked to the first promoter, where the shRNA silences expression of a target protein. The system also includes an expression nucleic acid molecule comprising a second promoter, an F-box hormone receptor operably linked to the second promoter, and a nucleic acid molecule encoding a fusion protein comprising a degron fused to the target protein, where the nucleic acid molecule encoding the fusion protein is operably linked to the second promoter. Also disclosed are vectors comprising the system of the present application and methods of use thereof.

Abstract: The present invention relates to a system for F-box hormone receptor regulated protein expression in mammalian cells. The system includes a silencing nucleic acid molecule comprising a first promoter and an shRNA operably linked to the first promoter, where the shRNA silences expression of a target protein. The system also includes an expression nucleic acid molecule comprising a second promoter, an F-box hormone receptor operably linked to the second promoter, and a nucleic acid molecule encoding a fusion protein comprising a degron fused to the target protein, where the nucleic acid molecule encoding the fusion protein is operably linked to the second promoter. Also disclosed are vectors comprising the system of the present application and methods of use thereof.

Abstract: Coronatine has been found to enhance binding of the JAZ1 degron to the Arabidopsis F-box protein COI1, and analysis of the JAZ1 degron sequence has resulted in the identification of specific peptide sequences that bind COI1 with high affinity in the presence of coronatine. Crystal structure analysis has determined that coronatine and JA-Ile enhance the interaction between COI1 and JAZ1 via binding to a specific binding pocket on COI1. Attachment of one or more JAZ1 peptide tags as disclosed herein to a target protein in a non-plant cell expressing Arabidopsis COI1 or a homolog thereof results in degradation of the target protein following addition of a molecule that binds the coronatine/JA-Ile binding pocket on COI1. Therefore, provided herein are compositions, methods, and kits for targeted protein degradation.