Abstract: The present invention relates to a method for the analysis of binding and cleavage sites followed by high-throughput sequencing. This method is called “ABC-seq”. The method is based on CUT&RUN (or CUT&Tag), originally developed for the detection of epigenetic marks, in combination with recombinant catalytically active or inactive Cas and a bioinformatics pipeline to identify off-site binding and off-site cleavage events in parallel.

Abstract: The invention relates to using CUT&RUN to validate CRISPR-Cas targeting. To improve the efficiency of CRISPR-based gene editing and delivery for in vivo applications, a method which may comprise: (a) expressing a catalytically inactive Cas protein (dCas) in target cells, (b) optional hypotonic lysis of the cells of step (a) to release nuclei, (c) immobilizing cells of step (a) or nuclei of step (b) with magnetic beads, (d) incubating the product of step (c) with an anti-CRISPR-dCas antibody, (e) incubating the product of step (d) with ProteinA-MNase (pAG-MNase), (f) adding of a Ca2+ ions-containing buffer to start MNase digestion and release of pAG-MNase-antibody-chromatin complexes, (g) adding a chelator-containing buffer to stop the reaction of step (f), (h) pelletizing the obtained oligonucleosome and obtaining pAG-MNasebound digested chromatin fragments from the supernatant, (i) extracting of DNA and RNA from the chromatin fragments of step (h), and (j) sequencing of DNA and RNA.

Abstract: Compositions, targeting reagents, modified cells, nucleic acid molecules, systems, and methods for identifying and selecting genomic safe harbor sites for transgene insertion and other genome engineering applications. These materials and methods can be used to develop desired genome engineering applications, such as transgene insertion and expression or genome modification, that take into account the application-specific needs for safety, functional silence, and accessibility and other factors that vary with a desired application's goals and target population. Representative examples of desired genome engineering applications include, but are not limited to, transgene insertion, such as therapeutic transgene insertion, functional gene editing, gene or chromosomal location-specific structural modification, cell marking, gene activation, and/or gene repression.

Abstract: Isolated DNAs encoding the enzyme I-SpomI and its recognition and cutting site are provided. The DNA sequences can be incorporated in cloning and expression vectors, transformed cell lines and transgenic animals. The vectors are useful in gene mapping and site-directed insertion of genes.

Abstract: Isolated DNAs encoding the enzyme I-Spoml and its recognition and cutting site are provided. The DNA sequences can be incorporated in cloning and expression vectors, transformed cell lines and transgenic animals. The vectors are useful in gene mapping and site-directed insertion of genes.

Abstract: Isolated DNAs encoding the enzyme I-SpomI and its recognition and cutting site are provided. The DNA sequences can be incorporated in cloning and expression vectors, transformed cell lines and transgenic animals. The vectors are useful in gene mapping and site-directed insertion of genes.

Abstract: Isolated DNAs encoding the enzyme I-SpomI and its recognition and cutting site are provided. The DNA sequences can be incorporated in cloning and expression vectors, transformed cell lines and transgenic animals. The vectors are useful in gene mapping and site-directed insertion of genes.

Abstract: Isolated DNAs encoding the enzyme I-SpomI and its recognition and cutting site are provided. The DNA sequences can be incorporated in cloning and expression vectors, transformed cell lines and transgenic animals. The vectors are useful in gene mapping and site-directed insertion of genes.