Abstract: The present disclosure provides compositions, kits, genetically modified cells, non-human transgenic organisms, and methods for binding and/or cleaving a single stranded target nucleic acid. A method of cleaving includes contacting a single stranded target nucleic acid with (e.g., introducing into a cell) a subject argonaute (Ago) polypeptide and a guide RNA (e.g., having a 5?-OH). In some embodiments, a subject Ago polypeptide includes an amino acid sequence having 70% or more sequence identity with amino acids 282-430 and/or 431-639 of the Marinitoga piezophila argonaute (MpAgo) protein set forth in SEQ ID NO: 1. The present disclosure provides variant Ago polypeptides; and methods of use of same.

Abstract: The present disclosure provides atomic structures of Cas9 with and without polynucleotides bound thereto. Also provided is a computer-readable medium comprising atomic coordinates for Cas9 polypeptides in both an unbound configuration and a configuration wherein the Cas9 polypeptide is bound to one or more polynucleotides. The present disclosure provides crystals comprising Cas9 polypeptides; and compositions comprising the crystals. The present disclosure provides methods for the engineering of Cas9 polypeptides wherein Cas9 activity has been altered, ablated, or preserved and amended with additional activities.

Abstract: The present disclosure provides compositions, kits, genetically modified cells, non-human transgenic organisms, and methods for binding and/or cleaving a single stranded target nucleic acid. A method of cleaving includes contacting a single stranded target nucleic acid with (e.g., introducing into a cell) a subject argonaute (Ago) polypeptide and a guide RNA (e.g., having a 5?-OH). In some embodiments, a subject Ago polypeptide includes an amino acid sequence having 70% or more sequence identity with amino acids 282-430 and/or 431-639 of the Marinitoga piezophila argonaute (MpAgo) protein set forth in SEQ ID NO: 1. The present disclosure provides variant Ago polypeptides; and methods of use of same.

Abstract: The present disclosure provides atomic structures of Cas9 with and without polynucleotides bound thereto. Also provided is a computer-readable medium comprising atomic coordinates for Cas9 polypeptides in both an unbound configuration and a configuration wherein the Cas9 polypeptide is bound to one or more polynucleotides. The present disclosure provides crystals comprising Cas9 polypeptides; and compositions comprising the crystals. The present disclosure provides methods for the engineering of Cas9 polypeptides wherein Cas9 activity has been altered, ablated, or preserved and amended with additional activities.