No abstract

Silencing of transposable elements occurs during fetal gametogenesis in males viaArgonaute proteins, also known as PAZ Piwi domain (PPD) proteins, are members of a well-conserved family that is expressed in a variety of organisms, from fission yeasts to humans. The family can be divided into two subfamilies, Piwi and Ago, based on the primary sequence homology and expression pattern of each member. Piwi subfamily members are expressed only in germ lineage cells, whereas members of the Ago subfamily are expressed ubiquitously. The PPD proteins were initially characterized as essential molecules for stem cell selfrenewal and maintenance in Drosophila, Caenorhabditis elegans, and certain plant species (Cox et al. 1998;Moussian et al. 1998), and a member of the family is essential for stem cell function during regeneration in planaria (Reddien et al. 2005). There are three Piwi subfamily genes in the mouse genome: Miwi (mouse piwi), Miwi2,, which are termed Piwil1 (piwi-like homolog 1), Piwil4, and Piwil2, respectively, in the official nomenclature. Although they are ex-

Representing the 60 trillion cells that build a human body, a sperm and an egg meet, recognize each other, and fuse to form a new generation of life. The factors involved in this important membrane fusion event, fertilization, have been sought for a long time. Recently, CD9 on the egg membrane was found to be essential for fusion, but sperm-related fusion factors remain unknown. Here, by using a fusion-inhibiting monoclonal antibody and gene cloning, we identify a mouse sperm fusion-related antigen and show that the antigen is a novel immunoglobulin superfamily protein. We have termed the gene Izumo and produced a gene-disrupted mouse line. Izumo-/- mice were healthy but males were sterile. They produced normal-looking sperm that bound to and penetrated the zona pellucida but were incapable of fusing with eggs. Human sperm also contain Izumo and addition of the antibody against human Izumo left the sperm unable to fuse with zona-free hamster eggs.

The RNA-guided endonuclease Cas9 cleaves its target DNA and is a powerful genome-editing tool. However, the widely used Cas9 enzyme (SpCas9) requires an NGG protospacer adjacent motif (PAM) for target recognition, thereby restricting the targetable genomic loci. Here, we report a rationally engineered SpCas9 variant (SpCas9-NG) that can recognize relaxed NG PAMs. The crystal structure revealed that the loss of the base-specific interaction with the third nucleobase is compensated by newly introduced non-base-specific interactions, thereby enabling the NG PAM recognition. We showed that SpCas9-NG induces indels at endogenous target sites bearing NG PAMs in human cells. Furthermore, we found that the fusion of SpCas9-NG and the activation-induced cytidine deaminase (AID) mediates the C-to-T conversion at target sites with NG PAMs in human cells.