Summary A key limitation to the use of CRISPR-Cas9 proteins for genome editing and other applications is the requirement that a protospacer adjacent motif (PAM) be present at the target site. For the most commonly used Cas9 from Streptococcus pyogenes (SpCas9), this PAM requirement is NGG. No natural or engineered Cas9 variants shown to function efficiently in mammalian cells offer a PAM less restrictive than NGG. Here we used phage-assisted continuous evolution (PACE) to evolve an expanded PAM SpCas9 variant (xCas9) that can recognize a broad range of PAM sequences including NG, GAA, and GAT. The PAM compatibility of xCas9 is the broadest reported to date among Cas9s active in mammalian cells, and supports applications in human cells including targeted transcriptional activation, nuclease-mediated gene disruption, and both cytidine and adenine base editing. Remarkably, despite its broadened PAM compatibility, xCas9 has much greater DNA specificity than SpCas9, with substantially lower genome-wide off-target activity at all NGG target sites tested, as well as minimal off-target activity when targeting genomic sites with non-NGG PAMs. These findings expand the DNA targeting scope of CRISPR systems and establish that there is no necessary trade-off between Cas9 editing efficiency, PAM compatibility, and DNA specificity.

Normal human retinal development involves orderly generation of rods and cones by complex mechanisms. Cell-fate specification involves progenitor cell lineage and external signals such as soluble factors and cell-cell interactions. In most inherited human retinal degenerations, including retinitis pigmentosa, a mutant gene causes loss of visual function, death of mature rods, and eventually death of all cone subtypes. Only one inherited retinal disorder, the enhanced S cone syndrome (ESCS), shows increased visual function, involving the minority S (blue) cones, and decreased rod and L͞M (red͞green) cone function. This autosomal recessive disease is caused by mutations in NR2E3, a photoreceptor nuclear receptor transcription factor, and may result from abnormal cell-fate determination, leading to excess S cones at the expense of other photoreceptor subtypes. In 16 ESCS patients with the most common NR2E3 mutation, R311Q, we documented an abnormal ratio of S to L͞M cone function and progressive retinal degeneration. We studied the postmortem retina of an ESCS patient homozygous for NR2E3 R311Q. No rods were identified, but cones were increased approximately 2-fold, and 92% were S cones. Only 15% of the cones expressed L͞M cone opsin, and some coexpressed S cone opsin. The retina was disorganized, with densely packed cones intermixed with inner retinal neurons. The retina was also degenerate, retaining photoreceptors in only the central and far peripheral regions. These observations suggest a key role for NR2E3 in regulation of human photoreceptor development. Degeneration of the NR2E3 retina may result from defective development, known S cone fragility, or abnormal maintenance of mature photoreceptors. T he molecular events during development of invertebrate and vertebrate retinas are incompletely understood. The specification of rods and cones involves interactions of both intrinsic and extrinsic factors, including cell lineage, soluble factors, and local cell-cell interactions (1-3). These complex signaling pathways involve nuclear receptors, a superfamily of transcription factors that share structural features, including DNA and ligandbinding domains (4, 5). A human photoreceptor-specific nuclear receptor (PNR), NR2E3, is a member of the nuclear receptor subfamily II (6). Coexpression of NR2E3, CRX (cone-rod homeobox; ref. 7), and NRL (nuclear retina leucine zipper; ref. 8) in human Y79 retinoblastoma cells suggests that NR2E3 is a component of a photoreceptor transcription cascade in human retinal development (6).Mutations in NR2E3 (9, 10) cause a unique human retinal disease, the enhanced S cone syndrome (ESCS), initially described using psychophysical and electrophysiological methods (11). ESCS patients lack rod photoreceptor function, like other inherited retinal degenerations such as retinitis pigmentosa (12), but are unique because their S (blue) cones, normally a minority population (13), are more responsive to light than the usually more populous L͞M (red͞green) cones (11, 14-16). The retinal pathology ...