A PAMless base editor CRISPR-Cas DNA base editing typically requires a specific motif for targeting known as a protospacer-adjacent motif (PAM). This requirement limits the sequences within a genome that can be targeted. Walton et al. engineered specific variants of the Streptococcus pyogenes Cas9 enzyme named SpG and SpRY that could recognize and edit a wider array of PAMs. Using SpRY, the authors were able to correct previously uneditable mutations associated with human disease. Although off-target effects were observed for these engineered Cas enzymes at levels similar to those of the wild-type enzyme, depending on the context, these engineered enzymes widen the potential applications of precision genome editing. Science, this issue p. 290 Engineered Cas variants nearly eliminate the protospacer-adjacent motif requirement for DNA-targeting CRISPR enzymes. Manipulation of DNA by CRISPR-Cas enzymes requires the recognition of a protospacer-adjacent motif (PAM), limiting target site recognition to a subset of sequences. To remove this constraint, we engineered variants of Streptococcus pyogenes Cas9 (SpCas9) to eliminate the NGG PAM requirement. We developed a variant named SpG that is capable of targeting an expanded set of NGN PAMs, and we further optimized this enzyme to develop a near-PAMless SpCas9 variant named SpRY (NRN and to a lesser extent NYN PAMs). SpRY nuclease and base-editor variants can target almost all PAMs, exhibiting robust activities on a wide range of sites with NRN PAMs in human cells and lower but substantial activity on those with NYN PAMs. Using SpG and SpRY, we generated previously inaccessible disease-relevant genetic variants, supporting the utility of high-resolution targeting across genome editing applications.

中文翻译：

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使用近乎无 PAM 的工程化 CRISPR-Cas9 变体进行无约束的基因组靶向

无 PAM 碱基编辑器 CRISPR-Cas DNA 碱基编辑器通常需要一个特定的基序进行靶向，称为原型间隔区相邻基序 (PAM)。这一要求限制了可以靶向的基因组内的序列。沃尔顿等人。设计了化脓性链球菌 Cas9 酶的特定变体，名为 SpG 和 SpRY，可以识别和编辑更广泛的 PAM。使用 SpRY，作者能够纠正与人类疾病相关的以前不可编辑的突变。尽管在与野生型酶相似的水平上观察到这些工程化 Cas 酶的脱靶效应，但根据上下文，这些工程化酶拓宽了精确基因组编辑的潜在应用。科学，这个问题 p。290 种工程化 Cas 变体几乎消除了靶向 DNA 的 CRISPR 酶对原型间隔区相邻基序的要求。CRISPR-Cas 酶对 DNA 的操纵需要识别原始间隔区相邻基序 (PAM)，将目标位点识别限制为序列子集。为了消除这种限制，我们设计了化脓性链球菌 Cas9 (SpCas9) 的变体，以消除 NGG PAM 要求。我们开发了一种名为 SpG 的变体，它能够靶向一组扩展的 NGN PAM，并且我们进一步优化了这种酶以开发一种名为 SpRY（NRN 和在较小程度上是 NYN PAM）的接近 PAM 的 SpCas9 变体。SpRY 核酸酶和碱基编辑器变体几乎可以靶向所有 PAM，在人类细胞中具有 NRN PAMs 的广泛位点上表现出强大的活性，而对具有 NYN PAMs 的那些位点具有较低但显着的活性。