Expanding the Genome-Editing Toolbox with Abyssicoccus albus Cas9 Using a Unique Protospacer Adjacent Motif Sequence.

IF 3.7 4区生物学 Q2 GENETICS & HEREDITY CRISPR Journal Pub Date : 2024-08-01 Epub Date: 2024-08-07 DOI:10.1089/crispr.2024.0013

Akiyoshi Nakamura, Hiroshi Yamamoto, Tsubasa Yano, Reika Hasegawa, Yoichi Makino, Nobutaka Mitsuda, Teruhiko Terakawa, Seiichiro Ito, Shigeo S Sugano

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Abstract

The genome-editing efficiency of the CRISPR-Cas9 system hinges on the recognition of the protospacer adjacent motif (PAM) sequence, which is essential for Cas9 binding to DNA. The commonly used Streptococcus pyogenes (SpyCas9) targets the 5'-NGG-3' PAM sequence, which does not cover all the potential genomic-editing sites. To expand the toolbox for genome editing, SpyCas9 has been engineered to recognize flexible PAM sequences and Cas9 orthologs have been used to recognize novel PAM sequences. In this study, Abyssicoccus albus Cas9 (AalCas9, 1059 aa), which is smaller than SpyCas9, was found to recognize a unique 5'-NNACR-3' PAM sequence. Modification of the guide RNA sequence improved the efficiency of AalCas9-mediated genome editing in both plant and human cells. Predicted structure-assisted introduction of a point mutation in the putative PAM recognition site shifted the sequence preference of AalCas9. These results provide insights into Cas9 diversity and novel tools for genome editing.

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利用独特的原位相邻位点序列扩展 Abyssicoccus albus Cas9 的基因组编辑工具箱

CRISPR-Cas9系统的基因组编辑效率取决于对原位相邻基序（PAM）的识别，这是Cas9与DNA结合的关键。常用的化脓性链球菌（SpyCas9）以5'-NGG-3' PAM序列为目标，但这一序列并不能覆盖所有潜在的基因组编辑位点。为了扩大基因组编辑的工具箱，SpyCas9 已被设计成能识别灵活的 PAM 序列，Cas9 的同源物也被用来识别新的 PAM 序列。本研究发现，比SpyCas9小的Abyssicoccus albus Cas9（AalCas9，1059 aa）能识别独特的5'-NNACR-3'PAM序列。对引导 RNA 序列的修改提高了 AalCas9 介导的植物和人类细胞基因组编辑的效率。在假定的 PAM 识别位点上引入结构辅助点突变改变了 AalCas9 的序列偏好。这些结果提供了对 Cas9 多样性和基因组编辑新工具的见解。

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来源期刊

CRISPR Journal Biochemistry, Genetics and Molecular Biology-Biotechnology

CiteScore

6.30

自引率

2.70%

发文量

期刊介绍： In recognition of this extraordinary scientific and technological era, Mary Ann Liebert, Inc., publishers recently announced the creation of The CRISPR Journal -- an international, multidisciplinary peer-reviewed journal publishing outstanding research on the myriad applications and underlying technology of CRISPR. Debuting in 2018, The CRISPR Journal will be published online and in print with flexible open access options, providing a high-profile venue for groundbreaking research, as well as lively and provocative commentary, analysis, and debate. The CRISPR Journal adds an exciting and dynamic component to the Mary Ann Liebert, Inc. portfolio, which includes GEN (Genetic Engineering & Biotechnology News) and more than 80 leading peer-reviewed journals.