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巴斯德氏菌属 PpCas9-一种紧凑的 II-C 型 Cas9 直系同源物，可在人细胞中发挥作用。

PpCas9 from Pasteurella pneumotropica - a compact Type II-C Cas9 ortholog active in human cells.

机构信息

Skolkovo Institute of Science and Technology, Center of Life Sciences, Moscow, 121205, Russia.

Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences, Moscow, 119334, Russia.

出版信息

Nucleic Acids Res. 2020 Dec 2;48(21):12297-12309. doi: 10.1093/nar/gkaa998.

DOI:10.1093/nar/gkaa998
PMID:33152077
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7708072/
Abstract

CRISPR-Cas defense systems opened up the field of genome editing due to the ease with which effector Cas nucleases can be programmed with guide RNAs to access desirable genomic sites. Type II-A SpCas9 from Streptococcus pyogenes was the first Cas9 nuclease used for genome editing and it remains the most popular enzyme of its class. Nevertheless, SpCas9 has some drawbacks including a relatively large size and restriction to targets flanked by an 'NGG' PAM sequence. The more compact Type II-C Cas9 orthologs can help to overcome the size limitation of SpCas9. Yet, only a few Type II-C nucleases were fully characterized to date. Here, we characterized two Cas9 II-C orthologs, DfCas9 from Defluviimonas sp.20V17 and PpCas9 from Pasteurella pneumotropica. Both DfCas9 and PpCas9 cleave DNA in vitro and have novel PAM requirements. Unlike DfCas9, the PpCas9 nuclease is active in human cells. This small nuclease requires an 'NNNNRTT' PAM orthogonal to that of SpCas9 and thus potentially can broaden the range of Cas9 applications in biomedicine and biotechnology.

摘要

CRISPR-Cas 防御系统因其易于利用效应 Cas 核酸酶与向导 RNA 结合而成为基因组编辑领域的研究热点，从而能够靶向理想的基因组位点。来自酿脓链球菌的 II-A 型 SpCas9 是最早用于基因组编辑的 Cas9 核酸酶，也是目前最受欢迎的 Cas9 酶。然而，SpCas9 存在一些缺点，包括相对较大的尺寸和只能靶向侧翼为“NGG”PAM 序列的靶标。更为紧凑的 II-C 型 Cas9 同源物有助于克服 SpCas9 的尺寸限制。然而，迄今为止，只有少数 II-C 型核酸酶得到了充分的表征。在这里，我们对来自 Defluviimonas sp.20V17 的 DfCas9 和来自巴氏嗜血杆菌的 PpCas9 这两种 Cas9 II-C 同源物进行了表征。DfCas9 和 PpCas9 都能在体外切割 DNA，并且具有新的 PAM 需求。与 DfCas9 不同，PpCas9 核酸酶在人细胞中具有活性。这种小的核酸酶需要与 SpCas9 正交的“NNNNRTT”PAM，因此有可能拓宽 Cas9 在生物医学和生物技术中的应用范围。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7fd9/7708072/67377c505194/gkaa998fig8.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7fd9/7708072/6ece2c6a593f/gkaa998fig1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7fd9/7708072/95d45d3c5ee3/gkaa998fig2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7fd9/7708072/a7f7d78ca899/gkaa998fig3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7fd9/7708072/24a67df84786/gkaa998fig4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7fd9/7708072/140f87762ff8/gkaa998fig5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7fd9/7708072/923116b69afb/gkaa998fig7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7fd9/7708072/67377c505194/gkaa998fig8.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7fd9/7708072/6ece2c6a593f/gkaa998fig1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7fd9/7708072/95d45d3c5ee3/gkaa998fig2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7fd9/7708072/a7f7d78ca899/gkaa998fig3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7fd9/7708072/24a67df84786/gkaa998fig4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7fd9/7708072/140f87762ff8/gkaa998fig5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7fd9/7708072/923116b69afb/gkaa998fig7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7fd9/7708072/67377c505194/gkaa998fig8.jpg

相似文献

[1]
PpCas9 from Pasteurella pneumotropica - a compact Type II-C Cas9 ortholog active in human cells.

Nucleic Acids Res. 2020-12-2

[2]
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[3]
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[4]
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[5]
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[6]
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[7]
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[8]
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[9]
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[10]
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Biochemistry. 2021-12-14

引用本文的文献

[1]
Insight into crRNA Processing in P42S and Application of SmutCas9 in Genome Editing.

Int J Mol Sci. 2025-2-25

[2]
Next-generation CRISPR technology for genome, epigenome and mitochondrial editing.

Transgenic Res. 2024-10

[3]
Advances in bread wheat production through CRISPR/Cas9 technology: a comprehensive review of quality and other aspects.

Planta. 2023-7-31

[4]
Identification of a novel type II-C Cas9 from the fish pathogen .

Front Microbiol. 2023-6-15

[5]
Massively parallel evaluation and computational prediction of the activities and specificities of 17 small Cas9s.

Nat Methods. 2023-7

[6]
Identification of the EH CRISPR-Cas9 system on a metagenome and its application to genome engineering.

Microb Biotechnol. 2023-7

[7]
Genome editing with natural and engineered CjCas9 orthologs.

Mol Ther. 2023-4-5

[8]
Compact Cas9d and HEARO enzymes for genome editing discovered from uncultivated microbes.

Nat Commun. 2022-12-15

[9]
Closely related type II-C Cas9 orthologs recognize diverse PAMs.

Elife. 2022-8-12

[10]
FrCas9 is a CRISPR/Cas9 system with high editing efficiency and fidelity.

Nat Commun. 2022-3-17

本文引用的文献

[1]
A compact Cas9 ortholog from Staphylococcus Auricularis (SauriCas9) expands the DNA targeting scope.

PLoS Biol. 2020-3-30

[2]
Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants.

Nat Rev Microbiol. 2019-12-19

[3]
Structural basis for the promiscuous PAM recognition by Corynebacterium diphtheriae Cas9.

Nat Commun. 2019-4-29

[4]
CRISPResso2 provides accurate and rapid genome editing sequence analysis.

Nat Biotechnol. 2019-3

[5]
Engineer chimeric Cas9 to expand PAM recognition based on evolutionary information.

Nat Commun. 2019-2-4

[6]
A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo Genome Editing.

Mol Cell. 2018-12-20

[7]
Systematic evaluation of error rates and causes in short samples in next-generation sequencing.

Sci Rep. 2018-7-19

[8]
genome editing in animals using AAV-CRISPR system: applications to translational research of human disease.

F1000Res. 2017-12-20

[9]
Type II-C CRISPR-Cas9 Biology, Mechanism, and Application.

ACS Chem Biol. 2017-12-20

[10]
A thermostable Cas9 with increased lifetime in human plasma.

Nat Commun. 2017-11-10

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