Francisella novicida Cas9 interrogates genomic DNA with very high specificity and can be used for mammalian genome editing

被引：69

作者：

Acharya, Sundaram ^{[1
,2
]}

Mishra, Arpit ^{[1
,5
]}

Paul, Deepanjan ^{[1
]}

Ansari, Asgar Hussain ^{[1
,2
]}

Azhar, Mohd ^{[1
,2
]}

Kumar, Manoj ^{[1
,2
]}

Rauthan, Riya ^{[1
,2
]}

Sharma, Namrata ^{[1
]}

Aich, Meghali ^{[1
,2
]}

Sinha, Dipanjali ^{[1
,2
]}

Sharma, Saumya ^{[1
,2
]}

Jain, Shivani ^{[1
]}

Ray, Arjun ^{[1
,6
]}

Jain, Suman ^{[3
]}

Ramalingam, Sivaprakash ^{[1
,2
]}

Maiti, Souvik ^{[1
,2
,4
]}

Chakraborty, Debojyoti ^{[1
,2
]}

机构：

[1] Inst Genom & Integrat Biol, Council Sci & Ind Res, Genom & Mol Med Unit, New Delhi 110025, India

[2] Acad Sci & Innovat Res, Ghaziabad 201002, India

[3] Thalassemia & Sickle Cell Soc, Kamala Hosp & Res Ctr, Hyderabad 500052, India

[4] Joint Ctr Council Sci & Ind Research, NCL, IGIB, Pune 411008, Maharashtra, India

[5] Univ Washington, Sch Med, Div Med Genet, Seattle, WA 98195 USA

[6] Indraprastha Inst Informat Technol, Ctr Computat Biol, New Delhi 110020, India

来源：

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA | 2019年 / 116卷 / 42期

关键词：

CRISPR Cas9; genome editing; gene therapy; iPSCs; sickle cell anemia; PLURIPOTENT STEM-CELLS; RNA; CRISPR-CAS9; ENDONUCLEASE; EFFICIENT; CPF1; TECHNOLOGIES; CRISPR/CAS9; MECHANISM; DYNAMICS;

D O I：

10.1073/pnas.1818461116

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

Genome editing using the CRISPR/Cas9 system has been used to make precise heritable changes in the DNA of organisms. Although the widely used Streptococcus pyogenes Cas9 (SpCas9) and its engineered variants have been efficiently harnessed for numerous gene-editing applications across different platforms, concerns remain regarding their putative off-targeting at multiple loci across the genome. Here we report that Francisella novicida Cas9 (FnCas9) shows a very high specificity of binding to its intended targets and negligible binding to off-target loci. The specificity is determined by its minimal binding affinity with DNA when mismatches to the target single-guide RNA (sgRNA) are present in the sgRNA:DNA heteroduplex. FnCas9 produces staggered cleavage, higher homology-directed repair rates, and very low nonspecific genome editing compared to SpCas9. We demonstrate FnCas9-mediated correction of the sickle cell mutation in patient-derived induced pluripotent stem cells and propose that it can be used for precise therapeutic genome editing for a wide variety of genetic disorders.

引用

页码：20959 / 20968

页数：10

共 58 条

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