CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity
Reads0
Chats0
TLDR
It is demonstrated that CRISPR/Cas9 systems targeting the human hemoglobin β and C-C chemokine receptor type 5 genes have substantial off-target cleavage, especially within the hemoglobin δ and C of 2 genes, respectively, causing gross chromosomal deletions.Abstract:
The ability to precisely modify endogenous genes can significantly facilitate biological studies and disease treatment, and the clustered regularly interspaced short palindromic repeats (CRISPR) systems have the potential to be powerful tools for genome engineering. However, the target specificity of CRISPR systems is largely unknown. Here we demonstrate that CRISPR/Cas9 systems targeting the human hemoglobin β and C-C chemokine receptor type 5 genes have substantial off-target cleavage, especially within the hemoglobin δ and C-C chemokine receptor type 2 genes, respectively, causing gross chromosomal deletions. The guide strands of the CRISPR/Cas9 systems were designed to have a range of mismatches with the sequences of potential off-target sites. Off-target analysis was performed using the T7 endonuclease I mutation detection assay and Sanger sequencing. We found that the repair of the on-and off-target cleavage resulted in a wide variety of insertions, deletions and point mutations. Therefore, CRISPR/Cas9 systems need to be carefully designed to avoid potential off-target cleavage sites, including those with mismatches to the 12-bases proximal to the guide strand protospacer-adjacent motif.read more
Citations
More filters
Journal ArticleDOI
Development of the CRISPR-Cas9 System for the Marine-Derived Fungi Spiromastix sp. SCSIO F190 and Aspergillus sp. SCSIO SX7S7
TL;DR: An effective PEG-mediated chemical transformation system for protoplasts in two marine-derived fungi and a versatile CRISPR-Cas9-based gene disruption method was established that will promote the discovery of novel natural products and further biological studies.
Journal ArticleDOI
Genome engineering in human pluripotent stem cells
TL;DR: The fundamentals of both technologies, and the wide-ranging present and future applications of genome engineering in hPSCs are reviewed.
Dissertation
Sistema CRISPR/Cas y su aplicación a la edición dirigida de genomas
TL;DR: In this review, the structure and functioning of CRISPR/Cas as a bacterial immune system is exposed, as well as its adaptation for genetic engineering, explaining some of its applications and possible issues that can appear in its use.
Book ChapterDOI
Editing Cultured Human Cells: From Cell Lines to iPS Cells
Hongmei Lisa Li,Akitsu Hotta +1 more
TL;DR: The chronological advances made in ZFNs, TALENs and CRISPR/Cas9 systems for gene disruption, locus-specific integration and gene correction are reviewed and the emerging applications of tailor-made DNA binding modules for controlling the transcriptional state, modifying epigenetic marks and for live-cell imaging are summarized.
References
More filters
Journal ArticleDOI
A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.
Martin Jinek,Krzysztof Chylinski,Krzysztof Chylinski,Ines Fonfara,Michael H. Hauer,Jennifer A. Doudna,Emmanuelle Charpentier +6 more
TL;DR: This study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.
Journal ArticleDOI
Multiplex Genome Engineering Using CRISPR/Cas Systems
Le Cong,Le Cong,F. Ann Ran,F. Ann Ran,David M. Cox,David M. Cox,Shuailiang Lin,Shuailiang Lin,Robert P. J. Barretto,Naomi Habib,Patrick D. Hsu,Patrick D. Hsu,Xuebing Wu,Wenyan Jiang,Luciano A. Marraffini,Feng Zhang +15 more
TL;DR: The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage as discussed by the authors.
Multiplex Genome Engineering Using CRISPR/Cas Systems
Le Cong,F. A. Ran,David Benjamin Turitz Cox,Shuailiang Lin,Robert P. J. Barretto,Naomi Habib,Patrick D. Hsu,Xuebing Wu,Wenyan Jiang,Luciano A. Marraffini,Feng Zhang +10 more
TL;DR: Two different type II CRISPR/Cas systems are engineered and it is demonstrated that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.
Journal ArticleDOI
RNA-Guided Human Genome Engineering via Cas9
Prashant Mali,Luhan Yang,Kevin M. Esvelt,John Aach,Marc Güell,James E. DiCarlo,Julie E. Norville,George M. Church,George M. Church +8 more
TL;DR: The type II bacterial CRISPR system is engineer to function with custom guide RNA (gRNA) in human cells to establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.
Journal ArticleDOI
DNA targeting specificity of RNA-guided Cas9 nucleases
Patrick D. Hsu,David A. Scott,David A. Scott,Joshua A. Weinstein,Joshua A. Weinstein,F. Ann Ran,F. Ann Ran,F. Ann Ran,Silvana Konermann,Silvana Konermann,Vineeta Agarwala,Vineeta Agarwala,Vineeta Agarwala,Yinqing Li,Yinqing Li,Eli J. Fine,Xuebing Wu,Ophir Shalem,Ophir Shalem,Thomas J. Cradick,Luciano A. Marraffini,Gang Bao,Feng Zhang,Feng Zhang +23 more
TL;DR: In this article, the Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing.
Related Papers (5)
DNA targeting specificity of RNA-guided Cas9 nucleases
Patrick D. Hsu,David A. Scott,David A. Scott,Joshua A. Weinstein,Joshua A. Weinstein,F. Ann Ran,F. Ann Ran,F. Ann Ran,Silvana Konermann,Silvana Konermann,Vineeta Agarwala,Vineeta Agarwala,Vineeta Agarwala,Yinqing Li,Yinqing Li,Eli J. Fine,Xuebing Wu,Ophir Shalem,Ophir Shalem,Thomas J. Cradick,Luciano A. Marraffini,Gang Bao,Feng Zhang,Feng Zhang +23 more