Improving CRISPR-Cas specificity with chemical modifications in single-guide RNAs.
Daniel E. Ryan,David Taussig,Israel Steinfeld,Smruti M Phadnis,Benjamin D. Lunstad,Madhurima Singh,Xuan Vuong,Kenji D Okochi,Ryan McCaffrey,Magdalena Olesiak,Subhadeep Roy,Chong Wing Yung,Bo Curry,Jeffrey R. Sampson,Laurakay Bruhn,Douglas J. Dellinger +15 more
TLDR
Results show that a chemical modification incorporated at select sites in the ribose-phosphate backbone of gRNAs can dramatically reduce off-target cleavage activities while maintaining high on-target performance, as demonstrated in clinically relevant genes.Abstract:
CRISPR systems have emerged as transformative tools for altering genomes in living cells with unprecedented ease, inspiring keen interest in increasing their specificity for perfectly matched targets. We have developed a novel approach for improving specificity by incorporating chemical modifications in guide RNAs (gRNAs) at specific sites in their DNA recognition sequence ('guide sequence') and systematically evaluating their on-target and off-target activities in biochemical DNA cleavage assays and cell-based assays. Our results show that a chemical modification (2'-O-methyl-3'-phosphonoacetate, or 'MP') incorporated at select sites in the ribose-phosphate backbone of gRNAs can dramatically reduce off-target cleavage activities while maintaining high on-target performance, as demonstrated in clinically relevant genes. These findings reveal a unique method for enhancing specificity by chemically modifying the guide sequence in gRNAs. Our approach introduces a versatile tool for augmenting the performance of CRISPR systems for research, industrial and therapeutic applications.read more
Citations
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Journal ArticleDOI
Latest Developed Strategies to Minimize the Off-Target Effects in CRISPR-Cas-Mediated Genome Editing.
TL;DR: Several latest approaches to reduce the off- target effects, including biased or unbiased off-target detection, cytosine or adenine base editors, prime editing, dCas9, Cas9 paired nickase, ribonucleoprotein (RNP) delivery and truncated gRNAs are reviewed.
Journal ArticleDOI
Inhibition of histone deacetylase 1 (HDAC1) and HDAC2 enhances CRISPR/Cas9 genome editing
Bin Liu,Siwei Chen,Anouk La Rose,Deng Chen,Fangyuan Cao,Martijn R. H. Zwinderman,Dominik Kiemel,Dominik Kiemel,Manon Aïssi,Frank J. Dekker,Hidde J. Haisma +10 more
TL;DR: It is demonstrated that attenuation of HDAC1, HDAC2 activity, but not other HDACs, enhances CRISPR/Cas9-mediated gene knockout frequencies by NHEJ as well as gene knock-in by HDR, and inhibition ofHDAC3 decreases gene editing frequencies.
Journal ArticleDOI
Delivery Aspects of CRISPR/Cas for in Vivo Genome Editing
TL;DR: This Account focuses on the delivery aspects of CRISPR/Cas for therapeutic applications in vivo, and suggests that current trends seem to favor the use of sgRNA/Cas ribonucleoprotein complexes delivered in vivo by synthetic particles.
Journal ArticleDOI
CRISPR technology incorporating amplification strategies: molecular assays for nucleic acids, proteins, and small molecules
Wei Feng,Ashley M. Newbigging,Jeffrey Tao,Yiren Cao,Hanyong Peng,Connie Le,Jinjun Wu,Bo Pang,Bo Pang,Juan Li,D. Lorne Tyrrell,Hongquan Zhang,X. Chris Le +12 more
TL;DR: Successful integrations of CRISPR technology with nucleic acid amplification techniques result in highly sensitive and rapid detection of SARS-CoV-2, the virus that causes the COVID-19 pandemic.
Journal ArticleDOI
Gene editing and CRISPR in the clinic: current and future perspectives
TL;DR: The current status and scientific basis of clinical trials featuring ZFNs, TALENs, andCRISPR-based genome editing, the known limitations of CRISPR use in humans, and the rapidly developing CRISpr engineering space that should lay the groundwork for further translation to clinical application are examined.
References
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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.
Journal ArticleDOI
Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system.
Bernd Zetsche,Jonathan S. Gootenberg,Omar O. Abudayyeh,Ian Slaymaker,Kira S. Makarova,Patrick Essletzbichler,Sara E. Volz,Julia Joung,John van der Oost,Aviv Regev,Aviv Regev,Eugene V. Koonin,Feng Zhang +12 more
TL;DR: In this paper, the authors characterized Cpf1, a putative class 2 CRISPR effector, which is a single RNA-guided endonuclease lacking tracrRNA and utilizes a T-rich protospacer-adjacent motif.
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: It is found that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches.
Journal ArticleDOI
Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity
F. Ann Ran,Patrick D. Hsu,Chie Yu Lin,Jonathan S. Gootenberg,Silvana Konermann,Alexandro E. Trevino,David A. Scott,Azusa Inoue,Shogo Matoba,Yi Zhang,Feng Zhang +10 more
TL;DR: In this paper, an approach that combines a Cas9 nickase mutant with paired guide RNAs to introduce targeted double-strand breaks is described. But the approach is limited to mouse zygotes.
Journal ArticleDOI
High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells.
Yanfang Fu,Jennifer A Foden,Cyd Khayter,Morgan L. Maeder,Deepak Reyon,J. Keith Joung,Jeffry D. Sander +6 more
TL;DR: It is found that single and double mismatches are tolerated to varying degrees depending on their position along the guide RNA (gRNA)-DNA interface, and off-target cleavage of CRISPR-associated (Cas)9-based RGNs is characterized.
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DNA targeting specificity of RNA-guided Cas9 nucleases
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