Improving CRISPR-Cas nuclease specificity using truncated guide RNAs

Fu, Yanfang; Sander, Jeffry D.; Reyon, Deepak; Cascio, Vincent; Joung, J. Keith

doi:10.1038/nbt.2808

Cited by 1,798 publications

(1,580 citation statements)

References 30 publications

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“…Mismatches between the sgRNA and genomic DNA that are close to the PAM are poorly tolerated by Cas9 10 , increasing the likelihood of selective editing of the Bth mutant allele. A fourth sgRNA, Tmc1-mut4, is a truncated version of Tmc1-mut3 designed to increase genome editing DNA specificity 22 . We evaluated the ability of these four sgRNAs when complexed with Cas9 to cleave either the wild-type Tmc1 or the Bth allele in vitro .…”

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confidence: 99%

Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents

Gao

Tao

Lamas

et al. 2017

Nature

446

346

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Although genetic factors contribute to almost half of all deafness cases, treatment options for genetic deafness are limited1–5. We developed a genome editing approach to target a dominantly inherited form of genetic deafness. Here we show that cationic lipid-mediated in vivo delivery of Cas9:guide RNA complexes can ameliorate hearing loss in a mouse model of human genetic deafness. We designed and validated in vitro and in primary fibroblasts genome editing agents that preferentially disrupt the dominant deafness-associated allele in the Tmc1 (transmembrane channel-like 1) Beethoven (Bth) mouse model, even though the mutant Bth allele differs from the wild-type allele at only a single base pair. Injection of Cas9:guide RNA:lipid complexes targeting the Bth allele into the cochlea of neonatal Bth/+ mice substantially reduced progressive hearing loss. We observed higher hair cell survival rates and lower auditory brainstem response (ABR) thresholds in injected ears compared with uninjected ears or ears injected with complexes that target an unrelated gene. Enhanced acoustic reflex responses were observed among injected compared to uninjected Bth/+ animals. These findings suggest protein:RNA complex delivery of target gene-disrupting agents in vivo as a potential strategy for the treatment of some autosomal dominant hearing loss diseases.

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confidence: 99%

Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents

Gao

Tao

Lamas

et al. 2017

Nature

446

346

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“…Although Cas9 boasts the highest ease of use among the targeted nuclease platforms, several reports have indicated that it could be prone to inducing off-target mutations (Cradick et al 2013;Fu et al 2013). To this end, considerable effort has been devoted to improving the specificity of this system, including using paired Cas9 nickases (Mali et al 2013a;Ran et al 2013), which increase gene-editing specificity by requiring the induction of two sequential and adjacent nicking events for DSB formation, or truncated gRNA that are more sensitive to mismatches at the genomic target site than a full-length gRNA (Fu et al 2014). Off-target cleavage has also been reduced by controlling the dosage of either the Cas9 protein or gRNA within the cell , or even by using Cas9 variants configured to enable conditional genome editing, such as a rapamycininducible split-Cas9 architecture (Zetsche et al 2015b) or a Cas9 variant that contains a strategically placed small-molecule-responsive intein domain .…”

Section: Crispr-cas9mentioning

confidence: 99%

Genome-Editing Technologies: Principles and Applications

Gaj

Sirk

Shui

et al. 2016

Cold Spring Harb Perspect Biol

297

142

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Targeted nucleases have provided researchers with the ability to manipulate virtually any genomic sequence, enabling the facile creation of isogenic cell lines and animal models for the study of human disease, and promoting exciting new possibilities for human gene therapy. Here we review three foundational technologies-clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9), transcription activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs). We discuss the engineering advances that facilitated their development and highlight several achievements in genome engineering that were made possible by these tools. We also consider artificial transcription factors, illustrating how this technology can complement targeted nucleases for synthetic biology and gene therapy.

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“…By extension, 'paired nickases', i.e. using two adjacent gRNAs with Cas9n, can efficiently introduce both indel mutations and HR events with a single-stranded DNA oligo-nucleotide donor template in mammalian cells [28,10,80]. Complete disruption of the endonuclease activities (RuvC D10A along with HNH H840A ) results in a catalytically inactive Cas9, or dead-Cas9 (dCas9) [78,79].…”

Section: Expanding Cas9 Features Through Enzyme Engineeringmentioning

confidence: 99%

“…These are scarce and more importantly, some of them contain idiosyncratic features, e.g. U6 mammalian promoter requires to have a G at the 5′ end of the transcript [28]. Transcriptional expression can be improved by inserting self-processing elements, such as HDV ribozyme and tRNAs, at the 5′ or 3′ end to prevent potential degradation of the transcript [49,83].…”

Section: The Grna Characteristics and Extensionsmentioning

confidence: 99%

Advancing biotechnology with CRISPR/Cas9: recent applications and patent landscape

Ferreira

David

Nielsen

2018

Journal of Industrial Microbiology and Biotechnology

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Clustered regularly interspaced short palindromic repeats (CRISPR) is poised to become one of the key scientific discoveries of the twenty-first century. Originating from prokaryotic and archaeal immune systems to counter phage invasions, CRISPRbased applications have been tailored for manipulating a broad range of living organisms. From the different elucidated types of CRISPR mechanisms, the type II system adapted from Streptococcus pyogenes has been the most exploited as a tool for genome engineering and gene regulation. In this review, we describe the different applications of CRISPR/Cas9 technology in the industrial biotechnology field. Next, we detail the current status of the patent landscape, highlighting its exploitation through different companies, and conclude with future perspectives of this technology.

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Improving CRISPR-Cas nuclease specificity using truncated guide RNAs

Cited by 1,798 publications

References 30 publications

Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents

Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents

Genome-Editing Technologies: Principles and Applications

Advancing biotechnology with CRISPR/Cas9: recent applications and patent landscape

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