Creating and evaluating accurate CRISPR-Cas9 scalpels for genomic surgery

Bolukbasi, Mehmet Fatih; Gupta, Ankit; Wolfe, Scot A.

doi:10.1038/nmeth.3684

Cited by 106 publications

(91 citation statements)

References 140 publications

(224 reference statements)

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“…Second, transcription activatorlike effector nucleases are also fusion proteins of FokI, but the specificity-conferring targeting domain in this case is derived from the Xanthomonas transcription activator-like effector proteins [24,25]. Third, and most powerful, is clustered regulatory interspaced short palindromic repeat (CRISPR)-associated 9 (Cas9), which is effective, highly specific, and incredibly versatile while providing an unprecedented degree of control over genome editing [23,[26][27][28]. This review will focus on the actual and potential applications of CRISPR/ Cas9 system against human neurological viral infections.…”

Section: Recent Advances In Gene Editing Methodologiesmentioning

confidence: 99%

Gene Editing for Treatment of Neurological Infections

et al. 2016

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The study of neurological infections by viruses defines the field of neurovirology, which has emerged in the last 30 years and was founded upon the discovery of a number of viruses capable of infecting the human nervous system. Studies have focused on the molecular and biological basis of viral neurological diseases with the aim of revealing new therapeutic options. The first studies of neurovirological infections can be traced back to the discovery that some viruses have an affinity for the nervous system with research into rabies by Louis Pasteur and others in the 1880s. Today, the immense public health impact of neurovirological infections is illustrated by diseases such as neuroAIDS, progressive multifocal leukoencephalopathy, and viral encephalitis. Recent research has seen the development of powerful new techniques for gene editing that promise revolutionary opportunities for the development of novel therapeutic options. In particular, clustered regulatory interspaced short palindromic repeat-associated 9 system provides an effective, highly specific and versatile tool for targeting DNA viruses that are beginning to allow the development of such new approaches. In this short review, we discuss these recent developments, how they pertain to neurological infections, and future prospects.

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Section: Recent Advances In Gene Editing Methodologiesmentioning

confidence: 99%

Gene Editing for Treatment of Neurological Infections

et al. 2016

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“…The most widely used Cas9 currently is SpCas9, which is isolated from Streptococcus pyogenes. Unfortunately, 98.4% of them possess at least one off-target site with three or less mismatches to the gRNA (Bolukbasi et al 2015;Tsai et al 2015). Therefore, it is important to increase Cas9 machinery specificity, due to the large size of the genome and high off-target possibility, in order to achieve an efficient and safe therapy before applying it as a therapy to human subjects.…”

Section: Specificitymentioning

confidence: 99%

Improving the Function of Crispr-Cas9 for Genome Editing Therapy: Editing the Editor

Supit

2017

J Bioteknol Biosains Indones

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ABSTRAK Kata kunci: CRISPR, Cas9, efektivitas, spesifisitas, terapi gen ABSTRACTGene editing has become reasonably easy since the discovery of clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9). Most genetic diseases cannot be treated causally, and currently available therapies are mainly symptom-based. To treat the etiology of genetic diseases, a firm gene editing therapy is necessary to be established. This posits Cas9-facilitated gene editing as a prospective modality to become a clinically approved therapy in the future to treat genetic disorders. However, until recently, Cas9-based genome editing is still facing several hurdles, including low specificity, low effectiveness, and difficult delivery. Currently available Cas9 nucleases are able to bind to non-specific DNA sequence and produce non-specific cleavage. The efficiency has been relatively low due to the preference of non-homologous end-joining (NHEJ) over homology-directed repair (HDR) by the host cell. Furthermore, in order to deliver Cas9 into the nucleus, multiple physiological barriers have to be overcome. This review discussed recent developments in tackling these three hurdles, ranging from designing the guide RNA using multiple bioinformatics tools, modifying Cas9 structure, as well as packaging the nuclease-guide RNA complex into viral vectors and nanoparticles. Considering the active research on this area, it is expected that CRISPR/Cas9 can be utilized as a clinical therapy in the near future.

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“…160 For megaTALs, additional TALE RVD recognition modules may improve effective specificity. 100 For CRISPR/Cas systems, a number of technical strategies have been explored, including short gRNAs (of 17-18 nt), nickase mutants of Cas9 coupled to paired gRNAs targeting opposing strands, dCas9-FokI dimers for enforced heterodimerization, mutant Cas9 coupled to a programmable DNA binding domain such as ZFP or TALE, 161 and engineered variants of Cas9 that reduce its interaction strength with the gRNA:target DNA heteroduplex, 162,163 each of which appear to improve specificity.…”

Section: Editing Efficiency and Specificitymentioning

confidence: 99%

A genome editing primer for the hematologist

Hoban

Bauer

2016

Blood

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Gene editing enables the site-specific modification of the genome. These technologies have rapidly advanced such that they have entered common use in experimental hematology to investigate genetic function. In addition, genome editing is becoming increasingly plausible as a treatment modality to rectify genetic blood disorders and improve cellular therapies. Genome modification typically ensues from site-specific double-strand breaks and may result in a myriad of outcomes. Even single-strand nicks and targeted biochemical modifications that do not permanently alter the DNA sequence (epigenome editing) may be powerful instruments. In this review, we examine the various technologies, describe their advantages and shortcomings for engendering useful genetic alterations, and consider future prospects for genome editing to impact hematology.

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Creating and evaluating accurate CRISPR-Cas9 scalpels for genomic surgery

Cited by 106 publications

References 140 publications

Gene Editing for Treatment of Neurological Infections

Gene Editing for Treatment of Neurological Infections

Improving the Function of Crispr-Cas9 for Genome Editing Therapy: Editing the Editor

A genome editing primer for the hematologist

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