Gene editing and CRISPR in the clinic: current and future perspectives

Hirakawa, Matthew P.; Krishnakumar, Raga; Timlin, Jerilyn A.; Carney, James; Butler, Kimberly S.

doi:10.1042/bsr20200127

Cited by 146 publications

(121 citation statements)

References 297 publications

(431 reference statements)

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“…2 Not only have these systems been rapidly deployed as research tools in studying all domains of life from bacteria to humans, but they have offered new routes to advanced therapies for many diseases, from engineered cell lines such as CAR-T cells to editing humans genomes, offering the potential of cures for numerous genetic diseases. [3][4][5][6] CRISPR/Cas systems and S. pyogenes (Cas9) in particular (the most well studied) work remarkably well across a diversity of species and applications and are easier to use than the tools previously available. 7 However, challenges remain in the use of CRISPR/Cas systems, even with S. pyogenes Cas9.…”

Section: Introductionmentioning

confidence: 99%

CRISPR/Cas “non-target” sites inhibit on-target cutting rates

Moreb¹,

Hutmacher²,

Lynch³

2020

Preprint

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CRISPR/Cas systems have become ubiquitous for genome editing in eukaryotic as well as bacterial systems. Cas9 associated with a guide RNA (gRNA) searches DNA for a matching sequence (target site) next to a protospacer adjacent motif (PAM) and once found, cuts the DNA.The number of PAM sites in the genome are effectively a non-target pool of inhibitory substrates, competing with the target site for the Cas9/gRNA complex. We demonstrate that increasing the number of non-target sites for a given gRNA reduces on-target activity in a dose dependent manner. Furthermore, we show that the use of Cas9 mutants with increased PAM specificity towards a smaller subset of PAMs (or smaller pool of competitive substrates) improves cutting rates. Decreasing the non-target pool by increasing PAM specificity provides a path towards improving on-target activity for slower high fidelity Cas9 variants. These results demonstrate the importance of competitive non-target sites on Cas9 activity and, in part, may help to explain sequence and context dependent activities of gRNAs. Engineering improved PAM specificity to reduce the competitive non-target pool offers an alternative strategy to engineer Cas9 variants with increased specificity and maintained on-target activity.

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Section: Introductionmentioning

confidence: 99%

CRISPR/Cas “non-target” sites inhibit on-target cutting rates

Moreb¹,

Hutmacher²,

Lynch³

2020

Preprint

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“…Precise detection of off-target activity is crucial if CRISPR technology is to be used more widely and especially in a clinical setting 78 . However, many existing methods have differing sensitivities 79 making comparisons between studies difficult (e.g.…”

Section: Challenges Inconsistent Off-target Detection Methodsmentioning

confidence: 99%

Advances in Engineering CRISPR-Cas9 as a Molecular Swiss Army Knife

Meaker

Hair

Gorochowski

2020

Preprint

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The RNA-guided endonuclease system CRISPR-Cas9 has been extensively modified since its discovery, allowing its capabilities to be extending far beyond double-stranded cleavage to high fidelity insertions, deletions, and single base edits. Such innovations have been possible due to the modular architecture of CRISPR-Cas9 and the robustness of its component parts to modifications and the fusion of new functional elements. Here, we review the broad toolkit of CRISPR-Cas9-based systems now available for diverse genome editing tasks. We provide an overview of their core molecular structure and mechanism and distil the design principles used to engineer their diverse functionalities. We end by looking beyond the biochemistry and towards the societal and ethical challenges that these CRISPR-Cas9 systems face if their transformative capabilities are to be deployed in a safe and acceptable manner.

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“…Multifunctional AuNPs carrying TNAs and/or gene editing molecular tools have been mostly evaluated in in vitro models, but several of these concepts have also been proven in vivo. However, it must be said that most of these concepts do not make into the systemic evaluation required for further clinical development and translation, mostly due to hurdles relating to scale-up of production and precise physical characterization of these nanoconjugates, and for the complexity of assessing chronic and whole organism's nanotoxicology [152,153].…”

Section: Translation To the Clinicmentioning

confidence: 99%

Gold Nanoparticles for Vectorization of Nucleic Acids for Cancer Therapeutics

et al. 2020

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Cancer remains a complex medical challenge and one of the leading causes of death worldwide. Nanomedicines have been proposed as innovative platforms to tackle these complex diseases, where the combination of several treatment strategies might enhance therapy success. Among these nanomedicines, nanoparticle mediated delivery of nucleic acids has been put forward as key instrument to modulate gene expression, be it targeted gene silencing, interference RNA mechanisms and/or gene edition. These novel delivery systems have strongly relied on nanoparticles and, in particular, gold nanoparticles (AuNPs) have paved the way for efficient delivery systems due to the possibility to fine-tune their size, shape and surface properties, coupled to the ease of functionalization with different biomolecules. Herein, we shall address the different molecular tools for modulation of expression of oncogenes and tumor suppressor genes and discuss the state-of-the-art of AuNP functionalization for nucleic acid delivery both in vitro and in vivo models. Furthermore, we shall highlight the clinical applications of these spherical AuNP based conjugates for gene delivery, current challenges, and future perspectives in nanomedicine.

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Gene editing and CRISPR in the clinic: current and future perspectives

Cited by 146 publications

References 297 publications

CRISPR/Cas “non-target” sites inhibit on-target cutting rates

CRISPR/Cas “non-target” sites inhibit on-target cutting rates

Advances in Engineering CRISPR-Cas9 as a Molecular Swiss Army Knife

Gold Nanoparticles for Vectorization of Nucleic Acids for Cancer Therapeutics

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