Harnessing type I CRISPR–Cas systems for genome engineering in human cells

Cameron, P. Simmons; Coons, Mary; Klompe, Sanne E.; Lied, Alexandra M.; Smith, Stephen C.; Vidal, Bastien; Donohoue, Paul D.; Rotstein, Tomer; Kohrs, Bryan; Nyer, David B.; Kennedy, Rachel; Banh, Lynda; Williams, Carolyn; Toh, Mckenzi S.; Irby, Matthew J.; Edwards, Leslie; Lin, Ching Yih; Owen, Arthur; Künne, Tim; Oost, John van der; Brouns, Stan J. J.; Slorach, Euan M.; Fuller, Christopher R.; Gradia, Scott; Kanner, Steven B.; May, Andrew P.; Sternberg, Samuel H.

doi:10.1038/s41587-019-0310-0

Cited by 96 publications

(75 citation statements)

References 43 publications

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“…In addition to class 2 nucleases, the Type I CRISPR-Cascade RNA-guided effector complexes are increasingly repurposed for mammalian genome editing [71][72][73] . These CRISPR systems also recognize targets via a two-step binding mechanism.…”

Section: Discussionmentioning

confidence: 99%

Inhibition of CRISPR-Cas12a DNA Targeting by Nucleosomes and Chromatin

Strohkendl

Saifuddin

Gibson

et al. 2020

Preprint

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Genome engineering nucleases, including CRISPR-Cas12a, must access chromatinized DNA. Here, we investigate how Acidaminococcus sp. Cas12a cleaves DNA within human nucleosomes and phase-condensed nucleosome arrays. Using quantitative kinetics approaches, we show that dynamic nucleosome unwrapping regulates DNA target accessibility to Cas12a. Nucleosome unwrapping determines the extent to which both steps of Cas12a binding-PAM recognition and R-loop formation-are inhibited by the nucleosome. Nucleosomes inhibit Cas12a binding even beyond the canonical core particle. Relaxing DNA wrapping within the nucleosome by reducing DNA bendability, adding histone modifications, or introducing a target-proximal nuclease-inactive Cas9 enhances DNA cleavage rates over 10-fold. Surprisingly, Cas12a readily cleaves DNA linking nucleosomes within chromatin-like phase separated nucleosome arrays-with DNA targeting reduced only ~4-fold. This work provides a mechanism for the observation that on-target cleavage within nucleosomes occurs less often than off-target cleavage within nucleosome-depleted regions of cells. We conclude that nucleosome wrapping restricts accessibility to CRISPR-Cas nucleases and anticipate that increasing nucleosome breathing dynamics will improve DNA binding and cleavage in eukaryotic cells.

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Section: Discussionmentioning

confidence: 99%

Inhibition of CRISPR-Cas12a DNA Targeting by Nucleosomes and Chromatin

Strohkendl

Saifuddin

Gibson

et al. 2020

Preprint

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“…By deleting Cas3 from E. coli cells (∆cas3) it has been possible to steer Cascade alone to DNA targets for non-destructive gene repression, targeting ectopic reporter genes (e.g., GFP) or native genes [63]. Bacterial Cas3 has been used alongside Cascade in various human cell types, giving 'proof-of-principle' that Cas3-Cascade is functional for targeted genetic insertions or deletions ('indels') when delivered into cells as proteins with nuclear localization signals, or expressed endogenously [64][65][66]. One encouraging trait arising from Cascade-Cas3 editing reactions, if compared with Cas9, is the low rate of off-target effects observed in a study using T. fusca Cascade-Cas3 [64], attributed to the stringency by which Cascade interrogates DNA for a target, and the requirement for it to 'lock-on' to the DNA target before Cas3 can be recruited to start its nuclease rampage.…”

Section: Characteristics Of Cas3-cascade Used For Genetic Editingmentioning

confidence: 99%

Cas3 Protein—A Review of a Multi-Tasking Machine

Liu

James

Radovčić

et al. 2020

Genes

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Cas3 has essential functions in CRISPR immunity but its other activities and roles, in vitro and in cells, are less widely known. We offer a concise review of the latest understanding and questions arising from studies of Cas3 mechanism during CRISPR immunity, and highlight recent attempts at using Cas3 for genetic editing. We then spotlight involvement of Cas3 in other aspects of cell biology, for which understanding is lacking—these focus on CRISPR systems as regulators of cellular processes in addition to defense against mobile genetic elements.

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“…As Class 2 systems have been used in the majority of neuronal gene editing experiments, they will therefore be the focus of this review. Class 1 systems and their uses are described elsewhere (Cameron et al, 2019;.…”

Section: Crispr-casmentioning

confidence: 99%

Genetically Engineering the Nervous System with CRISPR-Cas

Sandoval

Elahi

Ploski

2020

eNeuro

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The multitude of neuronal subtypes and extensive interconnectivity of the mammalian brain presents a substantial challenge to those seeking to decipher its functions. While the molecular mechanisms of several neuronal functions remain poorly characterized, advances in next-generation sequencing (NGS) and gene-editing technology have begun to close this gap. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (CRISPR-Cas) system has emerged as a powerful genetic tool capable of manipulating the genome of essentially any organism and cell type. This technology has advanced our understanding of complex neurologic diseases by enabling the rapid generation of novel, disease-relevant in vitro and transgenic animal models. In this review, we discuss recent developments in the rapidly accelerating field of CRISPR-mediated genome engineering. We begin with an overview of the canonical function of the CRISPR platform, followed by a functional review of its many adaptations, with an emphasis on its applications for

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Harnessing type I CRISPR–Cas systems for genome engineering in human cells

Cited by 96 publications

References 43 publications

Inhibition of CRISPR-Cas12a DNA Targeting by Nucleosomes and Chromatin

Inhibition of CRISPR-Cas12a DNA Targeting by Nucleosomes and Chromatin

Cas3 Protein—A Review of a Multi-Tasking Machine

Genetically Engineering the Nervous System with CRISPR-Cas

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