Welcome to the CRISPR zoo

Reardon, Sara

doi:10.1038/531160a

Cited by 80 publications

(70 citation statements)

References 15 publications

(10 reference statements)

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“…In the CRISPR age, targeted genomic lesions can be introduced with unprecedented ease, which facilitate either high-efficiency gene disruption by nonhomologous end joining (NHEJ) or, in the presence of donor DNA, precise genome editing (PGE) by homology-directed repair (HDR) (Carroll 2014). Research models of the desired genotype can be expeditiously generated for most genetically tractable organisms (Bassett et al 2013;DiCarlo et al 2013;Liu et al 2014;Yang et al 2014;Reardon 2016) and within one round of subcloning of cultured cells (Ran et al 2013b;Byrne et al 2014). Although HDR is the more desirable pathway for genome editing and gene therapy, its activity is much lower than NHEJ and dependent on the cell cycle status in human cells (Mao et al 2008a,b;Lin et al 2014;Orthwein et al 2015).…”

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confidence: 99%

Mechanisms of precise genome editing using oligonucleotide donors

et al. 2017

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The use of programmable meganucleases is transforming genome editing and functional genomics. CRISPR/Cas9 was developed such that targeted genomic lesions could be introduced in vivo with unprecedented ease. In the presence of homology donors, these lesions facilitate high-efficiency precise genome editing (PGE) via homology-directed repair (HDR) pathways. However, the identity and hierarchy of the HDR (sub)pathways leading to the formation of PGE products remain elusive. Here, we established a green to blue fluorescent protein conversion system to systematically characterize oligodeoxynucleotide (ODN)-mediated PGE using Cas9 and its nickase variants in human cells. We demonstrate that, unlike double-stranded DNA (dsDNA) donors with central heterologies, ODNs generated short conversion tracts with Gaussianlike distributions. Interestingly, single-nick-induced PGE using ODN donors produced conversion tracts biased either mostly uni-or bidirectional depending on the relative strandedness of the ODNs and the nick. Moreover, the ODNs were physically incorporated into the genome only in the bidirectional, but not in the unidirectional, conversion pathway. In the presence of double-stranded genomic lesions, the unidirectional conversion pathway was preferentially utilized even though the knock-in mutation could theoretically have been converted by both pathways. Collectively, our results suggest that ODN-mediated PGE utilizes synthesis-dependent strand annealing and single-stranded DNA incorporation pathways. Both of these pathways generate short conversion tracts with Gaussian-like distributions. Although synthesis-dependent strand annealing is preferentially utilized, our work unequivocally establishes the existence of a single-stranded DNA incorporation pathway in human cells. This work extends the paradigms of HDR-mediated gene conversion and establishes guidelines for PGE in human cells.

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confidence: 99%

Mechanisms of precise genome editing using oligonucleotide donors

et al. 2017

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“…Parasites encompass a variety of human pathogens [63]. Parasitic worms, such as roundworms and flatworms, are amongst the most complex representing the sixth leading cause of morbidity worldwide and treatment options are limited [64]. Several investigators have employed CRISPR to interrupt essential genes in parasitic worms.…”

Section: Parasitesmentioning

confidence: 99%

Harnessing the potential of CRISPR-based platforms to advance the field of hospital medicine

McCarthy

2020

Expert Review of Anti-infective Therapy

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“…The rapid development of genome editing techniques is giving rise to what has been referred to as the ''CRISPR zoo'' (Reardon 2016), with the promise of a wide array of manipulated organisms coming into being. Because 'GMO' is a category term used to separate on the basis of technique (modern biotechnology is as modern biotechnology does), the uptake of a range of new biotechnological techniques raises serious questions about the identity, moral and regulatory status, and conservation value, of all the organisms that will be generated through them.…”

Section: It Is Not Clear How or Where To Draw The Boundaries Of Distimentioning

confidence: 99%

Do We Care About Synbiodiversity? Questions Arising from an Investigation into Whether There are GM Crops in the Svalbard Global Seed Vault

Wickson

2016

J Agric Environ Ethics

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The Svalbard Global Seed Vault provides a backup of seed collections from genebanks around the world. It's unique character has made it iconic in the public imagination as a 'Noah's Ark' for crop plants. Its remote location and strict controls on access have, however, also lent it an air of mystery, swirling with conspiracy theories. In this paper, I first clarify the aims of the Vault, the history of its development and the policies and practices of its current operation. Given concerns around its potential links to the biotechnology industry, I go on to ask whether GM crops are currently stored in the Vault. Presenting several reasons for why GM crops are formally excluded, while indicating the potential for both change and unintentional contamination, I am compelled to question whether GM crops should be excluded. Answering this requires an interrogation of their potential conservation value as modern contributors to crop biodiversity. In exploring this issue, I suggest that there has been surprisingly little discussion of the moral status and conservation value of bio-technological crop plants and indeed, of how we care for all the techno-lifeforms we are currently engaged in co-creating. I suggest that these are becoming important issues as biotechnological techniques and applications begin to rapidly evolve and diversify (e.g. through genome editing and synthetic biology). Emphasizing the scope for a refreshed interdisciplinary research agenda exploring the interface between biotechnology and biodiversity conservation, I conclude the article by proposing new concepts of synbiodiversity and symbiodiversity to encourage further debate.

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Welcome to the CRISPR zoo

Cited by 80 publications

References 15 publications

Mechanisms of precise genome editing using oligonucleotide donors

Mechanisms of precise genome editing using oligonucleotide donors

Harnessing the potential of CRISPR-based platforms to advance the field of hospital medicine

Do We Care About Synbiodiversity? Questions Arising from an Investigation into Whether There are GM Crops in the Svalbard Global Seed Vault

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