Pervasive head-to-tail insertions of DNA templates mask desired CRISPR-Cas9–mediated genome editing events

Skryabin, Boris V.; Kummerfeld, Delf-Magnus; Gubar, Leonid; Seeger, Birte; Kaiser, Helena; Stegemann, Agatha; Roth, Johannes; Meuth, Sven G.; Pavenstädt, Hermann; Sherwood, J.; Pap, Thomas; Wedlich‐Söldner, Roland; Sunderkötter, Cord; Schwartz, Yuri B.; Brosius, Juergen; Rozhdestvensky, Timofey S.

doi:10.1126/sciadv.aax2941

Cited by 75 publications

(80 citation statements)

References 36 publications

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“…There are still no reliable data on the molecular mechanism of the appearance of large deletions. This is especially important for genome editing in zygotes because several recent studies, including ours, show that major DSB repair pathways may have unusual high activity in early embryos that lead to the formation of concatemers from injected DNA molecules [84,85]. It is possible that such peculiar properties of DSB repair in zygotes will have a role in the formation of large on-target deletion during genome editing.…”

Section: Discussionmentioning

confidence: 94%

On-Target CRISPR/Cas9 Activity Can Cause Undesigned Large Deletion in Mouse Zygotes

Korablev

Lukyanchikova

Serova

et al. 2020

IJMS

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Genome engineering has been tremendously affected by the appearance of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9)-based approach. Initially discovered as an adaptive immune system for prokaryotes, the method has rapidly evolved over the last decade, overtaking multiple technical challenges and scientific tasks and becoming one of the most effective, reliable, and easy-to-use technologies for precise genomic manipulations. Despite its undoubtable advantages, CRISPR/Cas9 technology cannot ensure absolute accuracy and predictability of genomic editing results. One of the major concerns, especially for clinical applications, is mutations resulting from error-prone repairs of CRISPR/Cas9-induced double-strand DNA breaks. In some cases, such error-prone repairs can cause unpredicted and unplanned large genomic modifications within the CRISPR/Cas9 on-target site. Here we describe the largest, to the best of our knowledge, undesigned on-target deletion with a size of ~293 kb that occurred after the cytoplasmic injection of CRISPR/Cas9 system components into mouse zygotes and speculate about its origin. We suppose that deletion occurred as a result of the truncation of one of the ends of a double-strand break during the repair.

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Section: Discussionmentioning

confidence: 94%

On-Target CRISPR/Cas9 Activity Can Cause Undesigned Large Deletion in Mouse Zygotes

Korablev

Lukyanchikova

Serova

et al. 2020

IJMS

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“…Using the Cas9 protein/sgRNA ribonucleoproteins (RNPs) to perform genome editing has several advantages compared with co-transforming of Cas9 expression plasmid and sgRNA. A major advantage is transformation of Cas9 RNPs alleviates the possibility of integration of genetic material to a nontargeted region of the genome [23][24]. Additionally, Cas9 and sgRNA are able to form a stable ribonucleoprotein in vitro, so there is less likelihood of RNA degradation compared with Cas9 mRNA/sgRNA transformation.…”

Section: Discussionmentioning

confidence: 99%

A Strategy for Scleroglucan Production in Sclerotium Rolfsii: Lowering pH in Fermentation Process by Manipulating Oxalate Metabolic Pathway With CRISPR/Cas9 Tool

Bai¹,

Wang²,

Li³

et al. 2020

Preprint

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Background: Sclerotium rolfsii is a potent producer of many secondary metabolites, one of which like scleroglucan is an exopolysaccharide (EPS) appreciated as a multipurpose compound applicable in many industrial fields. Results: We chose AAT1 gene in oxalate metabolic pathway as target of CRISPR/Cas9. When the AAT1 gene is disrupted, oxalate was not converted to α-ketoglutarate (AKG), but accumulated. So AAT1-mutant serves to lower the pH (pH 3-4) to increase the production of the pH-sensitive metabolite scleroglucan to be 21.03 g l-1 with productivity reached 0.25 g/(L·h).Conclusions: We established a platform for gene editing to rapidly generate and select mutants, and provide a new beneficial strain of S. rolfsii as a scleroglucan hyper-producer which could also reduce the cost of controlling optimum pH condition in fermentation industry.

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“…Both HDR and NHEJ-mediated repair have been shown to cause multiple unwanted head-to-tail insertions of DNA donor templates at the target site during the generation of conditional knockout mice models [130]. Conventionally applied PCR analysis failed to identify these insertions.…”

Section: Unintended On-target Effects Associated With Crispr/cas Applmentioning

confidence: 99%

“…Conventionally applied PCR analysis failed to identify these insertions. Thus, it is essential to validate the integrity of the target DNA regions after applying CRISPR/Cas9 using a combination of suitably sensitive analytical techniques such as qPCR, digital droplet PCR and southern blotting [130].…”

Section: Unintended On-target Effects Associated With Crispr/cas Applmentioning

confidence: 99%

“…When CRISPR/Cas components are delivered as plasmids into the cells, both unintended additional integration events of this DNA or its fragments have been reported in both plants and animals, and where the plasmid was intended to enact genome editing without any DNA integration, unintentionally integrated [74,76,130,135]. This can refer to the CRISPR/Cas components as well as the plasmid backbone.…”

Section: Unintended Integration Of Crispr/cas Components or Plasmid Dnamentioning

confidence: 99%

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Broadening the GMO risk assessment in the EU for genome editing technologies in agriculture

Kawall¹,

Cotter²,

Then³

2020

Environ Sci Eur

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Genome editing techniques, especially the CRISPR/Cas technology, increase the possibilities and the speed of altering genetic material in organisms. So-called genome editing is increasingly being used to achieve agriculturally relevant novel traits and/or genetic combinations in both plants and animals, although predominantly as proof of concept studies, with commercial growing or rearing so far limited to the U.S. and Canada. However, there are numerous reports of unintended effects such as off-target effects, unintended on-target effects and other unintended consequences arising from genome editing, summarised under the term genomic irregularities. Despite this, the searching for genomic irregularities is far from routine in these studies and protocols vary widely, particularly for off-target effects, leading to differences in the efficacy of detection of off-target effects. Here, we describe the range of specific unintended effects associated with genome editing. We examine the considerable possibilities to change the genome of plants and animals with SDN-1 and SDN-2 genome editing (i.e. without the insertion of genes conferring the novel trait) and show that genome editing techniques are able to produce a broad spectrum of novel traits that, thus far, were not possible to be obtained using conventional breeding techniques. We consider that the current EU risk assessment guidance for GMOs requires revision and broadening to capture all potential genomic irregularities arising from genome editing and suggest additional tools to assist the risk assessment of genome-edited plants and animals for the environment and food/animal feed in the EU.

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Pervasive head-to-tail insertions of DNA templates mask desired CRISPR-Cas9–mediated genome editing events

Cited by 75 publications

References 36 publications

On-Target CRISPR/Cas9 Activity Can Cause Undesigned Large Deletion in Mouse Zygotes

On-Target CRISPR/Cas9 Activity Can Cause Undesigned Large Deletion in Mouse Zygotes

A Strategy for Scleroglucan Production in Sclerotium Rolfsii: Lowering pH in Fermentation Process by Manipulating Oxalate Metabolic Pathway With CRISPR/Cas9 Tool

Broadening the GMO risk assessment in the EU for genome editing technologies in agriculture

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