No unexpected CRISPR-Cas9 off-target activity revealed by trio sequencing of gene-edited mice

Iyer, Vivek; Boroviak, Katharina; Thomas, Mark; Doe, Brendan; Ryder, Edward J.; Adams, David J.

doi:10.1101/263129

Cited by 41 publications

(38 citation statements)

References 13 publications

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“…The sheep study did reveal a single 2.4 kb inversion in one of 54 founder animals, which the authors postulated was due to a double-stranded cleavage at two single gRNA target sites. These findings are consistent with previous CRISPR/Cas9 off-target studies in humans 19,20 , monkeys 21 and rodents [22][23][24] , which suggest the rate of Cas9-mediated mutagenesis is not distinguishable from the background de novo mutation rate.…”

Section: Discussionsupporting

confidence: 92%

Genomic and phenotypic analyses of six offspring of a genome-edited hornless bull

et al. 2019

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I n the modern US dairy cattle industry, destruction of hornproducing cells before they grow and attach to the skull (disbudding) is a routine practice to prevent horn growth. Animals that do not have horns do not injure other animals, require less feeding trough space, are less dangerous to handle and transport than horned animals and have fewer aggressive behaviors 1. Disbudding is an unpleasant process that has important implications for animal welfare, and many stakeholder groups have campaigned for alternative, humane solutions. One option is to select and breed animals that do not have horns, a phenotype referred to as polled. In 2016, Carlson et al. 2 reported the introgression of the P C Celtic POLLED allele into two male dairy bulls by genome editing using transcription activator-like effector nucleases (TALENs). Bulls RCI001 and RCI002 originated at Recombinetics, Inc., where the researchers genome-edited donor cells from a University of Minnesota crossbred dairy bull and then used reproductive cloning. Whole-genome sequencing (WGS) did not reveal any off-target alterations 2 , and both bulls reached maturity without developing horns. These genome-edited polled bulls were transferred to the University of California (UC), Davis and generated widespread interest. However, further work needs to be done in characterizing these animals if genome editing is to seamlessly integrate into livestock genetic improvement programs. Edits will likely need to be introduced into multiple elite founder animals to prevent genetic bottlenecks 3. Perhaps as importantly, appropriate regulatory frameworks that are risk-and evidencebased, proportionate and globally harmonized will be essential to allow research to occur, and to foster the development of useful applications 4. Others have reported on WGS of trios of genomeedited (CRISPR/Cas9) knockout livestock produced through cytoplasmic injection (CPI) of guide RNA (gRNA) and Cas9 into one-cell-stage zygotes. Genome-edited sheep were compared to their parents 5 and genome-edited goats were compared to their offspring 6 , and both trio-based studies concluded that de novo mutation rates were comparable to those observed in nonedited trios. A third study used an unbiased WGS on two genome-edited calves produced by a targeted gene knockout of beta-lactoglobulin using CPI of a homology-directed repair (HDR) donor plasmid and TALENs into early zygotes 7. These calves were free of any TALENmediated off-target mutations or donor plasmid integration events. To provide data to guide emerging regulatory frameworks and benefit future applications of genome editing in livestock, we set up a breeding experiment to investigate whether the POLLED genome edit was faithfully passed to offspring and whether there were any unique phenotypic or genotypic changes in those offspring. The calves produced as part of the current study are, to our knowledge, the first reported offspring of a genome-edited bull. These data will help inform regulatory agencies as they formulate processes to regulate g...

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Section: Discussionsupporting

confidence: 92%

Genomic and phenotypic analyses of six offspring of a genome-edited hornless bull

et al. 2019

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“…These candidate DNMs are most likely noise, not true DNMs, which may be caused by the technical bias of next-generation sequencing. The same scenario was also seen in the mouse data 13 . We filtered out these candidate DNMs by the DNM filtration procedure ("allele filtering", Methods).…”

supporting

confidence: 74%

“…However, previous studies mainly focused on the potential off-target loci predicted by sgRNA binding, not on a genome-wide evaluation of de novo mutations (DNMs). Recently, Schaefer et al found plenty unexpected mutations using WGS of Cas9-edited mice 12 though the claim was challenged by several groups [13][14][15][16][17][18] , and the latest trio sequencing of Cas9-edited mice did not see unexpected off-target activity 13 . To evaluate the situation in monkeys, we performed trio WGS of Cas9-edited rhesus monkeys (Macaca mulatta).…”

mentioning

confidence: 99%

Trio deep-sequencing does not reveal unexpected mutations in Cas9-edited monkeys

Luo

Zhang³

et al. 2018

Preprint

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CRISPR-Cas9 is a widely-used genome editing tool, but its off-target effect remains a concern, especially in view of future clinical applications. Non-human primates (NHPs) share close genetic and physiological similarities with humans, making them an ideal preclinical model for developing Cas9-based therapies. However, no comprehensive in vivo off-target assessment has been conducted in NHPs. Here we performed whole genome trio sequencing of Cas9-treated monkeys. We found they only carried a small number of de novo mutations that can be explained by expected spontaneous mutations, and no unexpected mutations were detected.

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“…This new communication problem even affected the stock value of publicly traded companies that work with genetic editing, whose shares plummeted after such negative results. However, the findings were not true; just a few days later, evidence and alternative explanations started to pile up proving that the mice used by the authors were different to those initially used in the genetic editing experiment to analyse the DNA sequences (Iyer et al, 2018). Because of this difference, all the changes they manifested, and which this publication attributed to specificity problems derived from the use of CRISPR tools, had a much simpler explanation: the wrong genetic information had been used.…”

Section: ■ ■ Crispr Communicationmentioning

confidence: 98%

Edición genética y comunicación

Montoliu¹

2018

Mètode

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Communication is essential in all areas of society, but communication in science is inescapable. Communicating means sharing, showing, teaching, and transferring knowledge about discoveries, observations, and findings both to colleagues and to society in general. That is why good communication must always accompany good science. CRISPR genetic editing tools allow us to modify, at will, the genome of any living organism, including our own species. In this text I review the different relevant communicative events in the short but intense life of these «molecular scissors», so called for their ability to cut the DNA molecule effectively and with precision.

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No unexpected CRISPR-Cas9 off-target activity revealed by trio sequencing of gene-edited mice

Cited by 41 publications

References 13 publications

Genomic and phenotypic analyses of six offspring of a genome-edited hornless bull

Genomic and phenotypic analyses of six offspring of a genome-edited hornless bull

Trio deep-sequencing does not reveal unexpected mutations in Cas9-edited monkeys

Edición genética y comunicación

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