Unexpected CRISPR on-target effects

Lee, Hyunji; Kim, Jin‐Soo

doi:10.1038/nbt.4207

Cited by 43 publications

(34 citation statements)

References 10 publications

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“…The affected pig (pig 116-1) had both one expected size deletion and a second that was larger than expected, reflecting the robust propensity of NHEJ and MMEJ mechanisms of DNA repair of dual CRISPR/Cas9-induced DSBs to generate unexpected mutations at target sites in vivo ( 67 , 68 ). Indeed, 2 genotyped blastocysts had an aberrantly sized deletion fragment on 1 allele but no WT band, suggesting the second allele had larger deletions than were detectable, indicating that aberrant DNA repair events are not uncommon.…”

Section: Discussionmentioning

confidence: 99%

A porcine model of phenylketonuria generated by CRISPR/Cas9 genome editing

et al. 2020

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Phenylalanine hydroxylase–deficient (PAH-deficient) phenylketonuria (PKU) results in systemic hyperphenylalaninemia, leading to neurotoxicity with severe developmental disabilities. Dietary phenylalanine (Phe) restriction prevents the most deleterious effects of hyperphenylalaninemia, but adherence to diet is poor in adult and adolescent patients, resulting in characteristic neurobehavioral phenotypes. Thus, an urgent need exists for new treatments. Additionally, rodent models of PKU do not adequately reflect neurocognitive phenotypes, and thus there is a need for improved animal models. To this end, we have developed PAH -null pigs. After selection of optimal CRISPR/Cas9 genome-editing reagents by using an in vitro cell model, zygote injection of 2 sgRNAs and Cas9 mRNA demonstrated deletions in preimplantation embryos, with embryo transfer to a surrogate leading to 2 founder animals. One pig was heterozygous for a PAH exon 6 deletion allele, while the other was compound heterozygous for deletions of exon 6 and of exons 6–7. The affected pig exhibited hyperphenylalaninemia (2000–5000 μM) that was treatable by dietary Phe restriction, consistent with classical PKU, along with juvenile growth retardation, hypopigmentation, ventriculomegaly, and decreased brain gray matter volume. In conclusion, we have established a large-animal preclinical model of PKU to investigate pathophysiology and to assess new therapeutic interventions.

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Section: Discussionmentioning

confidence: 99%

A porcine model of phenylketonuria generated by CRISPR/Cas9 genome editing

et al. 2020

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“…Another potential application area is in rare disease genetics, where scTRIP may help resolve "unclear cases" by widening the spectrum of accessible SVs leading to somatic mosaicism 56 . Furthermore, our framework can be used to assess genome integrity in conjunction with cell therapy, gene therapy, and therapeutic CRISPR-Cas9 editing, which can result in unanticipated (potentially pathogenic) SVs 63,64 . The ability of scTRIP to generate high-resolution karyotypes could be employed to detect the presence of such unwanted SVs to address safety concerns pertaining to these future therapies.…”

Section: Discussionmentioning

confidence: 99%

Single cell tri-channel-processing reveals structural variation landscapes and complex rearrangement processes

Sanders¹,

Meiers²,

Ghareghani

et al. 2019

Preprint

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Structural variation (SV), where rearrangements delete, duplicate, invert or translocate DNA segments, is a major source of somatic cell variation. It can arise in rapid bursts, mediate genetic heterogenity, and dysregulate cancer-related pathways. The challenge to systematically discover SVs in single cells remains unsolved, with copy-neutral and complex variants typically escaping detection. We developed single cell tri-channel-processing (scTRIP), a computational framework that jointly integrates read depth, template strand and haplotype phase to comprehensively discover SVs in single cells. We surveyed SV landscapes of 565 single cell genomes, including transformed epithelial cells and patient-derived leukemic samples, and discovered abundant SV classes including inversions, translocations and large-scale genomic rearrangements mediating oncogenic dysregulation. We dissected the 'molecular karyotype' of the leukemic samples and examined their clonal structure. Different from prior methods, scTRIP also enabled direct detection and discrimination of SV mutational processes in individual cells, including breakage-fusion-bridge cycles. scTRIP will facilitate studies of clonal evolution, genetic mosaicism and somatic SV formation, and could improve disease classification for precision medicine.

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“…The rapid advancement of CRISPR-Cas-based genome editing technologies has made gene therapy increasingly promising. However, several obstacles remain, including safety concerns due to both off-target and on-target mutations (Adikusuma et al, 2018; Fu et al, 2013; Hsu et al, 2013; Kosicki et al, 2018; Lee and Kim, 2018) and the requirement of proper PAM sequences for efficient and precise cleavage by the commonly used endonucleases (Komor et al, 2017), e.g., Cas9 and Cas12a/Cpf1. The Cas12a endonuclease has several advantages over Cas9.…”

Section: Introductionmentioning

confidence: 99%

“…Third, Cas12a has much reduced off-target effects compared to SpCas9 due to its irreversible binding to the target region and strong discrimination against the off-target sequences (Kim et al, 2017; Kleinstiver et al, 2016; Strohkendl et al, 2018). Finally, Cas9 has been shown to cause on-target mutations including large deletions and insertions (Adikusuma et al, 2018; Kosicki et al, 2018; Lee and Kim, 2018), whereas Cas12a only generates staggered DNA overhangs, which may lead to much lower rate of on-target mutations due to the so-called preferred microhomology-mediated end joining (MMEJ) repair mechanism (Zetsche et al, 2015). However, practical applications of Cas12a have been severely hindered due, at least in part, to its strict requirement for the TTTV PAM sequence.…”

Section: Introductionmentioning

confidence: 99%

Efficient multiplex genome editing using CRISPR-Mb3Cas12a in mice

Wang

et al. 2019

Preprint

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statement 1 CRISPR-Mb3Cas12a can target a broader range of sequences in murine zygotes 2 compared to AsCas12a and LbCas12a, and has lower on-target effects than Cas9 and 3 high overall knock-in efficiency. 4 5 Abstract 6Despite many advantages over Cas9, Cas12a has not been widely used in genome 7 editing in mammalian cells largely due to its strict requirement of the TTTV protospacer 8 adjacent motif (PAM) sequence. Here, we report that Mb3Cas12a (Moraxella bovoculi 9 AAX11_00205) could edit the genome in murine zygotes independent of TTTV PAM 10 sequences and with minimal on-target mutations and close to 100% editing efficiency 11 when crRNAs of 23nt spacers were used.

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Unexpected CRISPR on-target effects

Cited by 43 publications

References 10 publications

A porcine model of phenylketonuria generated by CRISPR/Cas9 genome editing

A porcine model of phenylketonuria generated by CRISPR/Cas9 genome editing

Single cell tri-channel-processing reveals structural variation landscapes and complex rearrangement processes

Efficient multiplex genome editing using CRISPR-Mb3Cas12a in mice

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