Rapid, precise quantification of large DNA excisions and inversions by ddPCR

Watry, Hannah L.; Feliciano, Carissa M.; Gjoni, Ketrin; Takahashi, Gou; Miyaoka, Yuichiro; Conklin, Bruce R.; Judge, Luke M.

doi:10.1038/s41598-020-71742-z

“…Canver et al (2014) estimated an overall deletion rate, across 17 targets of varying size, to occur at approximately one-third the rate of deletions (5.8% vs. 14.2%, respectively). A more recent study found this ratio to be approximately one-half (Watry et al 2020). We evaluated how this might affect our results.…”

Section: Inhibition Of Dna-pkcs During Dsb Formation Increases Crisprmentioning

confidence: 88%

“…However, it is incapable of distinguishing deletion from inversion, the latter being a nonnegligible editing outcome. In the future, it will be important to use methods capable of distinguishing deletion and inversion events, such as digital droplet PCR (ddPCR) (Antoniani et al 2018;Watry et al 2020), although it might not be practical for testing large numbers of samples and conditions. Although the CiDER and qPCR assays used here could not explicitly distinguish the ratios of deletion and insertion arising from pgRNA targeting, we found that most of the measured iDNA-PKcs-dependent changes arise from increases in deletion.…”

Section: Discussionmentioning

confidence: 99%

“…Paired DSBs are repaired by the endogenous nonhomologous end-joining (NHEJ) pathway, which frequently ejects the intervening fragment, resulting in a genomic deletion (Yang et al 2013;Maddalo et al 2014;Ho et al 2015;Vidigal and Ventura 2015). Less frequently, the target region may be inverted without deletion (Canver et al 2014;Antoniani et al 2018;Watry et al 2020). Cas9 complexes are recruited to the two edges of the target region via a user-defined pair of single guide RNAs (sgRNAs, or "pgRNAs" for the pair) (Pulido-Quetglas et al 2017), delivered by transfection or viral transduction (Aparicio-Prat et al 2015;Vidigal and Ventura 2015).…”

mentioning

confidence: 99%

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Enhancing CRISPR deletion via pharmacological delay of DNA-PKcs

Bosch-Guiteras

¹

,

Uroda

²

,

Guillén-Ramírez

³

et al. 2021

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CRISPR-Cas9 deletion (CRISPR-del) is the leading approach for eliminating DNA from mammalian cells and underpins a variety of genome-editing applications. Target DNA, defined by a pair of double-strand breaks (DSBs), is removed during nonhomologous end-joining (NHEJ). However, the low efficiency of CRISPR-del results in laborious experiments and falsenegative results. By using an endogenous reporter system, we show that repression of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs)-an early step in NHEJ-yields substantial increases in DNA deletion. This is observed across diverse cell lines, gene delivery methods, commercial inhibitors, and guide RNAs, including those that otherwise display negligible activity. We further show that DNA-PKcs inhibition can be used to boost the sensitivity of pooled functional screens and detect true-positive hits that would otherwise be overlooked. Thus, delaying the kinetics of NHEJ relative to DSB formation is a simple and effective means of enhancing CRISPR-deletion.

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“…However, inducing frameshifts at the site of mutations in distal coding exons may not be therapeutic, as nonsense codons in that context can lead to expression of a pathologic truncated protein rather than nonsense-mediated decay (Lindeboom et al, 2016; Stone et al, 2019). Alternate editing strategies for these mutations include excision of mutant alleles using pairs of guide RNAs, or precise correction using non-nuclease editing technologies such as base editors or PRIME editing (Watry et al, 2020; Komor et al, 2016; Anzalone et al, 2019).…”

Section: Discussionmentioning

confidence: 99%

Allele-specific gene editing rescues pathology in a human model of Charcot-Marie-Tooth disease type 2E

Feliciano

¹

,

Wu

²

,

Watry

³

et al. 2021

Preprint

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Many neuromuscular disorders are caused by dominant missense mutations that lead to dominant-negative or gain-of-function pathology. This category of disease is challenging to address via drug treatment or gene augmentation therapy because these strategies may not eliminate the effects of the mutant protein or RNA. Thus, effective treatments are severely lacking for these dominant diseases, which often cause severe disability or death. The targeted inactivation of dominant disease alleles by gene editing is a promising approach with the potential to completely remove the cause of pathology with a single treatment. Here, we demonstrate that allele-specific CRISPR gene editing in a human model of axonal Charcot-Marie-Tooth (CMT) disease rescues pathology caused by a dominant missense mutation in the neurofilament light chain gene (NEFL, CMT type 2E). We utilized a rapid and efficient method for generating spinal motor neurons from human induced pluripotent stem cells (iPSCs) derived from a patient with CMT2E. Diseased motor neurons recapitulated known pathologic phenotypes at early time points of differentiation, including aberrant accumulation of neurofilament light chain protein in neuronal cell bodies. We selectively inactivated the disease NEFL allele in patient iPSCs using Cas9 enzymes to introduce a frameshift at the pathogenic N98S mutation. Motor neurons carrying this allele-specific frameshift demonstrated an amelioration of the disease phenotype comparable to that seen in an isogenic control with precise correction of the mutation. Our results validate allele-specific gene editing as a therapeutic approach for CMT2E and as a promising strategy to silence dominant mutations in any gene for which heterozygous loss-of-function is well tolerated. This highlights the potential for gene editing as a therapy for currently untreatable dominant neurologic diseases.

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“…To overcome this challenge, we engineered patient-derived iPSCs with integrated, inducible, and isogenic (i 3 ) transcription factors in order to rapidly and efficiently differentiate them into lower motor neurons (i 3 LMNs) (Fernandopulle et al, 2018). We inserted a doxycycline-inducible cassette containing NGN2, ISL1, and LHX3 into the AAVS1 safe-harbor locus (Supplementary Figure 1) of a previously characterized CMT2E iPSC line with the NEFL N98S mutation, referred to here as N98S (Saporta et al, 2015;Maciel et al, 2020;Watry et al, 2020). For comparison, we used healthy control (WT) iPSC lines containing the same doxycycline-inducible cassette integrated into AAVS1 or CLYBL, as previously described (Fernandopulle et al, 2018;Shi et al, 2018).…”

Section: Rapid Development Of Cmt2e Phenotypes In Ipsc-derived Motor Neuronsmentioning

confidence: 99%

Allele-Specific Gene Editing Rescues Pathology in a Human Model of Charcot-Marie-Tooth Disease Type 2E

Feliciano

¹

,

Wu

²

,

Watry

³

et al. 2021

Front. Cell Dev. Biol.

Self Cite

View full text Add to dashboard Cite

Many neuromuscular disorders are caused by dominant missense mutations that lead to dominant-negative or gain-of-function pathology. This category of disease is challenging to address via drug treatment or gene augmentation therapy because these strategies may not eliminate the effects of the mutant protein or RNA. Thus, effective treatments are severely lacking for these dominant diseases, which often cause severe disability or death. The targeted inactivation of dominant disease alleles by gene editing is a promising approach with the potential to completely remove the cause of pathology with a single treatment. Here, we demonstrate that allele-specific CRISPR gene editing in a human model of axonal Charcot-Marie-Tooth (CMT) disease rescues pathology caused by a dominant missense mutation in the neurofilament light chain gene (NEFL, CMT type 2E). We utilized a rapid and efficient method for generating spinal motor neurons from human induced pluripotent stem cells (iPSCs) derived from a patient with CMT2E. Diseased motor neurons recapitulated known pathologic phenotypes at early time points of differentiation, including aberrant accumulation of neurofilament light chain protein in neuronal cell bodies. We selectively inactivated the disease NEFL allele in patient iPSCs using Cas9 enzymes to introduce a frameshift at the pathogenic N98S mutation. Motor neurons carrying this allele-specific frameshift demonstrated an amelioration of the disease phenotype comparable to that seen in an isogenic control with precise correction of the mutation. Our results validate allele-specific gene editing as a therapeutic approach for CMT2E and as a promising strategy to silence dominant mutations in any gene for which heterozygous loss-of-function is well tolerated. This highlights the potential for gene editing as a therapy for currently untreatable dominant neurologic diseases.

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Rapid, precise quantification of large DNA excisions and inversions by ddPCR

Cited by 12 publications

References 39 publications

Enhancing CRISPR deletion via pharmacological delay of DNA-PKcs

Enhancing CRISPR deletion via pharmacological delay of DNA-PKcs

Allele-specific gene editing rescues pathology in a human model of Charcot-Marie-Tooth disease type 2E

Allele-Specific Gene Editing Rescues Pathology in a Human Model of Charcot-Marie-Tooth Disease Type 2E

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