CRISPECTOR provides accurate estimation of genome editing translocation and off-target activity from comparative NGS data

AbstractControlling off-target editing activity is one of the central challenges in making CRISPR technology accurate and applicable in medical practice. Current algorithms for analyzing off-target activity do not provide statistical quantification, are not sufficiently sensitive in separating signal from noise in experiments with low editing rates, and do not address the detection of translocations. Here we present CRISPECTOR, a software tool that supports the detection and quantification of on- and off-target genome-editing activity from NGS data using paired treatment/control CRISPR experiments. In particular, CRISPECTOR facilitates the statistical analysis of NGS data from multiplex-PCR comparative experiments to detect and quantify adverse translocation events. We validate the observed results and show independent evidence of the occurrence of translocations in human cell lines, after genome editing. Our methodology is based on a statistical model comparison approach leading to better false-negative rates in sites with weak yet significant off-target activity.

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NGSremix: A software tool for estimating pairwise relatedness between admixed individuals from next-generation sequencing data

G3 Genes|Genome|Genetics ◽

10.1093/g3journal/jkab174 ◽

2021 ◽

Author(s):

Anne Krogh Nøhr ◽

Kristian Hanghøj ◽

Genis Garcia Erill ◽

Zilong Li ◽

Ida Moltke ◽

...

Keyword(s):

Next Generation Sequencing ◽

Genetic Research ◽

Likelihood Estimation ◽

Software Tool ◽

Estimation Methods ◽

Next Generation Sequencing Data ◽

Next Generation ◽

Sequencing Data ◽

Ngs Data ◽

Generation Sequencing

Abstract Estimation of relatedness between pairs of individuals is important in many genetic research areas. When estimating relatedness, it is important to account for admixture if this is present. However, the methods that can account for admixture are all based on genotype data as input, which is a problem for low-depth next-generation sequencing (NGS) data from which genotypes are called with high uncertainty. Here we present a software tool, NGSremix, for maximum likelihood estimation of relatedness between pairs of admixed individuals from low-depth NGS data, which takes the uncertainty of the genotypes into account via genotype likelihoods. Using both simulated and real NGS data for admixed individuals with an average depth of 4x or below we show that our method works well and clearly outperforms all the commonly used state-of-the-art relatedness estimation methods PLINK, KING, relateAdmix, and ngsRelate that all perform quite poorly. Hence, NGSremix is a useful new tool for estimating relatedness in admixed populations from low-depth NGS data. NGSremix is implemented in C/C ++ in a multi-threaded software and is freely available on Github https://github.com/KHanghoj/NGSremix.

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A Bayesian model comparison approach to test the specificity of visual integration impairment in schizophrenia or psychosis

Psychiatry Research ◽

10.1016/j.psychres.2018.04.061 ◽

2018 ◽

Vol 265 ◽

pp. 271-278 ◽

Cited By ~ 4

Author(s):

Tyler B. Grove ◽

Beier Yao ◽

Savanna A. Mueller ◽

Merranda McLaughlin ◽

Vicki L. Ellingrod ◽

...

Keyword(s):

Bayesian Model ◽

Model Comparison ◽

Visual Integration ◽

Bayesian Model Comparison ◽

Comparison Approach

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On Testing the Random Walk Hypothesis: A Model-Comparison Approach

SSRN Electronic Journal ◽

10.2139/ssrn.285715 ◽

2001 ◽

Author(s):

Ali F. Darrat ◽

Maosen Zhong

Keyword(s):

Random Walk ◽

Model Comparison ◽

Random Walk Hypothesis ◽

Comparison Approach

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Cas12a is a dynamic and precise RNA-guided nuclease without off-target activity on λ-DNA

10.1101/2021.06.09.447528 ◽

2021 ◽

Author(s):

Bijoya Paul ◽

Loic Chaubet ◽

Emma Verver ◽

Guillermo Montoya

Keyword(s):

Dna Binding ◽

Genome Editing ◽

Optical Tweezers ◽

Target Site ◽

Target Dna ◽

Multiple Binding ◽

Target Sites ◽

Dna Binding And Cleavage ◽

Target Activity ◽

Insight Into

Cas12a is an RNA-guided endonuclease that is emerging as a powerful genome-editing tool. Here we combined optical tweezers with fluorescence to monitor Cas12a binding onto λ-DNA, providing insight into its DNA binding and cleavage mechanisms. At low forces Cas12a binds DNA specifically with two off-target sites, while at higher forces numerous binding events appear driven by the mechanical distortion of the DNA and partial matches to the crRNA. Despite the multiple binding events, cleavage is only observed on the target site at low forces, when the DNA is flexible. Activity assays show that the preferential off-target sites are not cleaved, and the λ-DNA is severed at the target site. This precision is also observed in Cas12a variants where the specific dsDNA and the unspecific ssDNA cleavage are dissociated or nick the target DNA. We propose that Cas12a and its variants are precise endonucleases that efficiently scan the DNA for its target but only cleave the selected site in the λ-DNA.

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Capturing Ordinal Theoretical Constraint in Psychological Science

10.31234/osf.io/a4xu9 ◽

2018 ◽

Cited By ~ 1

Author(s):

Julia M. Haaf ◽

Fayette Klaassen ◽

Jeffrey Rouder

Keyword(s):

Social Sciences ◽

Linear Model ◽

Bayesian Model ◽

Model Comparison ◽

Social Scientists ◽

Psychological Science ◽

Bayesian Model Comparison ◽

Comparison Approach ◽

The Social ◽

Ordinal Level

Most theories in the social sciences are verbal and provide ordinal-level predictions for data. For example, a theory might predict that performance is better in one condition than another, but not by how much. One way of gaining additional specificity is to posit many ordinal constraints that hold simultaneously. For example a theory might predict an effect in one condition, a larger effect in another, and none in a third. We show how common theoretical positions naturally lead to multiple ordinal constraints. To assess whether multiple ordinal constraints hold in data, we adopt a Bayesian model comparison approach. The result is an inferential system that is custom-tuned for the way social scientists conceptualize theory, and that is more intuitive and informative than current linear-model approaches.

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Effect of incomplete registration at general practices on the annual prevalence of treated asthma

International Journal for Population Data Science ◽

10.23889/ijpds.v1i1.233 ◽

2017 ◽

Vol 1 (1) ◽

Author(s):

Mohammad Al Sallakh ◽

Sarah Rodgers ◽

Aziz Sheikh ◽

Ronan Lyons ◽

Gwyneth Davies

Keyword(s):

Local Authority ◽

National Level ◽

False Negative ◽

Routine Data ◽

Correction Method ◽

Relative Increase ◽

Accurate Estimation ◽

Health Board ◽

Annual Prevalence ◽

General Practices

ABSTRACTBackgroundAnnual prevalence of 'treated asthma' can be estimated using primary care routine data. However, the denominator may include people with incomplete registration at general practices (GP) in the year of estimation. Patients with incomplete registrations who actually take medications but have no recorded prescriptions represent false-negative cases leading to underestimated prevalence. ObjectivesTo estimate the effect of incomplete GP registrations on the annual prevalence of treated asthma in Wales. MethodsUsing the GP dataset of the Secure Anonymised Information Linkage (SAIL) Databank, we created a denominator of people who lived in Wales and registered at Welsh GPs for any period during 2010. For the uncorrected prevalence, the numerator included people who ever had a recorded asthma diagnosis and any current recorded asthma prescriptions. For the corrected prevalence, we estimated the number of false-negatives and added them to the numerator. To do that, we calculated, for asthma patients with incomplete registrations and no recorded prescriptions, the sum of probabilities of having prescriptions if they had complete registrations. We estimated the absolute and relative increases in the prevalence at national and local authority levels as well as for the subpopulation with incomplete registration. ResultsThe denominator included 2,221,967 people, of whom 94.8% had complete GP registration in 2010. Without correction, the numerator included 132,439 patients giving a prevalence of 5.96% [95% CI 5.93-5.99]. By adding estimated 1,801 false-negative cases to the numerator, the adjusted prevalence is 6.04% [95% CI 6.01-6.07] with absolute and relative increases of 0.08% and 1.36%, respectively. At the local authority level, the relative increase ranged from 0.47% for Blaenau Gwent to 3.94% for Monmouthshire. Among the subpopulation with incomplete registration (5.1%), the prevalence increased by 68.0% from 2.32% to 3.91%. ConclusionIn Wales, which has a highly stable population, incomplete GP registration has negligible effect on the prevalence of treated asthma at a national level, although it was more significant for sub-regions with less stable populations. This correction method could be useful for more accurate estimation of asthma burden and the prevalence of active disease in highly dynamic populations. FundingHealth and Care Research Wales and ABMU Health Board. Supported by Asthma UK Centre for Applied Research [AUK-AC-2012-01].

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Ultra-deep sequencing reveals no evidence of oncogenic mutations or enrichment by ex vivo CRISPR/Cas9 genome editing in human hematopoietic stem and progenitor cells

10.1101/2021.10.27.466166 ◽

2021 ◽

Author(s):

M. Kyle Cromer ◽

Valentin V. Barsan ◽

Erich Jaeger ◽

Mengchi Wang ◽

Jessica P. Hampton ◽

...

Keyword(s):

Genome Editing ◽

Deep Sequencing ◽

Limit Of Detection ◽

Ex Vivo ◽

High Fidelity ◽

Seed Region ◽

Hematopoietic Stem ◽

Oncogenic Mutations ◽

Ex Vivo Culture ◽

Target Activity

As CRISPR-based therapies enter the clinic, evaluation of the safety remains a critical and still active area of study. While whole genome sequencing is an unbiased method for identifying somatic mutations introduced by ex vivo culture and genome editing, this methodology is unable to attain sufficient read depth to detect extremely low frequency events that could result in clonal expansion. As a solution, we utilized an exon capture panel to facilitate ultra-deep sequencing of >500 tumor suppressors and oncogenes most frequently altered in human cancer. We used this panel to investigate whether transient delivery of high-fidelity Cas9 protein targeted to three different loci (using guide RNAs (gRNAs) corresponding to sites at AAVS1, HBB, and ZFPM2) at day 4 and day 10 timepoints post-editing resulted in the introduction or enrichment of oncogenic mutations. In three separate primary human HSPC donors, we identified a mean of 1,488 variants per Cas9 treatment (at <0.07% limit of detection). After filtering to remove germline and/or synonymous changes, a mean of 3.3 variants remained per condition, which were further reduced to six total mutations after removing variants in unedited treatments. Of these, four variants resided at the predicted off-target site in the myelodysplasia-associated EZH2 gene that were subject to ZFPM2 gRNA targeting in Donors 2 and 3 at day 4 and day 10 timepoints. While Donor 1 displayed on-target cleavage at ZFPM2, we found no off-target activity at EZH2. Sanger sequencing revealed a homozygous single nucleotide polymorphism (SNP) at position 14bp distal from the Cas9 protospacer adjacent motif in EZH2 that eliminated any detectable off-target activity. We found no evidence of exonic off-target INDELs with either of the AAVS1 or HBB gRNAs. These findings indicate that clinically relevant delivery of high-fidelity Cas9 to primary HSPCs and ex vivo culture up to 10 days does not introduce or enrich for tumorigenic variants and that even a single SNP outside the seed region of the gRNA protospacer is sufficient to eliminate Cas9 off-target activity with this method of delivery into primary, repair competent human HSPCs.

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