<tt>CRISPRroots</tt>: on- and off-target assessment of RNA-seq data in CRISPR–Cas9 edited cells

Corsi, Giulia I.; Gadekar, Veerendra P.; Gorodkin, Jan; Seemann, Stefan E.

doi:10.1093/nar/gkab1131

Cited by 17 publications

(22 citation statements)

References 49 publications

(66 reference statements)

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“…Additionally, the GC content of the sgRNA was also found to correlate with the off-target effect, enabling researchers to select more effective sgRNAs by their GC content [ 134 ]. Moreover, using Cas9 mRNA or protein instead of plasmids could also effectively reduce the off - target rate [ 151 , 152 ]. A mutant of Cas9, xCas9, is more precise than Cas9, can effectively reduce the off-target rate, and has been successfully used in gene therapy [ 153 ].…”

Section: Directions For Future Developments Of the Crispr/cas9 Systemmentioning

confidence: 99%

Advances in CRISPR/Cas9

Zhu

2022

BioMed Research International

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CRISPR/Cas9 technology has become the most examined gene editing technology in recent years due to its simple design, yet low cost, high efficiency, and simple operation, which can also achieve simultaneous editing of multiple loci. It can also be carried out without using plasmids, saving lots of troubles caused by plasmids. CRISPR/Cas9 has shown great potential in the study of genes or genomic functions in microorganisms, plants, animals, and human beings. In this review, we will examine the history, structure, and basic mechanisms of the CRISPR/Cas9 system, describe its great value in precision medicine and sgRNA library screening, and dig its great potential in a new field: DNA information storage.

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Section: Directions For Future Developments Of the Crispr/cas9 Systemmentioning

confidence: 99%

Advances in CRISPR/Cas9

Zhu

2022

BioMed Research International

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“…Differential expression analysis was carried out using DESeq2 v1.22.2 ( Love et al, 2014 ) and genes with Benjamini and Hochberg (1995) adjusted Wald test p -value < 0.05, absolute log2 fold change >1, and mean of normalized counts >10 were considered significant. The workflow includes RseQC v.4.0.0 ( Wang et al, 2012 ) for the confirmation of the library strandness and the verification of the editing status at on-target and potential off-target sites with CRISPRroots v.1.2 ( Corsi et al, 2022a ). Potential off-targets identified by CRISPRroots as high-risk were validate by sequencing, and no off-target edit was found ( Corsi et al, 2022b ).…”

Section: Methodsmentioning

confidence: 99%

Golgi fragmentation – One of the earliest organelle phenotypes in Alzheimer’s disease neurons

Haukedal

Corsi²,

Gadekar³

et al. 2023

Front. Neurosci.

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Alzheimer’s disease (AD) is the most common cause of dementia, with no current cure. Consequently, alternative approaches focusing on early pathological events in specific neuronal populations, besides targeting the well-studied amyloid beta (Aβ) accumulations and Tau tangles, are needed. In this study, we have investigated disease phenotypes specific to glutamatergic forebrain neurons and mapped the timeline of their occurrence, by implementing familial and sporadic human induced pluripotent stem cell models as well as the 5xFAD mouse model. We recapitulated characteristic late AD phenotypes, such as increased Aβ secretion and Tau hyperphosphorylation, as well as previously well documented mitochondrial and synaptic deficits. Intriguingly, we identified Golgi fragmentation as one of the earliest AD phenotypes, indicating potential impairments in protein processing and post-translational modifications. Computational analysis of RNA sequencing data revealed differentially expressed genes involved in glycosylation and glycan patterns, whilst total glycan profiling revealed minor glycosylation differences. This indicates general robustness of glycosylation besides the observed fragmented morphology. Importantly, we identified that genetic variants in Sortilin-related receptor 1 (SORL1) associated with AD could aggravate the Golgi fragmentation and subsequent glycosylation changes. In summary, we identified Golgi fragmentation as one of the earliest disease phenotypes in AD neurons in various in vivo and in vitro complementary disease models, which can be exacerbated via additional risk variants in SORL1.

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“…Detection of on/off-target sites in de novo transcriptome assembly was performed with Crisflash v.1.2.0( 43 ). Our off-target detection focused on up to 11nt mismatches and NRR PAM because the previous off-target reports indicated that off-target sites with ≥10 nt mismatches and NGG/NAG/NGA/NAA PAM exist 24,44 , although not with a high frequency.…”

Section: Methodsmentioning

confidence: 99%

“…Gorodkin and Seemann have previously reported that off-target sites affect the expression profile of LOF samples using reference-based RNA-seq analysis. Our study used the GRIN2B dataset to benchmark de novo transcriptome assembly based and referencebased RNA-seq analyses( 44 ). Moreover, we profiled the on/off-target assessment of GRIN2B-REV sgRNA.…”

Section: Methodsmentioning

confidence: 99%

Danger Analysis: Risk-Averse on/Off-Target Assessment for Crispr Editing Without a Reference Genome

Nakamae

Bono

2023

Preprint

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The CRISPR-Cas9 system has successfully achieved site-specific gene editing in organisms ranging from humans to bacteria. The technology efficiently generates mutants, allowing for phenotypic analysis of the on-target gene. However, some conventional studies did not investigate whether deleterious off-target effects partially affect the phenotype. Herein, we present a novel phenotypic assessment of CRISPR-mediated gene editing: Deleterious and ANticipatable Guides Evaluated by RNA-sequencing (DANGER) analysis. Using RNA-seq data, this bioinformatics pipeline can elucidate genomic on/off-target sites on mRNA-transcribed regions related to expression changes and then quantify phenotypic risk at the gene ontology (GO) term level. We demonstrated the risk-averse on/off-target assessment in RNA-seq data from gene-edited samples of human cells and zebrafish brains. Our DANGER analysis successfully detected off-target sites, and it quantitatively evaluated the potential contribution of deleterious off-targets to the transcriptome phenotypes of the edited mutants. Notably, DANGER analysis harnessed de novo transcriptome assembly to perform risk-averse on/off-target assessments without a reference genome. Thus, our resources would help assess genome editing in non-model organisms, individual human genomes, and atypical genomes from diseases and viruses. In conclusion, DANGER analysis facilitates the safer design of genome editing in all organisms with a transcriptome.

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`CRISPRroots`: on- and off-target assessment of RNA-seq data in CRISPR–Cas9 edited cells

Cited by 17 publications

References 49 publications

Advances in CRISPR/Cas9

Advances in CRISPR/Cas9

Golgi fragmentation – One of the earliest organelle phenotypes in Alzheimer’s disease neurons

Danger Analysis: Risk-Averse on/Off-Target Assessment for Crispr Editing Without a Reference Genome

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CRISPRroots: on- and off-target assessment of RNA-seq data in CRISPR–Cas9 edited cells

Cited by 17 publications

References 49 publications

Advances in CRISPR/Cas9

Advances in CRISPR/Cas9

Golgi fragmentation – One of the earliest organelle phenotypes in Alzheimer’s disease neurons

Danger Analysis: Risk-Averse on/Off-Target Assessment for Crispr Editing Without a Reference Genome

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Product

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`CRISPRroots`: on- and off-target assessment of RNA-seq data in CRISPR–Cas9 edited cells