CRISPR Start-Loss: A Novel and Practical Alternative for Gene Silencing through Base-Editing-Induced Start Codon Mutations

Chen, Siyu; Xie, Wanhua; Liu, Zhiquan; Shan, Huanhuan; Chen, Mao; Song, Yuning; Yu, Hao; Lai, Liangxue; Li, Zhanjun

doi:10.1016/j.omtn.2020.07.037

Cited by 21 publications

(13 citation statements)

References 70 publications

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“…Second, the start loss of CSNK2B was also recorded in the gnomAD database 4 with a relative high population frequencies. Considering that the start loss variant may theoretically affect the open reading frame and leads to gene silencing ( Chen et al, 2020 ), or re-initiate translation by another “ATG” triple nucleotide and mainly affects phenotypic penetrance ( Benkirane et al, 2021 ), whether those variants are pathogenic requires further study. There were eight individuals with c.G94 mutation, and detailed phenotypes were available for four individuals.…”

Section: Discussionmentioning

confidence: 99%

Splicing Interruption by Intron Variants in CSNK2B Causes Poirier–Bienvenu Neurodevelopmental Syndrome: A Focus on Genotype–Phenotype Correlations

Zhang

Ye²,

Chen³

et al. 2022

Front. Neurosci.

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CSNK2B has recently been identified as the causative gene for Poirier–Bienvenu neurodevelopmental syndrome (POBINDS). POBINDS is a rare neurodevelopmental disorder characterized by early-onset epilepsy, developmental delay, hypotonia, and dysmorphism. Limited by the scarcity of patients, the genotype–phenotype correlations in POBINDS are still unclear. In the present study, we describe the clinical and genetic characteristics of eight individuals with POBINDS, most of whom suffered developmental delay, generalized epilepsy, and hypotonia. Minigene experiments confirmed that two intron variants (c.367+5G>A and c.367+6T>C) resulted in the skipping of exon 5, leading to a premature termination of mRNA transcription. Combining our data with the available literature, the types of POBINDS-causing variants included missense, nonsense, frameshift, and splicing, but the variant types do not reflect the clinical severity. Reduced casein kinase 2 holoenzyme activity may represent a unifying pathogenesis. We also found that individuals with missense variants in the zinc finger domain had manageable seizures (p = 0.009) and milder intellectual disability (p = 0.003) than those with missense variants in other domains of CSNK2B. This is the first study of genotype–phenotype correlations in POBINDS, drawing attention to the pathogenicity of intron variants and expanding the understanding of neurodevelopmental disorders.

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Section: Discussionmentioning

confidence: 99%

Splicing Interruption by Intron Variants in CSNK2B Causes Poirier–Bienvenu Neurodevelopmental Syndrome: A Focus on Genotype–Phenotype Correlations

Zhang

Ye²,

Chen³

et al. 2022

Front. Neurosci.

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“…SUMO4 encodes one of four human SUMO isoforms that are attached to more than a thousand proteins in human cells and are essential for the regulation of several cellular processes such as transcription and mRNA processing [ 6 , 8 ]. As may be expected from a disrupted initiation codon shown to diminish gene expression in the case of an ATG to ATA conversion at the Fgf5 locus [ 17 ], the consequences of the heterozygous SUMO4 :c.2T>C variant were reduced SUMO4 mRNA and protein levels. This variant effect does not seem to be fully compensated by an increased expression of other SUMO isoforms consistent with SUMO4 haploinsufficiency as a contributor to ALS pathogenesis.…”

Section: Discussionmentioning

confidence: 99%

A SUMO4 initiator codon variant in amyotrophic lateral sclerosis reduces SUMO4 expression and alters stress granule dynamics

et al. 2022

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Background Recent evidence points toward a role of the small ubiquitin-like modifier (SUMO) system, including SUMO4, in protecting from stress insults and neurodegeneration, such as the progressive motor neuron disease amyotrophic lateral sclerosis (ALS), e.g., by regulating stress granule (SG) dynamics. Here, we investigated whether SUMO4 variants play a role in ALS pathogenesis. Methods Whole-exome or targeted SUMO4 sequencing was done in 222 unrelated European ALS patients. The consequences of the identified initiator codon variant were analyzed at the mRNA, protein and cellular level. SUMO4 expression was quantified in human tissues. All patients were subjected to clinical, electrophysiological, and neuroradiological characterization. Results A rare heterozygous SUMO4 variant, i.e., SUMO4:c.2T>C p.Met1?, was detected in four of 222 (1.8%) ALS patients, significantly more frequently than in two control cohorts (0.3% each). SUMO4 mRNA and protein expression was diminished in whole blood or fibroblasts of a SUMO4 variant carrier versus controls. Pertinent stress factors, i.e., head trauma or cancer (treated by radiochemotherapy), were significantly more frequent in SUMO4 variant carrier versus non-carrier ALS patients. The mean number of SGs per cell was significantly higher in fibroblasts of a SUMO4 variant carrier compared to controls at baseline, upon oxidative stress, and after recovery, and SUMOylation of ALS-associated valosin-containing protein by SUMO4 was decreased. SUMO4 mRNA expression was highest in brain of all human tissues analyzed. Conclusions Our results are consistent with SUMO4 haploinsufficiency as a contributor to ALS pathogenesis impacting SG dynamics and possibly acting in conjunction with environmental oxidative stress-related factors.

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“…Thus, altering a single base can lead to exon skipping into mature transcripts, removing exons permanently [ 348 ]. Ninth, CRISPR Start-Loss (CRISPR-SL); this method can abolish targeted gene expression by disturbing the start codon (ATG), using both CBEs and ABEs to convert ATG into ATA, ACG, or GTG [ 332 , 333 , 349 ]. Tenth, DNA-free genome editing; this innovation uses the delivery of pre-assembled gRNA/Cas9 ribonucleoproteins (RNPs), which facilitates gene editing without the use of transgenes, avoiding their integration in the genome [ 350 , 351 , 352 , 353 , 354 , 355 ].…”

Section: Molecular Toolsmentioning

confidence: 99%

Biotechnological Advances to Improve Abiotic Stress Tolerance in Crops

Villalobos-López

Arroyo-Becerra

Quintero-Jiménez

et al. 2022

IJMS

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The major challenges that agriculture is facing in the twenty-first century are increasing droughts, water scarcity, flooding, poorer soils, and extreme temperatures due to climate change. However, most crops are not tolerant to extreme climatic environments. The aim in the near future, in a world with hunger and an increasing population, is to breed and/or engineer crops to tolerate abiotic stress with a higher yield. Some crop varieties display a certain degree of tolerance, which has been exploited by plant breeders to develop varieties that thrive under stress conditions. Moreover, a long list of genes involved in abiotic stress tolerance have been identified and characterized by molecular techniques and overexpressed individually in plant transformation experiments. Nevertheless, stress tolerance phenotypes are polygenetic traits, which current genomic tools are dissecting to exploit their use by accelerating genetic introgression using molecular markers or site-directed mutagenesis such as CRISPR-Cas9. In this review, we describe plant mechanisms to sense and tolerate adverse climate conditions and examine and discuss classic and new molecular tools to select and improve abiotic stress tolerance in major crops.

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CRISPR Start-Loss: A Novel and Practical Alternative for Gene Silencing through Base-Editing-Induced Start Codon Mutations

Cited by 21 publications

References 70 publications

Splicing Interruption by Intron Variants in CSNK2B Causes Poirier–Bienvenu Neurodevelopmental Syndrome: A Focus on Genotype–Phenotype Correlations

Splicing Interruption by Intron Variants in CSNK2B Causes Poirier–Bienvenu Neurodevelopmental Syndrome: A Focus on Genotype–Phenotype Correlations

A SUMO4 initiator codon variant in amyotrophic lateral sclerosis reduces SUMO4 expression and alters stress granule dynamics

Biotechnological Advances to Improve Abiotic Stress Tolerance in Crops

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