Human genetic diversity alters off-target outcomes of therapeutic gene editing

Cancellieri, Samuele; Zeng, Jing; Lin, Linda Yingqi; Tognon, Manuel; Nguyen, Minh-Doan; Lin, Jiecong; Bombieri, Nicola; Maitland, Stacy; Ciuculescu, Marioara-Felicia; Katta, Varun; Tsai, Shengdar Q.; Armant, Myriam; Wolfe, Scot A.; Giugno, Rosalba; Bauer, Daniel E.; Pinello, Luca

doi:10.1038/s41588-022-01257-y

Cited by 44 publications

(22 citation statements)

References 49 publications

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“…The presence of single nucleotide polymorphism (SNP) in a population underrepresented in the human reference genomes could lead to disruption of the targeted sequence or cause unintended genome modifications/off-targets 55,56 . Recently, Cancellieri et al have developed a computational method to predict the impact of SNP and off-target sequences across diverse human populations; this method is aptly termed CRISPRme 57 . However, it is currently limited to the prediction of S. pyogenes- derived Cas9 targets, and the recognition sequence of S. aureus -derived Cas9 was not yet included at the time of preparation of this manuscript.…”

Section: Discussionmentioning

confidence: 99%

CRISPRa-induced upregulation of humanLAMA1compensates forLAMA2-deficiency in Merosin-deficient congenital muscular dystrophy

Arockiaraj

Johnson

Munir

et al. 2023

Preprint

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Merosin-deficient congenital muscular dystrophy (MDC1A) is an autosomal recessive disorder caused by mutations in the LAMA2 gene, resulting in a defective form of the extracellular matrix protein laminin-alpha2 (LAMA2). Individuals diagnosed with MDC1A exhibit progressive muscle wasting and declining neuromuscular functions. No treatments for this disorder are currently available. We previously showed that postnatal Lama1 upregulation, achieved through CRISPR activation (CRISPRa), compensates for Lama2 deficiency and prevents neuromuscular pathophysiology in a mouse model of MDC1A. In this study, we assessed the feasibility of upregulating human LAMA1 as a potential therapeutic strategy for individuals with MDC1A, regardless of their mutations. We hypothesized that CRISPRa-mediated upregulation of human LAMA1 would compensate for the lack of LAMA2 and rescue cellular abnormalities in MDC1A fibroblasts. Global transcriptomic and pathway enrichment analyses of fibroblasts collected from individuals carrying pathogenic LAMA2 mutations, compared with healthy controls, indicated higher expression of transcripts encoding proteins that contribute to wound healing, including Transforming Growth Factor-beta (TGF-beta) and Fibroblast Growth Factor (FGF). These findings were supported by wound-healing assays indicating that MDC1A fibroblasts migrated significantly more rapidly than the controls. Subsequently, we treated the MDC1A fibroblasts with SadCas9-2XVP64 and sgRNAs targeting the LAMA1 promoter. We observed robust LAMA1 expression, which was accompanied by significant decreases in cell migration and expression of FGFR2, TGF-beta, and ACTA2, which are involved in the wound-healing mechanism in MDC1A fibroblasts. Collectively, our data suggest that CRISPRa-mediated LAMA1 upregulation may be a feasible mutation-independent therapeutic approach for MDC1A. This strategy might be adapted to address other neuromuscular diseases and inherited conditions in which strong compensatory mechanisms have been identified.

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Section: Discussionmentioning

confidence: 99%

CRISPRa-induced upregulation of humanLAMA1compensates forLAMA2-deficiency in Merosin-deficient congenital muscular dystrophy

Arockiaraj

Johnson

Munir

et al. 2023

Preprint

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“…The prevalence distribution of HF impairment with thalassemias varies depending on the severity and type of the disorder as well as other factors such as age, gender and comorbidities 5 . Thalassemias are more common in populations of Mediterranean, African and Southeast Asian ancestry, where the gene mutations that cause the disorder are more prevalent 6 . The prevalence of thalassemias varies among different populations, with estimates ranging from .2% to 15% 7 .…”

Section: Introductionmentioning

confidence: 99%

“…5 Thalassemias are more common in populations of Mediterranean, African and Southeast Asian ancestry, where the gene mutations that cause the disorder are more prevalent. 6 The prevalence of thalassemias varies among different populations, with estimates ranging from .2% to 15%. 7 Left ventricular diastolic dysfunction has been reported in patients with thalassemia major in the early stage of myocardial damage.…”

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confidence: 99%

Changing trends in the prevalence of heart failure impairment with Thalassemias over three decades

Tang,

Zhang,

Deng

et al. 2023

Eur J Clin Investigation

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BackgroundTo assess the prevalence trend and contributing factors of heart failure (HF) impairment with thalassemias at global, regional and national levels.MethodsData on HF impairment with thalassemias was collected from the Global Burden of Disease study. The absolute number and prevalence of the disease were systematically collected for each year, and the estimated annual percentage changes (EAPC) in HF impairment were calculated by gender, region and country to measure temporal trends.ResultsThalassemias have caused a significant global burden since 1990, and the case number of HF related to thalassemias has been steadily increasing. The highest case number of HF impairments with thalassemias is observed in China (7739 cases) and the highest prevalence is in Pakistan (1.61 per 100,000) currently. Besides, the middle sociodemographic index (SDI) region carries the highest burden of comorbid disease yet exhibits the most evident trend for improvement across the five regions (EAPC = −.98). The burden of thalassemias and comorbid HF is generally higher in males than females with the gender gap growing chasm in the future. Besides, the hotspots of HF impairment with thalassemias have gradually shifted to low SDI regions, though middle SDI regions still hold a relatively higher prevalence (.37 per 100,000) across different regions.ConclusionsThe burden of thalassemias and accompanying HF, as well as their temporal trends, vary greatly across countries and regions. These findings can improve understanding of these conditions and guide policymakers in developing appropriate policies to address disparities between countries.

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“…It has been revealed that naturally occurring gene variants can lead to potentially cancerous off-target therapeutic CRISPR/Cas9 editing. These findings have led to the development of online tools like CRISPRme to facilitate variant-aware off-target gRNA assessments (17,18). To further minimize the risks of treatment failure or adverse outcomes in CRISPR/Cas9-based therapeutics related to human genetic diversity, we need to broadly assess how genetic diversity affects on-target CRISPR/Cas9 editing outcomes after a DSB.…”

Section: Introductionmentioning

confidence: 99%

Widespread impact of natural genetic variations in CRISPR/Cas9 outcomes

Li,

Tadych,

Wong

et al. 2023

Preprint

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CRISPR/Cas9 is a genome editing tool widely used in biological research and clinical therapeutics. Naturally occurring human genomic variations, through altering the sequence context of CRISPR/Cas9 target regions, can significantly affect its DNA repair outcomes and ultimately lead to different editing efficiencies. However, these effects have not been systematically studied or documented, even as CRISPR/Cas9 is broadly applied to primary cells and patient samples that harbor such genetic diversity. Herein, we present CROTONdb (https://croton.princeton.edu/), a publicly available database that enables fast and comprehensive investigations of single nucleotide variant (SNV) effects on CRISPR/Cas9 outcomes across the human genome. CROTONdb provides predictions for all possible CRISPR/Cas9 target sites in the coding region, spanning over 5.38 million gRNA targets, 32.32 million predicted editing outcomes, and 90.82 million estimated variant effects. Critically, variant effects presented in CROTONdb accurately cover both common and rare/understudied variants. We illustrate the utility of CROTONdb through two case studies, showcasing its impact on both preclinical discoveries and recent clinical trials. Our case studies demonstrate pitfalls of failing to account for genetic variations in Cas9 target selection, and how they can be effectively examined and avoided using our tool. We anticipate CROTONdb having broad clinical utilities in gene and cellular therapies.

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Human genetic diversity alters off-target outcomes of therapeutic gene editing

Cited by 44 publications

References 49 publications

CRISPRa-induced upregulation of humanLAMA1compensates forLAMA2-deficiency in Merosin-deficient congenital muscular dystrophy

CRISPRa-induced upregulation of humanLAMA1compensates forLAMA2-deficiency in Merosin-deficient congenital muscular dystrophy

Changing trends in the prevalence of heart failure impairment with Thalassemias over three decades

Widespread impact of natural genetic variations in CRISPR/Cas9 outcomes

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