CRISPR/Cas9-mediated tissue-specific knockout and cDNA rescue using sgRNAs that target exon-intron junctions in Drosophila melanogaster

Chilian, Madison; Parra, Karen Vargas; Sandoval, Abigail; Ramírez, Juan David; Yoon, Wan Hee

doi:10.1016/j.xpro.2022.101465

Cited by 8 publications

(6 citation statements)

References 15 publications

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“…A closer look at these deletions showed that most of them encompassed all three target sites. Our analysis illustrates the advantage of applying multiplexed gRNA with the 100% efficiency (this study) compared to single gRNA with the reported 23% efficiency of mutagenesis [ 33 ]. Taken together, the sequencing data and the lethal phenotype of the tub-Cas9 > RNZ KO larvae, support the expectation that multiple gRNAs targeting the same locus within a few hundred base pairs from each other produce large deficiencies resulting in knockout mutations.…”

Section: Discussionmentioning

confidence: 78%

“…To assess the cell autonomous action of RNase Z variants, we developed a new approach of spatially restricted mutagenesis that coupled tissue-specific CRISPR-Cas9 mediated KO with transgenic rescue. A similar protocol was recently proposed by Chilian and co-authors [ 33 ]. An important distinguishing feature of our approach is that instead of Gal4/UAS overexpression as a rescue system, we supply disease-associated protein variants in amounts defined by the activity of the natural RNase Z promoter.…”

Section: Resultsmentioning

confidence: 99%

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Cardiac RNase Z edited via CRISPR-Cas9 drives heart hypertrophy in Drosophila

et al. 2023

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Cardiomyopathy (CM) is a group of diseases distinguished by morphological and functional abnormalities in the myocardium. It is etiologically heterogeneous and may develop via cell autonomous and/or non-autonomous mechanisms. One of the most severe forms of CM has been linked to the deficiency of the ubiquitously expressed RNase Z endoribonuclease. RNase Z cleaves off the 3’-trailer of both nuclear and mitochondrial primary tRNA (pre-tRNA) transcripts. Cells mutant for RNase Z accumulate unprocessed pre-tRNA molecules. Patients carrying RNase Z variants with reduced enzymatic activity display a plethora of symptoms including muscular hypotonia, microcephaly and severe heart hypertrophy; still, they die primarily due to acute heart decompensation. Determining whether the underlying mechanism of heart malfunction is cell autonomous or not will provide an opportunity to develop novel strategies of more efficient treatments for these patients. In this study, we used CRISPR-TRiM technology to create Drosophila models that carry cardiomyopathy-linked alleles of RNase Z only in the cardiomyocytes. We found that this modification is sufficient for flies to develop heart hypertrophy and systolic dysfunction. These observations support the idea that the RNase Z linked CM is driven by cell autonomous mechanisms.

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Section: Discussionmentioning

confidence: 78%

Section: Resultsmentioning

confidence: 99%

Cardiac RNase Z edited via CRISPR-Cas9 drives heart hypertrophy in Drosophila

et al. 2023

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“…To determine the effects of the variants in a tissue-specific manner, we decided to use a recently-developed CRISPR-Cas9-mediated system that permits both tissue-specific dOgdh knockout and rescue by dOgdh cDNA. 28 , 31 We designed a gRNA that is complementary to the exon-intron junction of dOgdh and created transgenic flies carrying this gRNA under the control of U6:3 promoter ( U6:3-gRNA dOgdh ). The U6:3 promoter leads to ubiquitous expression of the gRNA dOgdh .…”

Section: Resultsmentioning

confidence: 99%

Biallelic variants in OGDH encoding oxoglutarate dehydrogenase lead to a neurodevelopmental disorder characterized by global developmental delay, movement disorder, and metabolic abnormalities

Whittle¹,

Chilian²,

Karimiani³

et al. 2023

Genetics in Medicine

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“…However, most viral infection models in flies are not focused on neurological outcomes. Nonetheless, novel approaches in genetic manipulation [ 159 ], behavioural assessment (automated monitoring systems [ 160 ]) and neuropathology (FlyClear tissue clearing protocols [ 161 ]) are expanding experimental design beyond the conventional readouts used to profile neuroinfection and immunity outcomes in flies ( Figure 1 ). Below, we will highlight examples of the benefits and drawbacks of using virus-infected Drosophila to study related neurological sequelae.…”

Section: Neuroinfection and Neuroimmunity Models In Drosophilamentioning

confidence: 99%

“…While most viral transgenic models seek to insert novel viral protein genes into the fly genome to study viral pathology, CRISPR technology also allows for selective addition or removal of genes, even in existing GAL4/UAS models [ 159 ]. This approach has been used in flies to selectively knock-out genes in neural tissues and perform rescue experiments, through UAS-controlled Cas9 expression in conjunction with specific guide-RNAs [ 159 ]. CRISPR technology has also been used to ablate the expression of viral transposable elements in mammalian genomes [ 198 ], although this approach has not yet been used in flies.…”

Section: Neuroinfection and Neuroimmunity Models In Drosophilamentioning

confidence: 99%

Drosophila as a Model for Human Viral Neuroinfections

Benoit

Curzio

Civetta

et al. 2022

Cells

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The study of human neurological infection faces many technical and ethical challenges. While not as common as mammalian models, the use of Drosophila (fruit fly) in the investigation of virus–host dynamics is a powerful research tool. In this review, we focus on the benefits and caveats of using Drosophila as a model for neurological infections and neuroimmunity. Through the examination of in vitro, in vivo and transgenic systems, we highlight select examples to illustrate the use of flies for the study of exogenous and endogenous viruses associated with neurological disease. In each case, phenotypes in Drosophila are compared to those in human conditions. In addition, we discuss antiviral drug screening in flies and how investigating virus–host interactions may lead to novel antiviral drug targets. Together, we highlight standardized and reproducible readouts of fly behaviour, motor function and neurodegeneration that permit an accurate assessment of neurological outcomes for the study of viral infection in fly models. Adoption of Drosophila as a valuable model system for neurological infections has and will continue to guide the discovery of many novel virus–host interactions.

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CRISPR/Cas9-mediated tissue-specific knockout and cDNA rescue using sgRNAs that target exon-intron junctions in Drosophila melanogaster

Cited by 8 publications

References 15 publications

Cardiac RNase Z edited via CRISPR-Cas9 drives heart hypertrophy in Drosophila

Cardiac RNase Z edited via CRISPR-Cas9 drives heart hypertrophy in Drosophila

Biallelic variants in OGDH encoding oxoglutarate dehydrogenase lead to a neurodevelopmental disorder characterized by global developmental delay, movement disorder, and metabolic abnormalities

Drosophila as a Model for Human Viral Neuroinfections

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