Multiplexed CRISPR technologies for gene editing and transcriptional regulation

McCarty, Nicholas S.; Graham, Alicia E.; Studená, Lucie; Ledesma-Amaro, Rodrigo

doi:10.1038/s41467-020-15053-x

Cited by 306 publications

(233 citation statements)

References 148 publications

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“…The ability to rapidly and automatically validate plasmids by Illumina sequencing will be especially applicable to large-scale plasmid production projects associated with synthetic biology (42). The rapidly decreasing cost of DNA synthesis and sophisticated computer-aided design tools (43,44) have facilitated disciplined factorial experiments involving large libraries of plasmids with various genetic parts (45) for applications such as improved genetic circuit design (46), multiplexed CRISPR-Cas (47) or metabolic engineering (48). These workflows, which are often automated (49,50), necessitate an automated sequence verification pipeline like the one we've described.…”

Section: Discussionmentioning

confidence: 99%

Rapid, robust plasmid verification by de novo assembly of short sequencing reads

Gallegos

Rogers²,

Cialek

et al. 2020

Preprint

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AbstractPlasmids are a foundational tool for basic and applied research across all subfields of biology. Increasingly, researchers in synthetic biology are relying on and developing massive libraries of plasmids as vectors for directed evolution, combinatorial gene circuit tests, and for CRISPR multiplexing. Verification of plasmid sequences following synthesis is a crucial quality control step that creates a bottleneck in plasmid fabrication workflows. Crucially, researchers often elect to forego the cumbersome verification step, potentially leading to reproducibility and— depending on the application—security issues. In order to facilitate plasmid verification to improve the quality and reproducibility of life science research, we developed a fast, simple, and open source pipeline for assembly and verification of plasmid sequences from Illumina reads. We demonstrate that our pipeline, which relies on de novo assembly, can also be used to detect contaminating sequences in plasmid samples. In addition to presenting our pipeline, we discuss the role for verification and quality control in the increasingly complex life science workflows ushered in by synthetic biology.

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Section: Discussionmentioning

confidence: 99%

Rapid, robust plasmid verification by de novo assembly of short sequencing reads

Gallegos

Rogers²,

Cialek

et al. 2020

Preprint

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show abstract

“…For instance, the transcriptional activation of many plant-dominant resistance genes by CRISPR-Act2.0 would enhance the plant immunity to confer a broad range of resistance to different kinds of pathogens (Lowder et al, 2018), and the primer editor is also an interesting alternative applied into the antiviral engineering (Anzalone et al, 2019). The multiplexed CRISPR technologies, in which numerous gRNAs or Cas proteins are expressed at once, have vastly enhanced the scope and efficiency of genetic editing and transcriptional regulation (McCarty et al, 2020). However, the application of these multiplexed CRISPR technologies into the field to control plant viruses is still on the way, mainly because of the complexity of experimental manipulation, the limitations of the genetic transformation of several simultaneous Cas proteins into one plant, and the lack of validated and effective endogenous targets against different viruses, among other things (McCarty et al, 2020).…”

Section: Conclusion and Future Perspectivesmentioning

confidence: 99%

Control of Plant Viruses by CRISPR/Cas System-Mediated Adaptive Immunity

Cao

Zhou

et al. 2020

Front. Microbiol.

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Plant diseases caused by invading plant viruses pose serious threats to agricultural production in the world, and the antiviral engineering initiated by molecular biotechnology has been an effective strategy to prevent and control plant viruses. Recent advances in clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system-mediated DNA or RNA editing/interference in plants make them very attractive tools applicable to the plant protection field. Here, we review the development of CRISPR/Cas systems and summarize their applications in controlling different plant viruses by targeting viral sequences or host susceptibility genes. We list some potential recessive resistance genes that can be utilized in antiviral breeding and emphasize the importance and promise of recessive resistance gene-based antiviral breeding to generate transgene-free plants without developmental defects. Finally, we discuss the challenges and opportunities for the application of CRISPR/Cas techniques in the prevention and control of plant viruses in the field.

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“…In addition, given the potential for CRISPRi for multiplexed gene silencing [25], it would be interesting to knockdown TMEM97 together with other AMD-associated genes to determine if these genes have synergistic contribution to retinal degeneration and pathogenesis of AMD.…”

Section: To Enablementioning

confidence: 99%

Functional study of the AMD-associated geneTMEM97in retinal pigmented epithelium using CRISPR interference

Wang

Urrutia-Cabrera

Mora

et al. 2020

Preprint

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Age-related macular degeneration (AMD) is a blinding disease characterised by dysfunction of the retinal pigmented epithelium (RPE) which culminates in disruption or loss of the neurosensory retina. Genome-wide association studies have identified >65 genetic risk factors for AMD, including the TMEM97 locus. TMEM97 encodes the Sigma-2 receptor which is involved in apoptosis and cytotoxicity across a range of neurodegenerative diseases. However, the expression pattern of TMEM97 in the human retina and its functional role in retinal cells has remained elusive. Here we utilised CRISPR interference (CRISPRi) to investigate the functional role of TMEM97 in the retina. Transcriptome analysis of all major cell types within the human retina showed that TMEM97 is expressed in the RPE, retinal ganglion cells (RGCs) and amacrine cells. Using CRISPRi, we performed loss-of-function study of TMEM97 in the human RPE cell line, ARPE19. We generated a stable ARPE19 cell line expressing dCas9-KRAB which facilitated knockdown of TMEM97 using specific sgRNAs. Our results show that knockdown of TMEM97 in ARPE19 exerts a protective effect against oxidative stress-induced cell death. This work provides the first functional study of TMEM97 in RPE and supports the role of TMEM97 in AMD pathobiology. Our study highlights the potential for using CRISPRi to study AMD genetics, and the CRISPRi cell line generated here provided an useful in vitro tool for functional studies of other AMD-associated genes.

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Multiplexed CRISPR technologies for gene editing and transcriptional regulation

Cited by 306 publications

References 148 publications

Rapid, robust plasmid verification by de novo assembly of short sequencing reads

Rapid, robust plasmid verification by de novo assembly of short sequencing reads

Control of Plant Viruses by CRISPR/Cas System-Mediated Adaptive Immunity

Functional study of the AMD-associated geneTMEM97in retinal pigmented epithelium using CRISPR interference

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