Multiplexed CRISPR technologies, in which numerous gRNAs or Cas enzymes are expressed at once, have facilitated powerful biological engineering applications, vastly enhancing the scope and efficiencies of genetic editing and transcriptional regulation. In this review, we discuss multiplexed CRISPR technologies and describe methods for the assembly, expression and processing of synthetic guide RNA arrays in vivo. Applications that benefit from multiplexed CRISPR technologies, including cellular recorders, genetic circuits, biosensors, combinatorial genetic perturbations, large-scale genome engineering and the rewiring of metabolic pathways, are highlighted. We also offer a glimpse of emerging challenges and emphasize experimental considerations for future studies.

/pdf/multiplexed-crispr-technologies-for-gene-editing-and-3cvs0mh9ts.pdf

Multiplexed CRISPR technologies for gene editing and transcriptional regulation.

In precision oncology, two major strategies are being pursued for predicting clinically relevant tumour behaviours, such as treatment response and emergence of drug resistance: inference based on genomic, transcriptomic, epigenomic and/or proteomic analysis of patient samples, and phenotypic assays in personalized cancer avatars. The latter approach has historically relied on in vivo mouse xenografts and in vitro organoids or 2D cell cultures. Recent progress in rapid combinatorial genetic modelling, the development of a genetically immunocompromised strain for xenotransplantation of human patient samples in adult zebrafish and the first clinical trial using xenotransplantation in zebrafish larvae for phenotypic testing of drug response bring this tiny vertebrate to the forefront of the precision medicine arena. In this Review, we discuss advances in transgenic and transplantation-based zebrafish cancer avatars, and how these models compare with and complement mouse xenografts and human organoids. We also outline the unique opportunities that these different models present for prediction studies and current challenges they face for future clinical deployment.

Zebrafish patient avatars in cancer biology and precision cancer therapy.

groups, as these groups were defined based on the immune profile seen at T3. Consequently these patients were not included in the subset of analyses involving the CD8 high/low groups Mass cytometry on patient samples was not repeated due to the limited volume of clinical material. Biological replicates ranged from 5 to 13 independent samples per group per timepoint (see Suppl Table 2), depending on the number of samples available. Samples were stained across 8 batches containing a mix of patient groups and a healthy donor control. The study did not involve in vitro functional assays. Randomisation was not relevant to our study design as our goal was to compare immune reconstitution across four different patient groups, defined as follows: The patients were retrospectively divided into four groups according to pre-transplant HCMV serostatus and magnitude of post-transplant HCMV reactivation. The ‘Seronegative’ (SN; n=11) group were HCMV-seronegative recipients with seronegative donors (D-/R-), and had no detected HCMV DNAemia. The ‘Seropositive No Reactivation’ (SP-NR; n=5) group were HCMV-seropositive recipient (R+) and/or donor (D+) patients with no documented HCMV reactivation in the first 100 days post-HSCT. ‘Low-level reactivators’ (LR; n=6) developed HCMV reactivation to <250 peak copies/mL. ‘High-level reactivators’ (HR; n=13) developed HCMV reactivation to >830 peak copies/mL. No patients in the study had peak HCMV titres between 250-830 copies/mL. Blinding was not relevant to our study as we retrospectively assigned patients into four different groups, based upon their HCMV serostatus and any subsequent post-transplant HCMV reactivation. However, at the time of collection and initial processing of patient samples, we were blinded to these parameters. 175Lu-CD279 (PD-1): clone EH12.2H7, 8  g/mL, BioLegend, catalogue number: 329902 176Yb-CD56: clone NCAM16.2, 1  g/mL, Becton Dickinson, catalogue number: 559043 formalin-fixed

A model of the post-implantation human embryo derived from pluripotent stem cells.

Next-generation cell therapies: the emerging role of CAR-NK cells.

CRISPR-Cas systems have revolutionized genome engineering by facilitating a wide range of targeted DNA perturbations. These systems have resulted in new powerful screens to test gene functions at the genomic scale. While there is tremendous potential for CRISPR screens to map and interrogate gene regulatory networks at unprecedented speed and scale, their implementation in plants remains in its infancy. Here we discuss the general concepts, tools and workflows for establishing CRISPR screens in plants and analyze the handful of recent reports using this strategy to generate mutant knockout collections or diversify DNA sequences. In addition, we provide insight on how to design CRISPR knockout screens in plants given the current challenges and limitations and examine multiple design options. Finally, we discuss the unique multiplexing capabilities of CRISPR screens to investigate redundant gene function in highly duplicated plant genomes. Combinatorial mutant screens have the potential to routinely generate higher-order mutant collections and facilitate the characterization of gene networks. By integrating this approach with the large resource of genomic profiles that were generated in the last two decades, the implementation of CRISPR screens offers new opportunities to analyze plant genomes at deeper resolution and will greatly advance plant functional and synthetic biology.

https://biblio.ugent.be/publication/8674195/file/8674196.pdf

CRISPR screens in plants: approaches, guidelines, and future prospects

The CRISPR/Cas9 system is an RNA guided nuclease system that evolved as a mechanism of adaptive immunity in bacteria. This system has been adopted for numerous genome engineering applications in research and recently, therapeutics. The CRISPR/Cas9 system has been largely implemented by delivery of Cas9 as protein, RNA, or plasmid along with a chimeric crRNA-tracrRNA guide RNA (gRNA) under the expression of a pol III promoter, such as U6. Using this approach, multiplex genome engineering has been achieved by delivering several U6-gRNA plasmids targeting multiple loci. However, this approach is limiting due to the efficiently of delivering multiple plasmids to a single cell at one time. To augment the capability and accessibility of multiplexed genome engineering, we developed an efficient golden gate based method to assemble gRNAs linked by optimal Csy4 ribonuclease sequences to deliver up to 10 gRNAs as a single gRNA array transcript. Here we report the optimal expression of our guide RNA array under a strong pol II promoter. This system can be implemented alongside the myriad of CRISPR applications, allowing users to model complex biological processes requiring numerous gRNAs.

/pdf/highly-multiplexed-genome-engineering-using-crispr-cas9-grna-4iilej6qf6.pdf

Highly Multiplexed Genome Engineering Using CRISPR/Cas9 gRNA Arrays

The CRISPR/Cas9 system is an RNA guided nuclease system that evolved as a mechanism of adaptive immunity in bacteria. This system has been adopted for numerous genome engineering applications in research and recently, therapeutics. The CRISPR/Cas9 system has been largely implemented by delivery of Cas9 as protein, RNA, or plasmid along with a chimeric crRNA-tracrRNA guide RNA (gRNA) under the expression of a pol III promoter, such as U6. Using this approach, multiplex genome engineering has been achieved by delivering several U6-gRNA plasmids targeting multiple loci. However, this approach is limited due to the efficiently of delivering multiple plasmids to a single cell at one time. To augment the capability and accessibility of multiplexed genome engineering, we developed an efficient golden gate based method to assemble gRNAs linked by optimal Csy4 ribonuclease sequences to deliver up to 10 gRNAs as a single gRNA array transcript. Here we report the optimal expression of our guide RNA array under a strong pol II promoter. This system can be implemented alongside the myriad of CRISPR applications, allowing users to model complex biological processes requiring numerous gRNAs.

/pdf/highly-multiplexed-genome-engineering-using-crispr-cas9-grna-11ngj48wpu.pdf

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Highly Multiplexed Genome Engineering Using CRISPR/Cas9 gRNA Arrays

Highly Multiplexed Genome Engineering Using CRISPR/Cas9 gRNA Arrays