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1. Analyzing CRISPR screens in non-conventional microbes

   https://doi.org/10.1093/jimb/kuad006  

   Trivedi, Varun ; Ramesh, Adithya ; Wheeldon, Ian ( March 2023 , Journal of Industrial Microbiology and Biotechnology)

   The multifaceted nature of CRISPR screens has propelled advancements in the field of functional genomics. Pooled CRISPR screens involve creating programmed genetic perturbations across multiple genomic sites in a pool of host cells subjected to a challenge, empowering researchers to identify genetic causes of desirable phenotypes. These genome-wide screens have been widely used in mammalian cells to discover biological mechanisms of diseases and drive the development of targeted drugs and therapeutics. Their use in non-model organisms, especially in microbes to improve bioprocessing-relevant phenotypes, has been limited. Further compounding this issue is the lack of bioinformatic algorithms for analyzing microbial screening data with high accuracy. Here, we describe the general approach and underlying principles for conducting pooled CRISPR knockout screens in non-conventional yeasts and performing downstream analysis of the screening data, while also reviewing state-of-the-art algorithms for identification of CRISPR screening outcomes. Application of pooled CRISPR screens to non-model yeasts holds considerable potential to uncover novel metabolic engineering targets and improve industrial bioproduction.

   This mini-review describes experimental and computational approaches for functional genomic screening using CRISPR technologies in non-conventional microbes.

    

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2. Transcriptional and epigenetic regulators of human CD8+ T cell function identified through orthogonal CRISPR screens

   https://doi.org/10.1038/s41588-023-01554-0  

   McCutcheon, Sean R. ; Swartz, Adam M. ; Brown, Michael C. ; Barrera, Alejandro ; McRoberts Amador, Christian ; Siklenka, Keith ; Humayun, Lucas ; ter Weele, Maria A. ; Isaacs, James M. ; Reddy, Timothy E. ; et al ( November 2023 , Nature Genetics)

   Clinical response to adoptive T cell therapies is associated with the transcriptional and epigenetic state of the cell product. Thus, discovery of regulators of T cell gene networks and their corresponding phenotypes has potential to improve T cell therapies. Here we developed pooled, epigenetic CRISPR screening approaches to systematically profile the effects of activating or repressing 120 transcriptional and epigenetic regulators on human CD8⁺T cell state. We found that BATF3 overexpression promoted specific features of memory T cells and attenuated gene programs associated with cytotoxicity, regulatory T cell function, and exhaustion. Upon chronic antigen stimulation, BATF3 overexpression countered phenotypic and epigenetic signatures of T cell exhaustion. Moreover, BATF3 enhanced the potency of CAR T cells in both in vitro and in vivo tumor models and programmed a transcriptional profile that correlates with positive clinical response to adoptive T cell therapy. Finally, we performed CRISPR knockout screens that defined cofactors and downstream mediators of the BATF3 gene network.

    

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3. Engineering digitizer circuits for chemical and genetic screens in human cells

   https://doi.org/10.1038/s41467-021-26359-9  

   Wong, Nicole M. ; Frias, Elizabeth ; Sigoillot, Frederic D. ; Letendre, Justin H. ; Hild, Marc ; Wong, Wilson W. ( October 2021 , Nature Communications)

   Cell-based transcriptional reporters are invaluable in high-throughput compound and CRISPR screens for identifying compounds or genes that can impact a pathway of interest. However, many transcriptional reporters have weak activities and transient responses. This can result in overlooking therapeutic targets and compounds that are difficult to detect, necessitating the resource-consuming process of running multiple screens at various timepoints. Here, we present RADAR, a digitizer circuit for amplifying reporter activity and retaining memory of pathway activation. Reporting on the AP-1 pathway, our circuit identifies compounds with known activity against PKC-related pathways and shows an enhanced dynamic range with improved sensitivity compared to a classical reporter in compound screens. In the first genome-wide pooled CRISPR screen for the AP-1 pathway, RADAR identifies canonical genes from the MAPK and PKC pathways, as well as non-canonical regulators. Thus, our scalable system highlights the benefit and versatility of using genetic circuits in large-scale cell-based screening.

    

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4. Click chemistry–enabled CRISPR screening reveals GSK3 as a regulator of PLD signaling

   https://doi.org/10.1073/pnas.2025265118  

   Bumpus, Timothy W. ; Huang, Shiying ; Tei, Reika ; Baskin, Jeremy M. ( November 2021 , Proceedings of the National Academy of Sciences)

   Enzymes that produce second messengers are highly regulated. Revealing the mechanisms underlying such regulation is critical to understanding both how cells achieve specific signaling outcomes and return to homeostasis following a particular stimulus. Pooled genome-wide CRISPR screens are powerful unbiased approaches to elucidate regulatory networks, their principal limitation being the choice of phenotype selection. Here, we merge advances in bioorthogonal fluorescent labeling and CRISPR screening technologies to discover regulators of phospholipase D (PLD) signaling, which generates the potent lipid second messenger phosphatidic acid. Our results reveal glycogen synthase kinase 3 as a positive regulator of protein kinase C and PLD signaling. More generally, this work demonstrates how bioorthogonal, activity-based fluorescent tagging can expand the power of CRISPR screening to uncover mechanisms regulating specific enzyme-driven signaling pathways in mammalian cells. 

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5. acCRISPR: an activity-correction method for improving the accuracy of CRISPR screens

   https://doi.org/10.1038/s42003-023-04996-8  

   Ramesh, Adithya ; Trivedi, Varun ; Lee, Sangcheon ; Tafrishi, Aida ; Schwartz, Cory ; Mohseni, Amirsadra ; Li, Mengwan ; Lonardi, Stefano ; Wheeldon, Ian ( June 2023 , Communications Biology)

   High throughput CRISPR screens are revolutionizing the way scientists unravel the genetic underpinnings of engineered and evolved phenotypes. One of the critical challenges in accurately assessing screening outcomes is accounting for the variability in sgRNA cutting efficiency. Poorly active guides targeting genes essential to screening conditions obscure the growth defects that are expected from disrupting them. Here, we develop acCRISPR, an end-to-end pipeline that identifies essential genes in pooled CRISPR screens using sgRNA read counts obtained from next-generation sequencing. acCRISPR uses experimentally determined cutting efficiencies for each guide in the library to provide an activity correction to the screening outcomes via calculation of an optimization metric, thus determining the fitness effect of disrupted genes. CRISPR-Cas9 and -Cas12a screens were carried out in the non-conventional oleaginous yeastYarrowia lipolyticaand acCRISPR was used to determine a high-confidence set of essential genes for growth under glucose, a common carbon source used for the industrial production of oleochemicals. acCRISPR was also used in screens quantifying relative cellular fitness under high salt conditions to identify genes that were related to salt tolerance. Collectively, this work presents an experimental-computational framework for CRISPR-based functional genomics studies that may be expanded to other non-conventional organisms of interest.

    

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