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1. A CRISPRi-dCas9 System for Archaea and Its Use To Examine Gene Function during Nitrogen Fixation by Methanosarcina acetivorans

   https://doi.org/10.1128/AEM.01402-20  

   Dhamad, Ahmed E.; Lessner, Daniel J. (October 2020, Applied and Environmental Microbiology)

   Atomi, Haruyuki (Ed.)

   ABSTRACT CRISPR-based systems are emerging as the premier method to manipulate many cellular processes. In this study, a simple and efficient CRISPR interference (CRISPRi) system for targeted gene repression in archaea was developed. The Methanosarcina acetivorans CRISPR-Cas9 system was repurposed by replacing Cas9 with the catalytically dead Cas9 (dCas9) to generate a CRISPRi-dCas9 system for targeted gene repression. To test the utility of the system, genes involved in nitrogen (N 2 ) fixation were targeted for dCas9-mediated repression. First, the nif operon ( nifHI 1 I 2 DKEN ) that encodes molybdenum nitrogenase was targeted by separate guide RNAs (gRNAs), one targeting the promoter and the other targeting nifD . Remarkably, growth of M. acetivorans with N 2 was abolished by dCas9-mediated repression of the nif operon with each gRNA. The abundance of nif transcripts was >90% reduced in both strains expressing the gRNAs, and NifD was not detected in cell lysate. Next, we targeted NifB, which is required for nitrogenase cofactor biogenesis. Expression of a gRNA targeting the coding sequence of NifB decreased nifB transcript abundance >85% and impaired but did not abolish growth of M. acetivorans with N 2 . Finally, to ascertain the ability to study gene regulation using CRISPRi-dCas9, nrpR1 , encoding a subunit of the repressor of the nif operon, was targeted. The nrpR1 repression strain grew normally with N 2 but had increased nif operon transcript abundance, consistent with NrpR1 acting as a repressor. These results highlight the utility of the system, whereby a single gRNA when expressed with dCas9 can block transcription of targeted genes and operons in M. acetivorans . IMPORTANCE Genetic tools are needed to understand and manipulate the biology of archaea, which serve critical roles in the biosphere. Methanogenic archaea (methanogens) are essential for the biological production of methane, an intermediate in the global carbon cycle, an important greenhouse gas, and a biofuel. The CRISPRi-dCas9 system in the model methanogen Methanosarcina acetivorans is, to our knowledge, the first Cas9-based CRISPR interference system in archaea. Results demonstrate that the system is remarkably efficient in targeted gene repression and provide new insight into nitrogen fixation by methanogens, the only archaea with nitrogenase. Overall, the CRISPRi-dCas9 system provides a simple, yet powerful, genetic tool to control the expression of target genes and operons in methanogens. 

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2. Advances in CRISPR-enabled genome-wide screens in yeast

   https://doi.org/10.1093/femsyr/foaf013  

   Robertson, Nicholas_R; Lee, Sangcheon; Tafrishi, Aida; Wheeldon, Ian (March 2025, FEMS Yeast Research)

   Abstract Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas genome-wide screens are powerful tools for unraveling genotype–phenotype relationships, enabling precise manipulation of genes to study and engineer industrially useful traits. Traditional genetic methods, such as random mutagenesis or RNA interference, often lack the specificity and scalability required for large-scale functional genomic screens. CRISPR systems overcome these limitations by offering precision gene targeting and manipulation, allowing for high-throughput investigations into gene function and interactions. Recent work has shown that CRISPR genome editing is widely adaptable to several yeast species, many of which have natural traits suited for industrial biotechnology. In this review, we discuss recent advances in yeast functional genomics, emphasizing advancements made with CRISPR tools. We discuss how the development and optimization of CRISPR genome-wide screens have enabled a host-first approach to metabolic engineering, which takes advantage of the natural traits of nonconventional yeast—fast growth rates, high stress tolerance, and novel metabolism—to create new production hosts. Lastly, we discuss future directions, including automation and biosensor-driven screens, to enhance high-throughput CRISPR-enabled yeast engineering. 

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3. The CRISPR-Cas Mechanism for Adaptive Immunity and Alternate Bacterial Functions Fuels Diverse Biotechnologies

   https://doi.org/10.3389/fcimb.2020.619763  

   Newsom, Sydney; Parameshwaran, Hari Priya; Martin, Lindsie; Rajan, Rakhi (January 2021, Frontiers in Cellular and Infection Microbiology)

   null (Ed.)

   Bacterial and archaeal CRISPR-Cas systems offer adaptive immune protection against foreign mobile genetic elements (MGEs). This function is regulated by sequence specific binding of CRISPR RNA (crRNA) to target DNA/RNA, with an additional requirement of a flanking DNA motif called the protospacer adjacent motif (PAM) in certain CRISPR systems. In this review, we discuss how the same fundamental mechanism of RNA-DNA and/or RNA-RNA complementarity is utilized by bacteria to regulate two distinct functions: to ward off intruding genetic materials and to modulate diverse physiological functions. The best documented examples of alternate functions are bacterial virulence, biofilm formation, adherence, programmed cell death, and quorum sensing. While extensive complementarity between the crRNA and the targeted DNA and/or RNA seems to constitute an efficient phage protection system, partial complementarity seems to be the key for several of the characterized alternate functions. Cas proteins are also involved in sequence-specific and non-specific RNA cleavage and control of transcriptional regulator expression, the mechanisms of which are still elusive. Over the past decade, the mechanisms of RNA-guided targeting and auxiliary functions of several Cas proteins have been transformed into powerful gene editing and biotechnological tools. We provide a synopsis of CRISPR technologies in this review. Even with the abundant mechanistic insights and biotechnology tools that are currently available, the discovery of new and diverse CRISPR types holds promise for future technological innovations, which will pave the way for precision genome medicine. 

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4. CRISPR gene editing to improve crop resistance to parasitic plants

   https://doi.org/10.3389/fgeed.2023.1289416  

   Jhu, Min-Yao; Ellison, Evan E; Sinha, Neelima R (October 2023, Frontiers in Genome Editing)

   Parasitic plants pose a significant threat to global agriculture, causing substantial crop losses and hampering food security. In recent years, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) gene-editing technology has emerged as a promising tool for developing resistance against various plant pathogens. Its application in combating parasitic plants, however, remains largely unexplored. This review aims to summarise current knowledge and research gaps in utilising CRISPR to develop resistance against parasitic plants. First, we outline recent improvements in CRISPR gene editing tools, and what has been used to combat various plant pathogens. To realise the immense potential of CRISPR, a greater understanding of the genetic basis underlying parasitic plant-host interactions is critical to identify suitable target genes for modification. Therefore, we discuss the intricate interactions between parasitic plants and their hosts, highlighting essential genes and molecular mechanisms involved in defence response and multilayer resistance. These include host resistance responses directly repressing parasitic plant germination or growth and indirectly influencing parasitic plant development via manipulating environmental factors. Finally, we evaluate CRISPR-mediated effectiveness and long-term implications for host resistance and crop improvement, including inducible resistance response and tissue-specific activity. In conclusion, this review highlights the challenges and opportunities CRISPR technology provides to combat parasitic plants and provides insights for future research directions to safeguard global agricultural productivity. 

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5. Genome editing in rice and tomato with a small Un1Cas12f1 nuclease

   https://doi.org/10.1002/tpg2.20465  

   Tang, Xu; Eid, Ayman; Zhang, Rui; Cheng, Yanhao; Liu, Annan; Chen, Yurong; Chen, Pengxu; Zhang, Yong; Qi, Yiping (May 2024, The Plant Genome)

   Abstract The clustered regularly interspaced short palindromic repeats (CRISPR) systems have been demonstrated to be the foremost compelling genetic tools for manipulating prokaryotic and eukaryotic genomes. Despite the robustness and versatility of Cas9 and Cas12a/b nucleases in mammalian cells and plants, their large protein sizes may hinder downstream applications. Therefore, investigating compact CRISPR nucleases will unlock numerous genome editing and delivery challenges that constrain genetic engineering and crop development. In this study, we assessed the archaeal miniature Un1Cas12f1 type‐V CRISPR nuclease for genome editing in rice and tomato protoplasts. By adopting the reengineered guide RNA modifications ge4.1 and comparing polymerase II (Pol II) and polymerase III (Pol III) promoters, we demonstrated uncultured archaeon Cas12f1 (Un1Cas12f1) genome editing efficacy in rice and tomato protoplasts. We characterized the protospacer adjacent motif (PAM) requirements and mutation profiles of Un1Cas12f1 in both plant species. Interestingly, we found that Pol III promoters, not Pol II promoters, led to higher genome editing efficiency when they were used to drive guide RNA expression. Unlike in mammalian cells, the engineered Un1Cas12f1‐RRA variant did not perform better than the wild‐type Un1Cas12f1 nuclease, suggesting continued protein engineering and other innovative approaches are needed to further improve Un1Cas12f1 genome editing in plants. 

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