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1. Integrating Biomaterials and Genome Editing Approaches to Advance Biomedical Science

   https://doi.org/10.1146/annurev-bioeng-122019-121602  

   Abdeen, Amr A.; Cosgrove, Brian D.; Gersbach, Charles A.; Saha, Krishanu (July 2021, Annual Review of Biomedical Engineering)

   null (Ed.)

   The recent discovery and subsequent development of the CRISPR–Cas9 (clustered regularly interspaced short palindromic repeat–CRISPR-associated protein 9) platform as a precise genome editing tool have transformed biomedicine. As these CRISPR-based tools have matured, multiple stages of the gene editing process and the bioengineering of human cells and tissues have advanced. Here, we highlight recent intersections in the development of biomaterials and genome editing technologies. These intersections include the delivery of macromolecules, where biomaterial platforms have been harnessed to enable nonviral delivery of genome engineering tools to cells and tissues in vivo. Further, engineering native-like biomaterial platforms for cell culture facilitates complex modeling of human development and disease when combined with genome engineering tools. Deeper integration of biomaterial platforms in these fields could play a significant role in enabling new breakthroughs in the application of gene editing for the treatment of human disease. 

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2. A frugal CRISPR kit for equitable and accessible education in gene editing and synthetic biology

   https://doi.org/10.1038/s41467-024-50767-2  

   Collins, Marvin; Lau, Matthew B; Ma, William; Shen, Aidan; Wang, Brenda; Cai, Sa; La_Russa, Marie; Jewett, Michael C; Qi, Lei S (December 2024, Nature Communications)

   Equitable and accessible education in life sciences, bioengineering, and synthetic biology is crucial for training the next generation of scientists, fostering transparency in public decision-making, and ensuring biotechnology can benefit a wide-ranging population. As a groundbreaking technology for genome engineering, CRISPR has transformed research and therapeutics. However, hands-on exposure to this technology in educational settings remains limited due to the extensive resources required for CRISPR experiments. Here, we develop CRISPRkit, an affordable kit designed for gene editing and regulation in high school education. CRISPRkit eliminates the need for specialized equipment, prioritizes biosafety, and utilizes cost-effective reagents. By integrating CRISPRi gene regulation, colorful chromoproteins, cell-free transcription-translation systems, smartphone-based quantification, and an in-house automated algorithm (CRISPectra), our kit offers an inexpensive (~$2) and user-friendly approach to performing and analyzing CRISPR experiments, without the need for a traditional laboratory setup. Experiments conducted by high school students in classroom settings highlight the kit's utility for reliable CRISPRkit experiments. Furthermore, CRISPRkit provides a modular and expandable platform for genome engineering, and we demonstrate its applications for controlling fluorescent proteins and metabolic pathways such as melanin production. We envision CRISPRkit will facilitate biotechnology education for communities of diverse socioeconomic and geographic backgrounds. 

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3. Genome editing in ubiquitous freshwater Actinobacteria

   https://doi.org/10.1128/aem.00865-24  

   Bairagi, Nachiketa; Keffer, Jessica L; Heydt, Jordan C; Maresca, Julia A (November 2024, Applied and Environmental Microbiology)

   Bose, Arpita (Ed.)

   ABSTRACT Development of genome-editing tools in diverse microbial species is an important step both in understanding the roles of those microbes in different environments, and in engineering microbes for a variety of applications. Freshwater-specific clades of Actinobacteria are ubiquitous and abundant in surface freshwaters worldwide. Here, we show thatRhodoluna lacicolaandAurantimicrobium photophilum, which represent widespread clades of freshwater Actinobacteria, are naturally transformable. We also show that gene inactivation via double homologous recombination and replacement of the target gene with antibiotic selection markers can be used in both strains, making them convenient and broadly accessible model organisms for freshwater systems. We further show that in both strains, the predicted phytoene synthase is the only phytoene synthase, and its inactivation prevents the synthesis of all pigments. The tools developed here enable targeted modification of the genomes of some of the most abundant microbes in freshwater communities. These genome-editing tools will enable hypothesis testing about the genetics and (eco)physiology of freshwater Actinobacteria and broaden the available model systems for engineering freshwater microbial communities. IMPORTANCETo advance bioproduction or bioremediation in large, unsupervised environmental systems such as ponds, wastewater lagoons, or groundwater systems, it will be necessary to develop diverse genetically amenable microbial model organisms. Although we already genetically modify a few key species, tools for engineering more microbial taxa, with different natural phenotypes, will enable us to genetically engineer multispecies consortia or even complex communities. Developing genetic tools for modifying freshwater bacteria is particularly important, as wastewater, production ponds or raceways, and contaminated surface water are all freshwater systems where microbial communities are already deployed to do work, and the outputs could potentially be enhanced by genetic modifications. Here, we demonstrate that common tools for genome editing can be used to inactivate specific genes in two representatives of a very widespread, environmentally relevant group of Actinobacteria. These Actinobacteria are found in almost all tested surface freshwater environments, where they co-occur with primary producers, and genome-editing tools in these species are thus a step on the way to engineering microbial consortia in freshwater environments. 

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4. Development of a homeolog-specific gene editing system in an evolutionary model for the study of polyploidy in nature

   https://doi.org/10.3389/fgeed.2025.1645542  

   Shan, Shengchen; Pisias, Michael T; Mavrodiev, Evgeny V; Spoelhof, Jonathan P; Hauser, Bernard A; Barbazuk, W Brad; Soltis, Pamela S; Soltis, Douglas E; Yang, Bing (August 2025, Frontiers in Genome Editing)

   Polyploidy, or whole-genome duplication (WGD), is a significant evolutionary force. Following allopolyploidy, duplicate gene copies (homeologs) have divergent evolutionary trajectories: some genes are preferentially retained in duplicate, while others tend to revert to single-copy status. Examining the effect of homeolog loss (i.e., changes in gene dosage) on associated phenotypes is essential for unraveling the genetic mechanisms underlying polyploid genome evolution. However, homeolog-specific editing has been demonstrated in only a few crop species and remains unexplored beyond agricultural applications.Tragopogon(Asteraceae) includes an evolutionary model system for studying the immediate consequences of polyploidy in nature. In this study, we developed a CRISPR-mediated homeolog-specific editing platform in allotetraploidT. mirus. Using theMYB10andDFRgenes as examples, we successfully knocked out the targeted homeolog inT. mirus(4x) without editing the other homeolog (i.e., no off-target events). The editing efficiencies, defined as the percentage of plants with at least one allele of the targeted homeolog modified, were 35.7% and 45.5% forMYB10andDFR, respectively. Biallelic modification of the targeted homeolog occurred in the T₀generation. These results demonstrate the robustness of homeolog-specific editing in polyploidTragopogon, laying the foundation for future studies of genome evolution following WGD in nature. 

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5. CRISPR-Based Approaches for Gene Regulation in Non-Model Bacteria

   https://doi.org/10.3389/fgeed.2022.892304  

   Call, Stephanie N.; Andrews, Lauren B. (June 2022, Frontiers in Genome Editing)

   CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) have become ubiquitous approaches to control gene expression in bacteria due to their simple design and effectiveness. By regulating transcription of a target gene(s), CRISPRi/a can dynamically engineer cellular metabolism, implement transcriptional regulation circuitry, or elucidate genotype-phenotype relationships from smaller targeted libraries up to whole genome-wide libraries. While CRISPRi/a has been primarily established in the model bacteria Escherichia coli and Bacillus subtilis , a growing numbering of studies have demonstrated the extension of these tools to other species of bacteria (here broadly referred to as non-model bacteria). In this mini-review, we discuss the challenges that contribute to the slower creation of CRISPRi/a tools in diverse, non-model bacteria and summarize the current state of these approaches across bacterial phyla. We find that despite the potential difficulties in establishing novel CRISPRi/a in non-model microbes, over 190 recent examples across eight bacterial phyla have been reported in the literature. Most studies have focused on tool development or used these CRISPRi/a approaches to interrogate gene function, with fewer examples applying CRISPRi/a gene regulation for metabolic engineering or high-throughput screens and selections. To date, most CRISPRi/a reports have been developed for common strains of non-model bacterial species, suggesting barriers remain to establish these genetic tools in undomesticated bacteria. More efficient and generalizable methods will help realize the immense potential of programmable CRISPR-based transcriptional control in diverse bacteria. 

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