Advances In Genome Engineering Research Articles

Streamlined Generation of CRISPR/Cas9-Mediated Single-Cell Knockout Clones in Murine Cell Lines.

Clonal cell lines harboring loss-of-function mutations in genes of interest are crucial for studying the cellular functions of the encoded proteins. Recent advances in genome engineering have converged on the CRISPR/Cas9 technology to quickly and reliably generate frameshift mutations in the target genes across various cell lines and species. Although high on-target cleavage efficiencies can be obtained reproducibly, screening and identifying clones with loss-of-function alleles remains a major bottleneck. Here, we describe a single sgRNA strategy to generate CRISPR/Cas9-mediated frameshift mutations in target genes of mammalian cell lines that can be easily and cost-effectively identified. Given the proliferation of workhorse cell lines such as N2a cells and the resulting clonal expansion of the cell type, our protocol can facilitate the isolation of knockout clonal cell lines and their genetic validation within a period of down to 6-8 weeks.

Abstract NG03: Functional and computational approaches to uncover selection advantages of cancer aneuploidy

Abstract Aneuploidy, including the gain or loss of whole chromosomes or chromosome arms, is a near-universal feature of cancer. We previously applied methods that define chromosome arm aneuploidy to over 10,000 tumors in the Cancer Genome Atlas (TCGA). Cancer subtypes are often characterized by tumor specific patterns of chromosome arm copy number alterations and breakpoints; for example, squamous cell carcinomas (SCCs) from different tissues of origin are characterized by chromosome 3p (chr3p) loss and chromosome 3q (chr3q) gain. From the TCGA aneuploidy data, we developed an algorithm called BISCUT to distinguish peak regions of aneuploidy breakpoints on each chromosome arm. BISCUT identified loci affected by broad copy number alterations that provide fitness advantages or disadvantages both within individual cancer types and across cancers. Our analyses are consistent with selection being the primary driver of aneuploidy events in cancer. We next wanted to validate some BISCUT peaks without a known driver, to identify potential gene deletions that are beneficial in cancer cells. We focused on chromosome 8p (chr8p), as this arm is frequently deleted across cancer types, but no strong tumor suppressors have been identified. Recent advances in genome engineering allow generation of large chromosomal alterations and validation of findings from patient genomic data. For this study, we used our CRISPR-Cas9 arm-deletion system to delete chr8p in human immortalized epithelial cells. Cells with chr8p deletion showed lower amounts of cell death in culture. Knockdown of WRN, one of the two genes in the smallest chr8p BISCUT peak, was sufficient to reproduce this phenotype, suggesting that WRN haploinsufficiency may be beneficial to tumor development. As aneuploidy occurs in tissue-specific patterns, we also wanted to explore the role of chr3 arm aneuploidies in squamous cancers. We again used the CRISPR-Cas9 system to delete one copy of chr3p in a human immortalized lung epithelial cell line similar to the putative cell-of-origin in lung SCC. Consistent with patient data, expression of chr3p genes was decreased upon deletion, as well as increased expression of interferon response genes. Cells with chr3p deletion initially proliferated more slowly than their siblings. Interestingly, after several passages in culture, this proliferation defect was rescued in chr3p deleted cells. Genome sequencing and karyotype analyses suggested that this was partially the result of chr3 duplication, with cells transitioning to a state of chr3q gain. Culturing these cells as organoids also demonstrated that chr3p deletion and chr3q gain can affect basal cell differentiation, consistent with their high frequency in squamous cancers. Our genome engineering approach to model chromosome arm aneuploidies provides a robust model to validate drivers from aneuploidy events, which will be critical to address the gap in our understanding of aneuploidy in cancer. In addition, our methods have identified consequences of individual aneuploidy events, which will lead to new precision oncology targets for patients. Citation Format: Alison M. Taylor. Functional and computational approaches to uncover selection advantages of cancer aneuploidy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr NG03.

Recent advances in genome engineering by CRISPR technology

Recent advances in genome engineering by CRISPR technology

CRISPRi-Driven Genetic Screening for Designing Novel Microbial Phenotypes.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has enabled rapid advances in genomic engineering and transcriptional regulation. Specifically, CRISPR interference (CRISPRi) system has been used to systematically investigate the gene functions of microbial strains in a high-throughput manner. This method involves growth profiling using cells that have been transformed with the deactivated Cas9 (dCas9) and single-guide RNA (sgRNA) libraries that target individual genes. The fitness scores of each gene are calculated by measuring the abundance of individual sgRNAs during cell growth and represent gene essentiality. In this chapter, a process is described for functional genetic screening using CRISPRi at the whole-genome scale, starting from the synthesis of sgRNA libraries, construction of CRISPRi libraries, and identification of essential genes through growth profiling. The commensal bacterium Bacteroides thetaiotaomicron was used to implement the protocol. This method is expected to be applicable to a broader range of microorganisms to explore the novel phenotypic characteristics of microorganisms.

Context-dependent neocentromere activity in synthetic yeast chromosome VIII

Pioneering advances in genome engineering, and specifically in genome writing, have revolutionized the field of synthetic biology, propelling us toward the creation of synthetic genomes. The Sc2.0 project aims to build the first fully synthetic eukaryotic organism by assembling the genome of Saccharomyces cerevisiae. With the completion of synthetic chromosome VIII (synVIII) described here, this goal is within reach. In addition to writing the yeast genome, we sought to manipulate an essential functional element: the point centromere. By relocating the native centromere sequence to various positions along chromosome VIII, we discovered that the minimal 118-bp CEN8 sequence is insufficient for conferring chromosomal stability at ectopic locations. Expanding the transplanted sequence to include a small segment (∼500bp) of the CDEIII-proximal pericentromere improved chromosome stability, demonstrating that minimal centromeres display context-dependent functionality.

Rise of synthetic yeast: Charting courses to new applications

Microbes have long provided us with important capabilities, and the genome engineering of microbes has greatly empowered research and applications in biotechnology. This is especially true with the emergence of synthetic biology and recent advances in genome engineering to control microbial behavior. A fully synthetic, rationally designed genome promises opportunities for unprecedented control of cellular function. As a eukaryotic workhorse for research and industrial use, yeast is an organism at the forefront of synthetic biology; the tools and engineered cellular platform being delivered by the Sc2.0 consortium are enabling a new era of bespoke biology. This issue highlights recent advances delivered by this consortium, but hurdles remain to maximize the impact of engineered eukaryotic cells more broadly.

HIV-1 Remission: Accelerating the Path to Permanent HIV-1 Silencing.

Despite remarkable progress, a cure for HIV-1 infection remains elusive. Rebound competent latent and transcriptionally active reservoir cells persevere despite antiretroviral therapy and rekindle infection due to inefficient proviral silencing. We propose a novel "block-lock-stop" approach, entailing long term durable silencing of viral expression towards an irreversible transcriptionally inactive latent provirus to achieve long term antiretroviral free control of the virus. A graded transformation of remnant HIV-1 in PLWH from persistent into silent to permanently defective proviruses is proposed, emulating and accelerating the natural path that human endogenous retroviruses (HERVs) take over millions of years. This hypothesis was based on research into delineating the mechanisms of HIV-1 latency, lessons from latency reversing agents and advances of Tat inhibitors, as well as expertise in the biology of HERVs. Insights from elite controllers and the availability of advanced genome engineering technologies for the direct excision of remnant virus set the stage for a rapid path to an HIV-1 cure.

Sickle Cell Disease

Sickle cell disease and its variants constitute the most common inherited blood disorders affecting millions of individuals worldwide. Significant information regarding the nature of the genetic mutations and modifier genes that result in increased or decreased severity of the disease are available. In recent years, detailed data regarding molecular genetics, pathophysiology, mechanisms for the development of symptoms and side effects of sickle cell disease have been published. The relationship of physiological changes, cellular interactions, coexisting coagulation disorders, effects of association with other genetic disorders and a number of intervening factors have been explored. New techniques for pre-conception, prenatal, in utero, and neonatal screening are available. Means for prediction of the severity of the disease, clinical course of the disorder, and prevention of some of its major complications have been developed. The effects of psychosocial and environmental factors have been explored. Various therapeutic strategies including bone marrow and stem cell transplantation are currently employed in the treatment of patients with sickle cell disease. Recent progress in understanding the molecular pathways controlling mammalian erythropoiesis and globin switching, as well as advances in genome engineering, particularly the gene-editing techniques, have opened a venue for genetic-based treatment of the disease. Currently, sickle cell disease is often associated with a high rate of complications and mortality. The development of new pharmacological agents, methods for gene therapy, and alterations and modification of the coexisting genetic factors and modifiers for treatment of the disease are encouraging.

Multiplex genome engineering in Clostridium beijerinckii NCIMB 8052 using CRISPR-Cas12a

Clostridium species are re-emerging as biotechnological workhorses for industrial acetone–butanol–ethanol production. This re-emergence is largely due to advances in fermentation technologies but also due to advances in genome engineering and re-programming of the native metabolism. Several genome engineering techniques have been developed including the development of numerous CRISPR-Cas tools. Here, we expanded the CRISPR-Cas toolbox and developed a CRISPR-Cas12a genome engineering tool in Clostridium beijerinckii NCIMB 8052. By controlling the expression of FnCas12a with the xylose-inducible promoter, we achieved efficient (25–100%) single-gene knockout of five C. beijerinckii NCIMB 8052 genes (spo0A, upp, Cbei_1291, Cbei_3238, Cbei_3832). Moreover, we achieved multiplex genome engineering by simultaneously knocking out the spo0A and upp genes in a single step with an efficiency of 18%. Finally, we showed that the spacer sequence and position in the CRISPR array can affect the editing efficiency outcome.

Supporting crop plant resilience during climate change

AbstractThe changing climate poses significant threats to agriculture and the ability to ensure sufficient global food production. With the expanding population, there is an urgent demand to increase crop productivity to meet the rising food demand. Producing climate‐smart crop varieties together with developing new agronomic management strategies are strategies that may help address this issue. Recent advances in genomics‐assistant breeding, the use of high‐throughput DNA sequencing, high‐resolution phenotyping, and advanced genome engineering can support the development of advanced, climate resilient crops. Here, we assess the potential to enhance the resilience of crops under the changing climate. Through the use of big data, advanced breeding strategies, and advanced agriculture practices, crop varieties could be produced with enhanced resilience and increased productivity and nutrition, supporting future global food security.

Discovery and characterization of novel Cre-type tyrosine site-specific recombinases for advanced genome engineering.

Tyrosine-type site-specific recombinases (Y-SSRs) are versatile tools for genome engineering due to their ability to mediate excision, integration, inversion and exchange of genomic DNA with single nucleotide precision. The ever-increasing need for sophisticated genome engineering is driving efforts to identify novel SSR systems with intrinsic properties more suitable for particular applications. In this work, we develop a systematic computational workflow for annotation of putative Y-SSR systems and apply this pipeline to identify and characterize eight new naturally occurring Cre-type SSR systems. We test their activity in bacterial and mammalian cells and establish selectivity profiles for the new and already established Cre-type SSRs with regard to their ability to mutually recombine their target sites. These data form the basis for sophisticated genome engineering experiments using combinations of Y-SSRs in research fields including advanced genomics and synthetic biology. Finally, we identify putative pseudo-sites and potential off-targets for Y-SSRs in the human and mouse genome. Together with established methods for altering the DNA-binding specificity of this class of enzymes, this work should facilitate the use of Y-SSRs for future genome surgery applications.

Increasing Specificity of Targeted DNA Methylation Editing by Non-Enzymatic CRISPR/dCas9-Based Steric Hindrance.

As advances in genome engineering inch the technology towards wider clinical use-slowed by technical and ethical hurdles-a newer offshoot, termed "epigenome engineering", offers the ability to correct disease-causing changes in the DNA without changing its sequence and, thus, without some of the unfavorable correlates of doing so. In this review, we note some of the shortcomings of epigenetic editing technology-specifically the risks involved in the introduction of epigenetic enzymes-and highlight an alternative epigenetic editing strategy using physical occlusion to modify epigenetic marks at target sites without a requirement for any epigenetic enzyme. This may prove to be a safer alternative for more specific epigenetic editing.

CRISPR-Combo-mediated orthogonal genome editing and transcriptional activation for plant breeding.

CRISPR-Cas nuclease systems, base editors, and CRISPR activation have greatly advanced plant genome engineering. However, the combinatorial approaches for multiplexed orthogonal genome editing and transcriptional regulation were previously unexploited in plants. We have recently established a single Cas9 protein-based CRISPR-Combo platform, enabling efficient multiplexed orthogonal genome editing (double-strand break-mediated genome editing or base editing) and transcriptional activation in plants via engineering the single guide RNA (sgRNA) structure. Here, we provide step-by-step instructions for constructing CRISPR-Combo systems for speed breeding of transgene-free, genome-edited Arabidopsis plants and enhancing rice regeneration with more heritable targeted mutations in a hormone-free manner. We also provide guidance on designing efficient sgRNA, Agrobacterium-mediated transformation of Arabidopsis and rice, rice regeneration without exogenous plant hormones, gene editing evaluation and visual identification of transgene-free Arabidopsis plants with high editing activity. With the use of this protocol, it takes ~2 weeks to establish the CRISPR-Combo systems, 4 months to obtain transgene-free genome-edited Arabidopsis plants and 4 months to obtain rice plants with enrichment of heritable targeted mutations by hormone-free tissue culture.

Abstract 1783: High-throughput arrayed screening of logic-gated CARs enables the selection of candidates for ccRCC with optimal potency and fidelity traits

Abstract The development of clinically effective CAR-T cell products for solid tumors will require substantial cell engineering to confer sufficient specificity, potency, and persistence. Advances in genome engineering and synthetic biology have provided an increasingly complex set of features that can be introduced into CAR-T cells to augment their function. However, combining multiple features may result in unpredictable negative interactions between components. Here, we report the use of high-throughput screening to optimize the design of a highly-engineered Integrated Circuit T Cell (ICT) product for the treatment of clear cell renal cell carcinoma (ccRCC). ICT cells are CAR-T cells that contain an AND logic gate requiring two antigens to be present to trigger tumor cell killing together with multiple enhancement modules. First, to create the logic gate we generated hundreds of novel scFv and VH/VHH binders targeting PSMA (as a priming target) and CA9 (as a cytolytic target) via two parallel de novo binder discovery efforts: 1) transgenic mice immunizations and 2) internally-developed phage display panning campaigns. Two independent arrayed screens with 500 PSMA prime receptors (PrimeRTM) and 750 CA9 CARs were conducted to find PrimeRs with high inducibility and CARs with strong on-target potency. From these screens, the top 25 PSMA PrimeRs and 20 CA9 CARs were combined with an shRNA cassette for targeted knockdowns along with two variations of a persistence module. We used a fully-automated workcell to perform end-to-end arrayed screening of the resulting 1,000 member library in T cells engineered from four human donors. Non-viral editing techniques were used to electroporate primary CD4/CD8 cells and robotic handlers were used to set up co-cultures. Circuit specificity and potency were assessed by flow and cytokine secretion and resistance to exhaustion was assessed in a seven day killing assay. Although the library was built from components that functioned well independently, we found that when combined, many of the circuits displayed suboptimal function. Integrated screening identified 20 variants that each far exceeded the performance of a small set of initial prototypes built from “best-guess” selections of individual components. The final candidates are significantly superior to constitutive CAR-T cells in a long term killing assay, show potent cytotoxicity of low expressing antigen lines, and display background levels of cytotoxicity against single antigen targets. Engineering multiple features into T cell products is limited by unpredictable negative interactions between components. We have overcome this limitation by using high-throughput screening which generated development-ready candidates for ccRCC with finely tuned desirability criteria in <18 months. Citation Format: Nishant Mehta, Jamie Thomas, Edward Yashin, Andrea Fua, Jonathan Li, Jonathan Chen, Laura Lim, Je Chua, Andrew Cardozo, Marian Sandoval, Duy P. Nguyen, Ziyan Hong, Jimmy Wu, Catherine Sue, Gustavo Guzman, Li Wang, Sofia K. Panagiotopoulou, Sophie Xu, Angela C. Boroughs, W. Nicholas Haining. High-throughput arrayed screening of logic-gated CARs enables the selection of candidates for ccRCC with optimal potency and fidelity traits [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1783.

Plant Virus-Derived Vectors for Plant Genome Engineering.

Advances in genome engineering (GE) tools based on sequence-specific programmable nucleases have revolutionized precise genome editing in plants. However, only the traditional approaches are used to deliver these GE reagents, which mostly rely on Agrobacterium-mediated transformation or particle bombardment. These techniques have been successfully used for the past decades for the genetic engineering of plants with some limitations relating to lengthy time-taking protocols and transgenes integration-related regulatory concerns. Nevertheless, in the era of climate change, we require certain faster protocols for developing climate-smart resilient crops through GE to deal with global food security. Therefore, some alternative approaches are needed to robustly deliver the GE reagents. In this case, the plant viral vectors could be an excellent option for the delivery of GE reagents because they are efficient, effective, and precise. Additionally, these are autonomously replicating and considered as natural specialists for transient delivery. In the present review, we have discussed the potential use of these plant viral vectors for the efficient delivery of GE reagents. We have further described the different plant viral vectors, such as DNA and RNA viruses, which have been used as efficient gene targeting systems in model plants, and in other important crops including potato, tomato, wheat, and rice. The achievements gained so far in the use of viral vectors as a carrier for GE reagent delivery are depicted along with the benefits and limitations of each viral vector. Moreover, recent advances have been explored in employing viral vectors for GE and adapting this technology for future research.

Efficient Bacterial Genome Engineering throughout the Central Dogma Using the Dual-Selection Marker tetAOPT.

Engineering of bacterial genomes is a fundamental craft in contemporary biotechnology. The ability to precisely edit chromosomes allows for the development of cells with specific phenotypes for metabolic engineering and for the creation of minimized genomes. Genetic tools are needed to select for cells that underwent editing, and dual-selection markers that enable both positive and negative selection are highly useful. Here, we present an optimized and easy-to-use version of the tetA dual-selection marker and demonstrate how this tetAOPT can be used efficiently to engineer at different stages of the central dogma of molecular biology. On the DNA level, tetAOPT can be used to create scarless knockouts across the Escherichia coli genome with efficiency above 90%, whereas recombinant gene integrations can be achieved with approximately 50% efficiency. On the RNA and protein level, we show that tetAOPT enables advanced genome engineering of both gene translation and transcription by introducing sequence variation in the translation initiation region or by exchanging promoters. Finally, we demonstrate the use of tetAOPT for genome engineering in the industrially relevant probiotic strain E. coli Nissle.

Abstract LE03-01: Functional and computational approaches to uncover selection advantages of cancer aneuploidy

Abstract Aneuploidy, including the gain or loss of whole chromosomes or chromosome arms, is a near-universal feature of cancer. We previously applied methods that define chromosome arm aneuploidy to over 10,000 tumors in the Cancer Genome Atlas (TCGA). Cancer subtypes are often characterized by tumor specific patterns of chromosome arm copy number alterations and breakpoints; for example, squamous cell carcinomas (SCCs) from different tissues of origin are characterized by chromosome 3p (chr3p) loss and chromosome 3q (chr3q) gain. From the TCGA aneuploidy data, we developed an algorithm called BISCUT to distinguish peak regions of aneuploidy breakpoints on each chromosome arm. BISCUT identified loci affected by broad copy number alterations that provide fitness advantages or disadvantages both within individual cancer types and across cancers. Our analyses are consistent with selection being the primary driver of aneuploidy events in cancer. Recent advances in genome engineering allow generation of large chromosomal alterations and validation of findings from patient genomic data. We used the CRISPR-Cas9 system to delete one copy of chr3p in a human immortalized lung epithelial cell line similar to the putative cell-of-origin in lung SCC. Consistent with patient data, expression of chr3p genes was decreased upon deletion, as well as increased expression of interferon response genes. Cells with chr3p deletion initially proliferated more slowly than their siblings. Interestingly, after several passages in culture, this proliferation defect was rescued in chr3p deleted cells. Genome sequencing and karyotype analyses suggested that this was partially the result of chr3 duplication, with cells transitioning to a state of chr3q gain. Isolated cells with chr3q gain show deficiencies in squamous differentiation in organoid culture. Our genome engineering approach to model chromosome arm aneuploidies provides a robust model that will address a gap in our understanding of aneuploidy in cancer. Citation Format: Alison Marie Taylor. Functional and computational approaches to uncover selection advantages of cancer aneuploidy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LE03-01.

Sickle Cell Disease, a Review

Sickle cell disease and its variants constitute the most common inherited blood disorders affecting millions of individuals worldwide. Significant information regarding the nature of the genetic mutations and modifier genes that result in increased or decreased severity of the disease are available. In recent years, detailed data regarding molecular genetics, pathophysiology, mechanisms for the development of symptoms and side effects of sickle cell disease have been published. The relationship of physiological changes, cellular interactions, coexisting coagulation disorders, effects of association with other genetic disorders and a number of intervening factors have been explored. New techniques for pre-conception, prenatal, in utero, and neonatal screening are available. Means for prediction of the severity of the disease, clinical course of the disorder, and prevention of some of its major complications have been developed. The effects of psychosocial and environmental factors have been explored. Various therapeutic strategies including bone marrow and stem cell transplantation are currently employed in the treatment of patients with sickle cell disease. Recent progress in understanding the molecular pathways controlling mammalian erythropoiesis and globin switching, as well as advances in genome engineering, particularly the gene-editing techniques, have opened a venue for genetic-based treatment of the disease. Currently, sickle cell disease is often associated with a high rate of complications and mortality. The development of new pharmacological agents, methods for gene therapy, and alterations and modification of the coexisting genetic factors and modifiers for treatment of the disease are encouraging.

Development of a dedicated Golden Gate Assembly Platform (RtGGA) for Rhodotorula toruloides

Rhodotorula toruloides is a potential chassis for microbial cell factories as this yeast can metabolise different substrates into a diverse range of natural products, but the lack of efficient synthetic biology tools hinders its applicability. In this study, the modular, versatile and efficient Golden Gate DNA assembly system (RtGGA) was adapted to the first basidiomycete, an oleaginous yeast R. toruloides. R. toruloides CCT 0783 was sequenced, and used for the GGA design. The DNA fragments were assembled with predesigned 4-nt overhangs and a library of standardized parts was created containing promoters, genes, terminators, insertional regions, and resistance genes. The library was combined to create cassettes for the characterization of promoters strength and to overexpress the carotenoid production pathway. A variety of reagents, plasmids, and strategies were used and the RtGGA proved to be robust. The RtGGA was used to build three versions of the carotenoid overexpression cassette by using different promoter combinations. The cassettes were transformed into R. toruloides and the three new strains were characterized. Total carotenoid concentration increased by 41%. The dedicated GGA platform fills a gap in the advanced genome engineering toolkit for R. toruloides, enabling the efficient design of complex metabolic pathways.

Genome Engineering of the Fast-Growing Mycoplasma feriruminatoris toward a Live Vaccine Chassis.

Development of a new generation of vaccines is a key challenge for the control of infectious diseases affecting both humans and animals. Synthetic biology methods offer new ways to engineer bacterial chassis that can be used as vectors to present heterologous antigens and train the immune system against pathogens. Here, we describe the construction of a bacterial chassis based on the fast-growing Mycoplasma feriruminatoris, and the first steps toward its application as a live vaccine against contagious caprine pleuropneumonia (CCPP). To do so, the M. feriruminatoris genome was cloned in yeast, modified by iterative cycles of Cas9-mediated deletion of loci encoding virulence factors, and transplanted back in Mycoplasma capricolum subsp. capricolum recipient cells to produce the designed M. feriruminatoris chassis. Deleted genes encoded the glycerol transport and metabolism systems GtsABCD and GlpOKF and the Mycoplasma Ig binding protein-Mycoplasma Ig protease (MIB-MIP) immunoglobulin cleavage system. Phenotypic assays of the M. feriruminatoris chassis confirmed the corresponding loss of H2O2 production and IgG cleavage activities, while growth remained unaltered. The resulting mycoplasma chassis was further evaluated as a platform for the expression of heterologous surface proteins. A genome locus encoding an inactivated MIB-MIP system from the CCPP-causative agent Mycoplasma capricolum subsp. capripneumoniae was grafted in replacement of its homolog at the original locus in the chassis genome. Both heterologous proteins were detected in the resulting strain using proteomics, confirming their expression. This study demonstrates that advanced genome engineering methods are henceforth available for the fast-growing M. feriruminatoris, facilitating the development of novel vaccines, in particular against major mycoplasma diseases.