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Effects of CRISPR technology on agricultural sustainability: global applications and turkish perspective.

This review evaluates CRISPR/Cas applications in agriculture from a global perspective with explicit reference to Türkiye. Using a literature gap-matrix approach organised around four analytical dimensions-environmental, economic, social and policy, and scientific and technological-we synthesize the primary evidence on water and input use, productivity, disease resistance, and product quality. The literature concentrates on water and fertilizer use, productivity, and off-target accuracy, whereas soil health, biodiversity, consumer acceptance, ethical considerations and regulatory frameworks remain systematically under-represented. Global deployment of CRISPR is already delivering measurable advantages in food security, shelf life and nutritional value, while in Türkiye the research base is at an early stage but has clear potential in wheat, barley, tomato and olive. Translating CRISPR into Turkish agricultural sustainability requires (i) a domestic biosafety framework aligned with the emerging European New Genomic Techniques approach, (ii) sustained investment in multi-location primary field trials, and (iii) inclusive deployment mechanisms-particularly through producer cooperatives-that allow smallholder farmers to benefit from edited varieties.

Plants, Genetically Modified↗

The biology of kinetoplastid parasites: insights and challenges from genomics and post-genomics.

Kinetoplastid parasites exhibit a rich and diverse biology which mirrors many of the most interesting topics of current interest and study in the broader biological sciences. These evolutionarily ancient organisms possess intriguing mechanisms for control of gene expression, and exhibit complex patterns of cell morphogenesis orchestrated by an internal cytoskeleton. Their cell shapes change during a set of complex cell type differentiations in their life cycles. These differentiations are intimately linked to interactions with mammalian hosts or insect vectors, and often, these differentiations appear central to the successful transfer of the parasite between vector and host, and host and vector. The basics of this rich and complex cell and life cycle biology were described (with often rather forgotten clarity and prescience) in the early period of the last century. The last 30 years have seen major developments in our understanding of this biology. Ultrastructural differences in the various cells of the life cycle stages of Trypanosoma brucei, Trypanosoma cruzi and the various Leishmania species have been documented, and such studies have proven highly informative in defining important aspects of parasite adaptation. They have also proven to be a rich source of information for defining unusual aspects of parasite cell biology, novel organelles and cell architecture. This ultrastructural cell biology has been mirrored in a set of biochemical explanations defining unusual aspects of metabolism, surface molecules, and organelles. Finally, the application of molecular biology to these parasites revealed fascinating layers of complexity in the control of gene expression. These molecular studies have given us particular insights into polycistronic transcription, trans-splicing, RNA editing and gene rearrangements during antigenic variation. In contrast to other microbial systems, these cell biological, biochemical and molecular studies have not been greatly aided by insights gained from genetics--the diploid nature of the genome has discouraged the application of selectional genetics, mutant isolation and analysis. This is an important fact, since in general, it means that we have only recently started to analyse the phenotypes of mutants produced in the context of reverse genetics. In the following, I will argue that this lack of investment in the analysis of mutant phenotype is just one of the challenges that will need to be met if we are to gain the expected added value from the parasite genome projects. In this presentation, I will use some of the current areas of interest in the biology of T. brucei, T. cruzi and the Leishmania species to rehearse some of the insights and challenges that are likely to stem from the application of genomics and post-genomic studies to the kinetoplastid parasites. In some cases, I will exemplify points by illustrations from my laboratory's work, interests and hypotheses. The presentation slants therefore towards T. brucei biology, however, in each case the reader will, no doubt, see the generalities of application to other kinetoplastid parasites.

Animals↗

Targeted disruption of the mouse apobec-1 gene abolishes apolipoprotein B mRNA editing and eliminates apolipoprotein B48.

A site-specific C to U editing reaction modifies nuclear apolipoprotein B100 (apoB100) mRNA, producing apolipoprotein B48 in the mammalian small intestine. This reaction is mediated by a multicomponent enzyme complex, which contains a catalytic subunit, Apobec-1. We have used gene targeting to disrupt mouse apobec-1 in order to establish its requisite importance in apoB mRNA editing and also, in view of its widespread tissue distribution in rodents, as a preliminary indication of other potential roles. Both heterozygous (apobec-1+/-) and homozygous (apobec-1-/-) gene-targeted mice appear healthy and fertile with no alterations in serum cholesterol or triglyceride concentrations. The apobec-1+/- mice demonstrated reduced levels of hepatic apoB mRNA editing. By contrast, levels of small intestinal apoB mRNA editing were indistinguishable in wild-type and apobec-1+/- animals, suggesting that Apobec-1 is expressed in limited quantities in the liver but not in the small intestine. The apobec-1-/- mice lacked detectable levels of Apobec-1 mRNA, expressed only unedited apoB mRNA in all tissues, and contained no apoB48 in their serum, demonstrating that there is no functional duplication of this gene.

APOBEC-1 Deaminase↗

The organization and evolution of cloned globin genes.

The globin genes represent a complex set of sequences that are expressed in a coordinate fashion during the development of red blood cells. while this complex family of genes may consist of as many as ten to fourteen members [34], three of these genes have now been cloned and their entire nucleotide sequence determined. As was initially observed in the case of beta globin major gene, all are encoded in three distinct coding blocks separated by two intervening sequences of DNA. Their intervening sequences of DNA are preserved, with respect to location, but are widely divergent, with respect to size and sequence. The divided information in each gene is edited and spliced together at the level of its initial RNA transcript which is complementary to the entire gene sequence including its intervening sequences. Structural correlation analyses have allowed us to identify sites in all three genes that might be responsible for the initiation of transcription, RNA splicing, and poly A addition. The function of these sites has been tested by cloning these genes in an animal virus vector SV40. Such animal virus hybrids have been used to infect tissue culture cells and have directed the synthesis of both alpha and beta mouse globin in cells of monkey origin. These studies indicate that such signals operate across species barriers and further indicate that the animal virus vector system will be useful in elucidating their function.

Amino Acid Sequence↗

Differential transcription profiles in Trypanosoma cruzi associated with clinical forms of Chagas disease: Maxicircle NADH dehydrogenase subunit 7 gene truncation in asymptomatic patient isolates.

The majority of individuals in the chronic phase of Chagas disease are asymptomatic (indeterminate form). Every year 2-3% of these individuals develop severe clinical manifestations (cardiac and digestive forms). In this study a Trypanosoma cruzi DNA microarray was used to compare the transcript profiles of six human isolates: three from asymptomatic and three from cardiac patients. Seven signals were expressed differentially between the two classes of isolates, including tryparedoxin, surface protease GP63, cyclophilin, some hypothetical proteins and the pre-edited maxicircle gene NADH dehydrogenase subunit 7 (ND7). The approximately 30-fold greater signal in cardiac strains for ND7 was the most pronounced of the group, and differential levels of pre-edited ND7 transcript confirmed the microarray analysis. The ND7 gene from asymptomatic isolates showed a deletion of 455bp from nt 222 to nt 677 relative to ND7 of the CL Brener reference strain. The ND7 gene structure correlated with disease manifestation for 20 isolates from clinically characterised, chronic phase patients. The ND7 lesion produces a truncated product that could impair the function of mitochondrial complex I. Possible links between the integrity of the electron transport chain and symptom presentation are discussed. We propose that ND7 and other genes of the pathway constitute valuable targets for PCR assays in the differential diagnosis of the infective T. cruzi strain. While this hypothesis requires validation by the examination of additional recent parasite isolates from patients with defined pathologies, the identification of specific molecular markers represents a promising advance in the association between parasite genetics and disease pathology.

Adolescent↗

General and robust sample preparation strategies for cryo-EM studies of CRISPR-Cas9 and Cas12 enzymes.

Cas9 and Cas12 are RNA-guided DNA endonucleases derived from prokaryotic CRISPR-Cas adaptive immune systems that have been repurposed as versatile genome-engineering tools. Computational mining of genomes and metagenomes has expanded the diversity of Cas9 and Cas12 enzymes that can be used to develop versatile, orthogonal molecular toolboxes. Structural information is pivotal to uncovering the precise molecular mechanisms of newly discovered Cas enzymes and providing a foundation for their application in genome editing. In this chapter, we describe detailed protocols for the preparation of Cas9 and Cas12 enzymes for cryo-electron microscopy. These methods will enable fast and robust structural determination of newly discovered Cas9 and Cas12 enzymes, which will enhance the understanding of diverse CRISPR-Cas effectors and provide a molecular framework for expanding CRISPR-based genome-editing technologies.

Cryoelectron Microscopy↗

The P gene of bovine parainfluenza virus 3 expresses all three reading frames from a single mRNA editing site.

The P gene of bovine parainfluenza virus 3 (bPIV3) contains two downstream overlapping ORFs, called V and D. By comparison with the mRNA editing sites of other paramyxoviruses, two editing sites were predicted for bPIV3; site a to express the D protein, and site b to express the V protein. Examination of the bPIV3 mRNAs, however, indicates that site b is non-functional whereas site a operates frequently. Insertions at site a give rise to both V and D protein mRNAs, because a very broad distribution of Gs is added when insertions occur. This broad distribution is very different from the editing sites of Sendai virus or SV5, where predominantly one form of edited mRNA containing either a one or two G insertion respectively is created, to access the single overlapping ORF of these viruses. A model is proposed to explain how paramyxoviruses control the range of G insertions on that fraction of the mRNAs where insertions occur. The bPIV3 P gene is unique as far as we know, in that a sizeable portion of the gene expresses all 3 reading frames as protein. bPIV3 apparently does this from a single editing site by removing the constraints which control the number of slippage rounds which take place.

Animals↗

Molecular biology of Leishmania.

Leishmania is a trypanosomatid protozoa with a digenetic life cycle. Sandflies inject promastigotes, the free living form present in their salivary glands, into mammals where the parasite colonizes macrophages, transforming into intracellular amastigotes. The cycle is completed when during a blood meal the insect ingests infected macrophages, the amastigotes are released in the gut where they transform back into promastigotes. Leishmania has to adapt to the changing life conditions, from free-living forms in the poikilothermic insect vector to obligatory intracellular parasite in the homeothermic mammalian host. It also has to adapt to the acidic pH of the macrophage's phagolysosome where amastigotes multiply. The adaptative response of Leishmania includes morphological, physiological, and biochemical changes. Promastigotes can be grown in culture medium. Studies of changes taking place during adaptation have been facilitated by the establishment of in vitro conditions that allow the transformation of amastigotes into promastigotes and vice versa. The system is well suited for studying regulation of gene expression during adaptative differentiation. Some mechanisms of mRNA processing are unique to these protozoa: trans-splicing and RNA editing. Several genes that are differentially expressed in the two stages have been studied. No obvious cis regulatory motifs have been found in the DNA.

Amino Acid Sequence↗

RBP16 is a multifunctional gene regulatory protein involved in editing and stabilization of specific mitochondrial mRNAs in Trypanosoma brucei.

RBP16 is a Trypanosoma brucei mitochondrial RNA-binding protein that associates with guide RNAs (gRNAs), mRNAs, and ribosomal RNAs. Based on its inclusion in the multifunctional Y-box protein family and its ability to bind multiple RNA classes, we hypothesized that RBP16 plays a role in diverse aspects of mitochondrial gene regulation. To gain insight into RBP16 function, we generated cells expressing less than 10% of wild-type RBP16 levels by tetracycline-regulated RNA interference (RNAi). Poisoned primer extension analyses revealed that edited, but not unedited, CYb mRNA is reduced by approximately 98% in tetracycline-induced RBP16 RNAi cells, suggesting that RBP16 is critical for CYb RNA editing. The down-regulation of CYb editing in RBP16 RNAi transfectants apparently entails a defect in gRNA utilization, as gCYb[560] abundance is similar in uninduced and induced cells. We observed a surprising degree of specificity regarding the ability of RBP16 to modulate editing, as editing of mRNAs other than CYb is not significantly affected upon RBP16 disruption. However, the abundance of the never edited mitochondrial RNAs COI and ND4 is reduced by 70%-80% in RBP16 RNAi transfectants, indicating an additional role for RBP16 in the stabilization of these mRNAs. Analysis of RNAs bound to RBP16 immunoprecipitated from wild-type cells reveals that RBP16 is associated with multiple gRNA sequence classes in vivo, including those whose abundance and usage appear unaffected by RBP16 disruption. Overall, our results indicate that RBP16 is an accessory factor that regulates the editing and stability of specific populations of mitochondrial mRNAs.

Animals↗

Rapeseed mitochondrial ccb206, a gene involved in cytochrome c biogenesis, is co-transcribed with the nad3 and rps12 genes: organization, transcription, and RNA editing of the nad3/rps12/ccb206 locus.

We describe here the structure and expression of the nad3/rps12/ccb206 locus in the mitochondrial genomes of rapeseed (Brassica napus L.) and related species. In the rapeseed mitochondrial genome there are three tandemly located open reading frames for subunit 3 of NADH dehydrogenase (nad3), the S12 subunit protein of the mitochondrial ribosome (rps12), and a homologue of one subunit of the ABC-heme transporter (ccb206). The gene organization of the nad3/rps12/ccb206 locus found in rapeseed mitochondria is not conserved in Arabidopsis thaliana mitochondria, where the ccb206 and nad3/rps12 loci are separated from each other. We show here that the rapeseed gene-organization pattern is also present in other Brassica species, but not in radish, a species very closely related to Brassica. Northern hybridization and RT-PCR analyses indicate that these three mitochondrial genes are possibly co-transcribed. Ten, seven, and 39 C-to-U RNA editing events occur in the rapeseed nad3, rps12, and ccb206 transcripts, respectively. Finally we show that codon position 21 of nad3 is edited in a highly species-specific manner among related Brassica species, including rapeseed and radish.

ATP-Binding Cassette Transporters↗

Emerging techniques of CRISPR/Cas system in antiviral therapy and diagnostics: Applications, limitations, and translational perspectives.

The CRISPR/Cas (clustered regularly interspaced short palindromic repeats) system is a versatile technology for developing antiviral medicines and editing viral genomes in both diagnostics and vaccine synthesis. Emerging insights into class 2 effectors, such as Cas9, Cas12, and Cas13, which target viral DNA and RNA, have revolutionized vaccines against viruses such as HIV, HPV, HBV, and EBV. Innovative diagnostic techniques such as SHERLOCK, DETECTR, and FELUDA have demonstrated system's diversity and accuracy in detecting the virus markers, supporting clinical decision-making, indicating adaptability and precision of CRISPR. This review critically evaluates CRISPR's role in RNA editing, emphasizing its importance for functional genomics and development of recombinant vaccines. Translational challenges are critically discussed, including off-target effects, delivery limitations, and ethical issues, for which unique approaches such as high-fidelity Cas variants, non-viral delivery systems, and bioethical frameworks are evaluated to address these limitations. This review also covers other social implications, such as accessibility and biosecurity risks, associated with CRISPR technologies Collectively, these advances underscore the transformative potential of CRISPR technologies in shaping next-generation antiviral diagnostics and therapeutics.

CRISPR-Cas Systems↗

The absence of genes for cytochrome c oxidase and reductase subunits in maxicircle kinetoplast DNA of the respiration-deficient plant trypanosomatid Phytomonas serpens.

By completing the sequencing of the maxicircle conserved region in the kinetoplast DNA of Phytomonas serpens, we showed that the genes for subunits I and II (COI and COII) of cytochrome c oxidase in this organism were missing. We had previously shown that the genes for cytochrome c oxidase subunit III and apocytochrome b were also missing. These deletions did not affect the structure or expression of the remaining genes. Partial editing of the mRNA for NADH dehydrogenase subunit 8, previously found in strain IG from insects, was complete in two other strains isolated from plants. The appearance of a novel maxicircle gene for MURF2 block I gRNA, which substitutes for the gene missing due to the COII gene deletion, may illustrate a general mechanism for the origin of gRNAs.

Amino Acid Sequence↗

Prime editing in bacteria with BacPE.

Programmable genome editing technologies have revolutionized the ability of researchers to alter the genomes of microorganisms in a straightforward and efficient manner, significantly advancing the field of microbiology. To date, several CRISPR-Cas-based genome-editing systems have been developed for use in E. coli, including CRISPR/Cas9, base editing, and prime editing technologies. In this chapter, we describe the design and experimental application of BacPE, a variant of prime editing technology optimized for E. coli. BacPE facilitates the introduction of point mutations, insertions, and deletions without the need for double-strand DNA breaks. We demonstrate that BacPE is a powerful tool for genome editing in E. coli and highlight its potential applicability to other bacterial species.

Gene Editing↗

Experimental strategy for characterization of novel TnpB orthologs.

TnpB proteins encoded in IS200/IS605 and IS607 mobile genetic elements are among the most widespread proteins in the microbial world. They function as RNA-guided DNA nucleases that play a critical role in transposon proliferation and are the predecessors of CRISPR-Cas12 effector proteins of the type V CRISPR-Cas family. Small size of TnpB nucleases makes them an attractive alternative for larger Cas9 and Cas12 proteins in genome editing applications. However, only a small fraction of TnpB nucleases characterized to date are active in human cells, highlighting the need to identify new TnpB variants that can function as genome editors. Here, we present an experimental pipeline for the characterization of TnpB proteins by combining in silico analysis with in vitro assays. To validate it we determined guide RNA and identified TAM for a set of TnpB orthologs. The proposed workflow can be employed for rapid screening and characterization of the huge TnpB protein family to identify novel TnpB variants that might expand the genome editing toolbox.

Humans↗

Death by deamination: a novel host restriction system for HIV-1.

A human gene with potent antiretroviral activity has recently been found to encode a new member of the family of cytidine deaminases, previously known to be involved in mRNA editing, immunoglobulin gene class switching, and immunoglobulin gene hypermutation. The enzyme attacks viral DNA as it is synthesized in infected cells and prevents the formation of functional proviruses. The Vif gene of HIV-1 blocks this host restriction and so allows virus replication.

APOBEC-3G Deaminase↗

Transcripts from the frameshifted MURF3 gene from Crithidia fasciculata are edited by U insertion at multiple sites.

In trypanosome mitochondria an RNA editing process is operative, which co- or post-transcriptionally alters the nucleotide sequence of transcripts by insertion and/or deletion of U residues at specific sites. To increase our understanding of the mechanism of this process we have compared the nucleotide sequence of the frameshifted mitochondrial MURF3 gene from Crithidia fasciculata to that of a large number of MURF3 cDNAs. We found cDNAs derived from transcripts edited at two different sites in the protein coding sequence: (i) at the frameshift position five extra U residues connect the two reading frames and (ii) at the 5' terminus 22 inserted Us shift a putative initiator codon out of phase. The collection also contained cDNAs that were derived from non-edited transcripts. Partially edited sequences were not found, except in one cDNA, which contained an edited frameshift site in combination with a non-edited 5' terminus. The analysis further showed that MURF3 transcripts have a 3'-terminal poly(AU) extension, which varies in sequence. The implications of these results are discussed.

Amino Acid Sequence↗

A quick guide to evaluating prime editing efficiency in mammalian cells.

According to the Clinvar database, modeling the diseases associated with pathogenic mutations requires the installation of base substitutions, small insertions or deletions. Prime editor (PE) was recently developed to precisely install any base substitutions and/or small insertions/deletions (indels) in mammalian cells and animals without requiring DSBs or donor DNA templates. PE also offers greater editing and targeting flexibility compared to other precision CRISPR editing methods because the versatile editing information is encoded in the reverse-transcription template of its prime editing guide RNA. However, optimal PE system selection and experimental design can be complex, and there are various factors that can affect PE efficiency. This chapter serves as a rapid entry-level guideline for the application of PE, providing an experimental framework for using PE at a specific genomic locus. RUNX1 was selected as a representative target site to illustrate the detailed methodology for constructing PE plasmids and the process of transfecting these plasmids into 293FT cells. We further examined the efficiency of PE-mediated genome editing in mammalian cells by using next-generation sequencing.

Gene Editing↗