Frontiers in genome editing最新文献

{"title":"Genome editing in rice mediated by miniature size Cas nuclease SpCas12f.","authors":"Satoru Sukegawa,&nbsp;Osamu Nureki,&nbsp;Seiichi Toki,&nbsp;Hiroaki Saika","doi":"10.3389/fgeed.2023.1138843","DOIUrl":"https://doi.org/10.3389/fgeed.2023.1138843","url":null,"abstract":"<p><p>Cas9 derived from <i>Streptococcus pyogenes</i> (SpCas9) is used widely in genome editing using the CRISPR-Cas system due to its high activity, but is a relatively large molecule (1,368 amino acid (a.a.) residues). Recently, targeted mutagenesis in human cells and maize using Cas12f derived from <i>Syntrophomonas palmitatica</i> (SpCas12f)-a very small Cas of 497 a.a, which is a more suitable size for virus vectors-was reported. However, there are no reports of genome editing using SpCas12f in crops other than maize. In this study, we applied SpCas12f to genome editing in rice-one of the most important staple crops in the world. An expression vector encoding rice codon-optimized SpCas12f and sgRNA for <i>OsTubulin</i> as a target was introduced into rice calli by <i>Agrobacterium</i>-mediated transformation. Molecular analysis of SpCas12f-transformed calli showed that mutations were introduced successfully into the target region. Detailed analysis by amplicon sequencing revealed estimated mutation frequencies (a ratio of the number of mutated calli to that of SpCas12f-transformed calli) of 28.8% and 55.6% in two targets. Most mutation patterns were deletions, but base substitutions and insertions were also confirmed at low frequency. Moreover, off-target mutations by SpCas12f were not found. Furthermore, mutant plants were regenerated successfully from the mutated calli. It was confirmed that the mutations in the regenerated plants were inherited to the next-generation. In the previous report in maize, mutations were introduced by treatment with heat shock at 45°C for 4 h per day for 3 days; no mutations were introduced under normal growth conditions at 28°C. Surprisingly, however, mutations can be introduced without heat-shock treatment in rice. This might be due to the culture conditions, with relatively higher temperature (30°C or higher) and constant light during callus proliferation. Taken together, we demonstrated that SpCas12f can be used to achieve targeted mutagenesis in rice. SpCas12f is thus a useful tool for genome editing in rice and is suitable for virus vector-mediated genome editing due to its very small size.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"5 ","pages":"1138843"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10040665/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9226450","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cytosine base editors optimized for genome editing in potato protoplasts.","authors":"Ida Westberg,&nbsp;Frida Meijer Carlsen,&nbsp;Ida Elisabeth Johansen,&nbsp;Bent Larsen Petersen","doi":"10.3389/fgeed.2023.1247702","DOIUrl":"https://doi.org/10.3389/fgeed.2023.1247702","url":null,"abstract":"<p><p>In this study, we generated and compared three cytidine base editors (CBEs) tailor-made for potato (<i>Solanum tuberosum</i>), which conferred up to 43% C-to-T conversion of all alleles in the protoplast pool. Earlier, gene-edited potato plants were successfully generated by polyethylene glycol-mediated CRISPR/Cas9 transformation of protoplasts followed by explant regeneration. In one study, a 3-4-fold increase in editing efficiency was obtained by replacing the standard <i>Arabidopsis thaliana At</i>U6-1 promotor with endogenous potato <i>St</i>U6 promotors driving the expression of the gRNA. Here, we used this optimized construct (<i>Sp</i>Cas9/<i>St</i>U6-1::gRNA1, target gRNA sequence GGTC₄C₅TTGGAGC₁₂AAAAC₁₇TGG) for the generation of CBEs tailor-made for potato and tested for C-to-T base editing in the granule-bound starch synthase 1 gene in the cultivar Desiree. First, the <i>Streptococcus pyogenes</i> Cas9 was converted into a (D10A) nickase (nCas9). Next, one of three cytosine deaminases from human hAPOBEC3A (A3A), rat (evo_rAPOBEC1) (rA1), or sea lamprey (evo_<i>Pm</i>CDA1) (CDA1) was C-terminally fused to nCas9 and a uracil-DNA glycosylase inhibitor, with each module interspaced with flexible linkers. The CBEs were overall highly efficient, with A3A having the best overall base editing activity, with an average 34.5%, 34.5%, and 27% C-to-T conversion at C4, C5, and C12, respectively, whereas CDA1 showed an average base editing activity of 34.5%, 34%, and 14.25% C-to-T conversion at C4, C5, and C12, respectively. rA1 exhibited an average base editing activity of 18.75% and 19% at C4 and C5 and was the only base editor to show no C-to-T conversion at C12.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"5 ","pages":"1247702"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10502308/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10307996","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Editing efficiencies with Cas9 orthologs, Cas12a endonucleases, and temperature in rice.","authors":"Eudald Illa-Berenguer,&nbsp;Peter R LaFayette,&nbsp;Wayne A Parrott","doi":"10.3389/fgeed.2023.1074641","DOIUrl":"https://doi.org/10.3389/fgeed.2023.1074641","url":null,"abstract":"<p><p>The advent of CRISPR-Cas technology has made it the genome editing tool of choice in all kingdoms of life, including plants, which can have large, highly duplicated genomes. As a result, finding adequate target sequences that meet the specificities of a given Cas nuclease on any gene of interest remains challenging in many cases. To assess target site flexibility, we tested five different Cas9/Cas12a endonucleases (SpCas9, SaCas9, St1Cas9, Mb3Cas12a, and AsCas12a) in embryogenic rice calli from Taipei 309 at 37°C (optimal temperature for most Cas9/Cas12a proteins) and 27°C (optimal temperature for tissue culture) and measured their editing rates under regular tissue culture conditions using Illumina sequencing. StCas9 and AsCas12 were not functional as tested, regardless of the temperature used. SpCas9 was the most efficient endonuclease at either temperature, regardless of whether monoallelic or biallelic edits were considered. Mb3Cas12a at 37°C was the next most efficient endonuclease. Monoallelic edits prevailed for both SaCas9 and Mb3Cas12a at 27°C, but biallelic edits prevailed at 37°C. Overall, the use of other Cas9 orthologs, the use of Cas12a endonucleases, and the optimal temperature can expand the range of targetable sequences.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"5 ","pages":"1074641"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10080323/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9637386","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Regulation and safety measures for nanotechnology-based agri-products.","authors":"Ritika Kumari,&nbsp;Kalpana Suman,&nbsp;Swagata Karmakar,&nbsp;Vandana Mishra,&nbsp;Sameer Gunjan Lakra,&nbsp;Gunjan Kumar Saurav,&nbsp;Binod Kumar Mahto","doi":"10.3389/fgeed.2023.1200987","DOIUrl":"https://doi.org/10.3389/fgeed.2023.1200987","url":null,"abstract":"<p><p>There is a wide range of application for nanotechnology in agriculture, including fertilizers, aquaculture, irrigation, water filtration, animal feed, animal vaccines, food processing, and packaging. In recent decades, nanotechnology emerged as a prospective and promising approach for the advancement of Agri-sector such as pest/disease prevention, fertilizers, agrochemicals, biofertilizers, bio-stimulants, post-harvest storage, pheromones-, and nutrient-delivery, and genetic manipulation in plants for crop improvement by using nanomaterial as a carrier system. Exponential increase in global population has enhanced food demand, so to fulfil the demand markets already included nano-based product likewise nano-encapsulated nutrients/agrochemicals, antimicrobial and packaging of food. For the approval of nano-based product, applicants for a marketing approval must show that such novel items can be used safely without endangering the consumer and environment. Several nations throughout the world have been actively looking at whether their regulatory frameworks are suitable for handling nanotechnologies. As a result, many techniques to regulate nano-based products in agriculture, feed, and food have been used. Here, we have contextualized different regulatory measures of several countries for nano-based products in agriculture, from feed to food, including guidance and legislation for safety assessment worldwide.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"5 ","pages":"1200987"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10320728/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9805772","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Specific correction of pyruvate kinase deficiency-causing point mutations by CRISPR/Cas9 and single-stranded oligodeoxynucleotides.","authors":"Sara Fañanas-Baquero,&nbsp;Matías Morín,&nbsp;Sergio Fernández,&nbsp;Isabel Ojeda-Perez,&nbsp;Mercedes Dessy-Rodriguez,&nbsp;Miruna Giurgiu,&nbsp;Juan A Bueren,&nbsp;Miguel Angel Moreno-Pelayo,&nbsp;Jose Carlos Segovia,&nbsp;Oscar Quintana-Bustamante","doi":"10.3389/fgeed.2023.1104666","DOIUrl":"https://doi.org/10.3389/fgeed.2023.1104666","url":null,"abstract":"<p><p>Pyruvate kinase deficiency (PKD) is an autosomal recessive disorder caused by mutations in the <i>PKLR</i> gene. PKD-erythroid cells suffer from an energy imbalance caused by a reduction of erythroid pyruvate kinase (RPK) enzyme activity. PKD is associated with reticulocytosis, splenomegaly and iron overload, and may be life-threatening in severely affected patients. More than 300 disease-causing mutations have been identified as causing PKD. Most mutations are missense mutations, commonly present as compound heterozygous. Therefore, specific correction of these point mutations might be a promising therapy for the treatment of PKD patients. We have explored the potential of precise gene editing for the correction of different PKD-causing mutations, using a combination of single-stranded oligodeoxynucleotides (ssODN) with the CRISPR/Cas9 system. We have designed guide RNAs (gRNAs) and single-strand donor templates to target four different PKD-causing mutations in immortalized patient-derived lymphoblastic cell lines, and we have detected the precise correction in three of these mutations. The frequency of the precise gene editing is variable, while the presence of additional insertions/deletions (InDels) has also been detected. Significantly, we have identified high mutation-specificity for two of the PKD-causing mutations. Our results demonstrate the feasibility of a highly personalized gene-editing therapy to treat point mutations in cells derived from PKD patients.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"5 ","pages":"1104666"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10175809/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9829720","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Editorial: <i>Ex-vivo</i> and <i>in-vivo</i> genome engineering for metabolic and neurometabolic diseases.","authors":"Pasqualina Colella,&nbsp;Vasco Meneghini,&nbsp;Guilherme Baldo,&nbsp;Natalia Gomez-Ospina","doi":"10.3389/fgeed.2023.1248904","DOIUrl":"https://doi.org/10.3389/fgeed.2023.1248904","url":null,"abstract":"Recent advances in genome modification tools have led to a growing interest in using genome engineering as a therapeutic solution for many diseases. At the forefront of this revolution is the CRISPR-Cas9 technology, which made genome editing broadly accessible and engendered the development of chimeric genome editing tools like base editors and prime editors. To achieve the desired DNA modifications, nucleasebased platforms use cellular DNA repair pathways, such as Homology Directed Repair (HDR), Non-Homologous End Joining (NHEJ), and Microhomology-Mediated End Joining (MMEJ), while prime editors employ an RNA-based reverse transcription mechanism. For therapeutic applications, genome engineering platforms can be used ex vivo and in vivo and can either disrupt coding or regulatory sequences (therapeutic NHEJ) or make precise sequence changes (therapeutic HDR, Base editing, and Prime editing). The most advanced applications of genome editing for human monogenic diseases involve therapeutic NHEJ, which uses Cas9 endonuclease and guide RNAs (gRNAs) to create site-specific double-strand breaks (DSBs), which NHEJ then repairs. This process often results in the insertion/deletion of a few nucleotides (INDELs) or larger deletions, depending on the gRNA design, mostly disrupting, or inactivating the target gene. Therapeutic NHEJ has been successfully applied ex vivo to modify CD34 hematopoietic stem and progenitor cells (HSPCs) from individuals affected by beta-Thalassemia (b-Thal) and Sickle cell disease (SCD), both caused bymutations in the β-globin gene (HBB) (Ledford, 2020; Frangoul et al., 2021). In this strategy, Cas9/gRNAs are used to reactivate the expression of the fetal γ-globin by knocking down the erythroid expression of BCL11A, its key transcriptional repressor. Data from clinical trials confirmed that γ-globin could functionally complement the deficiency of β-globin in the hemoglobin tetramers and exert an anti-sickling function. This approach can be applied to β-Thal and SCD independently from the underlying beta-globin mutations. It is also proving to be safe and effective in OPEN ACCESS","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"5 ","pages":"1248904"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10359423/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10241065","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"On-site and visual detection of sorghum mosaic virus and rice stripe mosaic virus based on reverse transcription-recombinase-aided amplification and CRISPR/Cas12a.","authors":"Junkai Wang,&nbsp;Xiuqin Huang,&nbsp;Siping Chen,&nbsp;Jiahao Chen,&nbsp;Zhengyi Liang,&nbsp;Biao Chen,&nbsp;Xin Yang,&nbsp;Guohui Zhou,&nbsp;Tong Zhang","doi":"10.3389/fgeed.2023.1124794","DOIUrl":"https://doi.org/10.3389/fgeed.2023.1124794","url":null,"abstract":"<p><p>Rapid, sensitive and visual detection of plant viruses is conducive to effective prevention and control of plant viral diseases. Therefore, combined with reverse transcription and recombinase-aided amplification, we developed a CRISPR/Cas12a-based visual nucleic acid detection system targeting sorghum mosaic virus and rice stripe mosaic virus, which cause harm to crop production in field. When the RT-RAA products were recognized by crRNA and formed a complex with LbCas12a, the ssDNA labeled with a quenched green fluorescent molecule will be cleaved by LbCas12a, and then a significant green fluorescence signal will appear. The entire detection process can be completed within 30 min without using any sophisticated equipment and instruments. The detection system could detect samples at a dilution of 10⁷, about 10⁴-fold improvement over RT-PCR, so the system was successfully to detect rice stripe mosaic virus in a single leafhopper, which is the transmission vector of the virus. Finally, the CRISPR/Cas12a-based detection system was utilized to on-site detect the two viruses in the field, and the results were fully consistent with that we obtained by RT-PCR in laboratory, demonstrating that it has the application prospect of detecting important crop viruses in the field.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"5 ","pages":"1124794"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9895793/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10652547","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Squid express conserved ADAR orthologs that possess novel features.","authors":"Isabel C Vallecillo-Viejo,&nbsp;Gjendine Voss,&nbsp;Caroline B Albertin,&nbsp;Noa Liscovitch-Brauer,&nbsp;Eli Eisenberg,&nbsp;Joshua J C Rosenthal","doi":"10.3389/fgeed.2023.1181713","DOIUrl":"https://doi.org/10.3389/fgeed.2023.1181713","url":null,"abstract":"The coleoid cephalopods display unusually extensive mRNA recoding by adenosine deamination, yet the underlying mechanisms are not well understood. Because the adenosine deaminases that act on RNA (ADAR) enzymes catalyze this form of RNA editing, the structure and function of the cephalopod orthologs may provide clues. Recent genome sequencing projects have provided blueprints for the full complement of coleoid cephalopod ADARs. Previous results from our laboratory have shown that squid express an ADAR2 homolog, with two splice variants named sqADAR2a and sqADAR2b and that these messages are extensively edited. Based on octopus and squid genomes, transcriptomes, and cDNA cloning, we discovered that two additional ADAR homologs are expressed in coleoids. The first is orthologous to vertebrate ADAR1. Unlike other ADAR1s, however, it contains a novel N-terminal domain of 641 aa that is predicted to be disordered, contains 67 phosphorylation motifs, and has an amino acid composition that is unusually high in serines and basic amino acids. mRNAs encoding sqADAR1 are themselves extensively edited. A third ADAR-like enzyme, sqADAR/D-like, which is not orthologous to any of the vertebrate isoforms, is also present. Messages encoding sqADAR/D-like are not edited. Studies using recombinant sqADARs suggest that only sqADAR1 and sqADAR2 are active adenosine deaminases, both on perfect duplex dsRNA and on a squid potassium channel mRNA substrate known to be edited in vivo. sqADAR/D-like shows no activity on these substrates. Overall, these results reveal some unique features in sqADARs that may contribute to the high-level RNA recoding observed in cephalopods.","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"5 ","pages":"1181713"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10278661/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10067579","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"In search of an ideal template for therapeutic genome editing: A review of current developments for structure optimization.","authors":"Alena Shakirova,&nbsp;Timofey Karpov,&nbsp;Yaroslava Komarova,&nbsp;Kirill Lepik","doi":"10.3389/fgeed.2023.1068637","DOIUrl":"https://doi.org/10.3389/fgeed.2023.1068637","url":null,"abstract":"<p><p>Gene therapy is a fast developing field of medicine with hundreds of ongoing early-stage clinical trials and numerous preclinical studies. Genome editing (GE) now is an increasingly important technology for achieving stable therapeutic effect in gene correction, with hematopoietic cells representing a key target cell population for developing novel treatments for a number of hereditary diseases, infections and cancer. By introducing a double strand break (DSB) in the defined locus of genomic DNA, GE tools allow to knockout the desired gene or to knock-in the therapeutic gene if provided with an appropriate repair template. Currently, the efficiency of methods for GE-mediated knock-in is limited. Significant efforts were focused on improving the parameters and interaction of GE nuclease proteins. However, emerging data suggests that optimal characteristics of repair templates may play an important role in the knock-in mechanisms. While viral vectors with notable example of AAVs as a donor template carrier remain the mainstay in many preclinical trials, non-viral templates, including plasmid and linear dsDNA, long ssDNA templates, single and double-stranded ODNs, represent a promising alternative. Furthermore, tuning of editing conditions for the chosen template as well as its structure, length, sequence optimization, homology arm (HA) modifications may have paramount importance for achieving highly efficient knock-in with favorable safety profile. This review outlines the current developments in optimization of templates for the GE mediated therapeutic gene correction.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"5 ","pages":"1068637"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9992834/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9097000","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Editorial: Insights in genome editing tools and mechanisms: 2022.","authors":"Chanjuan Jiang,&nbsp;Qunxin She,&nbsp;Hailong Wang","doi":"10.3389/fgeed.2023.1240576","DOIUrl":"https://doi.org/10.3389/fgeed.2023.1240576","url":null,"abstract":"Genome editing technologies are important tools for studying the specific functions of individual genes or modulating the expression of important genes in organisms for biological research. CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated) is the prokaryotic adaptive immune system that protects hosts from invading viruses and plasmids. CRISPR/Cas9 systems are the most frequently used type of genome editing tool, which is composed of the Cas9 nuclease and the guide RNA which directs Cas9 to the target DNA site by sequence complementarity. In natural systems, guide RNAs are composed of two separate RNA molecules, the CRISPR RNA (crRNA) and the transactivating crRNA (tracrRNA), which are commonly artificially fused together to yield a single guide RNA for genome editing (Jinek et al., 2012). In addition to CRISPR/Cas9, a variety of other CRISPR-Cas systems, such as CRISPR/Cas12a (Cpf1), have been developed to overcome the difficulties of genome editing at different loci in different organisms. Recently, several new CRISPR/Cas systems have been identified and employed for genome editing, some of which are bacteriophage origin (Al-Shayeb et al., 2022). In addition, efforts have continuously been made to optimize genome editing efficiency by different CRISPR/ Cas systems belonging to all six known types. Furthermore, CRISPR/Cas9 systems have been optimized for reducing their toxicity and for boosting knock-in efficiency in genome editing of primary human cells by using long single-stranded DNA homology-directed repair templates with short regions of double-stranded DNA containing Cas9 target sequences on both ends (Shy et al., 2023). This Research Topic is aimed to further explore the application of CRISPR/Cas genome editing tools in more biological systems and it includes four research articles. Three articles are under the category of Original Research, and one belongs to the Brief Research Report. Peanut (Arachis hypogaea L.) seeds are the source of our daily edible oil and are rich in monounsaturated oleic acid and polyunsaturated linoleic acid. Fatty Acid Desaturase 2 (FAD2) catalyzes the conversion of oleic acid to linoleic acid. Compared with linoleic acid, oleic acid has better oxidative stability and health benefits, but increasing oleic acid content by knocking out the FAD2 gene can lead to poor plant stress tolerance. The RY repeat element and 2S seed protein motif cis-regulatory elements in the 5′UTR of FAD2 genes have been suggested to have enhancer activity. Neelakandan et al. targeted these two cisregulatory elements of the FAD2 gene promoter by CRISPR/Cas9 to downregulate the expression levels of two homologous FAD2 genes in seed while maintaining normal OPEN ACCESS","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"5 ","pages":"1240576"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10367544/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9872660","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}