Gene editing technology has proved its potential in transforming animal agriculture in the coming decades, especially pig husbandry, mainly due to its high programmability, precision and simplicity in generating pigs with desired traits. Bibliometrics is a method to evaluate and explore the advancement of a research area by applying statistical methods for linking and mapping the elementary bibliographic data of research publications, such as authors, co-author, citations, journals, co-words and keywords (Ferreira, 2018). In the present study we applied bibliometric mapping strategies to gain insights into the intellectual development of the genome editing technology vis-à-vis its application in pigs. The preliminary data analysis on the total number of documents on “genome editing in pigs” (n = 727), which includes different types of research articles, reviews, book chapters, notes, letter, editorial, short survey etc. in all publishing languages, shows a constant rise till year 2022 with a decline in year 2023. This depicts the constant interest of researchers, funding agencies and organizations in the application of gene editing technology in pigs, with a change in research priority with time. The majority of publication is in English language reflecting the dominancy of English as a global scientific language. The open access documents constitute the major publication mode showing an increasing trend among authors for sharing their research data to the scientific community (Tedersoo et al., 2021). The different metrics indicate that research organizations from China, followed by United States and Japan were most active and published majority of the scientific documents on genome editing in pigs. This may be due to the fact that majority of the funding agencies belongs to these countries and pigs constitute an import food animal for their nutritional and economic security (Schneider, 2020). The similar trend continues in the topmost publishing countries, with China (43.9 %) and United States (30.1 %) publishing more than 50 % of the documents, indicating their academic influence in the field of highly translational technology like genome editing (Cohen, 2019; Wei et al., 2022). When it comes to citations per publication, United States is in the leading position, followed by Spain and Poland, indicating the greater influence of research in these countries as compared to China which occupies eighth position (Wei et al., 2022).
Bibliometric mapping was conducted only on specified research articles (n = 407), which utilized pigs either in vivo or in vitro for accomplishing their research objectives, including the end application on pigs like disease diagnosis or drug design. Review and other document type were excluded to eliminate duplication in the reporting of research findings. The publication of research articles shows an increasing trend except the initial years and the year 2022, however, the trend differs from the total publication, which shows a publication peak in 2022. The decline in research articles during year 2022 may be due to the COVID19 pandemic during year 2020-2021, which affected the research’ activities, funding and shift in research priority (Sohrabi et al., 2021), nevertheless, researches continued to publish reviews, book chapters etc. on genome editing in pigs. Thus, the difference in the results may be due to the increased number of review and other articles types published during the year 2022. The ratio of publications between Y1 and Y2 is less than one, which indicates that the application of knowledge on genomic editing in pigs, is growing at a slower rate in comparison to its application in all fields. This indicates that this field is constantly evolving and the researchers have expanded the horizon of gene editing technology in diverse laboratory animals and livestock species like cattle, buffalo, sheep, goat, poultry etc. (Wray-Cahen et al., 2022), subsequently researches on pigs are shrinking.
There are greater number of CRISPR/Cas9 associated keywords and research publications during Y2, nevertheless, ZFNs and TALENs are found only in Y1. This clearly indicates that ZFNs and TALENs are outpaced by CRISPR based gene editing platform generating targeted edits in livestock, which is mainly due to its versatility, precision and simplicity that has made it popular among researchers (Randhawa and Sengar, 2021). The author keyword analysis revealed 87.7 % unique author keyword during Y2, with only 3.5 % common keywords between Y1 and Y2, indicating a drastic change in focus area of genome editing research between the time span Y1 and Y2. The differences in the keywords indicates shift in gene editing formats like epigenome regulation, prime editing and base editing (Yin et al., 2023). Investigations on optimization of procedure of gene editing like the format of dual programmable RNA and Cas9 endonuclease, methods of delivery and standardization of workflow to reduce off-target effects and increase the editing efficiency dominated the early phase of research (Tan et al., 2013; Whitworth et al., 2014). This trend shifted to application of the gene editing tools to generated KO or KI pigs in the recent years (Liu et al., 2023).
Co-authorship analysis depicts collaboration networks of scientists and research organizations, which are commonly used to assess trends in multidisciplinary or transdisciplinary teamwork between productive scientists and institutions (Fonseca et al., 2016). China is the leading contributor to scientific collaboration on genome editing in pigs, which is in contrast to reports by Wei et al. (Wei et al., 2022), which reported United States as the top collaborator. The difference in the results may be due to differing timelines and research specificity to “pigs”. Since, the inception of application of genome editing for developing modified traits in animals (Bharati et al., 2020), it is observed that investigators and research organizations continue to collaborate, to bring together skills and interdisciplinary approaches in research. The collaborative efforts have resulted in genome edited biallelic CMP-Neu5Ac hydroxylase KO pigs (Kwon et al., 2013), KI pigs as disease model for familial hypertrophic cardiomyopathy (HCM) mutation (Montag et al., 2018), KISS1 KO hypogonadotropic pigs (Flórez et al., 2023) and genome-wide CRISPR/Cas9 KO screen for ASF in porcine cells (Pannhorst et al., 2023) to name a few. However, the co-authorship network is highly localized in the high-income economies and upper-middle income economies (Farias, 2024) like United States, China, Germany, United Kingdom and Japan which may be indicative of the high-cost infrastructure and inputs required for genome editing research. One remarkable thing is that these are also the major pig producing countries, wherein, pork and its products are in great demand (Bharati et al., 2022). Two countries from lower-middle income economies, India and Vietnam have also figured in the list (Bharati et al., 2023), nevertheless they need to increase global collaboration on intellectual and scientific basis with the established labs, so as to share ideas, resources and information on genome editing. Intellectual collaborations between scientists, research institutions and countries can deliver solutions to tackle major emerging swine diseases like ASF, PRRS, PED etc. and produce innovations for pig husbandry with reduced cost and increased productivity. The co-occurrence analysis of all keywords indicates relevancy and application of genome editing in pigs for both agricultural and biomedical applications. The supporting technologies like somatic cell nuclear transfer (SCNT), microinjection and electroporation are commonly used for generating gene edited pigs (Wang et al., 2015).
The citation analysis revealed many intriguing facts like the research impact of gene editing in pigs including the facts on productive authors, journals, organizations and country, including. It is noteworthy that the top spots in the citation impact was occupied by publication on software for designing of SgRNA with reduced off-target effect, authored by (Naito et al., 2015), followed by inactivation of PERV in pigs using CRISPR/Cas9 by Niu et al. (Niu et al., 2017), development of interspecies chimerism by Wu et al. (Wu et al., 2017), generation of biallelic GGTA1 KO pigs by Hauschild et al. (Hauschild et al., 2011) and CD163 KO pigs by Whitworth et al. (Whitworth et al., 2014), indicating the importance of disease resistance trait for pig production and xenotransplantation for biomedical applications. The researches on genome editing in pigs were published across different reputed journals, the majority of which belonged to life sciences category. Scientific reports was the most active journal in terms of number of publications, citations and bibliographic coupling. However, Science was topmost co-cited journal, which has mainly published pioneering researches on programmable dual-RNA guided DNA nucleases for genome editing, describing the basic theories and methodologies for genome editing (Horvath and Barrangou, 2010; Jinek et al., 2012; Cong et al., 2013; Mali et al., 2013).
When it comes to productive authors, Fuminori Tanihara, Takeshige Otoi and Maki Hirata were the most prolific and bibliographically coupled authors publishing 27, 27 and 26 research articles, respectively, which comprises of 19.65 % of the total publications on genome editing in pigs. It is interesting to note that Fuminori Tanihara and Takeshige Otoi share the co-authorship of the publications and hence, have same values for indicators like citations, average citation and link strength. When it comes to maximum citations, Heng Zhao, Randall S. Prather and Huaqiang Yang are the most impactful authors with 810, 766 and 740 citations, respectively, whereas, Yong Wang along with Huaqiang Yang and Randall S. Prather were the top co-cited authors with link strength of 16016, 14762 and 13940, respectively. With regards to bibliographic coupling, the investigations on dual-fluorescent surrogate system for improved genome editing in mammalian cells (Zhou et al., 2016) had the maximum link strength, followed by the report on development of GGTA1 KO pigs by electroporating CRISPR/Cas9 into in vitro-fertilized zygotes (Tanihara et al., 2020). The bibliographically coupled publications belong to diverse focus areas of research like reporter system for KO detection, xenotransplantation, process optimization for precision gene editing, delivery system, creation of disease model etc. The publication on development of KO pigs by zygote injection of CRISPR/Cas in a single step using direct cytoplasmic injection of ribonucleoprotein into zygotes by Hai et al. (2014), was the most co-cited work, followed by the pioneering report on the CRISPR/Cas which explains its potential for programmable genome editing in the journal Science by Nobel prize recipients Jennifer A Doudna, Emmanuelle Charpentier and their team (Jinek et al., 2012).
While analysing the advancements in this field, the application of genome editing technology in pig research, has progressed manifold and has been dynamically evolving since 2010, when the first evidence of application of ZFNs for the development of KO pigs was reported (Watanabe et al., 2010). This study was followed by a research publication on successful genome editing in pigs using ZFNs to create biallelic α1,3-galactosyltransferase (GGTA1) gene KO pigs(Hauschild et al., 2011). The genome editing toolbox in pigs was subsequently expanded to TALEN and CRISPR/Cas9, which not only allowed unprecedented introgression of targeted alleles in pig genome through homology directed repair (Tan et al., 2013), but also enabled precision editing. The editing efficiency and off-targets screening were conducted on different cells/cell lines of porcine origin like porcine porcine fetal fibroblasts (PFF) (Gao et al., 2023). Application of CRISPR as genetic screening tool have been widely applied for understanding the replication of disease-causing viruses like Japanese encephalitis virus (JEV) in porcine cell lines and for exploring its possible antiviral strategies. A CRISPR-Cas9-mediated cytosine-base-editing point mutation screening method was developed in pigs, which acclaimed that CRISPR-mediated base editing can be another effective technique for detecting the antiviral role of coding and noncoding variants in the calreticulin gene (Xiong et al., 2023). Nevertheless, researchers continue to work on the process optimization and increasing efficiency of genomic edit like the development of a pig model containing doxycycline-inducible SpCas9-expressing (DIC) pigs for in vivo and/or in vitro genome and epigenome modification (Jin et al., 2023).
The summary of inferences drawn from the bibliometric analysis in the present study is shown graphically in Figure 9. The focus area of genome editing research for pig husbandry includes incorporation of traits related to greater muscling, lean meat, cold tolerance, development of pig resistance to diseases like ASF, PRRS, pseudorabies etc. (Whitworth et al., 2016; Burkard et al., 2017; Pannhorst et al., 2023). In the area of biomedical engineering xenotransplantation, development of human disease model and basic research has always been a priority. Newer formats like base and prime editors, CRISPR based genetic screens, diagnostics like SHERLOCK and DETECTOR which uses Cas13 and Cas12, respectively, including CRISPR based gene therapy are emerging areas in this field (Gootenberg et al., 2017; Harrington et al., 2018). The gene editing toolbox continues to expand with many variants of natural and engineered Cas nuclease (Gao et al., 2023; Yin et al., 2023). Scientists have continued the quest for identifying novel natural and developing engineered Cas9 nucleases, which can expand the scope of DNA target and enable efficient genome editing in diverse mammalian cells. In this direction, protospacer adjacent motif (PAM) regions of three orthologs of CjCas9 nuclease viz. Hsp1Cas9, Hsp2Cas9, CcuCas9 were investigated and a chimeric nuclease Hsp1-Hsp2Cas9 was generated, which could recognize a simple PAM N4CY (Y = C or T). The researchers developed a high-fidelity Hsp1-Hsp2Cas9-KY, which exhibited undetectable off-target effects as compared to SpCas9. This study employed PFFs for assessing indel efficiency of Hsp1-Hsp2Cas9-Y for B4GALNT2 and CMAH genes responsible for hyperacute rejection during organ transplantation due to natural antibodies (Gao et al., 2023).
Investigations on IGA of pigs for heat stress adaptation along with production traits for reducing the environmental footprint are most probably the research gaps that need to be addressed. Since, pig farming is highly susceptible to global warming induced thermal stress, hence pigs with improved climate resilient trait would be desired in the coming years. At the same time applying genome editing technology for decreasing environmental footprint of pig production system would be groundbreaking research. Nevertheless, an impact assessment studies on future farming of IGAs are critical to devise strategies for its practical utility and adoption by end users and stakeholders. However, in spite of significant advancements in this field, there are technical challenges like off-target effects, which have resulted in reduced viability and deformities in KO pigs (Chen et al., 2022). There exist regulatory, ethical and societal issues in channelizing the gene edited pigs from the boundaries of labs to the farmers field and then to the food plate (Meyer and Vergnaud, 2021; Martin-Collado et al., 2022), nevertheless the consumer acceptance for gene edited food will be a major factor in deciding the fate of gene edited pigs (Martin-Collado et al., 2022).
There are prospects of expansion of genome editing research on pigs, given the scientific community and leading countries endorses greater global collaboration on intellectual and resources sharing including promotion of cost reduction on inputs for genome editing research. At the same time harmonization of regulatory policies and consumer education on differences between “genome editing” and “genome modification” would change social perceptions and increase adoption of gene edited food animals.