The promiscuous biotin ligase TurboID reveals the proxisome of the T3SS chaperone IpgC in Shigella flexneri.

Promiscuous biotin ligases derived from the bacterial enzyme BirA are used to identify proteins vicinal to a bait protein, thereby defining its proxisome. Despite the popularity of this approach, surprisingly little is known about its use in prokaryotes. Here, we compared the activity of four widely used promiscuous biotin ligases in the cytoplasm of Shigella flexneri, a pathogenic subgroup of Escherichia coli. Our data indicate that the kinetics of TurboID's biotinylating activity is the highest of those tested. In addition, TurboID showed reduced interaction with the natural BirA binding partners, BccP and the biotin operator, when compared to its ancestor BioID. We therefore evaluated the ability of TurboID to probe the proxisome of the type III secretion system (T3SS) chaperone IpgC and the transcriptional activator MxiE. When the T3SS is inactive (off-state), these proteins are inhibited by forming complexes with the T3SS substrates OspD1 and IpaBC, respectively. In contrast, when the T3SS is active (on-state), OspD1 and IpaBC are secreted allowing MxiE and IpgC to interact together and activate their target genes. The results obtained with the IpgC and TurboID fusions capture a good fraction of these known interactions. It also suggests that the availability of IpgC increases in the on-state, resulting in a greater number of proteins detected in its vicinity. Among these is the T3SS ATPase SpaL (also known as Spa47 or SctN), further supporting the notion that chaperones escort their substrate to the T3SS. Interestingly, a specific subset of proteins conserved in E. coli completes the IpgC proxisome in the on-state.IMPORTANCEPromiscuous biotin ligases are widely used to study protein function in eukaryotes. Strikingly, their use in prokaryotes has been rare. Indeed, the small volume and the cytoplasmic location of the biotin ligase's natural binding partners in these organisms pose unique challenges that can interfere with the study of the proxisome of proteins of interest. Here, we evaluated four of the most common promiscuous biotin ligases and found TurboID was best suited for use in the cytoplasm of Shigella flexneri. Using this method, we extended the proxisome of IpgC beyond its known direct binding partners involved in the regulation of the type III secretion system (T3SS) signaling cascade. Of particular interest for further study are transcription factors and housekeeping proteins that are enriched around IpgC when the T3SS is active. We propose a model in which the increased availability of IpgC in the on-state may allow cross-talk of the T3SS with other cellular processes.

A novel Affi-Cova magnetic nanoparticles for one-step covalent immobilization of His-tagged enzyme directly from crude cell lysate.

Owing to the rapid advancement of in vitro synthetic biology, functional carriers capable of covalently binding target proteins from crude lysates under mild conditions have garnered escalating attention. Herein, a magnetic nanoparticle with affinity/covalent bifunction (MNP@Affi-Cova) was developed for the direct covalent immobilization of the recombinant enzyme of His-tagged birA (r-birA) from crude cell lysates in a single step. This innovative approach is attributed to the presence of chelated Ni2+ ions and epoxy groups on the surface of the beads. The fabricated magnetic nanoparticles were characterized by SEM, FT-IR spectrum, and zeta potential. The application conditions and stability of the MNP@Affi-Cova beads were systematically evaluated. Notably, the MNP@Affi-Cova beads exhibited a covalent capture efficiency of 91.25 µg r-birA/mg beads from a cell lysate supernatant containing 2.62 mg/mL crude protein. The immobilized r-birA exhibited significantly enhanced pH and thermal stability compared to the free counterpart. Additionally, the reusability of the immobilized r-birA on MNP@Affi-Cova demonstrated the retention of 76.1 % of its initial activity over ten cycles. These results suggest that the MNP@Affi-Cova presents considerable potential as a support for the covalent immobilization of recombinant His-tagged enzymes directly from crude lysates, thereby circumventing the labor-intensive purification process typically required before enzyme immobilization.

Structural insights into BirA from Haemophilus influenzae, a bifunctional protein as a biotin protein ligase and a transcriptional repressor.

Biotin is an essential coenzyme involved in various metabolic processes across all known organisms, with biotinylation being crucial for the activity of carboxylases. BirA from Haemophilus influenzae is a bifunctional protein that acts as a biotin protein ligase and a transcriptional repressor. This study reveals the crystal structures of Hin BirA in both its apo- and holo-(biotinyl-5'-AMP bound) forms. As a class II BirA, it consists of three domains: N-terminal DNA binding domain, central catalytic domain, and C-terminal SH3-like domain. The structural analysis shows that the biotin-binding loop forms an ordered structure upon biotinyl-5'-AMP binding. This facilitates its interaction with the ligand and promotes protein dimerization. Comparative studies with other BirA homologs from different organisms indicate that the residues responsible for binding biotinyl-5'-AMP are highly conserved. This study also utilized AlphaFold2 to model the potential heterodimeric interaction between Hin BirA and biotin carboxyl carrier protein, thereby providing insights into the structural basis for biotinylation. These findings enhance our understanding of the structural and functional characteristics of Hin BirA, highlighting its potential as a target for novel antibiotics that disrupt the bacterial biotin synthesis pathways.

[Progress in research and application of the biotin ligase BirA and its mutants in pathogen-host interaction].

Proteins serve as the primary executors of cellular activities in organisms, and thus investigating the subcellular localization and interactions of proteins is crucial for understanding protein functions and elucidating the molecular mechanisms in organisms. Proximity labeling is a recently developed effective method for detecting protein-protein interactions in live cells. Compared with the conventional methods for studying protein-protein interactions, proximity labeling demonstrates high sensitivity, strong specificity, and low background and is widely employed in the research of protein-protein interactions between pathogens and hosts. This article reviews the recent progress in the development and applications of the biotin ligase BirA and its mutants and elucidates the functioning principles of several classical biotin ligases. This review aims to clarify the role of proximity labeling based on BirA and its mutants in identifying protein-protein interactions between pathogens and hosts.

High-Throughput Protein Expression Screening of Cell-Surface Protein Ectodomains.

Cell-surface receptors can be difficult to express and purify for structural and biochemical studies due to low expression levels, misfolding, aggregation, and instability. Cell-surface receptor ectodomains are more amenable to large-scale production, but this requires designing and testing various truncation constructs. However, since each protein is unique, testing these constructs individually for many targets is a time-consuming process. In this context, we present a high-throughput ELISA fluorescence approach that allows the rapid assessment of numerous recombinant constructs simultaneously. Cell-surface ectodomains are expressed in small scale, enzymatically biotinylated, and detected using a C-terminal His-tag. As an example, we tested the expression of truncation constructs for the neurexin, neuroligin, and latrophilin families and show that the small-scale ELISA allowed us to prioritize well-expressing construct for large-scale production. By employing this method, one can efficiently detect clones with low expression levels, streamlining the process and saving valuable time in identifying optimal candidates for further study.

A Facile Method to Append a Bio-ID Tag to Endogenous Mutant Kras Alleles.

KRAS mutations occur in approximately ~50% of colorectal cancers (CRCs) and are associated with poor prognosis and resistance to therapy. While these most common mutations found at amino acids G12, G13, Q61, and A146 have long been considered oncogenic drivers of CRC, emerging clinical data suggest that each mutation may possess different biological functions, resulting in varying consequences in oncogenesis. Currently, the mechanistic underpinnings associated with each allelic variation remain unclear. Elucidating the unique effectors of each KRAS mutant could both increase the understanding of KRAS biology and provide a basis for allele-specific therapeutic opportunities. Biotinylation identification (BioID) is a method to label and identify proteins located in proximity of a protein of interest. These proteins are captured through the strong interaction between the biotin label and streptavidin bead and subsequently identified by mass spectrometry. Here, we developed a protocol using CRISPR-mediated gene editing to generate endogenous BioID2-tagged KrasG12D and KrasG12V isogenic murine colon epithelial cell lines to identify unique protein proximity partners by BioID.

A systematic proximity ligation approach to studying protein-substrate specificity identifies the substrate spectrum of the Ssh1 translocon.

Many cellular functions are carried out by protein pairs or families, providing robustness alongside functional diversity. For such processes, it remains a challenge to map the degree of specificity versus promiscuity. Protein-protein interactions (PPIs) can be used to inform on these matters as they highlight cellular locals, regulation and, in cases where proteins affect other proteins - substrate range. However, methods to systematically study transient PPIs are underutilized. In this study, we create a novel approach to systematically compare stable or transient PPIs between two yeast proteins. Our approach, Cel-lctiv (CELlular biotin-Ligation for Capturing Transient Interactions in vivo), uses high-throughput pairwise proximity biotin ligation for comparing PPIs systematically and in vivo. As a proof of concept, we studied the homologous translocation pores Sec61 and Ssh1. We show how Cel-lctiv can uncover the unique substrate range for each translocon allowing us to pinpoint a specificity determinator driving interaction preference. More generally, this demonstrates how Cel-lctiv can provide direct information on substrate specificity even for highly homologous proteins.

Proximity Mapping of CCP6 Reveals Its Association with Centrosome Organization and Cilium Assembly.

The cytosolic carboxypeptidase 6 (CCP6) catalyzes the deglutamylation of polyglutamate side chains, a post-translational modification that affects proteins such as tubulins or nucleosome assembly proteins. CCP6 is involved in several cell processes, such as spermatogenesis, antiviral activity, embryonic development, and pathologies like renal adenocarcinoma. In the present work, the cellular role of CCP6 has been assessed by BioID, a proximity labeling approach for mapping physiologically relevant protein-protein interactions (PPIs) and bait proximal proteins by mass spectrometry. We used HEK 293 cells stably expressing CCP6-BirA* to identify 37 putative interactors of this enzyme. This list of CCP6 proximal proteins displayed enrichment of proteins associated with the centrosome and centriolar satellites, indicating that CCP6 could be present in the pericentriolar material. In addition, we identified cilium assembly-related proteins as putative interactors of CCP6. In addition, the CCP6 proximal partner list included five proteins associated with the Joubert syndrome, a ciliopathy linked to defects in polyglutamylation. Using the proximity ligation assay (PLA), we show that PCM1, PIBF1, and NudC are true CCP6 physical interactors. Therefore, the BioID methodology confirms the location and possible functional role of CCP6 in centrosomes and centrioles, as well as in the formation and maintenance of primary cilia.

Molecular methods to study protein trafficking between organs.

Interorgan communication networks are key regulators of organismal homeostasis, and their dysregulation is associated with a variety of pathologies. While mass spectrometry proteomics identifies circulating proteins and can correlate their abundance with disease phenotypes, the tissues of origin and destinations of these secreted proteins remain largely unknown. In vitro approaches to study protein secretion are valuable, however, they may not mimic the complexity of in vivo environments. More recently, the development of engineered promiscuous BirA* biotin ligase derivatives has enabled tissue-specific tagging of cellular secreted proteomes in vivo. The use of biotin as a molecular tag provides information on the tissue of origin and destination, and enables the enrichment of low-abundance hormone proteins. Therefore, promiscuous protein biotinylation is a valuable tool to study protein secretion in vivo.

SUMO-ID: A Strategy for the Identification of SUMO-Dependent Proximal Interactors.

Posttranslational modifications by the ubiquitin-like family (UbL) of proteins determine the biological fate of a substrate, including new interaction partners. In the case of the small ubiquitin-like modifier (SUMO), this is achieved in part through its non-covalent interaction with SUMO-interacting motifs (SIMs) found in some proteins. Investigating such partner-complex formation is particularly challenging due to the fast dynamics and reversibility of SUMO modifications and the low affinity of SUMO-SIM interactions. Here, we present a detailed protocol of SUMO-ID, a technology that merges promiscuous proximity biotinylation by TurboID enzyme and protein-fragment complementation strategy to specifically biotinylate SUMO-dependent interactors of particular substrates. When coupled to streptavidin-affinity purification and mass spectrometry, SUMO-ID efficiently identifies SUMO-dependent interactors of a given protein. The methodology describes all the steps from SUMO-ID cell line generation to LC-MS sample preparation to study SUMO-dependent interactors of a particular protein. The protocol is generic and therefore adaptable to study other UbL-dependent interactors, such as ubiquitin.

An integrated gene network analysis to decode the multi-drug resistance mechanism in Klebsiella pneumoniae.

Antimicrobial resistance (AMR) among microorganisms has become one of the worldwide concerns of this century and continues to challenge us. To properly understand this problem, it is essential to know the genes that cause AMR and their resistance mechanisms. Our present study focused on Klebsiella pneumoniae, which possesses AMR genes conferring resistance against multiple antibiotics. A gene interaction network of 42 functional partners was constructed and analyzed to broaden our understanding. Three closely related clusters (C1-C3) having an association with multi-drug resistance mechanisms were identified by clustering analysis. The enrichment analysis illustrated 30 genes in biological processes, 24 genes in molecular function, and 25 genes in cellular components having a significant role. The analysis of the gene interaction network revealed genes birA2, folP, pabC, folA, gyrB, glmM, gyrA, thyA_2 had maximum no. of interactions with their functional partners viz. 26, 25, 25, 24, 23, 23, 23, 23 respectively and can be considered as hub genes. Analyzing the enriched pathways and Gene Ontologies provides insight into AMR's molecular basis. In addition, the proposed study could aid the researchers in developing new treatment options to combat multi-drug resistant K. pneumoniae.

In vivo Characterization of Endogenous Protein Interactomes in Drosophila Larval Brain, Using a CRISPR/Cas9-based Strategy and BioID-based Proximity Labeling.

Understanding protein-protein interactions (PPIs) and interactome networks is essential to reveal molecular mechanisms mediating various cellular processes. The most common method to study PPIs in vivo is affinity purification combined with mass spectrometry (AP-MS). Although AP-MS is a powerful method, loss of weak and transient interactions is still a major limitation. Proximity labeling (PL) techniques have been developed as alternatives to overcome these limitations. Proximity-dependent biotin identification (BioID) is one such widely used PL method. The first-generation BioID enzyme BirA*, a promiscuous bacterial biotin ligase, has been effectively used in cultured mammalian cells; however, relatively slow enzyme kinetics make it less effective for temporal analysis of protein interactions. In addition, BirA* exhibits reduced activity at temperatures below 37°C, further restricting its use in intact organisms cultured at lower optimal growth temperatures ( e.g., Drosophila melanogaster ). TurboID, miniTurbo, and BirA*-G3 are next generation BirA* variants with improved catalytic activity, allowing investigators to use this powerful tool in model systems such as flies. Here, we describe a detailed experimental workflow to efficiently identify the proximal proteome (proximitome) of a protein of interest (POI) in the Drosophila brain using CRISPR/Cas9-induced homology-directed repair (HDR) strategies to endogenously tag the POI with next generation BioID enzymes.

A genetic model for in vivo proximity labelling of the mammalian secretome.

Organ functions are highly specialized and interdependent. Secreted factors regulate organ development and mediate homeostasis through serum trafficking and inter-organ communication. Enzyme-catalysed proximity labelling enables the identification of proteins within a specific cellular compartment. Here, we report a BirA*G3 mouse strain that enables CRE-dependent promiscuous biotinylation of proteins trafficking through the endoplasmic reticulum. When broadly activated throughout the mouse, widespread labelling of proteins was observed within the secretory pathway. Streptavidin affinity purification and peptide mapping by quantitative mass spectrometry (MS) proteomics revealed organ-specific secretory profiles and serum trafficking. As expected, secretory proteomes were highly enriched for signal peptide-containing proteins, highlighting both conventional and non-conventional secretory processes, and ectodomain shedding. Lower-abundance proteins with hormone-like properties were recovered and validated using orthogonal approaches. Hepatocyte-specific activation of BirA*G3 highlighted liver-specific biotinylated secretome profiles. The BirA*G3 mouse model demonstrates enhanced labelling efficiency and tissue specificity over viral transduction approaches and will facilitate a deeper understanding of secretory protein interplay in development, and in healthy and diseased adult states.

Tag Thy Neighbour: Nanometre-Scale Insights Into Kinetoplastid Parasites With Proximity Dependent Biotinylation.

Proximity labelling is a powerful and rapidly developing technology for exploring the interaction space and molecular environment of a protein of interest at the nanometre scale. In proximity labelling, a promiscuous biotinylating enzyme is genetically fused to the protein of interest, initiation of labelling then results in the biotinylating enzyme generating reactive biotin which covalently 'tags' nearby molecules. Importantly, this labelling takes place in vivo whilst the protein of interest continues to perform its normal functions in the cell. Due to its unique advantageous characteristics, proximity labelling is driving discoveries in an ever increasing range of organisms. Here, we highlight the applications of proximity labelling to the study of kinetoplastids, a group of eukaryotic protozoa that includes trypanosomes and Leishmania which can cause serious disease in humans and livestock. We first provide a general overview of the proximity labelling experimental workflow including key labelling enzymes used, proper experimental design with appropriate controls and robust statistical analysis to maximise the amount of reliable spatial information that is generated. We discuss studies employing proximity labelling in kinetoplastid parasites to illustrate how these key principles of experimental design are applied. Finally, we highlight emerging trends in the development of proximity labelling methodology.

Studying the ubiquitin code through biotin-based labelling methods.

Post-translational modifications of cellular substrates by members of the ubiquitin (Ub) and ubiquitin-like (UbL) family are crucial for regulating protein homeostasis in organisms. The term "ubiquitin code" encapsulates how this diverse family of modifications, via adding single UbLs or different types of UbL chains, leads to specific fates for substrates. Cancer, neurodegeneration and other conditions are sometimes linked to underlying errors in this code. Studying these modifications in cells is particularly challenging since they are usually transient, scarce, and compartment-specific. Advances in the use of biotin-based methods to label modified proteins, as well as their proximally-located interactors, facilitate isolation and identification of substrates, modification sites, and the enzymes responsible for writing and erasing these modifications, as well as factors recruited as a consequence of the substrate being modified. In this review, we discuss site-specific and proximity biotinylation approaches being currently applied for studying modifications by UbLs, highlighting the pros and cons, with mention of complementary methods when possible. Future improvements may come from bioengineering and chemical biology but even now, biotin-based technology is uncovering new substrates and regulators, expanding potential therapeutic targets to manipulate the Ub code.

Proximity-dependent biotinylation approaches to study apicomplexan biology.

In the last 10 years, proximity-dependent biotinylation (PDB) techniques greatly expanded the ability to study protein environments in the living cell that range from specific protein complexes to entire compartments. This is achieved by using enzymes such as BirA* and APEX that are fused to proteins of interest and biotinylate proteins in their proximity. PDB techniques are now also increasingly used in apicomplexan parasites. In this review, we first give an overview of the main PDB approaches and how they compare with other techniques that address similar questions. PDB is particularly valuable to detect weak or transient protein associations under physiological conditions and to study cellular structures that are difficult to purify or have a poorly understood protein composition. We also highlight new developments such as novel smaller or faster-acting enzyme variants and conditional PDB approaches, providing improvements in both temporal and spatial resolution which may offer broader application possibilities useful in apicomplexan research. In the second part, we review work using PDB techniques in apicomplexan parasites and how this expanded our knowledge about these medically important parasites.

Lernaea cyprinacea Linnaeus, 1758 (Copepoda: Lernaeidae) infection on Betta rubra Perugia, 1893 (Anabantiformes: Osphronemidae) from Aceh Province, Indonesia / Infecção por Lernaea cyprinacea Linnaeus, 1758 (Copepoda: Lernaeidae) em Betta rubra Perugia, 1893 (Anabantiformes: Osphronemidae) provenientes da província de Aceh, Indonésia

Abstract Betta rubra is an ornamental freshwater fish endemic to northern Sumatra, Indonesia. The B. rubra population has decreased in recent decades, and is classified as an endangered species in the IUCN Red List. This study aims to report for the first time infection by L. cyprinacea in B. rubra harvested from the Aceh Besar region of Indonesia. The fish samples were obtained from the Cot Bira tributaries, Aceh Besar District, Indonesia from January to December 2020. The results showed that the parasite infected 6 out of 499 samples in August and September, with a prevalence and intensity rate of 1% and 2 parasites/fish, respectively. The eyes and pectoral fins were the common infection sites. Despite B. rubra is not an optimal host (small size) for the parasite, this parasite might serve as additional threatening factors for the endangered B. rubra fish population.

Resumo Betta rubra é um peixe de água doce ornamental endemico da região norte Sumatra, Indonesia. A população de Betta rubra diminuiu ao longo dos anos, sendo classificada como espécie em extinção na Lista Vermelha da IUCN. Este estudo tem como objetivo relatar pela primeira vez infecção por L. cyprinacea em B. rubra coletados na região de Aceh Besar na Indonésia. As amostras de peixes foram obtidas nos afluentes Cot Bira, distrito de Aceh Besar, Indonésia de janeiro a dezembro de 2020. Os resultados mostraram que o parasito infectou 6 das 499 amostras em agosto e setembro, com uma prevalência e taxa de intensidade de 1% e 2 parasitas/peixes, respectivamente. Os olhos e as nadadeiras peitorais foram os sítios de infecção mais comuns. Apesar de B. rubra não ser um hospedeiro ideal (pequeno tamanho) para o parasita, este parasita pode servir como fator de ameaça adicional para a população de peixes B. rubra, ameaçada de extinção.

Application of Skyline for Analysis of Protein-Protein Interactions In Vivo.

Quantitative and qualitative analyses of cell protein composition using liquid chromatography/tandem mass spectrometry are now standard techniques in biological and clinical research. However, the quantitative analysis of protein-protein interactions (PPIs) in cells is also important since these interactions are the bases of many processes, such as the cell cycle and signaling pathways. This paper describes the application of Skyline software for the identification and quantification of the biotinylated form of the biotin acceptor peptide (BAP) tag, which is a marker of in vivo PPIs. The tag was used in the Proximity Utilizing Biotinylation (PUB) method, which is based on the co-expression of BAP-X and BirA-Y in mammalian cells, where X or Y are interacting proteins of interest. A high level of biotinylation was detected in the model experiments where X and Y were pluripotency transcription factors Sox2 and Oct4, or heterochromatin protein HP1γ. MRM data processed by Skyline were normalized and recalculated. Ratios of biotinylation levels in experiment versus controls were 86 ± 6 (3 h biotinylation time) and 71 ± 5 (9 h biotinylation time) for BAP-Sox2 + BirA-Oct4 and 32 ± 3 (4 h biotinylation time) for BAP-HP1γ + BirA-HP1γ experiments. Skyline can also be applied for the analysis and identification of PPIs from shotgun proteomics data downloaded from publicly available datasets and repositories.

In vivo Profiling of the Alk Proximitome in the Developing Drosophila Brain.

Anaplastic lymphoma kinase (Alk) is an evolutionary conserved receptor tyrosine kinase belonging to the insulin receptor superfamily. In addition to its well-studied role in cancer, numerous studies have revealed that Alk signaling is associated with a variety of complex traits such as: regulation of growth and metabolism, hibernation, regulation of neurotransmitters, synaptic coupling, axon targeting, decision making, memory formation and learning, alcohol use disorder, as well as steroid hormone metabolism. In this study, we used BioID-based in vivo proximity labeling to identify molecules that interact with Alk in the Drosophila central nervous system (CNS). To do this, we used CRISPR/Cas9 induced homology-directed repair (HDR) to modify the endogenous Alk locus to produce first and next generation Alk::BioID chimeras. This approach allowed identification of Alk proximitomes under physiological conditions and without overexpression. Our results show that the next generation of BioID proteins (TurboID and miniTurbo) outperform the first generation BirA* fusion in terms of labeling speed and efficiency. LC-MS3-based BioID screening of AlkTurboID and AlkminiTurbo larval brains revealed an extensive neuronal Alk proximitome identifying numerous potential components of Alk signaling complexes. Validation of Alk proximitome candidates further revealed co-expression of Stardust (Sdt), Discs large 1 (Dlg1), Syntaxin (Syx) and Rugose (Rg) with Alk in the CNS and identified the protein-tyrosine-phosphatase Corkscrew (Csw) as a modulator of Alk signaling.

Optimal Method for Test and Repair Memories Using Redundancy Mechanism for SoC.

The current system-on-chip (SoC)-based devices uses embedded memories of enormous size. Most of these systems' area is dense with memories and promotes different types of faults appearance in memory. The memory faults become a severe issue when they affect the yield of the product. A memory-test and -repair scheme is an attractive solution to tackle this kind of problem. The built-in self-repair (BISR) scheme is a prominent method to handle this issue. The BISR scheme is widely used to repair the defective memories for an SoC-based system. It uses a built-in redundancy analysis (BIRA) circuit to allocate the redundancy when defects appear in the memory. The data are accessed from the redundancy allocation when the faulty memory is operative. Thus, this BIRA scheme affects the area overhead for the BISR circuit when it integrates to the SoC. The spare row and spare column-based BISR method is proposed to receive the optimal repair rate with a low area overhead. It tests the memories for almost all the fault types and repairs the memory by using spare rows and columns. The proposed BISR block's performance was measured for the optimal repair rate and the area overhead. The area overhead, timing, and repair rate were compared with the other approaches. Furthermore, the study noticed that the repair rate and area overhead would increase by increasing the spare-row/column allocation.