Clustered Regularly Interspaced Short Palindromic Repeats Cas Research Articles

A bioinformatic approach to identify confirmed and probable CRISPR-Cas systems in the Acinetobacter calcoaceticus-Acinetobacter baumannii complex genomes.

The Acinetobacter calcoaceticus-Acinetobacter baumannii complex, or Acb complex, consists of six species: Acinetobacter baumannii, Acinetobacter calcoaceticus, Acinetobacter nosocomialis, Acinetobacter pittii, Acinetobacter seifertii, and Acinetobacter lactucae. A. baumannii is the most clinically significant of these species and is frequently related to healthcare-associated infections (HCAIs). Clustered regularly interspaced short palindromic repeat (CRISPR) arrays and associated genes (cas) constitute bacterial adaptive immune systems and function as variable genetic elements. This study aimed to conduct a genomic analysis of Acb complex genomes available in databases to describe and characterize CRISPR systems and cas genes. Acb complex genomes available in the NCBI and BV-BRC databases, the identification and characterization of CRISPR-Cas systems were performed using CRISPRCasFinder, CRISPRminer, and CRISPRDetect. Sequence types (STs) were determined using the Oxford scheme and ribosomal multilocus sequence typing (rMLST). Prophages were identified using PHASTER and Prophage Hunter. A total of 293 genomes representing six Acb species exhibited CRISPR-related sequences. These genomes originate from various sources, including clinical specimens, animals, medical devices, and environmental samples. Sequence typing identified 145 ribosomal multilocus sequence types (rSTs). CRISPR-Cas systems were confirmed in 26.3% of the genomes, classified as subtypes I-Fa, I-Fb and I-Fv. Probable CRISPR arrays and cas genes associated with CRISPR-Cas subtypes III-A, I-B, and III-B were also detected. Some of the CRISPR-Cas systems are associated with genomic regions related to Cap4 proteins, and toxin-antitoxin systems. Moreover, prophage sequences were prevalent in 68.9% of the genomes. Analysis revealed a connection between these prophages and CRISPR-Cas systems, indicating an ongoing arms race between the bacteria and their bacteriophages. Furthermore, proteins associated with anti-CRISPR systems, such as AcrF11 and AcrF7, were identified in the A. baumannii and A. pittii genomes. This study elucidates CRISPR-Cas systems and defense mechanisms within the Acb complex, highlighting their diverse distribution and interactions with prophages and other genetic elements. This study also provides valuable insights into the evolution and adaptation of these microorganisms in various environments and clinical settings.

CRISPR-Cas systems feature and targeting phages diversity in Lacticaseibacillus rhamnosus strains.

One of the most important adaptive immune systems in bacteria against phages is clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (CAS) genes. In this investigation, an approach based on genome mining was employed to characterize the CRISPR-Cas systems of Lacticaseibacillus rhamnosus strains. The analysis involved retrieving complete genome sequences of L. rhamnosus strains, and assessing the diversity, prevalence, and evolution of their CRISPR-Cas systems. Following this, an analysis of homology in spacer sequences from identified CRISPR arrays was carried out to investigate and characterize the range of target phages. The findings revealed that 106 strains possessed valid CRISPR-Cas structures (comprising CRISPR loci and Cas genes), constituting 45% of the examined L. rhamnosus strains. The diversity observed in the CRISPR-Cas systems indicated that all identified systems belonged to subtype II-A. Analyzing the homology of spacer sequences with phage and prophage genomes discovered that strains possessing only CRISPR-Cas subtype II targeted a broader spectrum of foreign phages. In summary, this study suggests that while there is not significant diversity among the CRISPR-Cas systems identified in L. rhamnosus strains, there exists notable variation in subtype II-A systems between L. rhamnosus and other lactobacilli. The diverse nature of these CRISPR-Cas systems underscores their natural activity and importance in adaptive immunity.

Application of CRISPR/Cas9-mediated gene editing for abiotic stress management in crop plants.

Abiotic stresses, including drought, salinity, cold, heat, and heavy metals, extensively reducing global agricultural production. Traditional breeding approaches and transgenic technology have been widely used to mitigate the risks of these environmental stresses. The discovery of engineered nucleases as genetic scissors to carry out precise manipulation in crop stress-responsive genes and associated molecular network has paved the way for sustainable management of abiotic stress conditions. In this context, the clustered regularly interspaced short palindromic repeat-Cas (CRISPR/Cas)-based gene-editing tool has revolutionized due to its simplicity, accessibility, adaptability, flexibility, and wide applicability. This system has great potential to build up crop varieties with enhanced tolerance against abiotic stresses. In this review, we summarize the latest findings on understanding the mechanism of abiotic stress response in plants and the application of CRISPR/Cas-mediated gene-editing system towards enhanced tolerance to a multitude of stresses including drought, salinity, cold, heat, and heavy metals. We provide mechanistic insights on the CRISPR/Cas9-based genome editing technology. We also discuss applications of evolving genome editing techniques such as prime editing and base editing, mutant library production, transgene free and multiplexing to rapidly deliver modern crop cultivars adapted to abiotic stress conditions.

Comparative genomics reveals the diversity of CRISPR-Cas locus in Azotobacter organisms.

Clustered regularly interspaced short palindromic repeats (CRISPRs) are known to provide adaptive immunity to bacteria against invading bacteriophages. In recent years, CRISPR-based technologies have been used for creating improved plant varieties; however, the indigenous CRISPR-Cas elements of plant growth-promoting bacteria are usually neglected. These indigenous genetic cassettes have evolved over millions of years and have shaped the bacterial genome. Therefore, these genetic loci can be used to study the adaptive capability of the bacteria in the environment. This study aims to bioinformatically analyze the genomes of a common free-living nitrogen-fixing Azotobacter spp. to assess their CRISPR-Cas diversity. Strains of Azotobacter vinelandii and Azotobacter chroococcum were found to harbor a large number of spacers. The phylogeny of different Cas and Cse1 proteins revealed a close evolutionary relationship among A. chroococcum B3, A. chroococcum NCIMB 8003 locus II, and A. vinelandii DJ locus I. The secondary structure of the hairpin loop of the repeat was also analyzed, and a correlation was derived between the structural stability of the hairpin loop and the number of spacers acquired by the CRISPR loci. These findings revealed the diversity and evolution of the CRISPR sequences and Cas proteins in Azotobacter species. Although the adaptive immune system of bacteria against bacteriophage, CRISPR-Cas, has been identified in many bacteria, studies of plant growth-promoting bacteria have been neglected. These indigenous CRISPRs have shaped the genome over millions of years and their study can lead to the understanding of the genome composition of these organisms. Our results strengthen the idea of using A. chroococcum and A. vinelandii as biofertilizer strains as they possess more spacers with highly stable repeat sequences, thereby imparting them higher chance of survival against mobile genetic elements like phages and plasmids.

Genome editing in rice: New paths for an old crop

Abstract Rice is one of the most consumed cereals worldwide and feeds about 3 billion people. However, the occurrence of climate changes has influenced abiotic and biotic stresses and consequently affected the production and quality of rice grains. The development of new, higher yielding cultivars is necessary to ensure global food security. Although great progress has already been achieved by conventional breeding, biotechnological tools, such as transgenics and genome editing, can aid in meeting future demands. Gene editing is characterized by cutting and modifying target genes. Among the genome editing techniques, CRISPR/Cas (clustered regularly interspaced short palindromic repeats/Cas), has been proposed because of its ease of manipulation. Variants such as multiple Cas proteins, base editing and prime editing, which aim to increase editing efficiency have also been proposed. Edited plants are more accepted because they are transgene-free. Thus, the objective of this review is to describe the different gene editing techniques and their respective applications in rice breeding. It was observed that the CRISPR/Cas tool was efficient in gene editing in studies related to yield, tolerance to biotic and abiotic stresses, and rice grain quality. However, the impact of this approach in breeding programs depends on the cultivation of edited plants on a large scale in the field. Based on the progress made so far, it is believed that rice breeders can bet on CRISPR/Cas and its variants to help face the current and future challenges of climate change and high food demand.

Advances in CRISPR therapeutics.

The clustered regularly interspaced short palindromic repeats (CRISPR) renaissance was catalysed by the discovery that RNA-guided prokaryotic CRISPR-associated (Cas) proteins can create targeted double-strand breaks in mammalian genomes. This finding led to the development of CRISPR systems that harness natural DNA repair mechanisms to repair deficient genes more easily and precisely than ever before. CRISPR has been used to knock out harmful mutant genes and to fix errors in coding sequences to rescue disease phenotypes in preclinical studies and in several clinical trials. However, most genetic disorders result from combinations of mutations, deletions and duplications in the coding and non-coding regions of the genome and therefore require sophisticated genome engineering strategies beyond simple gene knockout. To overcome this limitation, the toolbox of natural and engineered CRISPR–Cas systems has been dramatically expanded to include diverse tools that function in human cells for precise genome editing and epigenome engineering. The application of CRISPR technology to edit the non-coding genome, modulate gene regulation, make precise genetic changes and target infectious diseases has the potential to lead to curative therapies for many previously untreatable diseases.

A comprehensive overview of CRISPR/Cas 9 technology and application thereof in drug discovery.

Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas technology possesses revolutionary potential to positively affect various domains of drug discovery. It has initiated a rise in the area of genetic engineering and its advantages range from classical science to translational medicine. These genome editing systems have given a new dimension to our capabilities to alter, detectand annotate specified gene sequences. Moreover, the ease, robustnessand adaptability of the CRISPR/Cas9 technology have led to its extensive utilization in research areas in such a short period of time. The applications include the development of model cell lines, understanding disease mechanisms, discovering disease targets, developing transgenic animals and plants, and transcriptional modulation. Further, the technology is rapidly growing; hence, an overlook of progressive success is crucial. This review presents the current status of the CRISPR-Cas technology in a tailor-made format from its discovery to several advancements for drug discovery alongwith future trends associated with possibilities and hurdles including ethical concerns.

Three strategies of transgenic manipulation for crop improvement.

Heterologous expression of exogenous genes, overexpression of endogenous genes, and suppressed expression of undesirable genes are the three strategies of transgenic manipulation for crop improvement. Up to 2020, most (227) of the singular transgenic events (265) of crops approved for commercial release worldwide have been developed by the first strategy. Thirty-eight of them have been transformed by synthetic sequences transcribing antisense or double-stranded RNAs and three by mutated copies for suppressed expression of undesirable genes (the third strategy). By the first and the third strategies, hundreds of transgenic events and thousands of varieties with significant improvement of resistance to herbicides and pesticides, as well as nutritional quality, have been developed and approved for commercial release. Their application has significantly decreased the use of synthetic pesticides and the cost of crop production and increased the yield of crops and the benefits to farmers. However, almost all the events overexpressing endogenous genes remain at the testing stage, except one for fertility restoration and another for pyramiding herbicide tolerance. The novel functions conferred by the heterologously expressing exogenous genes under the control of constitutive promoters are usually absent in the recipient crops themselves or perform in different pathways. However, the endogenous proteins encoded by the overexpressing endogenous genes are regulated in complex networks with functionally redundant and replaceable pathways and are difficult to confer the desirable phenotypes significantly. It is concluded that heterologous expression of exogenous genes and suppressed expression by RNA interference and clustered regularly interspaced short palindromic repeats-cas (CRISPR/Cas) of undesirable genes are superior to the overexpression of endogenous genes for transgenic improvement of crops.

Bio-informatic analysis of CRISPR protospacer adjacent motifs (PAMs) in T4 genome

BackgroundThe existence of protospacer adjacent motifs (PAMs) sequences in bacteriophage genome is critical for the recognition and function of the clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) machinery system. We further elucidate the significance of PAMs and their function, particularly as a part of transcriptional regulatory regions in T4 bacteriophages.MethodsA scripting language was used to analyze a sequence of T4 phage genome, and a list of few selected PAMs. Mann-Whitney Wilcoxon (MWW) test was used to compare the sequence hits for the PAMs versus the hits of all the possible sequences of equal lengths.ResultsThe results of MWW test show that certain PAMs such as: ‘NGG’ and ‘TATA’ are preferably located at the core of phage promoters: around -10 position, whereas the position around -35 appears to have no detectable count variation of any of the tested PAMs. Among all tested PAMs, the following three sequences: 5’-GCTV-3’, 5’-TTGAAT-3’ and 5’-TTGGGT-3’ have higher prevalence in essential genes. By analyzing all the possible ways of reading PAM sequences as codons for the corresponding amino acids, it was found that deduced amino acids of some PAMs have a significant tendency to prefer the surface of proteins.ConclusionThese results provide novel insights into the location and the subsequent identification of the role of PAMs as transcriptional regulatory elements. Also, CRISPR targeting certain PAM sequences is somehow likely to be connected to the hydrophilicity (water solubility) of amino acids translated from PAM’s triplets. Therefore, these amino acids are found at the interacting unit at protein-protein interfaces.

CRISPR-Cas and restriction-modification team up to achieve long-term immunity.

Bacteria have been shown to harbor a growing arsenal of various defense systems against phages. Maguin et al. have uncovered how two of the most frequent defense systems interact: the clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) system recycles by-products of the restriction-modification (RM) system to increase bacterial defense in the long run.

CRISPR-CAS9 Sisteminde Kullanılan Polimer Bazlı Transfeksiyon Ajanları

Genome editing is a method used to make desired changes in the target gene. Today, various methods are used for genome-editing studies; among them, one of the most widely used methods is the clustered, regularly interspaced short palindromic repeats (CRISPR). CRISPR-associated (Cas) genes and their corresponding CRISPR sequences constitute CRISPR-Cas systems. Due to its simplicity, it is likely that the CRISPR–Cas system could be used effectively in ex vivo gene therapy studies in humans. If this happens, the importance of CRISPR carrier systems will gradually increase. Viral and non-viral systems are used as delivery modalities in genome-editing studies. It has been proven that nanoparticles are the most promising tools for gene therapy due to their adjustable size, surface, shape, and biological behaviours. The polymeric carrier system has become the main non-viral substitute for gene delivery due to its reduced immunogenicity and pathogenicity. In this review, information about current studies related to polymeric carriers used in non-viral CRISPR delivery systems is presented.

CRISPR-Cas9-mediated Large Cluster Deletion and Multiplex Genome Editing in Paenibacillus polymyxa.

The use of molecular tools based on the clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) systems has rapidly advanced genetic engineering. These molecular biological tools have been applied for different genetic engineering purposes in multiple organisms, including the quite rarely explored Paenibacillus polymyxa. However, only limited studies on large cluster deletion and multiplex genome editing have been described for this highly interesting and versatile bacterium. Here, we demonstrate the utilization of a Cas9-based system to realize targeted deletions of four biosynthetic gene clusters in the range of 12-41 kb by the use of a single targeting sgRNA. Furthermore, we also harnessed the system for multiplex editing of genes and large genomic regions. Multiplex deletion was achieved with more than 80% efficiency, while simultaneous integration at two distantly located sites was obtained with 58% efficiency. The findings reported in this study are anticipated to accelerate future research in P. polymyxa and related species.

Potential Use of CRISPR/Cas13 Machinery in Understanding Virus-Host Interaction.

Prokaryotes have evolutionarily acquired an immune system to fend off invading mobile genetic elements, including viral phages and plasmids. Through recognizing specific sequences of the invading nucleic acid, prokaryotes mediate a subsequent degradation process collectively referred to as the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)–CRISPR-associated (Cas) (CRISPR–Cas) system. The CRISPR–Cas systems are divided into two main classes depending on the structure of the effector Cas proteins. Class I systems have effector modules consisting of multiple proteins, while class II systems have a single multidomain effector. Additionally, the CRISPR–Cas systems can also be categorized into types depending on the spacer acquisition components and their evolutionary features, namely, types I–VI. Among CRISPR/Cas systems, Cas9 is one of the most common multidomain nucleases that identify, degrade, and modulate DNA. Importantly, variants of Cas proteins have recently been found to target RNA, especially the single-effector Cas13 nucleases. The Cas13 has revolutionized our ability to study and perturb RNAs in endogenous microenvironments. The Cas13 effectors offer an excellent candidate for developing novel research tools in virological and biotechnological fields. Herein, in this review, we aim to provide a comprehensive summary of the recent advances of Cas13s for targeting viral RNA for either RNA-mediated degradation or CRISPR–Cas13-based diagnostics. Additionally, we aim to provide an overview of the proposed applications that could revolutionize our understanding of viral–host interactions using Cas13-mediated approaches.

Harnessing CRISPR-Cas to Combat COVID-19: From Diagnostics to Therapeutics.

The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a global threat with an ever-increasing death toll even after a year on. Hence, the rapid identification of infected individuals with diagnostic tests continues to be crucial in the on-going effort to combat the spread of COVID-19. Viral nucleic acid detection via real-time reverse transcription polymerase chain reaction (rRT-PCR) or sequencing is regarded as the gold standard for COVID-19 diagnosis, but these technically intricate molecular tests are limited to centralized laboratories due to the highly specialized instrument and skilled personnel requirements. Based on the current development in the field of diagnostics, the programmable clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system appears to be a promising technology that can be further explored to create rapid, cost-effective, sensitive, and specific diagnostic tools for both laboratory and point-of-care (POC) testing. Other than diagnostics, the potential application of the CRISPR–Cas system as an antiviral agent has also been gaining attention. In this review, we highlight the recent advances in CRISPR–Cas-based nucleic acid detection strategies and the application of CRISPR–Cas as a potential antiviral agent in the context of COVID-19.

Comparison of the Feasibility, Efficiency, and Safety of Genome Editing Technologies.

Recent advances in programmable nucleases including meganucleases (MNs), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) have propelled genome editing from explorative research to clinical and industrial settings. Each technology, however, features distinct modes of action that unevenly impact their applicability across the entire genome and are often tested under significantly different conditions. While CRISPR-Cas is currently leading the field due to its versatility, quick adoption, and high degree of support, it is not without limitations. Currently, no technology can be regarded as ideal or even applicable to every case as the context dictates the best approach for genetic modification within a target organism. In this review, we implement a four-pillar framework (context, feasibility, efficiency, and safety) to assess the main genome editing platforms, as a basis for rational decision-making by an expanding base of users, regulators, and consumers. Beyond carefully considering their specific use case with the assessment framework proposed here, we urge stakeholders interested in genome editing to independently validate the parameters of their chosen platform prior to commitment. Furthermore, safety across all applications, particularly in clinical settings, is a paramount consideration and comprehensive off-target detection strategies should be incorporated within workflows to address this. Often neglected aspects such as immunogenicity and the inadvertent selection of mutants deficient for DNA repair pathways must also be considered.

Investigation into the prevalent CRISPR-Cas systems among the Aeromonas genus.

Clustered regularly interspaced short palindromic repeats (CRISPR) is a prokaryotic adaptive immune system that checks invasion by mobile genetic elements through nuclease targeting. In this study, we investigated the occurrence, diversity, and features of the CRISPR system in the genus Aeromonas using bioinformatics tools. Only 13 out of 122 complete genomes (10.66%) of the genus Aeromonas from the NCBI GenBank database harbored the CRISPR system. The Type I-F system was the most prevalent CRISPR system among the Aeromonads, followed by the Type I-E system. Only one strain harbored a Type I-C CRISPR system. Among the Aeromonads, Aeromonas caviae (22.7%) and Aeromonas veronii (20%) had a higher prevalence rate of the complete CRISPR system. The analysis of direct repeat (DR) sequences showed that all could form stable RNA secondary structures. A phylogenetic tree generated for the Cas1 protein classified CRISPR subtypes into three distinct clusters. Among the 748 spacers investigated, 41.98% and 17.25% showed perfect homology to phage and plasmid sequences, respectively. Some arrays had duplicated spacers. The CRISPR loci are closely linked to antibiotic resistance genes in most strains. Collectively, our results would contribute to research on antibiotic resistance in the Aeromonas group, and provide new insights into the diversity and evolution of the CRISPR-Cas system.

Recent Advances and Challenges in Gene Delivery Mediated by Polyester-Based Nanoparticles.

Gene therapy is a promising approach for the treatment of several diseases, such as chronic or viral infections, inherited disorders, and cancer. The cellular internalization of exogenous nucleic acids (NA) requires efficient delivery vehicles to overcome their inherent pharmacokinetic drawbacks, e.g. electrostatic repulsions, enzymatic degradation, limited cellular uptake, fast clearance, etc. Nanotechnological advancements have enabled the use of polymer-based nanostructured biomaterials as safe and effective gene delivery systems, in addition to viral vector delivery methods. Among the plethora of polymeric nanoparticles (NPs), this review will provide a comprehensive and in-depth summary of the polyester-based nanovehicles, including poly(lactic-co-glycolic acid) (PLGA) and polylactic acid (PLA) NPs, used to deliver a variety of foreign NA, e.g. short interfering RNA (siRNA), messenger RNA (mRNA), and plasmid DNA (pDNA). The article will review the versatility of polyester-based nanocarriers including their recent application in the delivery of the clustered, regularly‐interspaced, short palindromic repeats/Cas (CRISPR/Cas) genome editing system for treating gene-related diseases. The remaining challenges and future trend of the targeted delivery of this revolutionary genome-editing system will be discussed. Special attention will be given to the pivotal role of nanotechnology in tackling emerging infections such as coronavirus disease 2019 (COVID-19): ground-breaking mRNA vaccines delivered by NPs are currently used worldwide to fight the pandemic, pushing the boundaries of gene therapy.

CRISPR-Cas system: a precise tool for plant genome editing

Biotic and abiotic stress affects the productivity and quality of the plant; it is necessarily important to develop improved crops that can withstand the environmental conditions. Conventional methods of breeding are very tedious process that require some alternative. The molecular approach of genetic modification emerged out as a precise method for plant breeding in the past few years. Genome is altered by removing, adding or substituting DNA bases in the host genome. Different methods of genome editing have been developed, including transcription activator like effector nucleases (TALENs), zinc-finger nucleases (ZFNs) and Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas. These nucleases have enabled researchers to manipulate gene of interest in crop plants. Among these CRISPR-Cas has been developed recently and found to be a more efficient tool for genome editing because of its simple design and low cost. In recent years, CRISPR-Cas9 has been utilized for gene knockouts and knock-ins, multiplex gene targeting, and gene regulation in plants. In this review, we provide the use of CRISPR–Cas9 technology in genome editing and its fruitful applications in plants specifically in terms of improvement in quality and resistance to biotic and abiotic stress. Designing of an efficient CRISPR–Cas system using bioinformatic tools have also been discussed.

CRISPR/Cas-New Molecular Scissors in Diagnostics and Therapeutics of COVID-19.

The current pandemic of COVID-19, with its climbing number of cases and deaths, has us searching for tools for rapid, reliable, and affordable methods of detection on one hand, and novel, improved therapeutic strategies on the other. The currently employed RT-PCR method, despite its all-encompassing utility, has its shortcomings. Newer diagnostic tools, based on the Clustered Regularly Interspaced Short Palindromic Repeats/Cas(CRISPR-Cas) system, with its better diagnostic accuracy measures, have come up to fill that void. These assay platforms are expected to slowly take up the place of COVID-19 diagnostics. Further, the current therapeutic options focus mainly on counteracting the viral proteins and components and their entry into host cells. The CRISPR-based system, especially through the RNA-guided Cas13 approach, can identify the genomic characteristics of SARS-CoV-2 and provide a novel inhibition strategy for coronaviruses. In this mini-review, we have discussed the available and upcoming CRISPR-based diagnostic assays and the potential of the CRISPR/Cas system as a therapeutic or prevention strategy in COVID-19. CRISPR-Cas system shows promise in both diagnostics as well as therapeutics and may as well change the face of molecular diagnosis and precision medicine.

Survival of Salmonella Under Heat Stress is Associated with the Presence/Absence of CRISPR Cas Genes and Iron Levels.

Clustered regularly interspaced short palindromic repeats (CRISPR) cas genes have been linked to stress response in Salmonella. Our aim was to identify the presence of CRISPR cas in Salmonella and its response to heat in the presence of iron. Whole genomes of Salmonella (n = 50) of seven serovars were compared to identify the presence of CRISPR cas genes, direct-repeats and spacers. All Salmonella genomes had all cas genes present except S. Newport 2393 which lacked these genes. Gene-specific primers were used to confirm the absence of these genes in S. Newport 2393. The presence/absence of CRISPR cas genes was further investigated among 469 S. Newport genomes from PATRIC with 283 genomes selected for pan-genome analysis. The response of eleven Salmonella strains of various serovars to gradual heat in ferrous and ferric forms of iron was investigated. A total of 32/283 S. Newport genomes that lacked all CRISPR cas genes clustered together. S. Newport 2393 was the most heat-sensitive strain at higher iron levels (200 and 220 pm) in ferrous and ferric forms of iron. The absence of CRISPR cas genes in S. Newport 2393 may contribute to its increase in heat sensitivity and iron may play a role in this. The high reduction in numbers of most Salmonella strains exposed to heat makes it unfeasible to extract RNA and conduct transcription studies. Further studies should be conducted to validate the survival of Salmonella when exposed to heat in the presence/absence of CRISPR cas genes and different iron levels.