CRIPSR Case System: Biological Role in Bacterial Virulence, Genome Editing and in Antimicrobial Resistance

Muhamamd Rizwan1*, Muhammad Arshad2, Muhammad Kashif3, Aneela Zameer Durrani4, Asghar Abbas5, Tanveer Ahmad6, Muhammad Nadeem7 , Kinza Khan8

1College of Veterinary Sciences, Bahauddin Zakariya University, Subcampus Layyah, Pakistan.

2Department of Microbiology, University of Veterinary and Animal Sciences, Lahore, Pakistan.

3College of Veterinary and Animal Sciences, Jhang, Pakistan.

4Department of Veterinary Medicine, University of Veterinary and Animal Sciences, Lahore, Pakistan.

5Department of Veterinary and Animal Sciences, MNS-University of Agriculture, Multan, Pakistan.

6Department of Clinical Sciences, Bahauddin Zakariya University, Multan, Pakistan.

7Arid Agriculture University, Khushab Subcampus, Pakistan.

8Department of Pathobiology, Bahauddin Zakariya University, Multan, Pakistan.

Abstract | The aim of the present review was to study about the CRISPR Cas system and there role in genome editing, bacterial virulence and antibiotics resistance. CRISPR Cas system is an integral part of prokaryotic (Bacteria and Archaea) immune system that provides protection against viral infection. When bacteria recognize viral DNA inside it, bacteria incorporate small fragment of viral DNA into its genome at specific site termed as CRISPR locus. Insertion of viral DNA at CRISPR locus allows to remember, diagnose and clear the viral infection by the mechanism of sequence specific Adaptive Immunity. CRISPR Cas system is sustainable to combate with the mutation developed in viral genome that help viruses to escape from bacterial CRISPR Cas based immune system. CRISPR Cas system is a molecular mechanism of prokaryotic microorganism. It acts as bacterial natural adaptive immune system against phages, plasmids and foreign genomic elements. Mostly prokaryotes uses their CRIPR Cas system to enhance the integrity of their cell membrane that inhibit the permeability of antimicrobials from host body into the bacterial cells. CRISPR Cas system also help bacteria to evade from the host immune system by suppressing the activity of their immune receptors e.g. TLR. CRISPR Cas system also help bacteria to attach with the host body and replicate with in host body. It can develop antibiotic resistance in pathogenic bacteria, enhance their pathogenicity and can survival in host body.

Novelty Statement | CRISPR Cas system is an integral part of prokaryotic immune system that provides protection against viral infection and involved in genome editing, bacterial virulence and antibiotics resistance.

Article History

Received: May 10, 2021

Revised: May 25, 2021

Accepted: June 01, 2021

Published: June 20, 2021

Authors’ Contributions

MR, MA, MK and AZD designed the research and collected data. AA, TA, NM and KK wrote the manusccript.

Keywords

Pathogenic, DNA, CRISPR Cas, Prokaryotic, Plasmids

Corresponding Author: Muhamamd Rizwan

mrizwan@bzu.edu.pk

To cite this article: Rizwan, M., Arshad, M., Kashif, M., Durrani, A.Z., Abbas, A., Ahmad, T., Nadeem, M. and Khan, K., 2021. CRIPSR case system: Biological role in bacterial virulence, genome editing and in antimicrobial resistance. Punjab Univ. J. Zool., 36(1): 111-118. https://dx.doi.org/10.17582/journal.pujz/2021.36.1.111.118

Introduction

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) Cas system is an integral part of prokaryotic (Bacteria and Archaea) immune system that provides protection against viral infection. It is a molecular mechanism that consists of two major domains: DNA-binding domain that facilitates identification and attachment with sequence- specific DNA (Westra and Swarts, 2012). When bacteria recognize viral DNA inside it, bacteria incorporate small fragment of viral DNA into its genome at specific site termed as CRISPR locus. This short fragment of viral genome is transcribed into CRISPR RNA (crRNA) by bacteria. When crRNA combines with Trans activating CRISPR RNA (tracrRNA) a complex named as guide RNA (gRNA) is synthesized. gRNA guides Cas9 (a protein acting as endonuclease enzyme known as Cas9 to detect viral DNA for double strand breakage (DSB) (Figure 2c). When PM (Protospacer Adjacent Motif) sequences of viral DNA that matches with bacteria are recognized by gRNA, Cas9 nucleases cut viral DNA. Hence, viral attack is inhibited (Wang and Zhang, 2016). Use of Cas9 of streptococcus pyogenes the sequence 5’- NGG-3’ is very common due to their wide acceptance by PMA. However, variation reported in Cas9 PMA sequences such as 5’-NGG-3’ and 5’-YNT-3’ affects the specificity of gRNA attachment with PAM (Esvelt and Mali, 2013; Kleinstiver and Prew, 2015).

Repairing of double strand breakages (DSBs) made either by the Cas9 enzyme or nucleases is done by Non-Homologous End Joining (NHEJ) method (Figure 2b). In this method small insertions or deletions (InDels) are presented at that site. Another method used to repair these Double Strand Breakages (DSBs) Homology directed repair (HDR) or Homologous Recombination (HR) method (Figure 2C) (Khan et al., 2018). In HR method a donor DNA fragment that is homologous to the flanking sequences is used as template for repairing of DSBs (Cai and Fisher, 2016).

AIMS of this study

The present review was written to collect adapt from published authentic researches about the key role of CRISPR Cas system in bacterial immune system against viral infection (Horvath and Barrangou, 2010), development of antimicrobials resistance in prokaryotes (Horvath and Barrangou, 2010), bacterial sustainability with in host body by evading host immune system (Sampson and Napier, 2014), and pathogenicity of bacteria (Louwen and Staals, 2014).

History of CRISPR cas system

CRISPR Cas system was first described by Yoshizumi Ishino and his colleagues from of Osaka University in 1987. They found that bacteria encounter a phage DNA they insert a 32-nt (nucleotide) spacer sequence in into 29 nt repeated sequences in CRISPR loci (Cai and Fisher, 2016). After that, these repeated sequences were discovered in 90% of Archaea and 40% of sequenced bacterial genome. Function of these repeat sequences was still unknown (Horvath and Barrangou, 2010). Later on, many types of CRISPR associated genes adjacent to these repeats were discovered (Horvath and Barrangou, 2010). Since 2005, it is well known that actual origin of these special sequences is phage genome (Barrangou and Fremaux, 2007). However, it was hypothesized that this CRISPR Cas system can work as adaptive immune system for prokaryotes. Addition or deletion of spacer DNA that is homologous to the phage DNA can change the resistance of Streptococcus thermophiles to inhibit phage attack (Bolotin and Quinquis, 2005). Various facts of CRISPR Cas system were rapidly revealed by the discovery of crRNA, (Cas genes) CRISPR –associated genes, tracrRNA, and protospacer adjacent motif (PAM) (Mojica et al., 2005).

 

 

Composition of CRISPR cas system

CRISPR Cas system is composed of Clustered Regularly interspaced short palindromic repeats and linked Cas proteins. This system act as adaptive defense mechanism in prokaryotes (Briner and Donohoue, 2014). CRISPR loci of this system consist on following three components:

Direct repeated sequences

Variation among the size (from 23 to 55 bps in length) of this region of CRISPR loci has been observed in different organisms. Clusters of these repeats are present on one or more loci of the same chromosome (Grissa and Vergunud, 2007). They are partially palindromic and have ability to construct hair pin. Usually, bacterial genome has three CRISPR arrays. While Archaeal genome contain five CRISPR arrays. A considerable feature of CRISPR array is the potential of their transcript to construct RNA secondary Structure (Kunin et al., 2007).

 

 

Non-repetitive spacer sequences

Size of spacer sequences varies within a range of 26-72bps. Length of spacers present in a specific CRISPR array may be similar but identical spacer cannot be found within one specific CRIPSR array (Lillestøl and Redder, 2006). It is considered that these spacer sequences are of foreign mobile genetic elements origin, but sequence map of all known spacer sequences indicates that they contain very small portion of extrachromosomal DNA i.e. phage or plasmid DNA (Shah and Hansen, 2009). CRISPR analysis studies indicate polymorphism is number as well as kind of spacer sequences of various strains of specific specie. Hence, these spacer sequences can be used as a tool in epidemiological studies. CRISPRs can affect auto immunity because of spacers that contain ability to target self-genes. However, CRISPR can acquire autoimmune fitness that explain the plenty of degraded CRISPR system within prokaryotes (Stern and Keren, 2010).

Leader sequences

Leader sequences are AT rich and non-coding extend nucleotides region. These are situated at 5’-end between First CRISPR Loci and Last Cas gene (Jansen and Embden, 2002; Tang and Bachelleri, 2002). Leader sequences are similar in same prokaryotic species and different in different prokaryotic species (Jansen and Embden, 2002). It is said that transcription of CRISPR arrays begins in leader region because pre-crRNA of Pyrococcus furiosus is homologous to the 3’-end of the leader sequences (Hale and Kleppe, 2008). Modern researches provide evidences that leader sequences act as promotor for pre-crRNA. Leader sequences provide platform for attachment of Cas proteins needed for integrating spacer (Mojica and García-Martínez, 2005; He and Deem, 2010). Leader sequences have potential to construct an open transcriptional initiation complex and show their promoter activity in vitro as well as in vivo (Pul and Wum, 2010). Bioinformatics analysis of CRSIPR loci indicates that their ability to incorporate new spacer (He and Deem, 2010) and to execute the CRISPR expression and interference diminish if they don’t have leader sequences (Marraffini and Sontheimer, 2008). The direct repeats and leader sequences are both conserved within same bacterial species but diverse in different bacterial species. Cas Genes CAR (CRISPER-Associated) genes are situated adjacent to CRISPR loci CRISPR containing Bacteria (Horvath and Barrangou, 2010). It is stated that CRISPR loci and CAS genes function is correlated i.e. in gene expression or DNA metabolism (Barrangou, 2013). According to Haft and His Colleagues (2005), there are 45 CAS gene families creating 6 core CAS families (cas 1 to cas 6). Out of these six core families’ cas1 and cas2 are universal. They are the integral part of all CAS subtypes (Haft and Selenghut, 2005; Barrangou and Marraffini, 2014). Cas1 and 2 families contributes in the construction of repeat spacer insertion at leader end, new spacer acquisition and new repeat synthesis (Barrangou, 2013). Due to variation in molecular mechanism of action of CAS genes and their different phylogeny, three different types of CRISPR CAS system are present in prokaryots. Type one CRISPR system contain Cas3 gene. Type two CRISPR system contain Cas9 gene and Cas10 gene is present in type three CRISPR system (Makarova and Haft, 2011; Louwen and Staals, 2014). Some feature are common between type1 and type 3 CRISPR system but different from type 2 CRISPR system. Three novel subtypes of CRSIPR system has been discovered that contain addition cas genes along with conserved cas genes (e.g. cas1, cas2, and cas9). Cas2 is present as additional gene in type II-A subtype of CRISPR

 

Table 1: Bacterial species, type of CRISPR CAS system used and CAS protein.

Bacterial species	Type of CRISPR CAS system	CAS protein	References
Enterococcus species (E. faecium, E. durans, E. hirae)	Type II CRISPR Cas system	CRSIPR Cas 1 (Nuclease)	[42], [29]
Francisella, Parasutterella, Sutterella, Legionella	Type II-B system		[15]
Tistrella mobilis, Aizospirillum	Subtype III-A, I-C, II-C		[17]
Mycoplasmas, Planococcs, Antarcticus, Staphyloccus, Pseudintermedius, Staphylococcus lugdunensis	Type II-A	Cas9 genes	[14]
E. coli, Salmonella species, M. xanthus, C.curves, C. ractus, C. concisus, Y. pestis, Pacnes C. fetus	Type I CRIPR system	Cas3 (signature gene), Cas6(key protein)	[9], [18], [26], [44], [60], [45], [34],
N. meningitides, C. jejuni, S.thermophiles, L.monocytogenes, F.nivicida, S.pyogenes, M. gallisepticum, S. mutants	Type II CRISPR system	Cas9 (signature gene)	[22], [17], [35], [16], [34],
P. furiosus, S. epidermidis, M. tuberculosis	Type III CRSIPR system	Cas10 (signature gene)	[46], [1],

 

system. Type II-B has additional Cas4. While type II-C doesn’t has any additional CAS gene (Makarova and Haft, 2011). Genes of Cas proteins are highly polymorphic. CAS proteins perform different functions at different stages of CRISPR mediated immunity. CAS proteins are classified into different families because of different numbers, arrangement and distribution (Marraffini and Sontheimer, 2010; Makarova and Haft, 2011). RRM (RNA Recognition Motif) is major functional domain of Cas protein. Sequence analysis of Cas protein indicates that it contain integrase, nuclease, polymerase and helicase domains. Presence of these domains in Cas protein predicts their involvement in DNA metabolism (Barrangou and Marraffini, 2014).

Classification of CRISPR Cas system

Classification of CRISPR Cas system is based on Cas signature genes and other related genes that mediate CRISPR activity. Each set of CRISPR system contain a different set of adaptation (spacer insertion), expression (pre-crRNA processing), interference (crRNA and target binding-cleavage) and Cas proteins i.e. Cas6 perform pre-crRNA processing; Cas8, Cas5, Cas7, and Csfi mediate crRNA attachment with target; Cas4, Cas1, and Cas2 mediate spacer insertion; target binding and cleavage is mediated by Cpf1, Cas 9, and Cas 10; Cas3 mediate target cleavage; while regulation of mechanism is controlled by Csn2. CRISPR Cas-system is divided into following types:

Type 1 CRISPR-Cas system

Cas3 signature protein or its mutants are encoded by the loci of type 1 CRISPR system. These signature proteins perform function of both nucleases as well as helicases. Type 1 system is divided into seven subtypes i.e. I-U and I-A to I-F. This system contains Cas1 to Cas8 proteins along with Cas3 protein. Various subtypes of Type 1 CRISPR system contain unique combination of Cas genes (Chaudhary, 2018).

Type 2 CRISPR system

This system is very simple because of its set of genes present in it. In this system crRNA binding with target and DNA cleavage is mediated by its signature protein called multidomain Cas9. 2-A, 2-B, and 2-C are the subtypes of Type 2 CRISPR system. All subtypes of type 2 CRISPR system also contain the set of Cas1, Cas2, and Cas4, and Csn2 genes (Dhawan and Sharma, 2015).

Type 3 CRISPR system

Cas 10 is the signature gene of this system. It also contains Cas7 and Cas5 genes. It has 2 subtypes: 3-A and 3-B. these subtypes may contain Cas6, Cas 2 and Cas1 genes (Dhawan and Sharma, 2015; Zhu and Klompe, 2018).

Other variants

Mostly bacteria contain type 4 CRISPR Cas system which contain a set of Cas7, Cas5 and Csf1 genes. Function of this system is still unknown (Zhu and Klompe, 2018).

The most advanced CRISPR Cas system is type 5 system that contain Cpf1 protein. This protein combine crRNA and form a complex that cut DNA and simplify the editing process (Luo and Lenay, 2016).

Biological role of CRISPR Cas system in bacterial virulence

It is suggested that CRISPR Cas system protects bacteria from foreign invaders and regulate bacterial pathogenicity by controlling their endogenous transcription. Bacteria (Francisella novicida) bypass host immune system because they contain Cas9 protein that inhibit the activation of TLR2 (Tall Like Receptor 2) (Shabbir ans Shabbir, 2019). TLR2 can recognize Bacterial lipoprotein, provoke pro-inflammatory response in host body and activate host immune cells to excrete bacteria out from host body (Mukherjee and Kamakar, 2016). It is suggested that type II CRISPR system have ability to suppress the BLP genes of bacteria (Barrangou, 2015). Cas9, tracrRNA and small CRISPR associated RNA (sacRNA) regulate the suppression of BLP in F. novicida (Shabbir ans Shabbir, 2019).

Cytotoxic Chromosomal Targeting by CRISPR/Cas Systems Can Reshape Bacterial Genomes and Expel or Remodel Pathogenicity Islands

Many pathogenic and commensals bacteria possess Cas9 in abundance. Involvement of Cas9 in attachment of bacteria (Neisseria meningitides and C. jejuni) to host cell surface and replication with in host cells has been reported. It is suggested that virulence of Legionella pneuomphila, causative agent of Ligeonarries’ disease, is due to Cas2 component of its CRISPR-Cas system.

Viral escape from CRISPR cas system

Bacteriophages have mutated their genome by deletion or insertion to escape from CRISPR Cas mediated immunity of Bacteria. According to Steel et al. (2013) acquisition and use of Bacterial CRISPR system to target antiviral defense mechanism by Bacteriophage is an evolutionary importance of this system (Qaisar et al., 2017). It is said that mutation in viral genome and sustainability of bacterial genome has been achieved by CRISPR Cas immune system.

Genome editing by CRISPR CAS technology

Group of technologies used by scientists to mutate the DNA of an organisms are termed as gene editing. These technologies permit genetic material to be inserted, deleted or changed at a specific location in genome (Hartung and Schiemann, 2014). CRISPR Cas9 is the best approach to be used for genome editing (Rizwan et al., 2016). It is naturally developed genome editing system of bacteria (Liu and Robinson, 2020). Bacteria recognize small fragment of phage genome and use them as a template to prepare CRISPR array (small fragment of DNA) (Nussenzweig and Marraffini, 2020).

These CRISPR array develop memory in bacteria against viral genome for protection in future invasion (Westra and Levin, 2020). Cas9 act as nuclease. CRSIPR Cas technology can be used for in vitro genome editing (Schulze and Lammers, 2020). Scientists synthesize a small fragment of RNA with short ‘guide’ sequence to that binds with a particular target sequence in genome. This guided RNA has ability to attach with Cas9 protein (Rizwan et al., 2016). These guided RNA helps Cas9 enzyme to recognize targeted DNA both in vitro as well as in vivo. Cas9 or Cpf1 enzyme act as nuclease and create Double breakages in targeted genome. After cutting DNA, Scientists use cellular DNA repair machinery to insert or remove fragments of genetic material (Huynh and Depner, 2020).

Genome editing capability of CRISPR Cas system has been adapted in bacteria e.g. E. coli, Lactobacillus reuteri (use in probiotics synthesis), Streptococcus pneumonia, Clostridium begrinckii (use in alcohol production at industrial leve) and Streptomyces species (use in production of antimicrobials) (Barrangou and van Pijkeren 2016).

The CRISPR-cas system promotes antimicrobial resistance in Campylobacter jejuni

Survival of bacterial pathogens with in host body depends upon integrity of bacterial envelope that counters act damage caused by membrane targeting Antibiotics (Hurdle and Onell, 2011). It is well known fact that CRISPR Cas system enhances the integrity of bacterial envelope and regulates its permeability. CRISPR Cas system of Prokaryotes provides them protection against invader viruses and foreign nucleic acids. It regulates the permeability of bacterial membrane is controlled by their CRISPR Cas system (Chen and Guan, 2016). It makes bacteria resistant to membrane targeting Antibiotics (Ahmad et al. 2019). Regulation of membrane permeability helps bacteria to resist recognition by multiple host receptors to enhance pathogenicity (Chen and Guan, 2016).

Conclusions and Recommendations

CRISPR Cas system is a molecular mechanism of prokaryotic microorganism. It acts as bacterial natural adaptive immune system against phages, plasmids and foreign genomic elements. It can develop antibiotic resistance in pathogenic bacteria, enhance their pathogenicity and can survival in host body. All these functions are performed by this system due to its genome editing capability. We can use this system (CRISPR CAS technology) to remove antibiotic resistance developed in bacteria. This technology can be used to treat several genetic disease i.e. cystic fibrosis, hemophilia, AIDS, etc. With the help of CRISPR Cas system we can edit genome of any organism.

Conflict of interest

The authors have declared no conflict of interest.

References

Abadia, E. and Zhang, J., 2010. Resolving lineage assignation on Mycobacterium tuberculosis clinical isolates classified by spoligotyping with a new high-throughput 3R SNPs based method. Infect. Genet. Evol., 10: 1066-1074. https://doi.org/10.1016/j.meegid.2010.07.006

Ahmad, S., Alia, S., Abbas, A., Abbas, R.Z., Zubair, M., Raza, M.A., Bano, N., Shafi, M.U., Badar, S.N., Hussain, K., Rizwan, M. and Ahmad, T., 2019. Effects of dietary supplementation of linseed oil (Omega-3) on quality parameters of Nili Ravi bull spermatozoa. 224: 57-59. https://doi.org/10.1016/j.livsci.2019.04.007

Barrangou, R., 2013. CRISPR-Cas systems and RNA-guided interference. Wiley Interdisciplinary Reviews: RNA, 4: 267-278. https://doi.org/10.1002/wrna.1159

Barrangou, R., 2015. The roles of CRISPR–Cas systems in adaptive immunity and beyond. Curr. Opin. Immunol., 32: 36-41. https://doi.org/10.1016/j.coi.2014.12.008

Barrangou, R. and van Pijkeren, J.-P., 2016. Exploiting CRISPR–Cas immune systems for genome editing in bacteria. Curr. Opin. Biotechnol., 37: 61-68. https://doi.org/10.1016/j.copbio.2015.10.003

Barrangou, R. and Marraffini, L.A., 2014. CRISPR-Cas systems: Prokaryotes upgrade to adaptive immunity. Mol. Cell, 54: 234-244. https://doi.org/10.1016/j.molcel.2014.03.011

Barrangou, R. and Fremaux, C., 2007. CRISPR provides acquired resistance against viruses in prokaryotes. Science, 315: 1709-1712. https://doi.org/10.1126/science.1138140

Bolotin, A. and Quinquis, B., 2005. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology, 151: 2551-2561. https://doi.org/10.1099/mic.0.28048-0

Briner, A.E. and Donohoue, P.D., 2014. Guide RNA functional modules direct Cas9 activity and orthogonality. Mol. Cell, 56: 333-339. https://doi.org/10.1016/j.molcel.2014.09.019

Brouns, S.J. and Jore, M.M., 2008. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science, 321: 960-964. https://doi.org/10.1126/science.1159689

Cai, L. and Fisher, A.L., 2016. CRISPR-mediated genome editing and human diseases. Genes Dis., 3: 244-251. https://doi.org/10.1016/j.gendis.2016.07.003

Chadwick, A.C. and Musunuru, K., 2018. CRISPR-Cas9 genome editing for treatment of atherogenic dyslipidemia. Arterioscl. Thromb. Vascul. Biol., 38: 12-18. https://doi.org/10.1161/ATVBAHA.117.309326

Chaudhary, K., 2018. CRISPR/Cas13a targeting of RNA virus in plants. Plant Cell Rep., 37: 1707-1712. https://doi.org/10.1007/s00299-018-2297-2

Chen, B. and Guan, J., 2016. Imaging specific genomic DNA in living cells. 2: 20-24.

Chylinski, K. and Le Rhun, A., 2013. The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems. RNA Biol., 10: 726-737. https://doi.org/10.4161/rna.24321

Chylinski, K. and Makarova, K.S., 2014. Classification and evolution of type II CRISPR-Cas systems. Nucl. Acids Res., 42: 6091-6105. https://doi.org/10.1093/nar/gku241

Delaney, N.F. and Balenger, S., 2012. Ultrafast evolution and loss of CRISPRs following a host shift in a novel wildlife pathogen, Mycoplasma gallisepticum. PLoS Genet., 8: e1002511. https://doi.org/10.1371/journal.pgen.1002511

Deltcheva, E. and Chylinski, K., 2011. CRISPR RNA maturation by trans-encoded small R https://doi.org/10.1038/nature09886 NA and host factor RNase III. Nature, 471: 602.

Deveau, H. and Garneau, J.E., 2010. CRISPR/Cas system and its role in phage-bacteria interactions. Ann. Rev. Microbiol., 64: 475-493. https://doi.org/10.1146/annurev.micro.112408.134123

Dhawan, M. and Sharma, M., 2015. CRISPR Systems: RNA-Guided defence mechanisms in Bacteria and Archaea. Int. J. Curr. Microbiol. Appl. Sci., 4: 187-200.

Esvelt, K.M. and Mali, P., 2013. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat. Methods, 10: 1116. https://doi.org/10.1038/nmeth.2681

Grissa, I. and Vergnaud, G., 2007. The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats. BMC Bioinf., 8: 172. https://doi.org/10.1186/1471-2105-8-172

Gunderson, F.F. and Cianciotto, N.P., 2013. The CRISPR-associated gene cas2 of Legionella pneumophila is required for intracellular infection of amoebae. MBiol., 4: e00074-00013. https://doi.org/10.1128/mBio.00074-13

Haft, D.H. and Selengut, J., 2005. A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes. PLoS Comput. Biol., 1: e60. https://doi.org/10.1371/journal.pcbi.0010060

Hale, C.R. and Zhao, P., 2009. RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell, 139: 945-956. https://doi.org/10.1016/j.cell.2009.07.040

Hale, C. and Kleppe, K., 2008. Prokaryotic silencing (psi) RNAs in Pyrococcus furiosus. RNA, 14: 2572-2579. https://doi.org/10.1261/rna.1246808

Hartung, F. and Schiemann, J., 2014. Precise plant breeding using new genome editing techniques: Opportunities, safety and regulation in the EU. Plant J., 78: 742-752. https://doi.org/10.1111/tpj.12413

Haurwitz, R.E. and Jinek, M., 2010. Sequence-and structure-specific RNA processing by a CRISPR endonuclease. Science, 329: 1355-1358. https://doi.org/10.1126/science.1192272

He, J. and Deem, M.W., 2010. Heterogeneous diversity of spacers within CRISPR (clustered regularly interspaced short palindromic repeats). Phys. Rev. Lett., 105: 128102. https://doi.org/10.1103/PhysRevLett.105.128102

Horvath, P. and Barrangou, R., 2010. CRISPR/Cas, the immune system of bacteria and archaea. Science, 327: 167-170. https://doi.org/10.1126/science.1179555

Horvath, P. and Romero, D.A., 2008. Diversity, activity, and evolution of CRISPR loci in Streptococcus thermophilus. J. Bacteriol., 190: 1401-1412. https://doi.org/10.1128/JB.01415-07

Hurdle, J.G. and O’neill, A.J., 2011. Targeting bacterial membrane function: An underexploited mechanism for treating persistent infections. Nat. Rev. Microbiol., 9: 62-75. https://doi.org/10.1038/nrmicro2474

Huynh, N. and Depner, N., 2020. A versatile toolkit for CRISPR-Cas13-based RNA manipulation in Drosophila. Genome Biol., 21: 1-29. https://doi.org/10.1186/s13059-020-02193-y

Jansen, R. and Embden, J.D.V., 2002. Identification of genes that are associated with DNA repeats in prokaryotes. Mol. Microbiol., 43: 1565-1575. https://doi.org/10.1046/j.1365-2958.2002.02839.x

Javed, M.R. and Sadaf, M., 2018. CRISPR-Cas system: History and prospects as a genome editing tool in microorganisms. Curr. Microbiol., 75: 1675-1683. https://doi.org/10.1007/s00284-018-1547-4

Karimi, Z. and Ahmadi, A., 2018. Bacterial CRISPR regions: General features and their potential for epidemiological molecular typing studies. Open Microbiol. J., 12: 59. https://doi.org/10.2174/1874285801812010059

Khan, A., Ahmed, T., Rizwan, M. and Khan, N., 2018. Comparative therapeutic efficacy of Phyllanthus emblica (Amla) fruit extract and procaine penicillin in the treatment of subclinical mastitis in dairy buffaloes. Microb. Pathog., 115: 8-11. https://doi.org/10.1016/j.micpath.2017.12.038

Kinnevey, P.M. and Shore, A.C., 2013. Emergence of sequence type 779 methicillin-resistant Staphylococcus aureus harboring a novel pseudo staphylococcal cassette chromosome mec (SCCmec)-SCC-SCCCRISPR composite element in Irish hospitals. Antimicrob. Agents Chemother., 57: 524-531. https://doi.org/10.1128/AAC.01689-12

Kleinstiver, B.P. and Prew, M.S., 2015. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature, 523: 481. https://doi.org/10.1038/nature14592

Kunin, V. and Sorek, R., 2007. Evolutionary conservation of sequence and secondary structures in CRISPR repeats. Genome Biol., 8: R61. https://doi.org/10.1186/gb-2007-8-4-r61

Lillestøl, R. and Redder, P., 2006. A putative viral defence mechanism in archaeal cells. Archaea, 2: 59-72. https://doi.org/10.1155/2006/542818

Liu, H. and Robinson, D.S., 2020. Bacterial genome editing by coupling Cre-lox and CRISPR-Cas9 systems. PLoS One, 15: e0241867. https://doi.org/10.1371/journal.pone.0241867

Louwen, R. and Staals, R.H., 2014. The role of CRISPR-Cas systems in virulence of pathogenic bacteria. Microbiol. Mol. Biol. Rev., 78: 74-88. https://doi.org/10.1128/MMBR.00039-13

Luo, M.L. and Leenay, R.T., 2016. Current and future prospects for CRISPR-based tools in bacteria. Biotechnol. Bioeng., 113: 930-943. https://doi.org/10.1002/bit.25851

Lyons, C. and Raustad, N., 2015. Incidence of Type II CRISPR1-Cas systems in Enterococcus is species-dependent. PLoS One, 10: e0143544. https://doi.org/10.1371/journal.pone.0143544

Makarova, K.S. and Haft, D.H., 2011. Evolution and classification of the CRISPR–Cas systems. Nat. Rev. Microbiol., 9: 467. https://doi.org/10.1038/nrmicro2577

Makarova, K.S. and Wolf, Y.I., 2015. An updated evolutionary classification of CRISPR–Cas systems. Nat. Rev. Microbiol., 13: 722.

Marinelli, L.J. and Fitz-Gibbon, S., 2012. Propionibacterium acnes bacteriophages display limited genetic diversity and broad killing activity against bacterial skin isolates. MBiol., 3: e00279-00212. https://doi.org/10.1128/mBio.00279-12

Marraffini, L.A. and Sontheimer, E.J., 2008. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science, 322: 1843-1845. https://doi.org/10.1126/science.1165771

Marraffini, L.A. and Sontheimer, E.J., 2010. CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nat. Rev. Genet., 11: 181. https://doi.org/10.1038/nrg2749

Mojica, F.J. and García-Martínez, J., 2005. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J. Mol. Evolut., 60: 174-182. https://doi.org/10.1007/s00239-004-0046-3

Mukherjee, S. and Karmakar, S., 2016. TLR2 and TLR4 mediated host immune responses in major infectious diseases: A review. Braz. J. Infect. Dis., 20: 193-204. https://doi.org/10.1016/j.bjid.2015.10.011

Nussenzweig, P.M. and Marraffini, L.A., 2020. Molecular Mechanisms of CRISPR-Cas Immunity in Bacteria. Ann. Rev. Genet., 54: 93-120. https://doi.org/10.1146/annurev-genet-022120-112523

Pul, Ü. and Wurm, R., 2010. Identification and characterization of E. coli CRISPR-cas promoters and their silencing by H-NS. Mol. Microbiol., 75: 1495-1512. https://doi.org/10.1111/j.1365-2958.2010.07073.x

Qaisar, H.M.U., Ahmad, T., Rizwan, M. and Saqib, M., 2017. Antimicrobial efficacy of combination of lincomycin and spiramycin (LispiracinTM) as systemic dry cow therapy for controlling bovine mastitis. Punjab Univ. J. Zool., 32: 197-201.

Rizwan, M., Ali, M., Khan, O.A., Ahmed, T. and Durrani, A.Z., 2016. Swayback disease in ruminants. Veterinaria,

Rizwan, M., Durrani, A.Z., Ijaz, M., Kashif, M. and Firyal, S., 2016. Clinio-bacterialogical investigation of sub-clinical and clinical mastitis in dairy goats. Veterinaria, 4: 4-6

Sampson, T.R. and Weiss, D.S., 2013. Alternative roles for CRISPR/Cas systems in bacterial pathogenesis. PLoS Pathog., 9: e1003621. https://doi.org/10.1371/journal.ppat.1003621

Sampson, T.R. and Napier, B.A., 2014. A CRISPR-Cas system enhances envelope integrity mediating antibiotic resistance and inflammasome evasion. Proc. Natl. Acad. Sci., 111: 11163-11168. https://doi.org/10.1073/pnas.1323025111

Schulze, S. and Lammers, M., 2020. The development of genome editing tools as powerful techniques with versatile applications in biotechnology and medicine: CRISPR/Cas9, ZnF and TALE nucleases, RNA interference, and Cre/loxP. Chem. Texts, 7: 1-18. https://doi.org/10.1007/s40828-020-00126-7

Shabbir, M.A.B. and Shabbir, M.Z., 2019. CRISPR-cas system: biological function in microbes and its use to treat antimicrobial resistant pathogens. Ann. Clin. Microbiol. Antimicrob., 18: 21. https://doi.org/10.1186/s12941-019-0317-x

Shah, S.A. and Hansen, N.R., 2009. Distribution of CRISPR spacer matches in viruses and plasmids of crenarchaeal acidothermophiles and implications for their inhibitory mechanism, Portland Press Ltd. https://doi.org/10.1042/BST0370023

Steel, Z., Marnane, C., Iranpour, C. Chey, T. Jackson, J.W. Patel, V. and Silove, D., 2013. The global prevalence of common mental disorders: A systematic review and meta-analysis 1980-2013. Int. J. Epidemiol., 43: 476-93.

Stern, A. and Keren, L., 2010. Self-targeting by CRISPR: Gene regulation or autoimmunity? Trends Genet., 26: 335-340. https://doi.org/10.1016/j.tig.2010.05.008

Tang, T.-H. and Bachellerie, J.-P., 2002. Identification of 86 candidates for small non-messenger RNAs from the archaeon Archaeoglobus fulgidus. Proc. Natl. Acad. Sci., 99: 7536-7541. https://doi.org/10.1073/pnas.112047299

Vergnaud, G. and Zhou, D., 2007. Analysis of the three Yersinia pestis CRISPR loci provides new tools for phylogenetic studies and possibly for the investigation of ancient DNA. Genus Yersinia, Springer, pp. 327-338. https://doi.org/10.1007/978-0-387-72124-8_30

Wang, Y. and Zhang, Z.-T.. 2016. Bacterial genome editing with CRISPR-Cas9: Deletion, integration, single nucleotide modification, and desirable “clean” mutant selection in Clostridium beijerinckii as an example. ACS Synthet. Biol., 5: 721-732. https://doi.org/10.1021/acssynbio.6b00060

Westra, E.R. and Levin, B.R., 2020. It is unclear how important CRISPR-Cas systems are for protecting natural populations of bacteria against infections by mobile genetic elements. Proc. Natl. Acad. Sci., 117: 27777-27785. https://doi.org/10.1073/pnas.1915966117

Westra, E.R. and Swarts, D.C., 2012. The CRISPRs, they are a-changin: How prokaryotes generate adaptive immunity. Ann. Rev. Genet., 46: 311-339. https://doi.org/10.1146/annurev-genet-110711-155447

Zhu, Y. and Klompe, S.E., 2018. Shooting the messenger: RNA-targetting CRISPR-Cas systems. Biosci. Rep., 38. https://doi.org/10.1042/BSR20170788