CRISPR-Based Gene Editing in Acinetobacter baumannii to Combat Antimicrobial Resistance

Antimicrobial resistance (AMR) poses a significant threat to the health, social, environment, and economic sectors on a global scale and requires serious attention to addressing this issue. Acinetobacter baumannii was given top priority among infectious bacteria because of its extensive resistance to nearly all antibiotic classes and treatment options. Carbapenem-resistant A. baumannii is classified as one of the critical-priority pathogens on the World Health Organization (WHO) priority list of antibiotic-resistant bacteria for effective drug development. Although available genetic manipulation approaches are successful in A. baumannii laboratory strains, they are limited when employed on newly acquired clinical strains since such strains have higher levels of AMR than those used to select them for genetic manipulation. Recently, the CRISPR-Cas (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) system has emerged as one of the most effective, efficient, and precise methods of genome editing and offers target-specific gene editing of AMR genes in a specific bacterial strain. CRISPR-based genome editing has been successfully applied in various bacterial strains to combat AMR; however, this strategy has not yet been extensively explored in A. baumannii. This review provides detailed insight into the progress, current scenario, and future potential of CRISPR-Cas usage for AMR-related gene manipulation in A. baumannii.


Introduction
The behavioral change in bacterial pathogenicity leading to AMR is one of the major constraints hindering several public health policies globally. It has been estimated that there might be a global economic loss of USD 100 trillion by the year 2050 if this threatening issue of antibiotic resistance continues [1]. Although there are several reasons for antibiotic resistance, the major one is due to a bottleneck in innovative research with a focus on the exploitation of the diversity of antibiotics against Gram-negative bacteria, while the existing antibiotics are in clinical trials mostly under phase II or phase III. This lack of research is an alarming situation for research scientists along with economic loss. The lives of millions of people are projected to be vulnerable to death by 2050 due to the emerging situation of AMR [2]. Other serious health issues related to AMR include the outbreak of different infectious diseases, the risk of common infectious diseases such as immunosuppression, intubation, catheterization, and other such procedures related to antibiotics [3]. In view of the above situation, it is very important to revolutionize therapeutic strategies to prevent AMR bacterial infections [4,5]. It is critical to focus on the most commonly occurring pathogenic bacteria, which have acquired AMR for most antibiotics.
RNA-based strategies such as RNA silencing and interference, antisense oligonucleotides, and steric-blocking oligonucleotides are also proven to be effective against AMR bacteria. Resistance genes can be eliminated by enzymatically targeting the mRNA with these oligonucleotides [179]. Translation can be ceased in bacteria by RNA silencing, which is a built-in process in many bacteria. The cisand trans-regions bind to the complementary regulatory regions present on a single mRNA strand (also known as antisense sequences) to halt the translation of certain genes [180]. Furthermore, this technique utilizes antisense RNA sequences to monitor resistant genes and mutations by creating antisense oligonucleotides that are continually redesigned to ensure that resistance is not encountered [181,182]. It also helps to identify and knock-down the AMR genes and to detect the mode of action of novel antibiotics [181]. Two important drawbacks of the use of RNA as a therapeutic measure are the limited intracellular absorptions and chemistry-dependent toxicities [179].
Genome-editing tools can be utilized to combat AMR in bacteria. These tools use restriction enzymes to target and cut a specific DNA sequence; for example, restriction nucleases, zinc finger nucleases (ZFN), and transcription-activator-like effector nucleases (TALENS), are the initially developed genome-editing technologies that can be specifically engineered for target-specific DNA cleavage. These enzymes produce target-specific double-strand breaks (DSBs) in the genome and thus help to obtain knock-down, knock-in, and/or knock-out mutants. The cleavage domain present in these enzymes can bind to a customized DNA binding domain, which allows the DNA cleavage at the targeted binding site. Several studies have shown the utilization of both these enzymes in genome editing; yet, they are costly, laborious, time-consuming, and error-prone due to higher ratios of off-target mutations [183]. Moreover, TALENs are much larger in size than ZFNs, making them difficult to deliver and express in the host cells [184,185].
Many of the above-discussed genetic manipulation approaches are effective in genome editing, providing desirable gene deletions and mutations for antimicrobials; however, the limitations associated with these approaches necessitate the development and discoverer of novel alternative strategies with a more precise and target-oriented approach for genetic manipulation [186]. Recently, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR-Cas) systems have become an effective tool to target drug-resistant bacteria and genes by targeting the specific genome sequences.
CRISPR-Cas systems are being utilized to develop precise and effective antimicrobials for various infections; however, CRISPR-based approaches for AMR are still needed to be explored on a wider scale.

Clustered Regularly Interspaced Palindromic Repeats/CRISPR-Associated Protein (CRISPR-Cas) System
CRISPR-Cas constitutes an adaptive immune system as present in bacteria and archaea, which provides them with an effective shield against various viral and bacteriophagal attacks [187]. Discovered by Japanese scientists in 1987 and obtaining its name, CRIPSR, in 1990 by Francisco Mojica, the biological function and ability of these repetitive palindromic DNA sequences were long unknown [188]. Initially, in 2007, CRISPR was experimentally attributed as a crucial component of a prokaryote's adaptive immune system in the fight against viruses [189]. Later, in 2012, Doudna and Charpentier discovered the role of CRISPR-Cas9 in DNA editing in a target-specific manner by using the appropriate template sequence. Moreover, they explained the processing mechanism of CRISPR-derived RNAs (crRNA) under the effect of transactivating CRISPR RNAs (tracrRNA) [190,191]. Since then, CRISPR-Cas has emerged as the most effective, precise, efficient, and powerful approach for editing the genome in all living cells and is used in a wide range of practical fields [190,192].
Three fundamental steps of the CRSIPR-Cas system, which are adaptation (spacer acquisition), crRNA synthesis (expression), and target interference, play a vital role in bacterial defense against viral attacks ( Figure 1). The cas gene encodes the Cas protein or nuclease protein, which is responsible for the cleavage and destruction of foreign viral DNA [193]. Its features, namely that it is an affordable, rapid, accurate, effective, and efficient method of gene editing, make CRISPR-Cas most researched genome-editing tool in recent years, where it has demonstrated the ability to get rid of bacterial infections [194,195], correct genetic flaws [196,197], and eradicate dangerous infectious viruses [198,199]. nuclease protein, which is responsible for the cleavage and destruction of foreign viral DNA [193]. Its features, namely that it is an affordable, rapid, accurate, effective, and efficient method of gene editing, make CRISPR-Cas most researched genome-editing tool in recent years, where it has demonstrated the ability to get rid of bacterial infections [194,195], correct genetic flaws [196,197], and eradicate dangerous infectious viruses [198,199].

Classification of CRISPR-Cas System
Two main classes of the CRISPR-Cas system, Class 1 and Class 2, are distinguished by their signature genes and the arrangement of the CRISPR loci [200]. Both classes are further divided into 6 types (I-VI) and 33 subtypes. Class 1 comprises three types (I, III,

Classification of CRISPR-Cas System
Two main classes of the CRISPR-Cas system, Class 1 and Class 2, are distinguished by their signature genes and the arrangement of the CRISPR loci [200]. Both classes are further divided into 6 types (I-VI) and 33 subtypes. Class 1 comprises three types (I, III, and IV) and sixteen subtypes, whereas Class 2 includes three types (II, V, and VI) and seventeen subtypes.
In Class 1, when complexed with the crRNA, multi-subunit Cas effector proteins (Cas5, Cas7, Cas8, and SS) carry out the processing and interference mechanisms (where type I, III, and IV use Cas3, Cas10, and Csf1, respectively) to degrade the foreign DNA [201]. Nearly 60% of the bacterial population contains the Type I CRISPR-Cas system, which carries a multi-subunit CRISPR-associated complex for antiviral defense (Cascade) [202]. Seven subtypes (A to G) exist for Type I, among which the Type I-F CRISPR-Cas system is most prevalent among bacteria [200,203]. In the case of Class 2, a single enzymatic crRNA-binding protein is responsible for the identification and cleavage of the target sequence [201,204,205]. These multidomain crRNA-binding effector proteins include Cas9, Cas12a (Cpf1), and Cas13, which are responsible for the interference and processing mechanisms in type II, V, and VI, respectively [206]. CRISPR-Cas9 is isolated from the bacterium Streptococcus pyogenes, and in Type II CRISPR-Cas systems, it has received substantial attention from researchers for gene editing, as it is the easiest and most effective, versatile, and specific system [200,204].

Role of CRISPR-Cas System against AMR Bacteria
Genome-editing technologies have been completely transformed by CRISPR-Cas due to its ability to be engineered to target almost any sequence of interest. As well as being repurposed for potential antimicrobials, CRISPR-Cas systems are also being used to reduce the level of undesirable genetic traits in bacteria [207,208]. Moreover, the ability of nucleic acid destruction by RNA enables the CRISPR-Cas system to develop next-generation antimicrobial mechanisms against infectious diseases, particularly those caused by AMR pathogens [209][210][211].
The CRISPR-Cas system based on the target gene locations can be employed as an antibacterial agent in two different ways, namely a pathogen-focused approach and a gene-focused approach [212]. In a pathogen-focused approach, particular bacterial chromosomal regions are targeted, where pathogen strain identification and bacterial cell death are achieved. On the other hand, targeting the plasmids that carry the AMR genes is part of the gene-focused approach. This method eliminates the plasmid and makes the bacterium susceptible to antibiotics [213,214]. The pathogen-focused approach is applied to cure distinct infections and can kill target-specific bacterial strains in a mixed culture, whereas the role of gene-focused approaches is still vague. These can be applied to the treatment of bacterial infections and can generally lessen the prevalence of the AMR gene in bacteria [206].
The CRISPR-Cas system, in contrast to conventional antimicrobials, operates in a target-specific manner, allowing the differentiation between friendly and harmful bacteria. Guide RNAs can be designed to target important virulence genes, antibiotic resistance, or pathogenicity [215,216]. According to the experimental designs and objectives, targeting effects can range from cell death to growth inhibition of the specific bacteria and at the genetic level can also result in gene deletions, transcriptional inhibition, and loss of antibiotic-resistant plasmids [217][218][219]. The CRISPR-Cas system utilizes the following three general strategies to address AMR:

1.
It can be used in target-specific cleavage of infection-causing genes, deploying the desired bacteria while leaving the host's microbiome unaffected [220,221]. For example, chromosomal genes for cell division and metabolism were removed from the mixed cell cultures of E. coli and S. enterica strains using the Type I CRISPR-Cas system [222];

2.
It can be applied to cleave drug-resistant genes by killing the pathogenic bacteria but not affecting wild-types [222,223]. Bikard et al. applied the RNA-guided nuclease Cas9 against the virulence genes in Staphylococcus aureus, which resulted in the specific killing of virulent strain without affecting avirulent staphylococci [224]; 3.
It can be engineered to modify or silence resistance genes, causing bacterial mutations where the functionality of resistance genes is halted, while bacterial viability is maintained, known as the re-sensitization process [225,226]. The re-sensitization of E. coli strains using ESBL-encoding plasmids was carried out by Kim et al. [220]. They used plasmids encoding for Cas9 and crRNAs against conserved areas in the ESBL genes to transform strains of E. coli that produce ESBLs. The CRISPR-Cas9 system effectively reduced the resistance in the transformants by targeting specific cleavage of resistant plasmids. The realization of the broad utility of the CRISPR-Cas system in gene editing accelerated the need to search for Cas protein variants with enhanced functions, including higher activity, potential for therapeutic delivery, nucleic acid detection, etc. [227]. Among various Cas proteins, the most frequently used Cas proteins are Cas9, which results in a double-strand break by specifically cleaving the targeted sequence [214]; dCas9, a catalytically "dead or defective" Cas9 protein that contrasts with Cas9 by not showing double-strand nuclease activity, but instead staying attached to the targeted sequence and obstructing the RNA polymerase binding to that specific region, thud hindering the transcription initiation [228]; nSpCas9:rAPOBEC1, a Cas9 protein without nuclease activity attached to a deaminase, resulting in the conversion of cytidine bases into thymine and hence forming a stop codon [229]; and Cas13a, an RNA-specific endonuclease that, when recognized by particular DNA sequence, causes the cleavage of RNA fragments [5]. Cas14 is also attracting scientists' attention as it is small, has single-stranded (ss) DNA-targeting activity, and does not require protospacer adjacent motif (PAM) sequences to bind, as compared to Cas9 and Cas12 proteins [227,230].

Recent Studies on the Application of CRISPR-Cas System in AMR Bacteria
Several academic studies have confirmed the application of the CRISPR-Cas system and its effectiveness in controlling and/or stopping of AMR in bacteria [231,232] (Table 2). The use of CRISPR-Cas13a to achieve cell death in E. coli strains against carbapenems and colistin-resistance genes was achieved through engineering and transfer of CRISPR-Cas13a via M13 phages [5]. Similarly, a trans-conjugative delivery system known as CRISPR Cas13a-based killing plasmids (CKPs) was applied to kill the endogenous AMR genes in S. enterica serotype Typhimurium, where colonies of S. typhimurium showed a substantial decrease through CRISPR-Cas13a [233]. More research can be performed on this strategy of employing Cas13a to target RNA transcripts to target the pathogenic bacterial strains depending on the presence of particular virulence genes.
AMR genes were successfully targeted in E. coli and S. aureus strains using a plasmid expressing a Cas9-driven RNA [212]. Clinical isolates of S. aureus were treated by manipulating the Cas9 and crRNA for the methicillin-resistance gene (mecA), which showed a significant (~50%) decrease in the disease as compared to the control [209,234,235]. Using the CRISPR-Cas system, a 20-time decrease in the number of viable cells of E. coli strain O157:H7 for the eae gene was observed, which is an essential gene for intestinal infections [219]. The application of the CRISPR-Cas9 system significantly reduced the S. aureus colonies on mouse skin in comparison to alternative treatment strategies [236]. A non-viral delivery was performed using a polymer, branched polyethyleneimine (bPEI), Cas9, and a single guide RNA (SgRNA) to combat the methicillin resistance gene, i.e., mecA, in S. aureus. The S. aureus strain treated with Cas9-bPEI did not grow in culture media, and growth was decreased by up to 32% compared to the control. These findings can lead to the development of novel CRISPR-based antimicrobial medications since the delivery of CRISPR via polymers can prevent risks of immunogenicity and off-target effects. It can also easily produce phenotypic alterations by editing and modifying the bacterial genome [237]. E. coli strains with resistance to colistin were developed by transforming the mcr-1 harboring E. coli strains with CRISPR-Cas9 plasmid, which not only eliminated the mcr-1 gene but also prohibited horizontal gene transfer after transformation with CRISPR-Cas9 plasmid [238]. In E. faecalis, the CRISPR-Cas9 system successfully targeted the erythromycin-resistance gene ermB and hampered growth, reducing the intestinal infections caused by this bacterium [239][240][241][242]. Many studies have shown the effectiveness of the CRISPR-Cas system for curing plasmids that present a resistant phenotype. This strategy can avoid horizontal gene transfer, target resistance genes to prevent AMR, and get rid of plasmid-carrying drug-resistant genes [234,[243][244][245].
The CRISPR-Cas system can be used to explore the functions of various genes that can contribute to the increased antibiotic resistance in bacteria. The role of different genes in K. pneumoniae against tigecycline and colistin resistance was identified via the CRISPR-Cas9 system [246]. The study showed the knock-out mutants for the tetA gene presented a decrease in minimum inhibitory concentration (MIC) for tigecycline, whereas when the mgrB gene was inactivated, it resulted in the activation of the PhoPQ two-component system, ultimately increasing the MIC for colistin. E. coli strain SE15, responsible for biofilm formation in urinary catheters, resulted in reduced biofilm formation by targeting the quorum-sensing (QS) gene luxS through a CRISPR-Cas9 plasmid [247,248]. The CRISPR interference (CRISPRi) system, based on dCas9 (lacks the endonuclease activity compared to Cas9), is also used to target AMR genes. dCas9 forms a complex with sgRNA to bind at the targeted DNA sequence to inactivate the transcription and lead to gene silencing. Instead of a gene knock-out, the CRISPRi method is utilized to knock-down the desired gene, allowing for reversibility [249]. This strategy has been utilized for gene silencing and knock-down for the AMR genes in E. coli [250][251][252][253], Enterococcus faecalis [254,255], Caulobacter crescentus [256], Campylobacter jejuni [257,258], and other bacteria.
Newer strategies are also being explored, such as creating bacteriophages with DNAencoding Cas9 and guide RNA and eliminating all phage sequences essential for phage replication. This approach will result in the cleavage and degradation of DNA in bacteriophagesinfected bacteria through the CRISPR-Cas9 system; however, a continuous evolution of bacterial cells against foreign DNA might lead to the development of resistance to these approaches. Hence, bacterial mutations need to be researched, and phages must be designed to specifically target those mutations using the same strategy [259]. Moreover, CRISPR-Cas systems can be employed to either target and remove the AMR pathogen or to eliminate the bacteria themselves, which harbor the drug-resistant genes. As the evolution of bacterial resistance cannot be prevented, various CRISPR-Cas system-usage strategies can be evaluated further because the system is practical and easily reprogrammable [260]. Genome editing and gene manipulation and deletions [262] A. baumannii AB43 AbaI Type I-F CRISPR-Cas system [263] A. baumannii AdvA and ftsZ CRISPRi, transposon mutagenesis, and gene editing [264]   C. botulinum Genome alteration and CRISPR-system presence analysis [282] C. cellulolyticum afp Genome editing and gene deletion and integration [283] C. difficile Multiple genome-editing applications [284] C. difficile JIR8094 selD Genome editing and~20-50% site-specific mutations [285] C. saccharoperbutylacetonicum N1-4 pta and buk Genomic modifications, gene deletions (~75%), and butanol production [286] C. pasteurianum cpa Genome editing and gene deletion and insertion [287] Corynebacterium Gram + C. glutamicum glgC, idsA, gltA, and pyc CRISPRi [288] C. glutamicum pyk and ldhA Base editor at different loci [289] C. glutamicum ldhA Genome modification, gene deletion and insertion (~60%), and 80% gene modification [290] C. glutamicum crtYf Genome editing and 86-100% successful deletions [291] C. glutamicum clpX, mepA, and porB Genome editing, deletion, insertion, and point mutation [292] C. glutamicum ATCC 13032 argR, gabT, and gabP Genome editing and gene knock-out for gamma-aminobutyric acid (GABA) over-production [293] C. glutamicum pgi, pck, and pyk CRISPRi (~98%) [   S. aureus 6538-GFP nuc [320] S. aureus AH1 mec Type III-A CRISPR-Cas system for gene editing [321] S. aureus ATCC 29213 rpoB Genome modifications and gene deletions [322] S. aureus USA300, USA300-∆mecA and RN4220 mecA Genome editing and gene inactivation [5] S. aureus USA300ϕ and S. aureus RNϕ mecA Genome editing [224] S. aureus ATCC 6538 tarH, tarG, and tarO Genome alteration and gene knock-out [228] S. aureus CTH96 Nuc Genome editing and genetic manipulation and deletion [323] Streptomyces Gram +

CRISPR-Cas System in A. baumannii
The existence of two endogenous CRISPR-Cas systems in the genomes of various Acinetobacter species has been verified by the analysis of the CRISPRCas database (CRISPRCasdb) [335][336][337]. Nearly 2500 genomes of A. baumannii subjected to a pangenome study also confirmed the presence of two CRISPR-Cas systems in Acinetobacter spp. [338]. The first system is present in the genome of clinical isolates of A. baumannii AYE, AB037, 4190, and AB0057, whereas the second system was discovered in A. baumannii type strain ATCC ® 19606 TM and A. baylyi ADP1 [339][340][341]. The CRISPR system is found in a variety of prokaryotes [337]. However, only 36% of the bacteria comprise both CRISPR arrays and Cas genes. The CRISPRCasdb analysis revealed the presence of CRISPR arrays and Cas genes in nearly 20% of organisms in the Acinetobacter genus and 18% of isolates of A. baumannii spp. [337,342]. The CRISPR system has been identified in different A. baumannii strains by analyzing the large volume of sequencing data and by application of bioinformatical tools [340]. The trailer and spacer regions of the CRISPR system are generally conserved among various bacterial isolates. This helps in the grouping of isolates and the identification of common ancestors based on the presence of sequence arrays [343]. Several studies have shown that the CRISPR-Cas system not only provides immunity in A. baumannii but also regulates various virulence gene expressions, controls group behaviors, provides DNA repair, and dictates genome evolution [342].
The Type I CRISPR-Cas system is the most common in nature, comprising a multisubunit effector complex [200,344]. This effector complex includes nine subtypes known as A, B, C, G, D, E, F1, F2, and F3 [200]. Type I-F CRISPR-Cas systems are the most common in A. baumannii [337], but the Type IV variant with genes csf3, csf4 (also named as dinG), and cas6e, along with CRISPR arrays at both ends, is also present in some Acinetobacter spp. [345]. Recently, several studies confirmed the presence of the Type I-F CRISPR-Cas system in various A. baumannii isolates from throughout the world [346][347][348][349][350]. Based on the presence or absence of the 14 common genes, the Acinetobacter genome can be divided into two groups. The first group comprises fewer common CRISPR genes and hence shows rarity in the presence of plasmids [338]. The existence of Type 1 and Type IV CRISPR-Cas systems in A. baumannii was also confirmed. In silico analysis of 4977 A. baumannii genomes from the NCBI Refseq database revealed nearly 14% of A. baumannii clinical isolates carried CRISPR-Cas systems [351][352][353]. Further classification of A. baumannii genomes presenting CRISPR-Cas systems showed that Type I-F1 CRISPR-Cas system was most abundant (~67%), followed by Type I-F2 (~28%), while both Type I-F1 and Type I-F2 were present in~4% genomes. Various studies have reported the coexistence of different types of CRISPR-Cas systems in other bacteria [345,[354][355][356]; however, the co-localization of Type I-F (I-F1 + I-F2) in A. baumannii was reported by Yadav and Singh [353].
The CRISPR-Cas system, specifically Type 1, is successfully employed for genome editing in various bacterial strains. There is still great potential to explore and implement it for genetic manipulation in A. baumannii by exploring the association of CRISPR-Cas systems with bacterial virulence and pathogenesis mechanisms [357,358]. Tyumentseva et al. confirmed the existence of CRISPR arrays and Cas genes related to Type I-F2 in clinical isolates of A. baumannii [342]. They also found a correlation between the AMR genotype/phenotype of A. baumannii with its type of CRISPR-Cas system. It was observed that a higher number of AMR genes was present in the isolates where both CRISPR arrays and active Cas genes were missing as compared to the isolates with only CRISPR arrays or both CRISPR arrays and Cas genes. This helps bacteria fight against phage infections and protect against the spreading of AMR genes in A. baumannii. Virulence factors were also found to be dependent on CRISPR-Cas systems in A. baumannii. Additionally, regarding the difference between Type I-F1 and Type I-F2 CRISPR-Cas systems among the isolates of A. baumannii, CRISPR arrays were lower in isolates with Type I-F1 CRISPR-Cas system as compared to Type I-F2 isolates, which have a stronger immune system. The presence of more AMR genes in Type I-F1 A. baumannii isolates supports easier adaptation to different environmental conditions, whereas Type I-F2 isolates may utilize the CRISPR-Cas system to control the distribution of AMR genes. Similarly, the Type I-F1 CRISPR-Cas system affects the acquisition of AMR plasmids in wild-type antimicrobial-susceptible E. coli isolates and sustains the susceptible profile of these E. coli isolates [359]. Other studies also confirmed the impact of the CRISPR-Cas system on the accretion of virulence and AMR-related genes, where it can prevent the accumulation of resistance genes but does not affect the mutations in cells to attain AMR. However, the A. baumannii genomes having CRISPR-Cas systems did not show any correlations with any specific antibiotic classes and with virulence genes [352], indicating no effect of this system on the acquisition of resistance and virulence genes in A. baumannii. The genomes possessing only co-localizing Type I-F1 + F2 CRISPR-Cas systems showed negative correlation for factors including biofilm-associated proteins (bap, bauA) and quorum-sensing (QS) genes (abaI and abaR) [353]. Similar results were also presented by other research groups [338,342,360]. These association analyses suggest that without affecting the phage-based memory, spacers can target plasmids through an unknown mechanism for the acquisition of virulence and resistance factors in the genomes of A. baumannii.

Recent Studies on the Application of the CRISPR-Cas System in A. baumannii
To apply an effective exogenous CRISPR-Cas system to A. baumannii, many researchers have generated and tested various models. An exogenous recombination system involving two plasmids carrying Cas9 from S. pyogenes and sgRNA was developed, where each plasmid could replicate in both E. coli and A. baumannii. The impact of the genes (blaOXA-23, blaADC-25, and blaTEM-1D) was reported on imipenem and sulbactam resistance in A. baumannii. Additionally, the researchers constructed single-, double-, and triple-gene mutants to explain the role of each gene in accomplishing AMR [262,361]. The application of CRISPR-Cas-mediated genome modifications in A. baumannii may be tricky, as the procurement of several resistance genes is related to mobile genetic elements such as plasmids and transposons. Hence, the loss of the plasmid carrying the CRISPR components and unwanted genetic combinations might be obtained [229]. In this regard, an alternate strategy to perform gene editing is cytidine-base editing (C to T replacement), which does not require a DSB and a donor template. In this process, single-base replacement (A/C to T) in CAA or CAG can produce stop codons (TAA or TAG). The successful application of cytidine-base editing was achieved in K. pneumoniae and A. baumannii ATCC ® 17978 TM by constructing the plasmid vector pBECAb-apr with a sgRNA and a fusion protein expression [229]. Moreover, a two-plasmid-based CRISPR-Cas system to perform gene editing in K. pneumoniae [262] was tested in A. baumannii.
The relationship between drug resistance and the CRISPR-Cas system was analyzed in A. baumannii strain AB43 using the whole-genome sequencing (WGS) technique [362]. The authors identified the presence of the Type I-Fb CRISPR-Cas system in the strain AB43 and found that the cas gene in the studied strain has a higher similarity index with the similar subtype cas genes. The role of the CRISPR-Cas system in the AB43 strain against invasive bacteriophage and plasmids was confirmed, as 28 out of 105 CRISPR spacers in the genome of this strain showed similarity with the genes present in the bacteriophage genome and with the plasmid database. However, no matches for CRISPR spacers were found for AMR genes for A. baumannii strain AB43. The drug resistance mechanism in A. baumannii strain AB43 via the CRISPR-Cas system is still unclear, as the endogenous CRISPR-Cas system might be responsible for inhibition of drug resistance gene expression, which requires further research [342,362]. Another study using WGS identified the CRISPR-Cas system Subtype I-F in A. baumannii strain ATCC BAA1605, with a high number of spacers present in the CRISPR loci [363]. The abaI gene responsible for biofilm formation through the quorum sensing in A. baumannii [364] was targeted to develop gene knockouts by designing sgRNAs using various bioinformatical tools [365]. To target the essential genes in A. baumannii ATCC 17978, a CRISPRi-system was applied to develop gene knock-down mutants using the anhydrotetracycline (aTc)-inducible dcas9 gene and a constitutive sgRNA for adc β-lactamases [264]. A significant (30-fold) decrease in β-lactamase synthesis was observed after the induction of aTc by dcas9, indicating successful silencing of the adc gene. The researchers also developed gene knockdowns for essential genes for cell replication, i.e., ftsZ and advA, using the CRISPRi approach and observed substantial decrease in cell growth as compared to the control. The CRISPRi approach was also used to characterize the transcriptional factor RS03245 encoding AraC in A. baumannii, which is important for bacterial growth. CRISPR-Cas12a array along with multiplex polymerase chain reaction (PCR) was implemented to detect MDR A. baumannii, enabling simultaneous amplification of essential genes and β-lactamase genes. This study showed the accuracy and specificity of the CRISPR-Cas12a system to detect important drug-resistance genes in A. baumannii [265].
An effective, convenient, and quick gene-manipulation system comprising pyrF-based suicide plasmids and pyrF-deleted uracil-auxotrophic hosts was developed and tested to successfully to delete the sequences of cas genes (cas1, cas3, and cascade) and the CRISPR sequence (except the leader and a single repeat structure) in the I-F CRISPR-Cas system in A. baumannii AYE∆F. This system is more efficient for developing knockouts in model strains than the clinical strains of A. baumannii due to lower transformation rates and biofilm formation [266]. The role of OxyR as an oxidative-stress-resistance regulator was also discovered using the CRISPR-Cas9 system. The CRISPR-Cas9-based genome-editing strategy (pCasAb-pSGAb) involved Cas9 and RecAb from A. baumannii. The amount and the length of the repair template were also optimized, which resulted in significant improvement in editing efficiency. The genome-editing efficiency of strains ATCC ® 17978 TM and ATCC ® 19606T in one clinical isolate, A. baumannii ABH2, was also tested. The researchers used multiple strains because of the anticipated variation in the effectiveness of CRISPR-Cas-based genome editing due to differences in genomic background. An exogenous CRISPR-Cas system was used to introduce point mutations into a clinical isolate of A. baumannii ABH2 to study the role of H 2 O 2 -sensing amino acid residues present in OxyR. As anticipated, the mutant strains did not exhibit any deficiencies with regard to H 2 O 2 sensitivity. However, a known residue (C 2 0 2 ) and three new residues (E130, S133, and S226) were identified to be important for OxyR function [262]. In another study, the use of RecAb from the A. baumannii IS-123 strain produced more colonies by deleting OxyR via CRISPR-Cas [366]. The CRISPR-based genome-editing system (pCasAb-pSGAb) in comparison to the gene-editing strategy (pyrF/5-FOA) developed by [266] showed higher deletion efficiency. However, the utilization of a single plasmid and its easier removal from cells in the pyrF/5-FOA system make it a more convenient method.

Conclusions
CRISPR-Cas-based gene editing has provided successful results for gene manipulation in various bacteria to combat AMR; affect various important physiological processes that cause infections and reduce virulence, pathogenicity, and biofilm formation; and can lead to bacterial death. However, the application of CRISPR-Cas in A. baumannii is quite recent and not fully explored. This revolutionary technique can be used to improve the genetic makeup of A. baumannii to combat AMR by targeting and disrupting specific genes associated with AMR, such as enzymes producing genes that protect the bacteria from antibiotics and biofilm-formation genes that provide antibiotic resistance. Moreover, the CRISPR can be utilized to modify the existing genes or introduce new genes into the genome of A. baumannii to reduce the bacteria's ability to resist antibiotics or to increase its sensitivity to antibiotics. For example, a specific gene associated with a receptor that regulates the activity of an antibiotic could be introduced into the bacteria to enhance its susceptibility to that specific antibiotic. Similarly, the introduction of genes into the bacterial genome that produce specific molecules that interfere with AMR mechanisms and bacterial growth can be achieved through CRISPR-based genetic manipulation. Overall, CRISPR-based gene editing could be effectively used to combat AMR in A. baumannii, and the method still requires more research in this regard in the future.