Phage Display as a Medium for Target Therapy Based Drug Discovery, Review and Update.

Phage libraries are now amongst the most prominent approaches for the identification of high-affinity antibodies/peptides from billions of displayed phages in a specific library through the biopanning process. Due to its ability to discover potential therapeutic candidates that bind specifically to targets, phage display has gained considerable attention in targeted therapy. Using this approach, peptides with high-affinity and specificity can be identified for potential therapeutic or diagnostic use. Furthermore, phage libraries can be used to rapidly screen and identify novel antibodies to develop immunotherapeutics. The Food and Drug Administration (FDA) has approved several phage display-derived peptides and antibodies for the treatment of different diseases. In the current review, we provided a comprehensive insight into the role of phage display-derived peptides and antibodies in the treatment of different diseases including cancers, infectious diseases and neurological disorders. We also explored the applications of phage display in targeted drug delivery, gene therapy, and CAR T-cell.

The Synthetic Antimicrobial Peptide Derived From Melittin Displays Low Toxicity and Anti-infectious Properties.

The low stability and nonspecific toxicity are the main limiting factors for the clinical applications of melittin (MLT). This study aimed to design and synthesize new analogs of MLT to increase stability, reduce toxicity, and retain their antimicrobial properties against bacterial pathogens. At first, peptide analogs were designed computationally by inducing single mutations in MLT peptides and evaluating their physicochemical properties. The stability of the analogs with the highest scores was determined by Gromacs software. In vitro assays were performed to examine the antimicrobial activity and toxicity of the selected analogs. Two peptide analogs, M1 and M2, were selected based on the SVM score in cell PPD. The M1 analog was created by replacing alanine with leucine on the 15th. The M2 analog was designed by substituting alanine with leucine and isoleucine with arginine at the 15th and 17th positions. According to the Gromacs results, the M2 peptide indicated more stability. RMSD and RMSF results showed no undesirable fluctuations during the 200 ns MD simulation. The MIC and MBC values for the M1 peptide were calculated in a range of 8-128 µg/ml, while the M2 peptide limited the bacterial growth to 32-128 µg/mL. Both peptides indicated less toxicity than natural MLT, based on MTT assay results. The hemolytic activity of the M1 analog was more than M2 at 16 µg/mL concentration. M1 peptide displayed the highest selectivity index against S. aureus and A. baumannii, which were approximately 5.27-fold improvements compared to MLT. In conclusion, we introduced two analogs of MLT with low toxicity, low hemolytic activity, and higher stability, along with retaining antimicrobial properties against gram-negative and positive bacteria.

Rational design of hybrid peptide with high antimicrobial property derived from Melittin and Lasioglossin.

Hybridization of Antimicrobial peptides (AMPs) with unique abilities is now considered to improve AMPs' function as promising therapeutic candidates. In the current research, Lasioglossin (LL-III) with a high antimicrobial effect on Acinetobacter (A.) baumanni and Melittin with a high antimicrobial effect against Staphylococcus (S.) aureus were selected for designing a hybrid peptide with modified properties. In the present study, a hybrid peptide with modified properties was designed. Molecular dynamic (MD) and coarse-grained (CG) simulations were done to evaluate the stability and interaction of the hybrid peptide with related membrane models. In this study, a truncated Melittin peptide (11 amino acids) was fused to an LL-III peptide (15 amino acids) to raise the antimicrobial activity. A new hybrid peptide analog (LM1) was selected by replacing the arginine with isoleucine in the fifth position of truncated Melittin to raise the antimicrobial rate of the peptide. The potential for binding of the LM1 to lipid membrane (D factor) was increased from 2.02 related to Melittin to 3.62. Based on VMD results, the N-terminal of LM1 peptide related to LL-III was the alpha helix during 200 ns. However, the C-terminal region related to the Melittin peptide only at 50 ns was in alpha helix form. The RMSD of the LM1 peptide was in the range of 0.2 to 0.8, which, after 160 ns, reached stability. RMSD and RMSF results indicated no unwanted fluctuations during the 200 ns MD simulation. A significant movement of LM1 peptide inside the S. aureus membrane(4.76 nm) and A. baumanni membrane (3.2 nm) was observed by CG simulation. Our findings highlight the high stability of the designed hybrid peptide and its antimicrobial potential to halter A. baumanii and S. aureus bacteria.Communicated by Ramaswamy H. Sarma.

The high cross-transmission in methicillin resistant Staphylococcus aureus between healthy and patient communities.

Background and Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) is one of the main causes of high mortality and morbidity in hospitals. This study was aimed to examine virulence factors, molecular typing, and the antibiotic resistance pattern of MRSA isolates in hemodialysis patients and healthy communities. Materials and Methods: Total of 231 and 400 nasal samples were obtained from hemodialysis patients and healthy communities, respectively. Virulence factors profile was examined in two groups by PCR reaction. Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR) was used as a molecular typing approach. Results: Overall, 35.49% (82/231) of hemodialysis patients were positive for S. aureus, and 47.56% (39/82) of isolates were positive for mecA. In a healthy community, 15% (60/400) of samples were positive for S. aureus, and 36.66% (22/60) were positive for mecA. The frequency of MDR was significantly higher in patients group (p-value < 0.00001). The frequency of pvl (p.value = 0.003932, P<0.05) and tsst-1 (p.value = 0.003173, p < .05) were significantly higher in patients group. The highest frequency virulence factors in healthy individuals were related to hla (68.33%, 41/60), hlb (53.33%, 32/60), and Acme/arcA (46.66%) genes. Two groups were clustered by the ERIC-PCR method into 7 clusters and 2 single isolate with a 0.74 similarity index. Based on the results, each cluster was combination with healthy and patient isolates. Conclusion: Our findings indicate a notable variation in the frequency of virulence factors between S. aureus isolates obtained from dialysis patients and the healthy community.

In silico Validation of Pseudomonas aeruginosa Exotoxin A Domain I Interaction with the Novel Human scFv Antibody.

BACKGROUND: Pseudomonas (P.) aeruginosa is one of the leading causes of nosocomial infections. The pathogenicity of P. aeruginosa is related to its inherent antimicrobial resistance and the diverse virulence factors of this bacterium. Owing to the specific role of exotoxin A in P. aeruginosa pathogenesis, it is known as a promising therapeutic candidate to develop antibodies as an alternative to antibiotics. OBJECTIVE: The present study aimed to validate the interaction between a single-chain fragment variable (scFv) antibody identified from an scFv phage library against domain I exotoxin A by bioinformatic tools. METHODS: For this, several bioinformatics tools, including Ligplot, Swiss PDB viewer (SPDBV), PyMOL, I-TASSER, Gromacs, and ClusPro servers were used to evaluate the interaction of scFv antibody with P. aeruginosa exotoxin A. The I-TASSER server was utilized to predict the function and structure of proteins. The interaction of two proteins was analyzed using ClusPro tools. The best docking results were further analyzed with Ligplot, Swiss PDB viewer, and PyMOL. Consequently, molecular dynamics simulation was utilized to predict the stability of the secondary structure of the antibody and the binding energy of the scFv antibody to the domain I of exotoxin A. RESULTS: As a result, we demonstrated that data from computational biology could provide proteinprotein interaction information between scFv antibody/domain I exotoxin A and offers new insights into antibody development and therapeutic expansion. CONCLUSION: In summary, a recombinant human scFv capable of neutralizing P. aeruginosa exotoxin A is recommended as a promising treatment for infections caused by P. aeruginosa.

MicroRNA-Based Biomarkers in Lung Cancer: Recent Advances and Potential Applications.

INTRODUCTION: MicroRNAs (miRNAs) are a group of small noncoding RNAs (ncRNAs) that post-transcriptionally control the expression of genes by binding and degrading their target mRNAs. miRNAs can function as possible tumor suppressors or oncogenes in various cancers. Lately, miRNAs application as a biomarker (prognosis and diagnosis) for different diseases has gained much attention. miRNAs exist in a stable form in several biological materials, including tissue, plasma, and serum. The noninvasive and easy screening of miRNAs in serum, blood, tissue, and other body fluids and acceptable stability make microRNA a noticeable factor as biomarkers in human malignancies. MATERIALS AND METHODS: In this review, we searched some online databases like Web of Science, Embase, and PubMed to find eligible manuscripts up to the end of 2021. RESULTS: Abnormal expressions of these molecules are associated with the incidence of many illnesses like cancer. Therefore, they are candidates as a molecular tool for noninvasive tumor prognosis and diagnosis. In the current study, we introduce important miRNAs that may be used as prognostic and diagnostic markers in lung cancer patients. CONCLUSION: We summarized the latest reports about critical miRNAs related to the diagnosis and prognosis in lung patients.

Rational design of an anti-cancer peptide inhibiting CD147 / Cyp A interaction.

The CD147 / Cyp A interaction is a critical pathway in cancer types and an essential factor in entering the COVID-19 virus into the host cell. Melittin acts as an inhibitory peptide in cancer types by blocking the CD147/ Cyp A interaction. The clinical application of Melittin is limited due to weak penetration into cancer cells. TAT is an arginine-rich peptide with high penetration ability into cells widely used in drug delivery systems. This study aimed to design a hybrid peptide derived from Melittin and TAT to inhibit CD147 /Cyp A interaction. An amino acid region with high anti-cancer activity in Melittin was selected based on the physicochemical properties. Based on the results, a truncated Melittin peptide with 15 amino acids by the GGGS linker was fused to a TAT peptide (nine amino acids) to increase the penetration rate into the cell. A new hybrid peptide analog(TM) was selected by replacing the glycine with serine based on random point mutation. Docking results indicated that the TM peptide acts as an inhibitory peptide with high binding energy when interacting with CD147 and the CypA proteins. RMSD and RMSF results confirmed the high stability of the TM peptide in interaction with CD147. Also, the coarse-grained simulation showed the penetration potential of TM peptide into the DOPS-DOPC model membrane. Our findings indicated that the designed multifunctional peptide could be an attractive therapeutic candidate to halter tumor types and COVID-19 infection.

Bio-nano scale modifications of melittin for improving therapeutic efficacy.

INTRODUCTION: Melittin (MLT), a natural membrane-active component, is the most prominent cytolytic peptide from bee venom. Remarkable biological properties of MLT, including anti-inflammatory, antimicrobial, anticancer, anti-protozoan, and antiarthritic activities, make it an up-and-coming therapeutic candidate for a wide variety of human diseases. Therapeutic applications of MLT may be hindered due to low stability, high toxicity, and weak tissue penetration. Different bio-nano scale modifications hold promise for improving its functionality and therapeutic efficacy. AREAS COVERED: In the current review, we aimed to provide a comprehensive insight into strategies used for MLT conjugations and modifications, cellular delivery of modified forms, and their clinical perspectives by reviewing the published literature on PubMed, Scopus, and Google Scholar databases. We also emphasized the MLT structure modifications, mechanism of action, and cellular toxicity. EXPERT OPINION: Developing new analogs and conjugates of MLT as a natural drug with improved functions and fewer side effects is crucial for the clinical translation of this approach worldwide, especially where the chemicals and synthetic drugs are more expensive or unavailable in the healthcare system. MLT-nanoconjugation may be one of the best-optimized strategies for improving peptide delivery, increasing its therapeutic efficacy, and providing minimal nonspecific cellular lytic activity. [Figure: see text].

The molecular characterization of colistin-resistant isolates of Acinetobacter baumannii from patients at intensive care units.

Background and Objectives: The objective of this study was to determine molecular characterization and genetic diversity of colistin-resistant A. baumannii clinical isolates in Intensive Care Unit hospitalized patients. Materials and Methods: A total of 127 A. baumannii clinical isolates were evaluated for antimicrobial susceptibility. PCR reaction and sequencing were performed for the detection of mutations in pmrAB and lpx ACD genes. Results: Based on antimicrobial susceptibility testing, 40.94% and 33.85% of the isolates were MDR and XDR respectively whereas 3.93% of them were found to be PDR. Results of agar dilution MIC and E-test indicated that 76% of the isolates were sensitive to colistin. All of the isolates were positive for bla OXA-51 and 50% of them were positive for both bla OXA-23 -like and bla OXA-143 -like genes while only 25% of the isolates were positive for bla OXA-72 . None of them were positive for the bla OXA-58 -like gene. There is no mutation in pmrA. The V162A substitution for pmrB gene was repeated in two isolates, and E394D and Y292H substitutions in lpxA were observed in two isolates; also, C120R and F165L substitutions in lpxC gene was repeated in two isolates. Analysis of phylogenetic tree based on alterations in lpxACD and pmrB genes indicated the appearance of new isolates compared to the reference strain ATCC17978 A. baumannii isolates. Conclusion: The present study indicated the prevalence of MDR and XDR A. baumannii isolates and the emergence of PDR isolates in the northwest portion of Iran. The appearance of colistin-resistant isolates with new mutations in pmrB, lpxACD genes indicates new resistance mechanisms.

Baricitinib combination therapy: a narrative review of repurposed Janus kinase inhibitor against severe SARS-CoV-2 infection.

PURPOSE: The Coronavirus disease 2019 (COVID-19) pandemic is one of the most devastating global problems. Regarding the lack of disease-specific treatments, repurposing drug therapy is currently considered a promising therapeutic approach in pandemic situations. Recently, the combination therapy of Janus kinase (JAK) inhibitor baricitinib has been authorized for emergency COVID-19 hospitalized patients; however, this strategy's safety, drug-drug interactions, and cellular signaling pathways remain a tremendous challenge. METHODS: In this study, we aimed to provide a deep insight into the baricitinib combination therapies in severe COVID-19 patients through reviewing the published literature on PubMed, Scopus, and Google scholar databases. We also focused on cellular and subcellular pathways related to the synergistic effects of baricitinib plus antiviral agents, virus entry, and cytokine storm (CS) induction. The safety and effectiveness of this strategy have also been discussed in moderate to severe forms of COVID-19 infection. RESULTS: The severity of COVID-19 is commonly associated with a dysregulated immune response and excessive release of pro-inflammatory agents, resulting in CS. It has been shown that baricitinib combined with antiviral agents could modulate the inflammatory response and provide a series of positive therapeutic outcomes in hospitalized adults and pediatric patients (age ≥ two years old). CONCLUSION: Baricitinib plus the standard of care treatment might be a potential strategy in hospitalized patients with severe COVID-19.

Rational Design of Anti-Angiogenic Peptides to Inhibit VEGF/VEGFR2 Interactions for Cancer Therapeutics.

BACKGROUND: Angiogenesis is a critical physiological process that plays a key role in tumor progression, metastatic dissemination, and invasion. In the last two decades, the vascular endothelial growth factor (VEGF) signaling pathway has been the area of extensive researches. VEGF executes its special effects by binding to vascular endothelial growth factor receptors (VEGFRs), particularly VEGFR-2. OBJECTIVE: The inhibition of VEGF/VEGFR2 interaction is known as an effective cancer therapy strategy. The current study pointed to design and model an anti-VEGF peptide based on VEGFR2 binding regions. METHOD: The large-scale peptide mutation screening was used to achieve a potent peptide with high binding affinity to VEGF for possible application in inhibition of VEGF/VEGFR2 interaction. The AntiCP and Peptide Ranker servers were used to generate the possible peptides library with anticancer activities and prediction of peptides bioactivity. Then, the interaction of VEGF and all library peptides were analyzed using Hex 8.0.0 and ClusPro tools. A number of six peptides with favorable docking scores were achieved. All of the best docking scores of peptides in complexes with VEGF were evaluated to confirm their stability, using molecular dynamics simulation (MD) with the help of the GROMACS software package. RESULTS: As a result, two antiangiogenic peptides with 13 residues of PepA (NGIDFNRDFFLGL) and PepC (NGIDFNRDKFLFL) were achieved and introduced to inhibit VEGF/VEGFR2 interactions. CONCLUSIONS: In summary, this study provided new insights into peptide-based therapeutics development for targeting VEGF signaling pathway in tumor cells. PepA and PepC are recommended as potentially promising anticancer agents for further experimental evaluations.

Micronutrient therapy and effective immune response: a promising approach for management of COVID-19.

The escalating prevalence of coronavirus disease 2019 (COVID-19) worldwide, with an increased rate of morbidity and mortality, highlights an urgent need to develop more effective therapeutic interventions. Despite the authorized treatment against COVID-19 by the European Union (EU), the safety and effectiveness of this therapeutic strategy for a wide variety of patients have remained a significant challenge. In this respect, micronutrients such as vitamins and minerals, as essential factors, can be considered for improving the function of the immune system and accelerating the treatment procedure. Dietary supplements can attenuate vascular and inflammatory manifestations related to infectious diseases in large part due to their anti-inflammatory and antioxidant properties. Recently, it has been revealed that poor nutritional status may be one of the notable risk factors in severe COVID-19 infections. In the current review, we focus on the micronutrient therapy of COVID-19 patients and provide a comprehensive insight into the essential vitamins/minerals and their role in controlling the severity of the COVID-19 infection. We also discuss the recent advancements, challenges, negative and positive outcomes in relevance to this approach.

Isolation and characterizations of a novel recombinant scFv antibody against exotoxin A of Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa is the leading cause of nosocomial infections, especially in people with a compromised immune system. Targeting virulence factors by neutralizing antibodies is a novel paradigm for the treatment of antibiotic-resistant pseudomonas infections. In this respect, exotoxin A is one of the most potent virulence factors in P. aeruginosa. The present study was carried out to identify a novel human scFv antibody against the P. aeruginosa exotoxin A domain I (ExoA-DI) from a human scFv phage library. METHODS: The recombinant ExoA-DI of P. aeruginosa was expressed in E. coli, purified by Ni-NTA column, and used for screening of human antibody phage library. A novel screening procedure was conducted to prevent the elimination of rare specific clones. The phage clone with high reactivity was evaluated by ELISA and western blot. RESULTS: Based on the results of polyclonal phage ELISA, the fifth round of biopanning leads to the isolation of several ExoA-DI reactive clones. One positive clone with high affinity was selected by monoclonal phage ELISA and used for antibody expression. The purified scFv showed high reactivity with the recombinant domain I and full-length native exotoxin A. CONCLUSIONS: The purified anti-exotoxin A scFv displayed high specificity against exotoxin A. The human scFv identified in this study could be the groundwork for developing a novel therapeutic agent to control P. aeruginosa infections.

Long non-coding RNAs as potential biomarkers in the prognosis and diagnosis of lung cancer: A review and target analysis.

Long non-coding RNAs (lncRNA) have been emerged as a novel class of molecular regulators in cancer. They are dysregulated in many types of cancer; however, there is not enough knowledge available on their expression and functional profiles. Lung cancer is the leading cause of the cancer deaths worldwide. Generally, lncRNAs may be associated with lung tumor pathogenesis and they may act as biomarkers for the cancer prognosis and diagnosis. Compared to other invasive prognostic and diagnostic methods, detection of lncRNAs might be a user-friendly and noninvasive method. In this review article, we selected 27 tumor-associated lncRNAs by literature reviewing to further discussing in detail for using as diagnostic and prognostic biomarkers in lung cancer. Also, in an in silico target analysis, the "Experimentally supported functional regulation" approach of the LncTarD web tool was used to identifying the target genes and regulatory mechanisms of the selected lncRNAs. The reports on diagnostic and prognostic potential of all selected lncRNAs were discussed. However, the target genes and regulatory mechanisms of the 22 lncRNAs were identified by in silico analysis and we found the pathways that are controlled by each target group of lncRNAs. They use epigenetic mechanisms, ceRNA mechanisms, protein interaction and sponge mechanism. Also, 10, 23, 5, and 28 target genes for each of these mechanisms were identified, respectively. Finally, each group of target genes controls 50, 12, 7, and 2 molecular pathways, respectively. In conclusion, LncRNAs could be used as biomarkers in lung cancer due to their roles in control of several signaling pathways related to lung tumors. Also, it seems that lncRNAs, which use epigenetic mechanisms for modulating a large number of pathways, could be considered as important subjects for lung cancer-related diagnostic and prognostic biomarkers.

New Developments in Anti-Angiogenic Therapy of Cancer, Review and Update.

BACKGROUND: Angiogenesis is one of the critical physiological processes, by which the new blood vessels are generated from the pre-existing vessels in the early stage of vasculogenesis. While normal angiogenesis is critical for development and tissue growth, pathologic angiogenesis is important for the growth and spread of cancers by supplying nutrients and oxygen as well as providing a conduit for distant metastasis. In the last two decades, angiogenesis has been the area of extensive researches, indicating antiangiogenic target therapy as an effective strategy for cancer therapy. At present, this field has become a major avenue for research and development of novel therapeutics. OBJECTIVE: This review is dedicated to an updated review of the most prominent antiangiogenic agents, emphasizing the novel advancements and their applications, in particular, in the fields of antibodies, peptides, vaccines, endogenous inhibitors, Nanoparticles (NPs), antiangiogenic oligonucleotides and small molecules. Also, the potential role of 3D microfluidic models as an affordable and time-saving tool for angiogenesis investigations are discussed. METHODS: Firstly, we collected and summarized new developments that have occurred in all review and research articles in databases. Then, we used important keywords related to antiangiogenic target therapy and their applications for retrieval of most relevant data. RESULTS: This review is based on recent research and review articles and intended to cover all newly discovered agents inhibiting tumor angiogenesis and in particular, VEGF. CONCLUSION: New research studies have shown that anti-angiogenesis agents especially, in the form of combination therapy are effective in various cancers treatment.

Anti-cancer Immunotoxins, Challenges, and Approaches.

The development of recombinant immunotoxins (RITs) as a novel therapeutic strategy has made a revolution in the treatment of cancer. RITs result from the fusion of antibodies to toxin proteins for targeting and eliminating cancerous cells by inhibiting protein synthesis. Despite indisputable outcomes of RITs regarding inhibition of multiple cancer types, high immunogenicity has been known as the main obstacle in the clinical use of RITs. Various strategies have been proposed to overcome these limitations, including immunosuppressive therapy, humanization of the antibody fragment moiety, generation of immunotoxins originated from endogenous human cytotoxic enzymes, and modification of the toxin moiety to escape the immune system. This paper is devoted to review recent advances in the design of immunotoxins with lower immunogenicity.

Cytoplasmic Chaperones Enhance Soluble Expression of Anti-EGFR huscFv in Escherichia coli.

BACKGROUND: Overexpression of EGFR is associated with carcinogenesis in more than 70% of head and neck cancers. Anti-EGFR monoclonal antibodies bind to the extracellular domain of EGFR and block the EGFR downstream signaling pathway, which results in the suppression of the growth of the tumor cells. Escherichia coli is the preferred system for expressing various recombinant proteins, including single chain antibodies, but the formation of inclusion bodies negatively affects the efficacy of this system. Several strategies have been suggested to solve this problem, notably the utilization of molecular chaperones. OBJECTIVES: In this study, we attempted to increase the soluble expression of huscfv antibody via co-expression with the cytoplasmic chaperones. MATERIALS AND METHODS: To achieve this purpose, chaperones plasmids pG-KJE8, pGro7, pKjE7, pTf16 and pG-Tf2 encoding cytoplasmic chaperones were co-expressed with the humanized anti-EGFR scFv construct in E. coli. Different temperatures, incubations times, and concentrations of IPTG were used to produce an active antibody with the highest solubility. Results were analyzed by SDS-PAGE. Soluble huscFv was purified by Ni-NTA column and the biologic activity of the recombinant protein was determined by ELISA. RESULT: The results indicated that the highest concentrations of humanized anti-EGFR scFv were obtained by co-expression of huscFv via chaperone plasmid pG-KJE8 with 0.2 mM concentration of inducer (IPTG), culture temperature of 25 °C, and 4 h incubation time after induction. CONCLUSION: In conclusion, co-expression with chaperones could be used as an efficient strategy to produce soluble active ScFvs in E. coli.

Current trends in targeted therapy for drug-resistant infections.

Escalating antibiotic resistance is now a serious menace to global public health. It may be led to the emergence of "postantibiotic age" in which most of infections are untreatable. At present, there is an essential need to explore novel therapeutic strategies as a strong and sustainable pipeline to combat antibiotic-resistant infections. This review focuses on recent advances in this area including therapeutic antibodies, antimicrobial peptides, vaccines, gene therapy, genome editing, and phage therapy for tackling drug-resistant infections.

CRISPR-Cas system: Toward a more efficient technology for genome editing and beyond.

Genome engineering technology is of great interest for biomedical research that enables scientists to make specific manipulation in the DNA sequence. Early methods for introducing double-stranded DNA breaks relies on protein-based systems. These platforms have enabled fascinating advances, but all are costly and time-consuming to engineer, preventing these from gaining high-throughput applications. The CRISPR-Cas9 system, co-opted from bacteria, has generated considerable excitement in gene targeting. In this review, we describe gene targeting techniques with an emphasis on recent strategies to improve the specificities of CRISPR-Cas systems for nuclease and non-nuclease applications.