CRISPR Mediated Genome Engineering and its Application in Industry.

The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated nuclease 9) method has been dramatically changing the field of genome engineering. It is a rapid, highly efficient and versatile tool for precise modification of genome that uses a guide RNA (gRNA) to target Cas9 to a specific sequence. This novel RNA-guided genome-editing technique has become a revolutionary tool in biomedical science and has many innovative applications in different fields. In this review, we briefly introduce the Cas9-mediated genome-editing tool, summarize the recent advances in CRISPR/Cas9 technology to engineer the genomes of a wide variety of organisms, and discuss their applications to treatment of fungal and viral disease. We also discuss advantageous of CRISPR/Cas9 technology to drug design, creation of animal model, and to food, agricultural and energy sciences. Adoption of the CRISPR/Cas9 technology in biomedical and biotechnological researches would create innovative applications of it not only for breeding of strains exhibiting desired traits for specific industrial and medical applications, but also for investigation of genome function.

Genome-wide mapping of unexplored essential regions in the Saccharomyces cerevisiae genome: evidence for hidden synthetic lethal combinations in a genetic interaction network.

Despite systematic approaches to mapping networks of genetic interactions in Saccharomyces cerevisiae, exploration of genetic interactions on a genome-wide scale has been limited. The S. cerevisiae haploid genome has 110 regions that are longer than 10 kb but harbor only non-essential genes. Here, we attempted to delete these regions by PCR-mediated chromosomal deletion technology (PCD), which enables chromosomal segments to be deleted by a one-step transformation. Thirty-three of the 110 regions could be deleted, but the remaining 77 regions could not. To determine whether the 77 undeletable regions are essential, we successfully converted 67 of them to mini-chromosomes marked with URA3 using PCR-mediated chromosome splitting technology and conducted a mitotic loss assay of the mini-chromosomes. Fifty-six of the 67 regions were found to be essential for cell growth, and 49 of these carried co-lethal gene pair(s) that were not previously been detected by synthetic genetic array analysis. This result implies that regions harboring only non-essential genes contain unidentified synthetic lethal combinations at an unexpectedly high frequency, revealing a novel landscape of genetic interactions in the S. cerevisiae genome. Furthermore, this study indicates that segmental deletion might be exploited for not only revealing genome function but also breeding stress-tolerant strains.

The Trend of CRISPR-Based Technologies in COVID-19 Disease: Beyond Genome Editing.

Biotechnological approaches have always sought to utilize novel and efficient methods in the prevention, diagnosis, and treatment of diseases. This science has consistently tried to revolutionize medical science by employing state-of-the-art technologies in genomic and proteomic engineering. CRISPR-Cas system is one of the emerging techniques in the field of biotechnology. To date, the CRISPR-Cas system has been extensively applied in gene editing, targeting genomic sequences for diagnosis, treatment of diseases through genomic manipulation, and in creating animal models for preclinical researches. With the emergence of the COVID-19 pandemic in 2019, there is need for the development and modification of novel tools such as the CRISPR-Cas system for use in diagnostic emergencies. This system can compete with other existing biotechnological methods in accuracy, precision, and wide performance that could guarantee its future in these conditions. In this article, we review the various platforms of the CRISPR-Cas system meant for SARS-CoV-2 diagnosis, anti-viral therapeutic procedures, producing animal models for preclinical studies, and genome-wide screening studies toward drug and vaccine development.

Reversing T Cell Exhaustion by Converting Membrane PD-1 to Its Soluble form in Jurkat Cells; Applying The CRISPR/Cas9 Exon Skipping Strategy.

OBJECTIVE: T-cells express two functional forms of the programmed cell death protein 1 (PD-1): membrane (mPD-1) and soluble (sPD-1). The binding of mPD-1 and its ligand (PD-L1) on tumor cells could lead activated lymphocytes toward exhaustion. Selective deletion of the transmembrane domain via alternative splicing of exon-3 in PD-1 mRNA could generate sPD-1. Overexpression of sPD-1 could disrupt the mPD-1/PD-L1 interaction in tumor-specific T cells. We investigated the effect of secreted sPD-1 from pooled engineered and non-engineered T cell supernatant on survival and proliferation of lymphocytes in the tumor microenvironment (TME). MATERIALS AND METHODS: In this experimental study, we designed two sgRNA sequences upstream and downstream of exon-3 in the PDCD1 gene. The lentiCRISPRv2 puro vector was used to clone the dual sgRNAs and produce lentiviral particles to transduce Jurkat T cells. Analysis assays were used to clarify the change in PD-1 expression pattern in the pooled (engineered and non-engineered) Jurkat cells. Co-culture conditions were established with PD-L1+ cancer cells and lymphocytes. RESULTS: CRISPR/Cas9 could delete exon-3 of the PDCD1 gene in the engineered cells based on the tracking of indels by decomposition (TIDE) and interference of CRISPR edit (ICE) sequencing analysis reports. Our results showed a 12% reduction in mPD-1 positive cell population after CRISPR manipulation and increment in sPD-1 concentration in the supernatant. The increased sPD-1 confirmed its positive effect on proliferation of lymphocytes co-cultured with PDL1+ cancer cells. The survival percent of lymphocytes co-cultured with the pooled cells supernatant was 12.5% more than the control. CONCLUSION: The CRISPR/Cas9 exon skipping approach could be used in adoptive cell immunotherapies to change PD-1 expression patterns and overcome exhaustion.

Plasmid-based CRISPR-Cas9 system efficacy for introducing targeted mutations in CD81 gene of MDA-MB-231 cell line.

INTRODUCTION: Breast cancer has been represented a challenging issue worldwide as it is one of the major leading causes of death among women. CD81 gene, a member of the tetraspanin protein family, has been associated with the development of human cancers. Genome editing technologies, particularly the CRISPR-Cas9 system, have shown rapid progress in gene function studies. In this study, we aimed to evaluate the ability of the CRISPR-Cas9 plasmid-based system to modify specific regions of the CD81 gene in the MDA-MB-231 breast cancer cell line. MATERIALS AND METHODS: Using bioinformatics database search, four different single guide RNAs (sgRNAs) to target exon 3 and exon 5 of the CD81 gene were designed. The intended sgRNAs sequences were cloned into the expression plasmid pSpCas9(BB)-2A-GFP (PX458) bearing sgRNA scaffold backbone, Cas9, and EGFP coding sequences, which was confirmed by colony PCR and sequencing. Transfection efficiency was determined by fluorescence microscopy and flow cytometry analysis. Gene editing efficiency was measured qualitatively and quantitatively using the T7E1 and TIDE software, respectively. RESULTS: Our data show that expression constructs were successfully introduced into MDA-MB-231 cells with an acceptable transfection efficiency. Two sgRNAs that were afforded to introduce significant mutations in their target regions were detected by TIDE software (p-value < 0.05). To the best of our knowledge, CD81 gene editing in these cells has been investigated for the first time in this study using the CRISPR/Cas9 technique. CONCLUSIONS: Taken together, our data show that the CRISPR-Cas9 system can change the genomic sequence in the target area of MDA-MB-231 cells. Along with previous studies, we propose forethought when using T7E1-based quantitative indel estimates, as comparing activities of multiple gRNAs with the T7E1 assay may lead to inaccurate conclusions. Instead, estimating non-homologous end-joining events (NHEJ) by Sanger sequencing and subsequent TIDE analysis is recommended.

A Whole Genome Sequencing-Based Approach to Track down Genomic Variants in Itraconazole-Resistant Species of Aspergillus from Iran.

The antifungal resistance in non-fumigatus Aspergillus spp., as well as Aspergillus fumigatus, poses a major therapeutic challenge which affects the entire healthcare community. Mutation occurrence of cyp51 gene paralogs is the major cause of azole resistance in Aspergillus spp. To obtain a full map of genomic changes, an accurate scan of the entire length of the Aspergillus genome is necessary. In this study, using whole genome sequencing (WGS) technique, we evaluated the mutation in cyp51A, cyp51B, Cdr1B, AtrR, Hmg1, HapE and FfmA genes in different clinical isolates of Aspergillus fumigatus, Aspergillus niger, Aspergillus tubingensis, Aspergillus welwitschiae and Aspergillus terreus which responded to minimum inhibitory concentrations of itraconazole above 16 µg mL-1. We found different nonsynonymous mutations in the cyp51A, cyp51B, Cdr1B, AtrR, Hmg1, HapE and FfmA gene loci. According to our findings, Aspergillus species isolated from different parts of the world may represent different pattern of resistance mechanisms which may be revealed by WGS.

Curcumin delivery by modified biosourced carbon-based nanoparticles.

Aim: To prepare a novel hybrid system for the controlled release and delivery of curcumin (CUR). Methods: A method for the ultrasound-assisted fabrication of protein-modified nanosized graphene oxide-like carbon-based nanoparticles (CBNPs) was developed. After being modified with bovine serum albumin (BSA), CUR was loaded onto the synthesized hybrid (labeled CBNPs@BSA-CUR). The structure and properties of the synthesized nanoparticles were elucidated using transmission electron microscopy (TEM), atomic force microscopy (AFM), ultraviolet-visible spectroscopy (UV-Vis), Fourier-transform infrared spectroscopy (FTIR) and x-ray photoelectron spectroscopy (XPS) methods. Results: CBNPs@BSA-CUR showed pH sensitivity and were calculated as controlled CUR release behavior. The drug-free system exhibited good biocompatibility and was nontoxic. However, CBNPs@BSA-CUR showed acceptable antiproliferative ability against MCF-7 breast cancer cells. Conclusion: CBNPs@BSA-CUR could be considered a highly promising nontoxic nanocarrier for the delivery of CUR with good biosafety.

BSA-PEI Nanoparticle Mediated Efficient Delivery of CRISPR/Cas9 into MDA-MB-231 Cells.

The discovery of bacterial-derived Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has revolutionized genome engineering and gene therapy due to its wide range of applications. One of the major challenging issues in CRISPR/Cas system is the lack of an efficient, safe, and clinically suitable delivery of the system's components into target cells. Here, we describe the development of polyethylenimine coated-bovine serum albumin nanoparticles (BSA-PEI NPs) for efficient delivery of CRISPR/Cas9 system in both DNA (px458 plasmid) and ribonucleoprotein (RNP) forms into MDA-MB-231 human breast cancer cell line. Our data showed that synthesized BSA-PEI (BP) NPs delivered plasmid px458 at concentrations of 0.15, 0.25, and 0.35 µg/µl with efficiencies of approximately 29.7, 54.8, and 84.1% into MDA-MB-231 cells, respectively. Our study demonstrated that Cas9/sgRNA RNP complex efficiently (~ 92.6%) delivered by BSA-PEI NPs into the same cells. Analysis of toxicity and biocompatibility of synthesized NPs on human red blood cells, MDA-MB-231 cells, and mice showed that the selected concentration (28 µg/µl) of BSA-PEI NPs for transfection had no remarkable toxicity effects. Thus, obtained results suggest BSA-PEI NPs as one of the most promising carrier for delivering CRISPR/Cas9 to target cells.

Recent Advances in Genome Editing Tools in Medical Mycology Research.

Manipulating fungal genomes is an important tool to understand the function of target genes, pathobiology of fungal infections, virulence potential, and pathogenicity of medically important fungi, and to develop novel diagnostics and therapeutic targets. Here, we provide an overview of recent advances in genetic manipulation techniques used in the field of medical mycology. Fungi use several strategies to cope with stress and adapt themselves against environmental effectors. For instance, mutations in the 14 alpha-demethylase gene may result in azole resistance in Aspergillusfumigatus strains and shield them against fungicide's effects. Over the past few decades, several genome editing methods have been introduced for genetic manipulations in pathogenic fungi. Application of restriction enzymes to target and cut a double-stranded DNA in a pre-defined sequence was the first technique used for cloning in Aspergillus and Candida. Genome editing technologies, including zinc-finger nucleases (ZFNs) and transcriptional activator-like effector nucleases (TALENs), have been also used to engineer a double-stranded DNA molecule. As a result, TALENs were considered more practical to identify single nucleotide polymorphisms. Recently, Class 2 type II Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 technology has emerged as a more useful tool for genome manipulation in fungal research.

CRISPR Systems for COVID-19 Diagnosis.

The emergence of the new coronavirus 2019 (COVID-19) was first seen in December 2019, which has spread rapidly and become a global pandemic. The number of cases of COVID-19 and its associated mortality have raised serious concerns worldwide. Early diagnosis of viral infection undoubtedly allows rapid intervention, disease management, and substantial control of the rapid spread of the disease. Currently, the standard approach for COVID-19 diagnosis globally is the RT-qPCR test; however, the limited access to kits and associated reagents, the need for specialized lab equipment, and the need for highly skilled personnel has led to a detection slowdown. Recently, the development of clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostic systems has reshaped molecular diagnosis. The benefits of the CRISPR system such as speed, precision, specificity, strength, efficiency, and versatility have inspired researchers to develop CRISPR-based diagnostic and therapeutic methods. With the global COVID-19 outbreak, different groups have begun to design and develop diagnostic and therapeutic programs based on the efficient CRISPR system. CRISPR-based COVID-19 diagnostic systems have advantages such as a high detection speed (i.e., 30 min from raw sample to reach a result), high sensitivity and precision, portability, and no need for specialized laboratory equipment. Here, we review contemporary studies on the detection of COVID-19 based on the CRISPR system.

A knockdown of the herpes simplex virus type-1 gene in all-in-one CRISPR vectors.

INTRODUCTION: Herpes simplex virus type 1 (HSV-1) is a virus that causes serious human disease and establishes a long-term latent infection. The latent form of this virus has shown to be resistant to antiviral drugs. Clustered Regularly Interspace Short Palindromic Repeats (CRISPR), is an important tool in genome engineering and composed of guide RNA (gRNA) and Cas9 nuclease that makes an RNA-protein complex to digest exclusive target sequences implementation of gRNA. Moreover, CRISPR-Cas9 system effectively suppresses HSV-1 infection by knockout of some viral genes. MATERIALS AND METHODS: To survey the efficacy of Cas9 system on HSV-1 genome destruction, we designed several guide RNAs (gRNAs) that all packaged in one vector. Additionally, we performed a one-step restriction using BamHI and Esp3I enzymes. RESULTS: CRISPR/Cas9 system targeted against the gD gene of HSV-1 was transfected into HEK-AD cells that showed a significant reduction of HSV-1 infection by plaque assay and real-time PCR. CONCLUSION: The pCas-Guide-EF1a-GFP CRISPR vector can create a fast and efficient method for gRNA cloning by restriction enzymes (Esp3I (BsmBI) and BamHI). Therefore, the CRISPR/Cas9 system may be utilized for the screening of genes critical for the HSV-1 infection and developing new strategies for targeted therapy of viral infections caused by HSV-1.

Anticancer effect of X-Ray triggered methotrexate conjugated albumin coated bismuth sulfide nanoparticles on SW480 colon cancer cell line.

The application of nanoparticles (NPs) as radio-sensitizers and carriers has opened up a new horizon to overcome the limitations of chemo and radiotherapy. In this study, bovine serum albumin-coated Bi2S3 NPs (Bi2S3@BSA NPs) were synthesized and evaluated in terms of their ability to be used as a radio-sensitizer and carrier for methotrexate (MTX). Physicochemical properties of MTX conjugated Bi2S3@BSA NPs (Bi2S3@BSA-MTX NPs) were characterized by DLS, TEM, FTIR, UV/Vis, and XRD analyses. After the evaluation of cellular uptake and intracellular localization, the cytotoxicity of the combination of Bi2S3@BSA-MTX NPs and X-Ray radiation was analyzed against the SW480 cell line. The synthesized NPs exhibited spherical-like shapes and homogenous morphology, possessing a hydrodynamic diameter of 140.2 ± 5.71 nm (mean ± SD) and zeta potential of -25 mV. Also, the release study showed that the release of MTX is faster and higher in the presence of the proteinase K enzyme than the absence of the enzyme. The results of in-vitro chemo-radiation therapy indicated that the viability of treated cells with Bi2S3@BSA-MTX NPs is significantly lower than the cells treated with Bi2S3@BSA NPs. Furthermore, cells treated with Bi2S3@BSA-MTX NPs showed a lower degree of viability when combined with X-Ray radiation in comparison with the absence of irradiation, which confirmed the ability of the Bi2S3@BSA-MTX NPs as radio-sensitizer.

An overview of applications of CRISPR-Cas technologies in biomedical engineering.

Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR) is one of the major genome editing systems and allows changing DNA levels of an organism. Among several CRISPR categories, the CRISPR-Cas9 system has shown a remarkable progression rate over its lifetime. Recently, other tools including CRISPR-Cas12 and CRISPR-Cas13 have been introduced. CRISPR-Cas9 system has played a key role in the industrial cell factory's production and improved our understanding of genome function. Additionally, this system has been used as one of the major genome editing systems for the diagnosis and treatment of several infectious and non-infectious diseases. In this review, we discuss CRISPR biology, its versatility, and its application in biomedical engineering.

Rapid detection of Candida species in bronchoalveolar lavage fluid from patients with pulmonary symptoms.

Candida species, especially C. albicans, are commensals on human mucosal surfaces, but are increasingly becoming one of the important invasive pathogens as seen by a rise in its prevalence in immunocompromised patients and in antibiotic consumption. Thus, an accurate identification of Candida species in patients with pulmonary symptoms can provide important information for effective treatment. A total of 75 clinical isolates of Candida species were obtained from the bronchoalveolar lavage fluid of both immunocompromised and immunocompetent patients with pulmonary symptoms. Candida cultures were identified based on nuclear ribosomal Internal Transcribed Spacer (ITS1-ITS2 rDNA) sequence analysis by polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP). Molecular identification indicated that the isolates belonged predominantly to C. albicans (52%), followed by C. tropicalis (24%), C. glabrata (14.7%), C. krusei (5.3%), C. parapsilosis (1.3%), C. kefyr (1.3%) and C. guilliermondii (1.3%). Given the increasing complexity of disease profiles and their management regimens in diverse patients, rapid and accurate identification of Candida species can lead to timely and appropriate antifungal therapy.

Improved stress resistance and ethanol production by segmental haploidization of the diploid genome in Saccharomyces cerevisiae.

Saccharomyces cerevisiae strains from industrial and natural geographical environments are reported to show great variation in copy number of chromosomal regions. Such variation contributes to the mechanisms underlying adaptation to different environments. Here, we created and phenotypically analyzed segmentally haploidized strains, each harboring a deletion of one copy of approximately 100-300 kb of the left or right terminal region of 16 chromosomes in a diploid strain by using a PCR-mediated chromosomal deletion method. No haploidized strain of the 158-kb deleted right terminal region of chromosome III or the 172-kb deleted right terminal region of chromosome VI was produced; however, segmentally haploidized strains of the remaining 30 terminal regions were obtained. Among these 30 strains, two exhibited higher lactic acid resistance and two displayed higher thermo-tolerance at 41°C versus the host diploid strain. By contrast, four and two segmentally haploidized strains showed sensitivity to 6% lactic acid and low temperature at 13°C, respectively. The effect of the decreased copy number of the chromosomal terminal regions on ethanol production was analyzed. As compared with the host diploid strain, a 3.8% and 4.3% improvement in ethanol production in 10% glucose medium was observed for two strains in which one of two copies of the 197-kb left terminal region of chromosome V and one of two copies of the 195-kb left terminal region of chromosome X was deleted, respectively. These results indicate that artificial segmental haploidization might contribute to improvement of industrially important phenotypes and provide a new approach to breeding superior yeast strains.

CRISPR-PCS: a powerful new approach to inducing multiple chromosome splitting in Saccharomyces cerevisiae.

PCR-mediated chromosome splitting (PCS) was developed in the yeast Saccharomyces cerevisiae. It is based on homologous recombination and enables division of a chromosome at any point to form two derived and functional chromosomes. However, because of low homologous recombination activity, PCS is limited to a single site at a time, which makes the splitting of multiple loci laborious and time-consuming. Here we have developed a highly efficient and versatile chromosome engineering technology named CRISPR-PCS that integrates PCS with the novel genome editing CRISPR/Cas9 system. This integration allows PCS to utilize induced double strand breaks to activate homologous recombination. CRISPR-PCS enhances the efficiency of chromosome splitting approximately 200-fold and enables generation of simultaneous multiple chromosome splits. We propose that CRISPR-PCS will be a powerful tool for breeding novel yeast strains with desirable traits for specific industrial applications and for investigating genome function.

Rapid detection of Candida species in bronchoalveolar lavage fluid from patients with pulmonary symptoms

Abstract Candida species, especially C. albicans, are commensals on human mucosal surfaces, but are increasingly becoming one of the important invasive pathogens as seen by a rise in its prevalence in immunocompromised patients and in antibiotic consumption. Thus, an accurate identification of Candida species in patients with pulmonary symptoms can provide important information for effective treatment. A total of 75 clinical isolates of Candida species were obtained from the bronchoalveolar lavage fluid of both immunocompromised and immunocompetent patients with pulmonary symptoms. Candida cultures were identified based on nuclear ribosomal Internal Transcribed Spacer (ITS1-ITS2 rDNA) sequence analysis by polymerase chain reaction–restriction fragment length polymorphisms (PCR-RFLP). Molecular identification indicated that the isolates belonged predominantly to C. albicans (52%), followed by C. tropicalis (24%), C. glabrata (14.7%), C. krusei (5.3%), C. parapsilosis (1.3%), C. kefyr (1.3%) and C. guilliermondii (1.3%). Given the increasing complexity of disease profiles and their management regimens in diverse patients, rapid and accurate identification of Candida species can lead to timely and appropriate antifungal therapy.