CRISPR-Cas9 Genome Editing System Research Articles

Manipulation of Proteostasis Networks in Transgenic ZAAT Zebrafish via CRISPR-Cas9 Gene Editing.

The CRISPR-Cas9 genome editing system is used to induce mutations in genes of interest resulting in the loss of functional protein. A transgenic zebrafish α1-antitrypsin deficiency (AATD) model displays an unusual phenotype, in that it lacks the hepatic accumulation of the misfolding Z α1-antitrypsin (ZAAT) evident in human and mouse models. Here we describe the application of the CRISPR-Cas9 system to generate mutant zebrafish with defects in key proteostasis networks likely to be involved in the hepatic processing of ZAAT in this model. We describe the targeting of the atf6a and man1b1 genes as examples.

Transplastomic plants — new approaches to solving “old” problems

Transplastomic plants are capable to accumulate the significant amounts (up to 70% of TSP) of target recombinant proteins in tissues. However, the production of such forms is severely limited by the low yield of initial transformants. This problem requires the development and optimization of new approaches to the delivery of transgenes into chloroplasts and an increase in the frequency of their integration into the plastome. Transplastomic tobacco plants expressing thegfpreporter gene and theaadAselectable marker under the control of thePrrnG10Lpromoter and theTpsbAterminator were obtained in the laboratory of plant bioengineering. It is known that the selected promoter and insertion region (between the tRNA genes of isoleucine and alanine) are capable to provide a high yield of recombinant proteins in the leaves of transplastomic plants [1]. However, the content of recombinant GFP in the leaves of the obtained transplastomic plants was determined at the level of 0.12%, and the variability for this trait was minimal and ranged from 0.09 to 0.16% of TSP. Insufficient accumulation of the target protein in transformants is not associated with transcription disorders or the presence of non-transgenic copies of the plastome. Probably, the low frequency of transformation and the lack of variability between the transformants are the reasons that make it difficult to select highly productive forms. It is proposed to increase the efficiency of targeted delivery of genetic constructs to plastids using single-walled carbon nanotubes loaded with recombinant DNA. This process can also be facilitated by our proposed approach to increase the frequency of DNA double-strand breaks in target regions of the plastome through the use of the CRISPR-Cas9 genome editing system.
 This work was supported by the Russian Science Foundation grant No. 23-24-00545.

The strong base for using base editing in plants

The most common application of CRISPR-Cas9 genome editing system is a gene knockout via indel mutations introducing. It is obvious, because this approach has minimum critical conditions in guide RNA design: available PAM sequence and conservative 19–25 nucleotides within all alleles of a target gene. Precise nucleotide changing with base editing systems has more limitations: target nucleotide should locate in editing window of adenine- or cytidine-deaminase, besides this, all undesired adenines or cytosines in editing window will be likely changed. However, there is a more fundamental issue — it is very difficult to find a single aminoacid substitution, which changes protein features in a desirable side. One of the good examples of base editing target will be considered in this work.
 Nicotiana tabacumL. is a plant fromSolanaceaefamily, the same as potato, tomato and pepper. All these plants are strongly affected by potato virus Y (PVY). It is known, that PVY recruits host translation initiation factor eIF4E by the viral protein VPg in order to start synthesis its proteins. If eIF4E can’t interact with VPg, plant will be resistant.
 In our work, we established an aminoacid substitution in tobacco eIF4E factor, which disrupted interaction with PVY VPg in yeast two-hybrid conditions, but didn’t influence the factor functionality. Then we designed two genetic constructions with different sgRNAs for introducing this mutation in tobacco plants using cytidine-deaminase system. These constructions were used to plant transformation and development of edited tobacco plants.
 This work is supported by State Task No. 0431-2022-0004.

The Influence of Trctf1 Gene Knockout by CRISPR–Cas9 on Cellulase Synthesis by Trichoderma reesei with Various Soluble Inducers

Knockout of the transcriptional repressor Trctf1 is known to enhance the yield of cellulose-induced cellulase synthesis in Trichoderma reesei. However, different inducers possess distinct induction mechanisms, and the effect of Trctf1 on cellulase synthesis with soluble inducers remains unknown. To evaluate the effect of the Trctf1 gene on cellulase synthesis and develop a high-yielding cellulase strain, we established a CRISPR–Cas9 genome editing system in T. reesei Rut C30 using codon-optimized Cas9 protein and in vitro transcribed RNA. This study demonstrated that T. reesei ΔTrctf1 with the Trctf1 gene knocked out showed no statistically significant differences in cellulase, cellobiohydrolase, endoglucanase, and β−glucosidase production when induced with MGD (the mixture of glucose and sophorose). However, when induced with lactose, the activities of these enzymes increased by 20.2%, 12.4%, and 12.9%, respectively, with no statistically significant differences in β−glucosidase activity. The hydrolysis efficiency on corn stover of cellulases produced by T. reesei ΔTrctf1 under different inducers was not significantly different from that of wild-type cellulases, indicating that Trctf1 gene deletion has little effect on the cellulase cocktail. These findings contribute to a better understanding of the molecular mechanisms underlying the regulation of T. reesei cellulase synthesis by different soluble inducers, as well as the construction of high-yield cellulase gene−engineered strains.

Recent advances in genome editing of bloodstream forms of Trypanosoma congolense using CRISPR-Cas9 ribonucleoproteins: Proof of concept

African Animal Trypanosomosis (AAT or Nagana) is a vector-borne disease caused by Trypanosomatidae, genus Trypanosoma. The disease is transmitted by the bite of infected hematophagous insects, mainly tsetse flies but also other blood-sucking insects including stomoxes and tabanids. Although many trypanosome species infect animals, the main agents responsible for this disease with a strong socio-economic and veterinary health impact are Trypanosoma congolense (T. congolense or Tc), Trypanosoma vivax (T.vivax), and to a lesser extent, Trypanosoma brucei brucei (T.brucei brucei or Tbb). These parasites mainly infect livestock, including cattle, in sub-Saharan Africa, with major repercussions in terms of animal productivity and poverty for populations which are often already very poor. As there is currently no vaccine, the fight against the disease is primarily based on diagnosis, treatment and vector control. To develop new tools (particularly therapeutic tools) to fight against the disease, we need to know both the biology and the genes involved in the pathogenicity and virulence of the parasites. To date, unlike for Trypanosoma brucei (T.brucei) or Trypanosoma cruzi (T.cruzi), genome editing tools has been relatively little used to study T. congolense. We present an efficient, reproducible and stable CRISPR-Cas9 genome editing system for use in Tc bloodstream forms (Tc-BSF). This plasmid-free system is based on transient expression of Cas9 protein and the use of a ribonucleoprotein formed by the Cas9 and sgRNA complex. This is the first proof of concept of genome editing using CRISPR-Cas9 ribonucleoproteins on Tc-BSF. This adapted protocol enriches the “toolbox” for the functional study of genes of interest in blood forms of the Trypanosoma congolense. This proof of concept is an important step for the scientific community working on the study of trypanosomes and opens up new perspectives for the control of and fight against animal trypanosomosis.

Design of high-monounsaturated fatty acid soybean seed oil using GmPDCTs knockout via a CRISPR-Cas9 system.

Design of high-monounsaturated fatty acid soybean seed oil using GmPDCTs knockout via a CRISPR-Cas9 system.

Current Applications and Future Perspective of CRISPR/Cas9 in the Diagnosis and Treatment of COVID 19: A Review

Since the outbreak of COVID-19, scientists have applied various techniques to diagnose and treat the viral disease. However, due to the limitations of other methods, they deployed Clustered-Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) protein (CRISPR/Cas) system that not just successfully diagnosed but also facilitated the therapeutic treatment of the COVID-19. CRISPR-Cas9 was first identified in the bacteria E. coli, which has a unique immune system for cutting the nucleic structures of invasive species. Scientists studied the bacterial system that led to the development of an identical model, generally called the CRISPR-Cas9 genome editing system. It has a guide RNA (gRNA) and Cas9 proteins; gRNA identifies and leads cas9 protein to cleave the specific sequence. This technique has dynamic applications, such as the ability to correct mutations by cleaving the mutant cells and to detect and develop optimal treatments for viral diseases like severe acute respiratory syndrome coronavirus-2 (SARS-CoV2). Apart from the extensive advantages of CRISPR-Cas technology, there are serious concerns regarding the commercialization of this technique. A rational suggestion would be to use it to resist a pandemic like COVID-19 rather than triggering another human race of genome enhancement. This article is aimed to review the background of CRISPR-Cas9, its mechanism as a diagnostic and therapeutic tool for COVID-19, whereas its limitations, future aspects, and ethical boundaries are discussed subsequently

Inhalable vaccine of bacterial culture supernatant extract mediates protection against fatal pulmonary anthrax

ABSTRACT Pulmonary anthrax is the most fatal clinical form of anthrax and currently available injectable vaccines do not provide adequate protection against it. Hence, next-generation vaccines that effectively induce immunity against pulmonary anthrax are urgently needed. In the present study, we prepared an attenuated and low protease activity Bacillus anthracis strain A16R-5.1 by deleting five of its extracellular protease activity-associated genes and its lef gene through the CRISPR-Cas9 genome editing system. This mutant strain was then used to formulate a lethal toxin (LeTx)-free culture supernatant extract (CSE) anthrax vaccine, of which half was protective antigen (PA). We generated liquid, powder, and powder reconstituted formulations that could be delivered by aerosolized intratracheal inoculation. All of them induced strong humoral, cellular, and mucosal immune responses. The vaccines also produced LeTx neutralizing antibodies and conferred full protection against the lethal aerosol challenges of B. anthracis Pasteur II spores in mice. Compared to the recombinant PA vaccine, the CSE anthrax vaccine with equal PA content provided superior immunoprotection against pulmonary anthrax. The preceding results suggest that the CSE anthrax vaccine developed herein is suitable and scalable for use in inhalational immunization against pulmonary anthrax.

A culture platform to study quiescent hematopoietic stem cells following genome editing

Other than genetically engineered mice, few reliable platforms are available for the study of hematopoietic stem cell (HSC) quiescence. Here we present a platform to analyze HSC cell cycle quiescence by combining culture conditions that maintain quiescence with a CRISPR-Cas9 genome editing system optimized for HSCs. We demonstrate that preculture of HSCs enhances editing efficiency by facilitating nuclear transport of ribonucleoprotein complexes. For post-editing culture, mouse and human HSCs edited based on non-homologous end joining and cultured under low-cytokine, low-oxygen, and high-albumin conditions retain their phenotypes and quiescence better than those cultured under the proliferative conditions. Using this approach, HSCs regain quiescence even after editing by homology-directed repair. Our results show that low-cytokine culture conditions for gene-edited HSCs are a useful approach for investigating HSC quiescence exvivo.

Repurposing the Endogenous CRISPR-Cas9 System for High-Efficiency Genome Editing in Lacticaseibacillus paracasei.

Lactobacilli such as Lacticaseibacillus (Lcb) paracasei are generally regarded as safe and health-promoting microbes, and have been widely applied in food and pharmaceutical industries. However, the genetic bases of their beneficial properties were mostly uncertain because of the lack of effective genetic manipulation tools. The type II CRISPR-Cas9 system is the largest family present in lactobacilli, but none of them yet have been developed for genetic modifications. Here, we establish the first endogenous CRISPR-Cas9 genome-editing system in lactobacilli. With a validated protospacer adjacent motif (PAM) and customized single guide RNA (sgRNA) expression cassette, the native CRISPR-Cas9 system was reprogrammed to achieve gene deletion and chromosomal insertion at over 90% efficiency, as well as nucleotide substitution at ≥50% efficiency. We also effectively accomplished deletions of large genomic fragments (5-10 kb) and simultaneous deletion of multiple genes at distal loci, both of which are the first cases in lactobacilli when either endogenous or exogenous CRISPR-Cas systems were employed. In addition, we designed a controllable plasmid-targeting sgRNA expression module and integrated it into the editing plasmid. The all-in-one vector realized gene deletion and plasmid curing at high efficiency (>90%). Collectively, the present study develops a convenient and precise genetic tool in Lcb. paracasei and contributes to the genetics and engineering of lactobacilli.

Control of λ Lysogenic Escherichia coli Cells by Synthetic λ Phage Carrying cIantisense.

Enterobacterial phage λ is a temperate phage that infects Escherichia coli and has a lytic-lysogenic life cycle. CI, a λ repressor, regulates the expression of lytic transcripts and acts as a major genetic switch that determines the lysogenic state. To manipulate the genome of phage λ, the CRISPR-Cas9 genome editing system was constructed in lysogenic E. coli MG1655 cells. For instance, we successfully changed cI857 to cIWT in the phage genome through Cas9-mediated single-nucleotide editing. A lytic phage was prepared by introducing an amber mutation in the middle of the cI gene, but it could not lyse lysogenic MG1655 cells. We prepared a phage expressing cI antisense mRNA by reverse substitution of the cI gene. Lysis of λ cI857 lysogenic cells occurred by the infection of the λ cIantisense. These results suggest an effective lysogenic cell control method by a synthetic phage expressing antisense mRNA of the genetic switch gene. It is expected to be applied as a tool to control harmful lysogenic microorganisms.

Sequential Accumulation of ‘Driver’ Pathway Mutations Induces the Upregulation of Hydrogen-Sulfide-Producing Enzymes in Human Colonic Epithelial Cell Organoids

Recently, a CRISPR-Cas9 genome-editing system was developed with introduced sequential ‘driver’ mutations in the WNT, MAPK, TGF-β, TP53 and PI3K pathways into organoids derived from normal human intestinal epithelial cells. Prior studies have demonstrated that isogenic organoids harboring mutations in the tumor suppressor genes APC, SMAD4 and TP53, as well as the oncogene KRAS, assumed more proliferative and invasive properties in vitro and in vivo. A separate body of studies implicates the role of various hydrogen sulfide (H2S)-producing enzymes in the pathogenesis of colon cancer. The current study was designed to determine if the sequential mutations in the above pathway affect the expression of various H2S producing enzymes. Western blotting was used to detect the expression of the H2S-producing enzymes cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST), as well as several key enzymes involved in H2S degradation such as thiosulfate sulfurtransferase/rhodanese (TST), ethylmalonic encephalopathy 1 protein/persulfide dioxygenase (ETHE1) and sulfide-quinone oxidoreductase (SQR). H2S levels were detected by live-cell imaging using a fluorescent H2S probe. Bioenergetic parameters were assessed by Extracellular Flux Analysis; markers of epithelial-mesenchymal transition (EMT) were assessed by Western blotting. The results show that the consecutive mutations produced gradual upregulations in CBS expression—in particular in its truncated (45 kDa) form—as well as in CSE and 3-MST expression. In more advanced organoids, when the upregulation of H2S-producing enzymes coincided with the downregulation of the H2S-degrading enzyme SQR, increased H2S generation was also detected. This effect coincided with the upregulation of cellular bioenergetics (mitochondrial respiration and/or glycolysis) and an upregulation of the Wnt/β-catenin pathway, a key effector of EMT. Thus sequential mutations in colon epithelial cells according to the Vogelstein sequence are associated with a gradual upregulation of multiple H2S generating pathways, which, in turn, translates into functional changes in cellular bioenergetics and dedifferentiation, producing more aggressive and more invasive colon cancer phenotypes.

Modeling Human Primary Microcephaly With hiPSC-Derived Brain Organoids Carrying CPAP-E1235V Disease-Associated Mutant Protein.

The centrosome is composed of a pair of centrioles and serves as the major microtubule-organizing center (MTOC) in cells. Centrosome dysfunction has been linked to autosomal recessive primary microcephaly (MCPH), which is a rare human neurodevelopmental disorder characterized by small brain size with intellectual disability. Recently, several mouse models carrying mutated genes encoding centrosomal proteins have been generated to address the genotype–phenotype relationships in MCPH. However, several human-specific features were not observed in the mouse models during brain development. Herein, we generated isogenic hiPSCs carrying the gene encoding centrosomal CPAP-E1235V mutant protein using the CRISPR-Cas9 genome editing system, and examined the phenotypic features of wild-type and mutant hiPSCs and their derived brain organoids. Our results showed that the CPAP-E1235V mutant perturbed the recruitment of several centriolar proteins involved in centriole elongation, including CEP120, CEP295, CENTROBIN, POC5, and POC1B, onto nascent centrioles, resulting in the production of short centrioles but long cilia. Importantly, our wild-type hiPSC-derived brain organoid recapitulated many cellular events seen in the developing human brain, including neuronal differentiation and cortical spatial lamination. Interestingly, hiPSC-CPAP-E1235V-derived brain organoids induced p53-dependent neuronal cell death, resulting in the production of smaller brain organoids that mimic the microcephaly phenotype. Furthermore, we observed that the CPAP-E1235V mutation altered the spindle orientation of neuronal progenitor cells and induced premature neuronal differentiation. In summary, we have shown that the hiPSC-derived brain organoid coupled with CRISPR/Cas9 gene editing technology can recapitulate the centrosome/centriole-associated MCPH pathological features. Possible mechanisms for MCPH with centriole/centrosome dysfunction are discussed.

Generation of CRISPR-Cas9-mediated genetic knockout human intestinal tissue-derived enteroid lines by lentivirus transduction and single-cell cloning.

Human intestinal tissue-derived enteroids (HIEs; also called organoids) are a powerful ex vivo model for gastrointestinal research. Genetic modification of these nontransformed cultures allows new insights into gene function and biological processes involved in intestinal diseases as well as gastrointestinal and donor segment-specific function. Here we provide a detailed technical pipeline and protocol for using the CRISPR-Cas9 genome editing system to knock out a gene of interest specifically in HIEs by lentiviral transduction and single-cell cloning. This protocol differs from a previously published alternative using electroporation of human colonoids to deliver piggyback transposons or CRISPR-Cas9 constructs, as this protocol uses a modified, fused LentiCRISPRv2-small-guiding RNA to express Cas9 and small-guiding RNA in a lentivirus. The protocol also includes the steps of gene delivery and subsequent single-cell cloning of the knockout cells as well as verification of clones and sequence identification of the mutation sites to establish knockout clones. An overview flowchart, step-by-step guidelines and troubleshooting suggestions are provided to aid the researcher in obtaining the genetic knockout HIE line within 2-3 months. In this protocol, we further describe how to use HIEs as an ex vivo model to assess host restriction factors for viral replication (using human norovirus replication as an example) by knocking out host attachment factors or innate immunity genes. Other applications are discussed to broaden the utility of this system, for example, to generate knockin or conditional knockout HIE lines to investigate the function of essential genes in many biological processes including other types of organoids.

DNA-free CRISPR-Cas9 gene editing of wild tetraploid tomato Solanum peruvianum using protoplast regeneration.

Wild tomatoes (Solanum peruvianum) are important genomic resources for tomato research and breeding. Development of a foreign DNA-free clustered regularly interspaced short palindromic repeat (CRISPR)-Cas delivery system has potential to mitigate public concern about genetically modified organisms. Here, we established a DNA-free CRISPR-Cas9 genome editing system based on an optimized protoplast regeneration protocol of S. peruvianum, an important resource for tomato introgression breeding. We generated mutants for genes involved in small interfering RNAs biogenesis, RNA-DEPENDENT RNA POLYMERASE 6 (SpRDR6), and SUPPRESSOR OF GENE SILENCING 3 (SpSGS3); pathogen-related peptide precursors, PATHOGENESIS-RELATED PROTEIN-1 (SpPR-1) and PROSYSTEMIN (SpProSys); and fungal resistance (MILDEW RESISTANT LOCUS O, SpMlo1) using diploid or tetraploid protoplasts derived from in vitro-grown shoots. The ploidy level of these regenerants was not affected by PEG-Ca2+-mediated transfection, CRISPR reagents, or the target genes. By karyotyping and whole genome sequencing analysis, we confirmed that CRISPR-Cas9 editing did not introduce chromosomal changes or unintended genome editing sites. All mutated genes in both diploid and tetraploid regenerants were heritable in the next generation. spsgs3 null T0 regenerants and sprdr6 null T1 progeny had wiry, sterile phenotypes in both diploid and tetraploid lines. The sterility of the spsgs3 null mutant was partially rescued, and fruits were obtained by grafting to wild-type (WT) stock and pollination with WT pollen. The resulting seeds contained the mutated alleles. Tomato yellow leaf curl virus proliferated at higher levels in spsgs3 and sprdr6 mutants than in the WT. Therefore, this protoplast regeneration technique should greatly facilitate tomato polyploidization and enable the use of CRISPR-Cas for S. peruvianum domestication and tomato breeding.

Multiplex CRISPR-Cas9 editing of DNA methyltransferases in rice uncovers a class of non-CG methylation specific for GC-rich regions.

DNA methylation in the non-CG context is widespread in the plant kingdom and abundant in mammalian tissues such as the brain and pluripotent cells. Non-CG methylation in Arabidopsis thaliana is coordinately regulated by DOMAINS REARRANGED METHYLTRANSFERASE (DRM) and CHROMOMETHYLASE (CMT) proteins but has yet to be systematically studied in major crops due to difficulties in obtaining genetic materials. Here, utilizing the highly efficient multiplex CRISPR-Cas9 genome-editing system, we created single- and multiple-knockout mutants for all the nine DNA methyltransferases in rice (Oryza sativa) and profiled their whole-genome methylation status at single-nucleotide resolution. Surprisingly, the simultaneous loss of DRM2, CHROMOMETHYLASE3 (CMT2), and CMT3 functions, which completely erases all non-CG methylation in Arabidopsis, only partially reduced it in rice. The regions that remained heavily methylated in non-CG contexts in the rice Os-dcc (Osdrm2/cmt2/cmt3a) triple mutant had high GC contents. Furthermore, the residual non-CG methylation in the Os-dcc mutant was eliminated in the Os-ddccc (Osdrm2/drm3/cmt2/cmt3a/cmt3b) quintuple mutant but retained in the Os-ddcc (Osdrm2/drm3/cmt2/cmt3a) quadruple mutant, demonstrating that OsCMT3b maintains non-CG methylation in the absence of other major methyltransferases. Our results showed that OsCMT3b is subfunctionalized to accommodate a distinct cluster of non-CG-methylated sites at highly GC-rich regions in the rice genome.

Efficient genome editing using endogenous U6 snRNA promoter-driven CRISPR/Cas9 sgRNA in Sclerotinia sclerotiorum

We previously reported on a CRISPR-Cas9 genome editing system for the necrotrophic fungal plant pathogen Sclerotinia sclerotiorum. This system (the TrpC-sgRNA system), based on an RNA polymerase II (RNA Pol II) promoter (TrpC) to drive sgRNA transcription in vivo, was successful in creating gene insertion mutants. However, relatively low efficiency targeted gene editing hampered the application of this method for functional genomic research in S. sclerotiorum. To further optimize the CRISPR-Cas9 system, a plasmid-free Cas9 protein/sgRNA ribonucleoprotein (RNP)-mediated system (the RNP system) and a plasmid-based RNA polymerase III promoter (U6)-driven sgRNA transcription system (the U6-sgRNA system) were established and evaluated. The previously characterized oxaloacetate acetylhydrolase (Ssoah1) locus and a new locus encoding polyketide synthase12 (Sspks12) were targeted in this study to create loss-of-function mutants. The RNP system, similar to the TrpC-sgRNA system we previously reported, creates mutations at the Ssoah1 gene locus with comparable efficiency. However, neither system successfully generated mutations at the Sspks12 gene locus. The U6-sgRNA system exhibited a significantly higher efficiency of genemutation at both loci. This technology provides a simple and efficient strategy for targeted gene mutation and thereby will accelerating the pace of research of pathogenicity and development in this economically important plant pathogen.

Optimization of CRISPR-Cas9 through promoter replacement and efficient production of L-homoserine in Corynebacterium glutamicum.

Corynebacterium glutamicum is an important chassis for industrial applications. The low efficiency of commonly used genome editing methods for C. glutamicum limits the rapid multiple engineering of the bacterium. In this study, chromosome-borne expression of cas9 and recET from Escherichia coli K12-MG1655 was achieved to avoid toxicity to the strain, increase the probability of homologous recombination, and reduce loss of viability caused by double-strand breaks. Constitutive strong promoters, such as P45 , Ptrc , and PH36 , were used to replace PglyA and to expand the application of the CRISPR-Cas9 system. By using this system, a C. glutamicum strain producing L-homoserine to 22.1g per L in a 5-L bioreactor after 96 h was obtained. Through the application of visualized fluorescent protein, the process of plasmid curing was optimized, obtain a continuous and rapid CRISPR-Cas9 genome editing system. The method described here could be useful to construct C. glutamicum mutant rapidly.

CRISPeering: Bioengineering the Host Cells through CRISPRCas9 Genome Editing System as the Next-generation of Cell Factories.

Nowadays, the CRISPR-Cas9 genome editing system has become a popular bioengineering-based tool for various applications. Owing to its high-target specificity, efficiency, versatility, and simplicity, it has gained attention as a robust tool for molecular biology research, which unveils the biological functions of unexplored genes and engineers the metabolic pathways. Chinese hamster ovary (CHO) cells and Escherichia coli are regarded as the most commonly used expression platforms for industrial- scale production of recombinant proteins. The emergence of the CRISPR-Cas9 genome editing system promotes the current status of expression hosts towards controllable and predictable strains. This paper presents the current status of expression hosts for biopharmaceutical production. Some major accomplishments in the utilization of the CRISPR-Cas9 genome editing tool in the different prokaryotic and eukaryotic systems are discussed, and more importantly, the future directions of this newly arrived technology to make the next-generation cell factories with improved or novel properties are suggested. Moreover, the challenges faced in recent patents in this field are also discussed. The CRISPR-Cas9 genome-editing tool has been adopted to be utilized in some major expression platforms. CRISPeering has been successfully employed for genome editing in different prokaryotic and eukaryotic host cells. The emergence of systems metabolic engineering, systems biology, and synthetic biology fortify the current situation of the CRISPR-Cas9 genome editing system.

Enhancing phytoremediation of hazardous metal(loid)s using genome engineering CRISPR–Cas9 technology

Rapid and drastic changes in the global climate today have given a strong impetus to developing newer climate-resilient phytoremediation approaches. These methods are of great public and scientific importance given the urgency of this environmental crisis. Climate change has adverse effects on the growth, outputs, phenology, and overall productivity of plants. Contamination of soil with metal(loid)s is a major worldwide problem. Some metal(loids) are carcinogenic pollutants that have a long half-life and are non-degradable in the environment. There are many instances of the potential link between chronic heavy metal exposure and human disease. The adaptation of plants in the changing environment is, however, a major concern in phytoremediation practice. The creation of climate-resistant metal hyperaccumulation plants using molecular techniques could provide new opportunities to mitigate these problems. Consequently, advancements in molecular science would accelerate our knowledge of adaptive plant remediation/resistance and plant production in the context of global warming. Genome modification using artificial nucleases has the potential to enhance phytoremediation by modifying genomes for a sustainable future. This review focuses on biotechnology to boost climate change tolerant metallicolous plants and the future prospects of such technology, particularly the CRISPR-Cas9 genome editing system, for enhancing phytoremediation of hazardous pollutants.