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04 Dec 2023 at 01:42
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Bibliography on: CRISPR-Cas


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Clustered regularly interspaced short palindromic repeats (CRISPR, pronounced crisper) are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of "spacer DNA" from previous exposures to foreign DNA (e.g a virus or plasmid). The CRISPR/Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present within plasmids and phages, and provides a form of acquired immunity. CRISPR associated proteins (Cas) use the CRISPR spacers to recognize and cut these exogenous genetic elements in a manner analogous to RNA interference in eukaryotic organisms. CRISPRs are found in approximately 40% of sequenced bacterial genomes and 90% of sequenced archaea. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added. The Cas9-gRNA complex corresponds with the CAS III crRNA complex in the above diagram. CRISPR/Cas genome editing techniques have many potential applications, including altering the germline of humans, animals, and food crops. The use of CRISPR Cas9-gRNA complex for genome editing was the AAAS's choice for breakthrough of the year in 2015.

Created with PubMed® Query: ( "CRISPR.CAS" OR "crispr/cas" ) NOT pmcbook NOT ispreviousversion

Citations The Papers (from PubMed®)


RevDate: 2023-12-01
CmpDate: 2023-12-01

Hao W, Chen Z, Tang J, et al (2023)

hnRNPA2B1 promotes the occurrence and progression of hepatocellular carcinoma by downregulating PCK1 mRNA via a m6A RNA methylation manner.

Journal of translational medicine, 21(1):861.

BACKGROUND: N6-methyladenosine (m6A) is the most prevalent RNA modification. Although hnRNPA2B1, as a reader of m6A modification, has been reported to promote tumorigenesis in a few types of tumors, its role in hepatocellular carcinoma (HCC) and the underlying molecular mechanism remains unclear.

METHODS: Multiple public databases were used to analyze the expression of hnRNPA2B1 in HCC and its correlation with survival prognosis. We employed a CRISPR-Cas9 sgRNA editing strategy to knockout hnRNPA2B1 expression in HCC cells. The biological function of hnRNPA2B1 in vitro in HCC cells was measured by CCK8, colony formation, migration, and invasion assay. The tumorigenic function of hnRNPA2B1 in vivo was determined by a subcutaneous tumor formation experiment and a HCC mouse model via tail injection of several plasmids into the mouse within 5s-7s. RNA binding protein immunoprecipitation (RIP) experiment using hnRNPA2B1 was performed to test the target genes of hnRNPA2B1 and methylated RNA immunoprecipitation (MeRIP) assay was performed to explore the m6A methylated mRNA of target genes.

RESULTS: hnRNPA2B1 highly expressed in HCC tissues, correlated with high grades and poor prognosis. Its knockout reduced HCC cell proliferation, migration, and invasion in vitro, while overexpression promoted these processes. hnRNPA2B1-knockout cells inhibited tumor formation in graft experiments. In HCC mice, endogenous knockout attenuated hepatocarcinogenesis. RNA-seq showed downregulated gluconeogenesis with high hnRNPA2B1 expression. hnRNPA2B1 negatively correlated with PCK1, a key enzyme. RIP assay revealed hnRNPA2B1 binding to PCK1 mRNA. hnRNPA2B1 knockout increased m6A-methylation of PCK1 mRNA. Interestingly, PCK1 knockout partially counteracted tumor inhibition by hnRNPA2B1 knockout in mice.

CONCLUSION: Our study indicated that hnRNPA2B1 is highly expressed in HCC and correlated with a poor prognosis. hnRNPA2B1 promotes the tumorigenesis and progression of HCC both in vitro and in vivo. Moreover, hnRNPA2B1 downregulates the expression of PCK1 mRNA via a m6A methylation manner. More importantly, the ability of hnRNPA2B1 to induce tumorigenesis and progression in HCC is dependent on its ability to decrease the expression of PCK1. Therefore, this study suggested that hnRNPA2B1 might be a diagnostic marker of poor prognosis of HCC and a potential therapeutic target for HCC patients.

RevDate: 2023-12-01
CmpDate: 2023-11-30

Yang Y, Mei H, Han X, et al (2023)

Synthetic CRISPR/dCas9-KRAB system driven by specific PSA promoter suppresses malignant biological behavior of prostate cancer cells through negative feedback inhibition of PSA expression.

Cellular & molecular biology letters, 28(1):96.

PSA is a type of proto-oncogene that is specifically and highly expressed in embryonic and prostate cancer cells, but not expressed in normal prostate tissue cells. The specific expression of prostate-specific antigen (PSA) is found to be related with the conditional transcriptional regulation of its promoter. Clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9-KRAB is a newly developed transcriptional regulatory system that inhibits gene expression by interupting the DNA transcription process. Induction of CRISPR-dCas9-KRAB expression through the PSA promoter may help feedback inhibition of cellular PSA gene expression via single guide RNA (sgRNA), thereby monitoring and suppressing the malignant state of tumor cells. In this study, we examined the transcriptional activity of the PSA promoter in different prostate cancer cells and normal prostate epithelial cells and determined that it is indeed a prostate cancer cell-specific promoter.Then we constructed the CRISPR-dCas9-KRAB system driven by the PSA promoter, which can inhibit PSA gene expression in the prostate cancer cells at the transcriptional level, and therefore supress the malignant growth and migration of prostate cancer cells and promote their apoptosis in vitro. This study provides a potentially effective anti-cancer strategy for gene therapy of prostate cancer.

RevDate: 2023-11-28

Tan J, Shen M, Chai N, et al (2023)

Genome editing for plant synthetic metabolic engineering and developmental regulation.

Journal of plant physiology, 291:154141 pii:S0176-1617(23)00235-3 [Epub ahead of print].

Plant metabolism and development are a reflection of the orderly expression of genetic information intertwined with the environment interactions. Genome editing is the cornerstone for scientists to modify endogenous genes or introduce exogenous functional genes and metabolic pathways, holding immense potential applications in molecular breeding and biosynthesis. Over the course of nearly a decade of development, genome editing has advanced significantly beyond the simple cutting of double-stranded DNA, now enabling precise base and fragment replacements, regulation of gene expression and translation, as well as epigenetic modifications. However, the utilization of genome editing in plant synthetic metabolic engineering and developmental regulation remains exploratory. Here, we provide an introduction and a comprehensive overview of the editing attributes associated with various CRISPR/Cas tools, along with diverse strategies for the meticulous control of plant metabolic pathways and developments. Furthermore, we discuss the limitations of current approaches and future prospects for genome editing-driven plant breeding.

RevDate: 2023-11-28

Wimmer F, Englert F, Wandera KG, et al (2023)

Interrogating two extensively self-targeting Type I CRISPR-Cas systems in Xanthomonas albilineans reveals distinct anti-CRISPR proteins that block DNA degradation.

Nucleic acids research pii:7453255 [Epub ahead of print].

CRISPR-Cas systems store fragments of invader DNA as spacers to recognize and clear those same invaders in the future. Spacers can also be acquired from the host's genomic DNA, leading to lethal self-targeting. While self-targeting can be circumvented through different mechanisms, natural examples remain poorly explored. Here, we investigate extensive self-targeting by two CRISPR-Cas systems encoding 24 self-targeting spacers in the plant pathogen Xanthomonas albilineans. We show that the native I-C and I-F1 systems are actively expressed and that CRISPR RNAs are properly processed. When expressed in Escherichia coli, each Cascade complex binds its PAM-flanked DNA target to block transcription, while the addition of Cas3 paired with genome targeting induces cell killing. While exploring how X. albilineans survives self-targeting, we predicted putative anti-CRISPR proteins (Acrs) encoded within the bacterium's genome. Screening of identified candidates with cell-free transcription-translation systems and in E. coli revealed two Acrs, which we named AcrIC11 and AcrIF12Xal, that inhibit the activity of Cas3 but not Cascade of the respective system. While AcrF12Xal is homologous to AcrIF12, AcrIC11 shares sequence and structural homology with the anti-restriction protein KlcA. These findings help explain tolerance of self-targeting through two CRISPR-Cas systems and expand the known suite of DNA degradation-inhibiting Acrs.

RevDate: 2023-12-01
CmpDate: 2023-12-01

Fadul SM, Arshad A, R Mehmood (2023)

CRISPR-based epigenome editing: mechanisms and applications.

Epigenomics, 15(21):1137-1155.

Epigenomic anomalies contribute significantly to the development of numerous human disorders. The development of epigenetic research tools is essential for understanding how epigenetic marks contribute to gene expression. A gene-editing technique known as CRISPR (clustered regularly interspaced short palindromic repeats) typically targets a particular DNA sequence using a guide RNA (gRNA). CRISPR/Cas9 technology has been remodeled for epigenome editing by generating a 'dead' Cas9 protein (dCas9) that lacks nuclease activity and juxtaposing it with an epigenetic effector domain. Based on fusion partners of dCas9, a specific epigenetic state can be achieved. CRISPR-based epigenome editing has widespread application in drug screening, cancer treatment and regenerative medicine. This paper discusses the tools developed for CRISPR-based epigenome editing and their applications.

RevDate: 2023-11-29

Deol P, Madhwal A, Sharma G, et al (2022)

CRISPR use in diagnosis and therapy for COVID-19.

Methods in microbiology, 50:123-150.

Since the beginning of the COVID-19 pandemic, many diagnostic approaches (RT-qPCR, RAPID, LFA) have been adopted, with RT-qPCR being the most popular/gold standard. But, one of the major problems of COVID-19 diagnostics is the presentation of a wide range of symptoms which varies among different patients and needs early diagnosis for better management. Even though RT-qPCR is a precise molecular technique false negative results may be obtained. On the other hand, CRISPR-based SARS-CoV-2 detection approaches are cost and time efficient, highly sensitive and specific, and do not require sophisticated instruments. Moreover, they also show promise for increased scalability and diagnostic tests can be carried out at the point-of-care (POC). The CRISPR can be customized to the target of any genomic region of interest within the desired genome possessing a broad range of other applications and has been efficiently implemented for diagnosis of SARS-CoV-2. The CRISPR/Cas systems provide the specific gene targeting with immense potential to develop new generation diagnostics and therapeutics. Moreover, with the CRISPR/Cas based therapeutics, multiplexing is possible, where different sgRNAs or crRNAs can be guided to more than one target within the same gene thus decreasing the possibility of viral escape mutants. As an exceptionally efficient tool CRISPR/Cas13 and CARVER (Cas13-assisted restriction of viral expression and readout) systems can be implemented to target a broad range of ssRNA viruses that can be used for both, diagnosis and treatment for a variety of viral diseases including SARS-CoV-2. However, the efficacy and safety of the CRISPR-based therapeutics needs to be assessed in pre-clinical and clinical settings. Although the CRISPR biotechnologies are not very helpful to control the present pandemic of COVID-19 it is hopeful that the limitations of the CRISPR/Cas system can be overcome in the near future. The CRISPR based strategies may lead to a new era in the field of disease diagnosis and therapeutic development that would make us better prepared for future viral threats.

RevDate: 2023-11-30
CmpDate: 2023-11-29

Javadi M, Sazegar H, A Doosti (2023)

Genome editing approaches with CRISPR/Cas9: the association of NOX4 expression in breast cancer patients and effectiveness evaluation of different strategies of CRISPR/Cas9 to knockout Nox4 in cancer cells.

BMC cancer, 23(1):1155.

BACKGROUND: The increasing prevalence of cancer detection necessitated practical strategies to deliver highly accurate, beneficial, and dependable processed information together with experimental results. We deleted the cancer biomarker NOX4 using three novel genetic knockout (KO) methods. Homology-directed repair (HDR), Dual allele HITI (Du-HITI) and CRISPR-excision were utilized in this study.

METHODS: The predictive value of the NOX4 expression profile was assessed using a combined hazard ratio (HR) with a 95% confidence interval (CI). With a 95% confidence interval, a pooled odd ratio (OR) was used to calculate the relationship between NOX4 expression patterns and cancer metastasis. There were 1060 tumor patients in all sixteen research that made up this meta-analysis. To stop the NOX4 from being transcribed, we employed three different CRISPR/Cas9-mediated knockdown methods. The expression of RNA was assessed using RT-PCR. We employed the CCK-8 assay, colony formation assays, and the invasion transwell test for our experiments measuring cell proliferation and invasion. Using a sphere-formation test, the stemness was determined. Luciferase reporter tests were carried out to verify molecular adhesion. Utilizing RT-qPCR, MTT, and a colony formation assay, the functional effects of NOX4 genetic mutation in CRISPR-excision, CRISPR-HDR, and CRISPR du-HITI knockdown cell lines of breast cancer were verified.

RESULTS: There were 1060 malignant tumors in the 16 studies that made up this meta-analysis. In the meta-analysis, higher NOX4 expression was linked to both a shorter overall survival rate (HR = 1.93, 95% CI 1.49-2.49, P < 0.001) and a higher percentage of lymph node metastases (OR = 3.22, 95% CI 2.18-4.29, P < 0.001). In breast carcinoma cells, it was discovered that NOX4 was overexpressed, and this increase was linked to a poor prognosis. The gain and loss-of-function assays showed enhanced NOX4 breast carcinoma cell proliferation, sphere-forming capacity, and tumor development. To activate transcription, the transcriptional factor E2F1 also attaches to the promoter region of the Nanog gene. The treatment group (NOX4 ablation) had substantially more significant levels of proapoptotic gene expression than the control group (P < 0.01). Additionally, compared to control cells, mutant cells expressed fewer antiapoptotic genes (P < 0.001). The du-HITI technique incorporated a reporter and a transcription termination marker into the two target alleles. Both donor vector preparation and cell selection were substantially simpler using this approach than with "CRISPR HDR" or "CRISPR excision." Furthermore, single-cell knockouts for both genotypes were created when this method was applied in the initial transfection experiment.

CONCLUSIONS: The NOX4 Knockout cell lines generated in this research may be used for additional analytical studies to reveal the entire spectrum of NOX4 activities. The du-HITI method described in this study was easy to employ and could produce homozygous individuals who were knockout for a specific protein of interest.

RevDate: 2023-11-30
CmpDate: 2023-11-29

Nan X, Hardinge P, Hoehn S, et al (2023)

VarLOCK: sequencing-independent, rapid detection of SARS-CoV-2 variants of concern for point-of-care testing, qPCR pipelines and national wastewater surveillance.

Scientific reports, 13(1):20832.

The COVID-19 pandemic demonstrated the need for rapid molecular diagnostics. Vaccination programs can provide protection and facilitate the opening of society, but newly emergent and existing viral variants capable of evading the immune system endanger their efficacy. Effective surveillance for Variants of Concern (VOC) is therefore important. Rapid and specific molecular diagnostics can provide speed and coverage advantages compared to genomic sequencing alone, benefitting the public health response and facilitating VOC containment. Here we expand the recently developed SARS-CoV-2 CRISPR-Cas detection technology (SHERLOCK) to provide rapid and sensitive discrimination of SARS-CoV-2 VOCs that can be used at point of care, implemented in the pipelines of small or large testing facilities, and even determine the proportion of VOCs in pooled population-level wastewater samples. This technology complements sequencing efforts to allow facile and rapid identification of individuals infected with VOCs to help break infection chains. We show the optimisation of our VarLOCK assays (Variant-specific SHERLOCK) for multiple specific mutations in the S gene of SARS-CoV-2 and validation with samples from the Cardiff University Testing Service. We also show the applicability of VarLOCK to national wastewater surveillance of SARS-CoV-2 variants and the rapid adaptability of the technique for new and emerging VOCs.

RevDate: 2023-11-27

Tripathi JN, Ntui VO, L Tripathi (2023)

Precision genetics tools for genetic improvement of banana.

The plant genome [Epub ahead of print].

Banana is an important food security crop for millions of people in the tropics but it faces challenges from diseases and pests. Traditional breeding methods have limitations, prompting the exploration of precision genetic tools like genetic modification and genome editing. Extensive efforts using transgenic approaches have been made to develop improved banana varieties with resistance to banana Xanthomonas wilt, Fusarium wilt, and nematodes. However, these efforts should be extended for other pests, diseases, and abiotic stresses. The commercialization of transgenic crops still faces continuous challenges with regulatory and public acceptance. Genome editing, particularly CRISPR/Cas, offers precise modifications to the banana genome and has been successfully applied in the improvement of banana. Targeting specific genes can contribute to the development of improved banana varieties with enhanced resistance to various biotic and abiotic constraints. This review discusses recent advances in banana improvement achieved through genetic modification and genome editing.

RevDate: 2023-11-27

He Y, Shao S, J Chen (2023)

High-Fidelity Identification of Single Nucleotide Polymorphism by Type V CRISPR Systems.

ACS sensors [Epub ahead of print].

Accurate and sensitive detection of single nucleotide polymorphism (SNP) holds significant clinical implications, especially in the field of cancer diagnosis. Leveraging its high accuracy and programmability, the CRISPR system emerges as a promising platform for advancing the identification of SNPs. In this study, we compared two type V CRISPR/Cas systems (Cas12a and Cas14a) for the identification of cancer-related SNP. Their identification performances were evaluated by characterizing their mismatch tolerance to the BRAF gene. We found that the CRISPR/Cas14a system exhibited superior accuracy and robustness over the CRISPR/Cas12a system for SNP detection. Furthermore, blocker displacement amplification (BDA) was combined with the CRISPR/Cas14a system to eliminate the interference of the wild type (WT) and increase the detection accuracy. In this strategy, we were able to detect BRAF V600E as low as 10[3] copies with a sensitivity of 0.1% variant allele frequency. Moreover, the BDA-assisted CRISPR/Cas14a system has been applied to identify the BRAF mutation from human colorectal carcinoma cells, achieving a high sensitivity of 0.5% variant allele frequency, which is comparable to or even superior to those of most commercially available products. This work has broadened the scope of the CRISPR system and provided a promising method for precision medicine.

RevDate: 2023-11-29
CmpDate: 2023-11-29

Thomsen MK (2023)

The double-edge sword of CRISPR application for in vivo studies.

Oncotarget, 14:919-920.

RevDate: 2023-11-27

Liu J, Jaffe AL, Chen L, et al (2023)

Host translation machinery is not a barrier to phages that interact with both CPR and non-CPR bacteria.

mBio [Epub ahead of print].

Here, we profiled putative phages of Saccharibacteria, which are of particular importance as Saccharibacteria influence some human oral diseases. We additionally profiled putative phages of Gracilibacteria and Absconditabacteria, two Candidate Phyla Radiation (CPR) lineages of interest given their use of an alternative genetic code. Among the phages identified in this study, some are targeted by spacers from both CPR and non-CPR bacteria and others by both bacteria that use the standard genetic code as well as bacteria that use an alternative genetic code. These findings represent new insights into possible phage replication strategies and have relevance for phage therapies that seek to manipulate microbiomes containing CPR bacteria.

RevDate: 2023-11-27

Muzyukina P, Shkaruta A, Guzman NM, et al (2023)

Identification of an anti-CRISPR protein that inhibits the CRISPR-Cas type I-B system in Clostridioides difficile.

mSphere [Epub ahead of print].

Clostridioides difficile is the widespread anaerobic spore-forming bacterium that is a major cause of potentially lethal nosocomial infections associated with antibiotic therapy worldwide. Due to the increase in severe forms associated with a strong inflammatory response and higher recurrence rates, a current imperative is to develop synergistic and alternative treatments for C. difficile infections. In particular, phage therapy is regarded as a potential substitute for existing antimicrobial treatments. However, it faces challenges because C. difficile has highly active CRISPR-Cas immunity, which may be a specific adaptation to phage-rich and highly crowded gut environment. To overcome this defense, C. difficile phages must employ anti-CRISPR mechanisms. Here, we present the first anti-CRISPR protein that inhibits the CRISPR-Cas defense system in this pathogen. Our work offers insights into the interactions between C. difficile and its phages, paving the way for future CRISPR-based applications and development of effective phage therapy strategies combined with the engineering of virulent C. difficile infecting phages.

RevDate: 2023-11-26

Wang Q, Ren Y, Meng T, et al (2023)

Cas14a1-advanced LAMP for ultrasensitive and visual Pathogen diagnostic.

Talanta, 269:125458 pii:S0039-9140(23)01209-2 [Epub ahead of print].

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas enzymes have been widely applied for biosensor development, combined with various isothermal amplification strategies (IAS) to boost sensitivity and specificity. Currently, the unstable assay and tedious manipulation usually hinder its practical applications. Here, a Cas14a1-advanced LAMP assay (CALA) combined with Rapid Extraction of Bacterial Genomic DNA (REBGD) is proposed for pathogen detection. For rapid CALA, a single stranded fluorescence reporter and ssDNA-gold nanoparticles (AuNPs) are used as signal indicators to establish ultrasensitive and visual platforms. This assay displays precise detection of bacteria, which can achieve an ultrasensitive limit of detection (LOD) 10 aM target genomic DNA. Furthermore, the high reliability of pathogen diagnostic for contrived samples is validated through the rapid visual CALA platform, demonstrating the promising practical testing availability of pathogen detection.

RevDate: 2023-11-28
CmpDate: 2023-11-28

Feng Q, J Li (2023)

Construction and Validation of a CRISPR/dCpf1-Based Transcriptional Regulatory System in Saccharomyces Cerevisiae.

Studies in health technology and informatics, 308:680-688.

Saccharomyces cerevisiae has been extensively studied and applied as a model microbial platform for efficient and sustainable production of biofuels, chemicals and natural products. In recent years, the application of CRISPR system in genome editing and transcriptional regulation has greatly improved the efficiency of microbial genome editing. To achieve transcriptional regulation the CRISPR/dCpf1-mediated CRISPRa/i transcriptional regulation system was constructed in this study. By constructing crRNA arrays and adding different activation proteins, repression efficiencies from 62% to 177% for the CRISPRi system and activation efficiencies from 154% to 320% for the CRISPRa system were achieved in the eGFP gene reporter system.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Chung T, Merrill JR, SK Lyons (2024)

CRISPR/Cas for PET Reporter Gene Engineering.

Methods in molecular biology (Clifton, N.J.), 2729:285-301.

The relatively recent discovery of CRISPR/Cas has led to a revolution in our ability to efficiently manipulate the genome of eukaryotic cells. We describe here a protocol that employs CRISPR technology to precisely knock-in a PET imaging reporter transgene into a specific genetic locus of interest. Resulting transcription of the targeted reporter will more accurately mimic physiologic expression of the endogenous allele than conventional approaches, and so this method has the potential to become an efficient way to generate a new generation of "gold-standard" reporter transgenes. We break down the protocol into three experimental stages: how to identify the genomic location that the reporter transgene will be inserted, how to practically insert the reporter transgene into the genome, and how to screen resultant clones for the correct targeted event.

RevDate: 2023-11-30
CmpDate: 2023-11-30

Cao H, Mao K, Zhang H, et al (2024)

Thermal stability and micrdose-based coupling CRISPR/Cas12a biosensor for amplification-free detection of hgcA gene in paddy soil.

The Science of the total environment, 909:168536.

The lack of point-of-use (POU) methods hinders the utilization of the hgcA gene to rapidly evaluate methylmercury risks. CRISPR/Cas12a is a promising technology, but shortcomings such as low sensitivity, a strict reaction temperature and high background signal limit its further utilization. Here, a thermally stable microsystem-based CRISPR/Cas12a biosensor was constructed to achieve POU analysis for hgcA. First, three target gRNAs were designed to recognize hgcA. Then, a microsystem was developed to eliminate the background signal. Next, the effect of temperature on the activity of the Cas12a-gRNA complex was explored and its thermal stability was discovered. After that, coupling gRNA assay was introduced to improve sensitivity, exhibiting a limit of detection as low as 0.49 pM with a linear range of 0.98-125 pM, and a recovery rate between 90 and 110 % for hgcA. The biosensor was finally utilized to assess hgcA abundance in paddy soil, and high abundance of hgcA was found in these paddy soil samples. This study not only systematically explored the influence of temperature and microsystem on CRISPR/Cas12a, providing vital references for other novel CRISPR-based detection methods, but also applied the CRISPR-based analytical method to the field of environmental geochemistry for the first time, demonstrating enormous potential for POU detection in this field.

RevDate: 2023-11-30
CmpDate: 2023-11-30

Sasaki-Honda M, Akatsuka K, T Sawai (2023)

Is epigenome editing non-inheritable? Implications for ethics and the regulation of human applications.

Stem cell reports, 18(11):2005-2009.

Epigenome editing offers ethical advantages with non-inheritable gene expression control. However, concerns arise regarding potential transgenerational effects in humans. Ethical and regulatory evaluation is crucial, considering recent advancements and enhanced understanding of transgenerational epigenetics in both mammals and humans.

RevDate: 2023-11-30
CmpDate: 2023-11-30

Crunkhorn S (2023)

Engineering a compact genome-editing tool.

Nature reviews. Drug discovery, 22(12):953.

RevDate: 2023-11-30
CmpDate: 2023-04-25

Diaz Quiroz JF, Ojha N, Shayhidin EE, et al (2023)

Development of a selection assay for small guide RNAs that drive efficient site-directed RNA editing.

Nucleic acids research, 51(7):e41.

A major challenge confronting the clinical application of site-directed RNA editing (SDRE) is the design of small guide RNAs (gRNAs) that can drive efficient editing. Although many gRNA designs have effectively recruited endogenous Adenosine Deaminases that Act on RNA (ADARs), most of them exceed the size of currently FDA-approved antisense oligos. We developed an unbiased in vitro selection assay to identify short gRNAs that promote superior RNA editing of a premature termination codon. The selection assay relies on hairpin substrates in which the target sequence is linked to partially randomized gRNAs in the same molecule, so that gRNA sequences that promote editing can be identified by sequencing. These RNA substrates were incubated in vitro with ADAR2 and the edited products were selected using amplification refractory mutation system PCR and used to regenerate the substrates for a new round of selection. After nine repetitions, hairpins which drove superior editing were identified. When gRNAs of these hairpins were delivered in trans, eight of the top ten short gRNAs drove superior editing both in vitro and in cellula. These results show that efficient small gRNAs can be selected using our approach, an important advancement for the clinical application of SDRE.

RevDate: 2023-11-30
CmpDate: 2022-12-19

Wang Y, Cottle WT, Wang H, et al (2022)

Achieving single nucleotide sensitivity in direct hybridization genome imaging.

Nature communications, 13(1):7776.

Direct visualization of point mutations in situ can be informative for studying genetic diseases and nuclear biology. We describe a direct hybridization genome imaging method with single-nucleotide sensitivity, single guide genome oligopaint via local denaturation fluorescence in situ hybridization (sgGOLDFISH), which leverages the high cleavage specificity of eSpCas9(1.1) variant combined with a rationally designed guide RNA to load a superhelicase and reveal probe binding sites through local denaturation. The guide RNA carries an intentionally introduced mismatch so that while wild-type target DNA sequence can be efficiently cleaved, a mutant sequence with an additional mismatch (e.g., caused by a point mutation) cannot be cleaved. Because sgGOLDFISH relies on genomic DNA being cleaved by Cas9 to reveal probe binding sites, the probes will only label the wild-type sequence but not the mutant sequence. Therefore, sgGOLDFISH has the sensitivity to differentiate the wild-type and mutant sequences differing by only a single base pair. Using sgGOLDFISH, we identify base-editor-modified and unmodified progeroid fibroblasts from a heterogeneous population, validate the identification through progerin immunofluorescence, and demonstrate accurate sub-nuclear localization of point mutations.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Kang KK, YG Cho (2023)

Genetic Analysis Based on CRISPR/Cas9 Technology in Plants.

International journal of molecular sciences, 24(22):.

Genome-editing technology is a type of genetic engineering in which DNA is inserted into, replaced in, or deleted from the genome using artificially engineered nucleases or genetic scissors [...].

RevDate: 2023-11-27
CmpDate: 2023-11-27

Ding S, Liu J, Han X, et al (2023)

CRISPR/Cas9-Mediated Genome Editing in Cancer Therapy.

International journal of molecular sciences, 24(22):.

The Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system, an RNA-based adaptive immune system found in bacteria and archaea, has catalyzed the development and application of a new generation of gene editing tools. Numerous studies have shown that this system can precisely target a wide range of human genes, including those associated with diseases such as cancer. In cancer research, the intricate genetic mutations in tumors have promoted extensive utilization of the CRISPR/Cas9 system due to its efficient and accurate gene editing capabilities. This includes improvements in Chimeric Antigen Receptor (CAR)-T-cell therapy, the establishment of tumor models, and gene and drug target screening. Such progress has propelled the investigation of cancer molecular mechanisms and the advancement of precision medicine. However, the therapeutic potential of genome editing remains underexplored, and lingering challenges could elevate the risk of additional genetic mutations. Here, we elucidate the fundamental principles of CRISPR/Cas9 gene editing and its practical applications in tumor research. We also briefly discuss the primary challenges faced by CRISPR technology and existing solutions, intending to enhance the efficacy of this gene editing therapy and shed light on the underlying mechanisms of tumors.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Movahedi A, Aghaei-Dargiri S, Li H, et al (2023)

CRISPR Variants for Gene Editing in Plants: Biosafety Risks and Future Directions.

International journal of molecular sciences, 24(22):.

The CRISPR genome editing technology is a crucial tool for enabling revolutionary advancements in plant genetic improvement. This review shows the latest developments in CRISPR/Cas9 genome editing system variants, discussing their benefits and limitations for plant improvement. While this technology presents immense opportunities for plant breeding, it also raises serious biosafety concerns that require careful consideration, including potential off-target effects and the unintended transfer of modified genes to other organisms. This paper highlights strategies to mitigate biosafety risks and explores innovative plant gene editing detection methods. Our review investigates the international biosafety guidelines for gene-edited crops, analyzing their broad implications for agricultural and biotechnology research and advancement. We hope to provide illuminating and refined perspectives for industry practitioners and policymakers by evaluating CRISPR genome enhancement in plants.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Davis-Anderson K, Micheva-Viteva S, Solomon E, et al (2023)

CRISPR/Cas9 Directed Reprogramming of iPSC for Accelerated Motor Neuron Differentiation Leads to Dysregulation of Neuronal Fate Patterning and Function.

International journal of molecular sciences, 24(22):.

Neurodegeneration causes a significant disease burden and there are few therapeutic interventions available for reversing or slowing the disease progression. Induced pluripotent stem cells (iPSCs) hold significant potential since they are sourced from adult tissue and have the capacity to be differentiated into numerous cell lineages, including motor neurons. This differentiation process traditionally relies on cell lineage patterning factors to be supplied in the differentiation media. Genetic engineering of iPSC with the introduction of recombinant master regulators of motor neuron (MN) differentiation has the potential to shorten and streamline cell developmental programs. We have established stable iPSC cell lines with transient induction of exogenous LHX3 and ISL1 from the Tet-activator regulatory region and have demonstrated that induction of the transgenes is not sufficient for the development of mature MNs in the absence of neuron patterning factors. Comparative global transcriptome analysis of MN development from native and Lhx-ISL1 modified iPSC cultures demonstrated that the genetic manipulation helped to streamline the neuronal patterning process. However, leaky gene expression of the exogenous MN master regulators in iPSC resulted in the premature activation of genetic pathways characteristic of the mature MN function. Dysregulation of metabolic and regulatory pathways within the developmental process affected the MN electrophysiological responses.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Nagy B, Öktem A, Ferenc G, et al (2023)

CRISPR/Cas9 Mutagenesis through Introducing a Nanoparticle Complex Made of a Cationic Polymer and Nucleic Acids into Maize Protoplasts.

International journal of molecular sciences, 24(22):.

Presently, targeted gene mutagenesis attracts increasing attention both in plant research and crop improvement. In these approaches, successes are largely dependent on the efficiency of the delivery of gene editing components into plant cells. Here, we report the optimization of the cationic polymer poly(2-hydroxypropylene imine) (PHPI)-mediated delivery of plasmid DNAs, or single-stranded oligonucleotides labelled with Cyanine3 (Cy3) or 6-Carboxyfluorescein (6-FAM)-fluorescent dyes into maize protoplasts. Co-delivery of the GFP-expressing plasmid and the Cy3-conjugated oligonucleotides has resulted in the cytoplasmic and nuclear accumulation of the green fluorescent protein and a preferential nuclear localization of oligonucleotides. We show the application of nanoparticle complexes, i.e., "polyplexes" that comprise cationic polymers and nucleic acids, for CRISPR/Cas9 editing of maize cells. Knocking out the functional EGFP gene in transgenic maize protoplasts was achieved through the co-delivery of plasmids encoding components of the editing factors Cas9 (pFGC-pcoCas9) and gRNA (pZmU3-gRNA) after complexing with a cationic polymer (PHPI). Several edited microcalli were identified based on the lack of a GFP fluorescence signal. Multi-base and single-base deletions in the EGFP gene were confirmed using Sanger sequencing. The presented results support the use of the PHPI cationic polymer in plant protoplast-mediated genome editing approaches.

RevDate: 2023-11-29
CmpDate: 2023-11-29

Mao K, Zhang H, Ran F, et al (2024)

Portable biosensor combining CRISPR/Cas12a and loop-mediated isothermal amplification for antibiotic resistance gene ermB in wastewater.

Journal of hazardous materials, 462:132793.

Wastewater is among the main sources of antibiotic resistance genes (ARGs) in the environment, but effective methods to quickly assess ARGs on-site in wastewater are lacking. Here, using the typical ARG ermB as the target, we report a portable biosensor combining CRISPR/Cas12a and loop-mediated isothermal amplification (LAMP) for the detection of ARGs. Six primers of LAMP and the crRNA of CRISPR/Cas12a were first designed to be preamplification with LAMP and lead Cas12a to recognize the ermB via base pairing. Due to the trans-cleavage activity of CRISPR/Cas12a after amplicon recognition, ssDNA probes modified with reporter molecules were used to implement a visual assay with lateral flow test strips and fluorescence. After a simple nucleic acid extraction with magnetic beads, the constructed biosensor possesses excellent sensitivity and selectivity as low as 2.75 × 10[3] copies/μL using fluorescence and later flow strips in wastewater. We further evaluated the community-wide prevalence of ermB in wastewater influent and found high mass loads of ermB during different months. This user-friendly and low-cost biosensor is applicable for rapid on-site ARG detection, providing a potential point-of-use method for rapid assessments of ARG abundance in wastewater from large city areas with many wastewater treatment plants and in resource-limited rural areas.

RevDate: 2023-11-29
CmpDate: 2023-11-29

Tulkens D, Boelens M, Naert T, et al (2023)

Mutations in the histone methyltransferase Ezh2 drive context-dependent leukemia in Xenopus tropicalis.

Leukemia, 37(12):2404-2413.

CRISPR-mediated simultaneous targeting of candidate tumor suppressor genes in Xenopus tropicalis allows fast functional assessment of co-driver genes for various solid tumors. Genotyping of tumors that emerge in the mosaic mutant animals rapidly exposes the gene mutations under positive selection for tumor establishment. However, applying this simple approach to the blood lineage has not been attempted. Multiple hematologic malignancies have mutations in EZH2, encoding the catalytic subunit of the Polycomb Repressive Complex 2. Interestingly, EZH2 can act as an oncogene or a tumor suppressor, depending on cellular context and disease stage. We show here that mosaic CRISPR/Cas9 mediated ezh2 disruption in the blood lineage resulted in early and penetrant acute myeloid leukemia (AML) induction. While animals were co-targeted with an sgRNA that induces notch1 gain-of-function mutations, sequencing of leukemias revealed positive selection towards biallelic ezh2 mutations regardless of notch1 mutational status. Co-targeting dnm2, recurrently mutated in T/ETP-ALL, induced a switch from myeloid towards acute T-cell leukemia. Both myeloid and T-cell leukemias engrafted in immunocompromised hosts. These data underline the potential of Xenopus tropicalis for modeling human leukemia, where mosaic gene disruption, combined with deep amplicon sequencing of the targeted genomic regions, can rapidly and efficiently expose co-operating driver gene mutations.

RevDate: 2023-11-29
CmpDate: 2023-11-29

Raban R, Marshall JM, Hay BA, et al (2023)

Manipulating the Destiny of Wild Populations Using CRISPR.

Annual review of genetics, 57:361-390.

Genetic biocontrol aims to suppress or modify populations of species to protect public health, agriculture, and biodiversity. Advancements in genome engineering technologies have fueled a surge in research in this field, with one gene editing technology, CRISPR, leading the charge. This review focuses on the current state of CRISPR technologies for genetic biocontrol of pests and highlights the progress and ongoing challenges of using these approaches.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Tyumentseva M, Tyumentsev A, V Akimkin (2023)

CRISPR/Cas9 Landscape: Current State and Future Perspectives.

International journal of molecular sciences, 24(22):.

CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is a unique genome editing tool that can be easily used in a wide range of applications, including functional genomics, transcriptomics, epigenetics, biotechnology, plant engineering, livestock breeding, gene therapy, diagnostics, and so on. This review is focused on the current CRISPR/Cas9 landscape, e.g., on Cas9 variants with improved properties, on Cas9-derived and fusion proteins, on Cas9 delivery methods, on pre-existing immunity against CRISPR/Cas9 proteins, anti-CRISPR proteins, and their possible roles in CRISPR/Cas9 function improvement. Moreover, this review presents a detailed outline of CRISPR/Cas9-based diagnostics and therapeutic approaches. Finally, the review addresses the future expansion of genome editors' toolbox with Cas9 orthologs and other CRISPR/Cas proteins.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Mohammadian Gol T, Kim M, Sinn R, et al (2023)

CRISPR-Cas9-Based Gene Knockout of Immune Checkpoints in Expanded NK Cells.

International journal of molecular sciences, 24(22):.

Natural killer (NK) cell immunotherapy has emerged as a novel treatment modality for various cancer types, including leukemia. The modulation of inhibitory signaling pathways in T cells and NK cells has been the subject of extensive investigation in both preclinical and clinical settings in recent years. Nonetheless, further research is imperative to optimize antileukemic activities, especially regarding NK-cell-based immunotherapies. The central scientific question of this study pertains to the potential for boosting cytotoxicity in expanded and activated NK cells through the inhibition of inhibitory receptors. To address this question, we employed the CRISPR-Cas9 system to target three distinct inhibitory signaling pathways in NK cells. Specifically, we examined the roles of A2AR within the metabolic purinergic signaling pathway, CBLB as an intracellular regulator in NK cells, and the surface receptors NKG2A and CD96 in enhancing the antileukemic efficacy of NK cells. Following the successful expansion of NK cells, they were transfected with Cas9+sgRNA RNP to knockout A2AR, CBLB, NKG2A, and CD96. The analysis of indel frequencies for all four targets revealed good knockout efficiencies in expanded NK cells, resulting in diminished protein expression as confirmed by flow cytometry and Western blot analysis. Our in vitro killing assays demonstrated that NKG2A and CBLB knockout led to only a marginal improvement in the cytotoxicity of NK cells against AML and B-ALL cells. Furthermore, the antileukemic activity of CD96 knockout NK cells did not yield significant enhancements, and the blockade of A2AR did not result in significant improvement in killing efficiency. In conclusion, our findings suggest that CRISPR-Cas9-based knockout strategies for immune checkpoints might not be sufficient to efficiently boost the antileukemic functions of expanded (and activated) NK cells and, at the same time, point to the need for strong cellular activating signals, as this can be achieved, for example, via transgenic chimeric antigen receptor expression.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Güngör B, Biró JB, Domonkos Á, et al (2023)

Targeted mutagenesis of Medicago truncatula Nodule-specific Cysteine-Rich (NCR) genes using the Agrobacterium rhizogenes-mediated CRISPR/Cas9 system.

Scientific reports, 13(1):20676.

The host-produced nodule specific cysteine-rich (NCR) peptides control the terminal differentiation of endosymbiotic rhizobia in the nodules of IRLC legumes. Although the Medicago truncatula genome encodes about 700 NCR peptides, only few of them have been proven to be crucial for nitrogen-fixing symbiosis. In this study, we applied the CRISPR/Cas9 gene editing technology to generate knockout mutants of NCR genes for which no genetic or functional data were previously available. We have developed a workflow to analyse the mutation and the symbiotic phenotype of individual nodules formed on Agrobacterium rhizogenes-mediated transgenic hairy roots. The selected NCR genes were successfully edited by the CRISPR/Cas9 system and nodules formed on knockout hairy roots showed wild type phenotype indicating that peptides NCR068, NCR089, NCR128 and NCR161 are not essential for symbiosis between M. truncatula Jemalong and Sinorhizobium medicae WSM419. We regenerated stable mutants edited for the NCR068 from hairy roots obtained by A. rhizogenes-mediated transformation. The analysis of the symbiotic phenotype of stable ncr068 mutants showed that peptide NCR068 is not required for symbiosis with S. meliloti strains 2011 and FSM-MA either. Our study reports that gene editing can help to elicit the role of certain NCRs in symbiotic nitrogen fixation.

RevDate: 2023-11-28
CmpDate: 2023-11-28

Witkowsky L, Norstad M, Glynn AR, et al (2023)

Towards affordable CRISPR genomic therapies: a task force convened by the Innovative Genomics Institute.

Gene therapy, 30(10-11):747-752.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Li Y, Wu Y, Xu R, et al (2023)

In vivo imaging of mitochondrial DNA mutations using an integrated nano Cas12a sensor.

Nature communications, 14(1):7722.

Mutations in mitochondrial DNA (mtDNA) play critical roles in many human diseases. In vivo visualization of cells bearing mtDNA mutations is important for resolving the complexity of these diseases, which remains challenging. Here we develop an integrated nano Cas12a sensor (InCasor) and show its utility for efficient imaging of mtDNA mutations in live cells and tumor-bearing mouse models. We co-deliver Cas12a/crRNA, fluorophore-quencher reporters and Mg[2+] into mitochondria. This process enables the activation of Cas12a's trans-cleavage by targeting mtDNA, which efficiently cleave reporters to generate fluorescent signals for robustly sensing and reporting single-nucleotide variations (SNVs) in cells. Since engineered crRNA significantly increase Cas12a's sensitivity to mismatches in mtDNA, we can identify tumor tissue and metastases by visualizing cells with mutant mtDNAs in vivo using InCasor. This CRISPR imaging nanoprobe holds potential for applications in mtDNA mutation-related basic research, diagnostics and gene therapies.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Kim Y, HM Lee (2023)

CRISPR-Cas System Is an Effective Tool for Identifying Drug Combinations That Provide Synergistic Therapeutic Potential in Cancers.

Cells, 12(22):.

Despite numerous efforts, the therapeutic advancement for neuroblastoma and other cancer treatments is still ongoing due to multiple challenges, such as the increasing prevalence of cancers and therapy resistance development in tumors. To overcome such obstacles, drug combinations are one of the promising applications. However, identifying and implementing effective drug combinations are critical for achieving favorable treatment outcomes. Given the enormous possibilities of combinations, a rational approach is required to predict the impact of drug combinations. Thus, CRISPR-Cas-based and other approaches, such as high-throughput pharmacological and genetic screening approaches, have been used to identify possible drug combinations. In particular, the CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats) is a powerful tool that enables us to efficiently identify possible drug combinations that can improve treatment outcomes by reducing the total search space. In this review, we discuss the rational approaches to identifying, examining, and predicting drug combinations and their impact.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Yun D, C Jung (2023)

MiRNA-Responsive CRISPR-Cas System via a DNA Regulator.

Biosensors, 13(11):.

Clustered regularly interspaced short palindromic repeats (CRISPR)- CRISPR-associated protein 9 (Cas9) genome editing technology is widely used for gene editing because it provides versatility in genetic manipulation. Several methods for regulating CRISPR activity already exist for accurate editing, but these require complex engineering. Thus, a simple and convenient regulatory system is required. In this study, we devised a CRISPR activation system using a DNA regulator that can be activated by miRNAs. The designed regulator was divided into two parts. The inhibition component consisted of the protospacer-adjacent motif (PAM) and seed sequence, which are important for Cas9 target recognition and bind to the ribonucleoprotein (RNP) complex for inhibition. The miRNA recognition component has a single-stranded toehold DNA for target miRNA binding and a partial double-stranded DNA complementary to the remaining miRNA sequence. In the presence of target miRNAs, the structure of the regulator is disrupted by the miRNAs, leading to its dissociation from the RNP complex and subsequent restoration of CRISPR activity. This method is easy to design and can be applied to various miRNAs via simple sequence manipulation. Therefore, this strategy provides a general platform for controlled genome editing.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Jeung JH, Han H, Lee CY, et al (2023)

CRISPR/Cas12a Collateral Cleavage Activity for Sensitive 3'-5' Exonuclease Assay.

Biosensors, 13(11):.

This study presents a technique for detecting 3'-5' exonuclease activity through the use of CRISPR/Cas12a. These enzymes, including 3'-5' exonuclease (Exo III), perform crucial roles in various cellular processes and are associated with life expectancy. However, imbalances in their expression can increase susceptibility to diseases such as cancer, particularly under prolonged stress. In this study, an activator sequence of CRISPR/Cas12a was constructed on the 5'-end of a hairpin probe (HP), forming a blunt end. When the 3'-end of the HP was hydrolyzed with Exo III activity, the activator sequence of Cas12a was exposed, which led to collateral cleavage of the DNA signal probe and generated a fluorescent signal, allowing sensitive and highly specific Exo III detection. This detection principle relied on the fact that Exo III exclusively cleaves the 3'-end mononucleotide of dsDNA and does not affect ssDNA. Based on this strategy, Exo III activity was successfully assayed at 0.0073 U/mL, demonstrating high sensitivity. In addition, this technique was used to screen candidate inhibitors of Exo III activity.

RevDate: 2023-11-26

Joshi A, Yang SY, Song HG, et al (2023)

Genetic Databases and Gene Editing Tools for Enhancing Crop Resistance against Abiotic Stress.

Biology, 12(11):.

Abiotic stresses extensively reduce agricultural crop production globally. Traditional breeding technology has been the fundamental approach used to cope with abiotic stresses. The development of gene editing technology for modifying genes responsible for the stresses and the related genetic networks has established the foundation for sustainable agriculture against environmental stress. Integrated approaches based on functional genomics and transcriptomics are now expanding the opportunities to elucidate the molecular mechanisms underlying abiotic stress responses. This review summarizes some of the features and weblinks of plant genome databases related to abiotic stress genes utilized for improving crops. The gene-editing tool based on clustered, regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has revolutionized stress tolerance research due to its simplicity, versatility, adaptability, flexibility, and broader applications. However, off-target and low cleavage efficiency hinder the successful application of CRISPR/Cas systems. Computational tools have been developed for designing highly competent gRNA with better cleavage efficiency. This powerful genome editing tool offers tremendous crop improvement opportunities, overcoming conventional breeding techniques' shortcomings. Furthermore, we also discuss the mechanistic insights of the CRISPR/Cas9-based genome editing technology. This review focused on the current advances in understanding plant species' abiotic stress response mechanism and applying the CRISPR/Cas system genome editing technology to develop crop resilience against drought, salinity, temperature, heavy metals, and herbicides.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Cercenado E (2023)

What are the most relevant publications in Clinical Microbiology in the last two years?.

Revista espanola de quimioterapia : publicacion oficial de la Sociedad Espanola de Quimioterapia, 36 Suppl 1:64-67.

This minireview describes some of the articles published in the last two years related to innovative technologies including CRISPR-Cas, surface-enhanced Raman spectroscopy, microfluidics, flow cytometry, Fourier transform infrared spectroscopy, and artificial intelligence and their application to microbiological diagnosis, molecular typing and antimicrobial susceptibility testing. In addition, some articles related to resistance to new antimicrobials (ceftazidime-avibactam, meropenem-vaborbactam, imipenem-relebactam, and cefiderocol) are also described.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Qiao Z, Xue L, Sun M, et al (2023)

Highly sensitive detection of Salmonella based on dual-functional HCR-mediated multivalent aptamer and amplification-free CRISPR/Cas12a system.

Analytica chimica acta, 1284:341998.

BACKGROUND: Salmonella infection severely threatens human health and causes substantial medical and financial concerns. Sensitive and specific detection of Salmonella in food samples is crucial but remains challenging. While some traditional assays for S. typhimurium are reliable, they suffer from various limitations, such as being time-consuming (culture-based methods), involving intricate nucleic molecular extraction (polymerization chain reaction, PCR), and exhibiting inadequate sensitivity (enzyme-linked immunosorbent assay, ELISA). In this case, it is essential to establish a rapid, simple-operation, and sensitive method for monitoring S. typhimurium to preserve food quality and prevent contamination.

RESULT: Herein, an amplification-free detection method for Salmonella was developed by coupling the aptamer magnetic separation with dual-functional HCR (hybridization chain reaction)-scaffold multivalent aptamer and the activity of CRISPR/Cas12a. In the detection system, the dual-functional HCR-scaffold multivalent aptamer with high binding affinity and specificity was fabricated in advance by assembling numerous Salmonella specific aptamers on the long HCR products. In addition to the enhanced affinity, the HCR-multiApt also contains a massive amount of repeated CRISPR-targetable DNA units in its HCR scaffold, which could trigger the trans-cleavage activity of Cas12a. In the presence of target bacteria, the HCR-scaffold multivalent aptamer could attach on the surface of bacteria effectively and amplified the signal of bacteria into CRISPR/Cas12a based fluorescent readout. The proposed detection system allowed for ultrasensitive detection of Salmonella in a linear range from 10[0] to 10[7] cfu mL[-1] with a LOD (limit of detection) of 2 cfu mL[-1].

SIGNIFICANCE: The novel dual-functional HCR-multiApt presents a simple and powerful strategy for improving the aptamer binding affinity toward Salmonella. Simultaneously, integrating this dual-functional HCR-multiApt with the CRISPR/Cas12a system significantly enhances the sensitivity by cascade signal amplification in a nucleic acids amplification-free way. Finally, leveraging the versatility of the aptamer, this highly sensitive method can be further extended for application in the detection of other bacteria, food safety monitoring, or clinical diagnostics.

RevDate: 2023-11-23

Tu W, Hu X, Wan R, et al (2023)

Effective delivery of miR-511-3p with mannose-decorated exosomes with RNA nanoparticles confers protection against asthma.

Journal of controlled release : official journal of the Controlled Release Society pii:S0168-3659(23)00751-4 [Epub ahead of print].

Our previous studies have shown that miR-511-3p treatment has a beneficial effect in alleviating allergic airway inflammation. Here, we sought to explore its therapeutic potential in animal models and gain a deeper understanding of its therapeutic value for asthma. miR-511-3p knockout mice (miR-511-3p[-/-]) were generated by CRISPR/Cas and showed exacerbated airway hyper-responsiveness and Th2-associated allergic airway inflammation compared with wild-type (WT) mice after exposed to cockroach allergen. RNA nanoparticles with mannose decorated EV-miR-511-3p were also created by loading miR-511-3p mimics into the mannose decorated EVs with engineered RNA nanoparticle PRNA-3WJ (Man-EV-miR-511-3p). Intra-tracheal inhalation of Man-EV-miR-511-3p, which could effectively penetrate the airway mucus barrier and deliver functional miR-511-3p to lung macrophages, successfully reversed the increased airway inflammation observed in miR-511-3p[-/-] mice. Through microarray analysis, complement C3 (C3) was identified as one of the major targets of miR-511-3p. C3 was increased in LPS-treated macrophages but decreased after miR-511-3p treatment. Consistent with these findings, C3 expression was elevated in the lung macrophages of an asthma mouse model but decreased in mice treated with miR-511-3p. Further experiments, including miRNA-mRNA pulldown and luciferase reporter assays, confirmed that miR-511-3p directly binds to C3 and activates the C3 gene. Thus, miR-511-3p represents a promising therapeutic target for asthma, and RNA nanotechnology reprogrammed EVs are efficient carriers for miRNA delivery for disease treatment.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Altae-Tran H, Kannan S, Suberski AJ, et al (2023)

Uncovering the functional diversity of rare CRISPR-Cas systems with deep terascale clustering.

Science (New York, N.Y.), 382(6673):eadi1910.

Microbial systems underpin many biotechnologies, including CRISPR, but the exponential growth of sequence databases makes it difficult to find previously unidentified systems. In this work, we develop the fast locality-sensitive hashing-based clustering (FLSHclust) algorithm, which performs deep clustering on massive datasets in linearithmic time. We incorporated FLSHclust into a CRISPR discovery pipeline and identified 188 previously unreported CRISPR-linked gene modules, revealing many additional biochemical functions coupled to adaptive immunity. We experimentally characterized three HNH nuclease-containing CRISPR systems, including the first type IV system with a specified interference mechanism, and engineered them for genome editing. We also identified and characterized a candidate type VII system, which we show acts on RNA. This work opens new avenues for harnessing CRISPR and for the broader exploration of the vast functional diversity of microbial proteins.

RevDate: 2023-11-23

Jibrilla M, Raji H, MI Okeke (2023)

Survey of attitude to human genome modification in Nigeria.

Journal of community genetics [Epub ahead of print].

Gene editing and mitochondrial replacement therapy (MRT) are biotechnologies used to modify the host nuclear and mitochondrial DNA, respectively. Gene editing is the modification of a region of the host genome using site-specific nucleases, in particular the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system. Heritable and somatic genome editing (HGE and SGE) are used in gene therapy. MRT is a technique used to substitute the defective mitochondria in the recipient embryo with a female donor healthy mitochondrion in order to prevent the inheritance of mothers' defective mitochondria resulting in the change of mitochondria of the entire generation to come. To evaluate the perception of the Nigerian citizens on human genome modification, two survey forms were created and distributed in-person and majorly online. There was a total of 268 responses, 188 from the public and 80 from health workers and bio-scientists. The results showed poor knowledge about gene editing and MRT by the Nigerian public, but its use to prevent and cure inherited diseases was supported. Morality and religion have great influence on the attitude of Nigerians towards genome modification, but the influence of religion and morality is not unequivocal. Multiple regression analysis of Nigerian public responses shows that gender (females), age (19-30 years), monthly income (NGN 0 to 30,000), and level of education (tertiary) are significantly associated with approval of human genome editing, but the survey of health workers and bio-scientists shows no significant association except for females who approve and Muslims who disapprove of human genome editing.

RevDate: 2023-11-25
CmpDate: 2023-11-24

Hu Y, Liu L, Jiang Q, et al (2023)

CRISPR/Cas9: a powerful tool in colorectal cancer research.

Journal of experimental & clinical cancer research : CR, 42(1):308.

Colorectal cancer (CRC) is one of the most common malignant cancers worldwide and seriously threatens human health. The clustered regulatory interspaced short palindromic repeat/CRISPR-associate nuclease 9 (CRISPR/Cas9) system is an adaptive immune system of bacteria or archaea. Since its introduction, research into various aspects of treatment approaches for CRC has been accelerated, including investigation of the oncogenes, tumor suppressor genes (TSGs), drug resistance genes, target genes, mouse model construction, and especially in genome-wide library screening. Furthermore, the CRISPR/Cas9 system can be utilized for gene therapy for CRC, specifically involving in the molecular targeted drug delivery or targeted knockout in vivo. In this review, we elucidate the mechanism of the CRISPR/Cas9 system and its comprehensive applications in CRC. Additionally, we discussed the issue of off-target effects associated with CRISPR/Cas9, which serves to restrict its practical application. Future research on CRC should in-depth and systematically utilize the CRISPR/Cas9 system thereby achieving clinical practice.

RevDate: 2023-11-25
CmpDate: 2023-11-24

Kouroukli AG, Rajaram N, Bashtrykov P, et al (2023)

Targeting oncogenic TERT promoter variants by allele-specific epigenome editing.

Clinical epigenetics, 15(1):183.

BACKGROUND: Activation of dominant oncogenes by small or structural genomic alterations is a common driver mechanism in many cancers. Silencing of such dominantly activated oncogenic alleles, thus, is a promising strategy to treat cancer. Recently, allele-specific epigenome editing (ASEE) has been described as a means to reduce transcription of genes in an allele-specific manner. In cancer, specificity to an oncogenic allele can be reached by either targeting directly a pathogenic single-nucleotide variant or a polymorphic single-nucleotide variant linked to the oncogenic allele. To investigate the potential of ASEE in cancer, we here explored this approach by targeting variants at the TERT promoter region. The TERT promoter region has been described as one of the most frequently mutated non-coding cancer drivers.

RESULTS: Sequencing of the TERT promoter in cancer cell lines showed 53% (41/77) to contain at least one heterozygous sequence variant allowing allele distinction. We chose the hepatoblastoma cell line Hep-G2 and the lung cancer cell line A-549 for this proof-of-principle study, as they contained two different kinds of variants, namely the activating mutation C228T in the TERT core promoter and the common SNP rs2853669 in the THOR region, respectively. These variants were targeted in an allele-specific manner using sgRNA-guided dCas9-DNMT3A-3L complexes. In both cell lines, we successfully introduced DNA methylation specifically to the on-target allele of the TERT promoter with limited background methylation on the off-target allele or an off-target locus (VEGFA), respectively. We observed a maximum CpG methylation gain of 39% and 76% on the target allele when targeting the activating mutation and the common SNP, respectively. The epigenome editing translated into reduced TERT RNA expression in Hep-G2.

CONCLUSIONS: We applied an ASEE-mediated approach to silence TERT allele specifically. Our results show that the concept of dominant oncogene inactivation by allele-specific epigenome editing can be successfully translated into cancer models. This new strategy may have important advantages in comparison with existing therapeutic approaches, e.g., targeting telomerase, especially with regard to reducing adverse side effects.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Song Y (2023)

RNA-based anti-CRISPRs.

Nature chemical biology, 19(12):1433.

RevDate: 2023-11-22

Zaman QU, Raza A, Lozano-Juste J, et al (2023)

Engineering plants using diverse CRISPR-associated proteins and deregulation of genome-edited crops.

Trends in biotechnology pii:S0167-7799(23)00298-6 [Epub ahead of print].

The CRISPR/Cas system comprises RNA-guided nucleases, the target specificity of which is directed by Watson-Crick base pairing of target loci with single guide (sg)RNA to induce the desired edits. CRISPR-associated proteins and other engineered nucleases are opening new avenues of research in crops to induce heritable mutations. Here, we review the diversity of CRISPR-associated proteins and strategies to deregulate genome-edited (GEd) crops by considering them to be close to natural processes. This technology ensures yield without penalties, advances plant breeding, and guarantees manipulation of the genome for desirable traits. DNA-free and off-target-free GEd crops with defined characteristics can help to achieve sustainable global food security under a changing climate, but need alignment of international regulations to operate in existing supply chains.

RevDate: 2023-11-25

Wen J, Deng H, He D, et al (2023)

Dual-functional DNAzyme powered CRISPR-Cas12a sensor for ultrasensitive and high-throughput detection of Pb[2+] in freshwater.

The Science of the total environment, 911:168708 pii:S0048-9697(23)07336-9 [Epub ahead of print].

Freshwater lead pollution has posed severe threat to the environment and human health, underscoring the urgent necessity for accurate and user-friendly detection methods. Herein, we introduce a novel Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-Cas) sensor for highly sensitive Pb[2+] detection. To accomplish this, we designed a dual-functional deoxyribozyme (df-DNAzyme) probe that functions as an activator for the CRISPR-Cas12a system while also recognizing Pb[2+]. The df-DNAzyme probe was subsequently combined with gold nanoparticles (AuNPs) to fabricate a DNAzyme/AuNP nanoprobe, facilitating the activation of CRISPR-Cas12a in a one-to-multiple manner. Upon exposure to Pb[2+], the df-DNAzyme is cleaved, causing disintegration of the DNAzyme/AuNP nanoprobe from magnetic beads. The degraded DNAzyme/AuNP containing multiple double-stranded DNA activators efficiently triggers CRISPR-Cas12a activity, initiating cleavage of fluorescence-quenched reporter DNA and generating amplified signals accordingly. The amplified fluorescence signal is accurately quantified using a quantitative polymerase chain reaction (qPCR) instrument capable of measuring 96 or 384 samples simultaneously at the microliter scale. This technique demonstrates ultra-sensitive detection capability for Pb[2+] at concentrations as low as 1 pg/L within a range from 1 pg/L to 10 μg/L, surpassing limits set by World Health Organization (WHO) and United States Environmental Protection Agency (US EPA) guidelines. This study offers an ultrasensitive and high-throughput method for the detection of Pb[2+] in freshwater, thereby advancing a novel approach towards the development of precise and convenient techniques for detecting harmful contaminants.

RevDate: 2023-11-22

Yirmiya E, Leavitt A, Lu A, et al (2023)

Phages overcome bacterial immunity via diverse anti-defence proteins.

Nature pii:10.1038/s41586-023-06869-w [Epub ahead of print].

It was recently shown that bacteria employ, apart from CRISPR-Cas and restriction systems, a considerable diversity of phage resistance systems[1-4], but it is largely unknown how phages cope with this multilayered bacterial immunity. Here, we analyzed groups of closely related Bacillus phages that showed differential sensitivity to bacterial defense systems, and discovered four distinct families of anti-defense proteins that inhibit the Gabija, Thoeris, and Hachiman systems. We show that these proteins Gad1, Gad2, Tad2, and Had1 efficiently cancel the defensive activity when co-expressed with the respective defense system or introduced into phage genomes. Homologs of these anti-defense proteins are found in hundreds of phages that infect taxonomically diverse bacterial species. We show that the anti-Gabija protein Gad1 blocks the ability of the Gabija defense complex to cleave phage-derived DNA. Our data further reveal an anti-Thoeris protein, denoted Tad2, which is a "sponge" that sequesters the immune signaling molecules produced by Thoeris TIR-domain proteins in response to phage. Our results demonstrate that phages encode an arsenal of anti-defense proteins that can disable a variety of bacterial defense mechanisms.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Limaye A, Cho K, Hall B, et al (2023)

Genotyping Protocols for Genetically Engineered Mice.

Current protocols, 3(11):e929.

Historically, the laboratory mouse has been the mammalian species of choice for studying gene function and for modeling diseases in humans. This was mainly due to their availability from mouse fanciers. In addition, their short generation time, small size, and minimal food consumption compared to that of larger mammals were definite advantages. This led to the establishment of large hubs for the development of genetically modified mouse models, such as the Jackson Laboratory. Initial research into inbred mouse strains in the early 1900s revolved around coat color genetics and cancer studies, but gene targeting in embryonic stem cells and the introduction of transgenes through pronuclear injection of a mouse zygote, along with current clustered regularly interspaced short palindromic repeat (CRISPR) RNA gene editing, have allowed easy manipulation of the mouse genome. Originally, to distribute a mouse model to other facilities, standard methods had to be developed to ensure that each modified mouse trait could be consistently identified no matter which laboratory requested it. The task of establishing uniform protocols became easier with the development of the polymerase chain reaction (PCR). This chapter will provide guidelines for identifying genetically modified mouse models, mainly using endpoint PCR. In addition, we will discuss strategies to identify genetically modified mouse models that have been established using newer gene-editing technology such as CRISPR. Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Digestion with proteinase K followed by purification of genomic DNA using phenol/chloroform Alternate Protocol: Digestion with proteinase K followed by crude isopropanol extraction of genomic DNA for tail biopsy and ear punch samples Basic Protocol 2: Purification of genomic DNA using a semi-automated system Basic Protocol 3: Purification of genomic DNA from semen, blood, or buccal swabs Basic Protocol 4: Purification of genomic DNA from mouse blastocysts to assess CRISPR gene editing Basic Protocol 5: Routine endpoint-PCR-based genotyping using DNA polymerase and thermal cycler Basic Protocol 6: T7E1/Surveyor assays to detect insertion or deletions following CRISPR editing Basic Protocol 7: Detecting off-target mutations following CRISPR editing Basic Protocol 8: Detecting genomic sequence deletion after CRISPR editing using a pair of guide RNAs Basic Protocol 9: Detecting gene knock-in events following CRISPR editing Basic Protocol 10: Screening of conditional knockout floxed mice.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Rueff AS, van Raaphorst R, Aggarwal SD, et al (2023)

Synthetic genetic oscillators demonstrate the functional importance of phenotypic variation in pneumococcal-host interactions.

Nature communications, 14(1):7454.

Phenotypic variation is the phenomenon in which clonal cells display different traits even under identical environmental conditions. This plasticity is thought to be important for processes including bacterial virulence, but direct evidence for its relevance is often lacking. For instance, variation in capsule production in the human pathogen Streptococcus pneumoniae has been linked to different clinical outcomes, but the exact relationship between variation and pathogenesis is not well understood due to complex natural regulation. In this study, we use synthetic oscillatory gene regulatory networks (GRNs) based on CRISPR interference (CRISPRi) together with live cell imaging and cell tracking within microfluidics devices to mimic and test the biological function of bacterial phenotypic variation. We provide a universally applicable approach for engineering intricate GRNs using only two components: dCas9 and extended sgRNAs (ext-sgRNAs). Our findings demonstrate that variation in capsule production is beneficial for pneumococcal fitness in traits associated with pathogenesis providing conclusive evidence for this longstanding question.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Liang P, Lv B, Chen K, et al (2023)

An ultrasensitive Cd[2+] detection biosensor based on DNAzyme and CRISPR/Cas12a coupled with hybridization chain reaction.

Analytica chimica acta, 1283:341950.

The detection of cadmium is essential because it poses a significant threat to human health and the environment. Recent advancements in biosensors that detect nonnucleic-acid targets using CRISPR/Cas12a in combination with aptamers or DNAzymes show promising performance. Herein, we integrated DNAzyme, hybridization chain reaction (HCR) and CRISPR/Cas12a into a single biosensor for the first time and realized the ultrasensitive detection of Cd[2+]. A single phosphorothioate ribonucleobase (rA)-containing oligonucleotide (PS substrate) and a Cd[2+]-specific DNAzyme (Cdzyme) are used for Cd[2][+] recognition, releasing short single-stranded DNA. Then, the HCR is triggered by the cleavage products for signal transduction and amplification. Next, the trans-cleavage activity of Cas12a is activated due to the presence of crRNA complementary strands and PAM sites in the HCR products. As a result, FQ-reporters are cleaved, and the fluorescence values can be easily read using a fluorometer, allowing Cd[2][[+]] quantification by measuring the fluorescent signal. The Cd[2][[+]] detection biosensor is ultrasensitive with a detection limit of 1.25 pM. Moreover, the biosensor shows great stability under different pH and various anion conditions. The proposed sensor was utilized for environmental water sample detection, demonstrating the dependability of the detection system. Considering the high sensitivity and reliable performance of the assay, it could be further used in environmental monitoring. In addition, the design strategy reported in this study could extend the application of CRISPR/Cas12a in heavy metal detection.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Benarroch L (2023)

[CRIPSR-Cas9: A therapeutic strategy for laminopathies?].

Medecine sciences : M/S, 39 Hors série n° 1:65.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Naddaf M (2023)

First trial of 'base editing' in humans lowers cholesterol - but raises safety concerns.

Nature, 623(7988):671-672.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Yudin Kharismasari C, Irkham , Zein MIHL, et al (2024)

CRISPR/Cas12-based electrochemical biosensors for clinical diagnostic and food monitoring.

Bioelectrochemistry (Amsterdam, Netherlands), 155:108600.

Each organism has a unique sequence of nitrogenous bases in in the form of DNA or RNA which distinguish them from other organisms. This characteristic makes nucleic acid-based detection extremely selective and compare to other molecular techniques. In recent years, several nucleic acid-based detection technology methods have been developed, one of which is the electrochemical biosensor. Electrochemical biosensors are known to have high sensitivity and accuracy. In addition, the ease of miniaturization of this electrochemical technique has garnered interest from many researchers. On the other hand, the CRISPR/Cas12 method has been widely used in detecting nucleic acids due to its highly selective nature. The CRISPR/Cas12 method is also reported to increase the sensitivity of electrochemical biosensors through the utilization of modified electrodes. The electrodes can be modified according to detection needs so that the biosensor's performance can be improved. This review discusses the application of CRISPR/Cas12-based electrochemical biosensors, as well as various electrode modifications that have been successfully used to improve the performance of these biosensors in the clinical and food monitoring fields.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Shaw WM, Khalil AS, T Ellis (2023)

A Multiplex MoClo Toolkit for Extensive and Flexible Engineering of Saccharomyces cerevisiae.

ACS synthetic biology, 12(11):3393-3405.

Synthetic biology toolkits are one of the core foundations on which the field has been built, facilitating and accelerating efforts to reprogram cells and organisms for diverse biotechnological applications. The yeast Saccharomyces cerevisiae, an important model and industrial organism, has benefited from a wide range of toolkits. In particular, the MoClo Yeast Toolkit (YTK) enables the fast and straightforward construction of multigene plasmids from a library of highly characterized parts for programming new cellular behavior in a more predictable manner. While YTK has cultivated a strong parts ecosystem and excels in plasmid construction, it is limited in the extent and flexibility with which it can create new strains of yeast. Here, we describe a new and improved toolkit, the Multiplex Yeast Toolkit (MYT), that extends the capabilities of YTK and addresses strain engineering limitations. MYT provides a set of new integration vectors and selectable markers usable across common laboratory strains, as well as additional assembly cassettes to increase the number of transcriptional units in multigene constructs, CRISPR-Cas9 tools for highly efficient multiplexed vector integration, and three orthogonal and inducible promoter systems for conditional programming of gene expression. With these tools, we provide yeast synthetic biologists with a powerful platform to take their engineering ambitions to exciting new levels.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Chen X, Huang C, Zhang J, et al (2024)

Mini crRNA-mediated CRISPR/Cas12a system (MCM-CRISPR/Cas12a) and its application in RNA detection.

Talanta, 268(Pt 1):125350.

Some non-coding RNAs are abnormally expressed during the occurrence and development of diseases, so it is necessary to develop analytical methods that can specifically and sensitively detect them. In typical CRISPR/Cas12a system, a complete crRNA that can recognize single-stranded or double-stranded DNA is necessary to activate its trans-cleavage activity, which limits its direct application in RNA detection. Here, we prospectively find that slicing the facilitated crRNA in the typical CRISPR/Cas12a system at a fitted site did not affect its trans-cleavage activity, and a mini crRNA-mediated CRISPR/Cas12a system (MCM-CRISPR/Cas12a) was proposed based on this. This system can detect non-coding RNA to pM-level (10 pM for miRNA-21). To expand the application of this system, we combined it with HCR and CHA to establish a detection platform for non-coding RNA. The results show that the proposed method can specifically detect RNA to fM-level (2.5 fM for miRNA-21, 8.98 fM for miR-128-3p, and 81.6 fM for lncRNA PACER). The spiked recovery rates of miRNA-21, miR-128-3p, and lncRNA PACER in normal human serum were in range from 104.7 to 109.4 %, indicating the proposed method owns good applicability. In general, this MCM-CRISPR/Cas12a system further breaks the limitations of the typical CRISPR/Cas12a system that cannot be directly used for non-coding RNA detection. Besides, its combination with HCR and CHA achieves highly sensitive detection of non-coding RNA.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Feng Q, Hao S, Fang P, et al (2023)

Role of GPX4 inhibition-mediated ferroptosis in the chemoresistance of ovarian cancer to Taxol in vitro.

Molecular biology reports, 50(12):10189-10198.

BACKGROUND: Ovarian cancer remains a common gynecological tumor and the fifth leading cause of death worldwide. Taxol-based chemotherapy is a standard approach to the treatment of ovarian cancer. Glutathione peroxidase 4 (GPX4) is the key regulator of ferroptosis, which is an important form of cell death. Here, we investigate the effect of GPX4 inhibition-mediated ferroptosis on the sensitivity of ovarian cancer cells to Taxol.

METHODS AND RESULTS: A2780/PTX and OVCAR-3/PTX Taxol-resistant ovarian cancer cells were established, and stable GPX4 knockout cell lines were generated via lentivirus GPX4-sgRNA. The GPX4 expression level, the apoptosis rate and cell viability were analyzed. The levels of ferroptosis-related factor indicators such as malondialdehyde (MDA) and reactive oxygen species (ROS) were measured. The results showed that the GPX4 protein and mRNA levels were increased in the Taxol-resistant cells. Moreover, GPX4 knockout reduced cell viability and inhibited the colony formation rate. In addition, we found that GPX4 inhibition increased Taxol sensitivity by inducing ferroptosis.

CONCLUSIONS: In summary, our studies reveal that GPX4 inhibition promotes ferroptosis and increases the sensitivity of ovarian cancer cells to Taxol in vitro.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Li T, N Cheng (2023)

Sensitive and Portable Signal Readout Strategies Boost Point-of-Care CRISPR/Cas12a Biosensors.

ACS sensors, 8(11):3988-4007.

Point-of-care (POC) detection is getting more and more attention in many fields due to its accuracy and on-site test property. The CRISPR/Cas12a system is endowed with excellent sensitivity, target identification specificity, and signal amplification ability in biosensing because of its unique trans-cleavage ability. As a result, a lot of research has been made to develop CRISPR/Cas12a-based biosensors. In this review, we focused on signal readout strategies and summarized recent sensitivity-improving strategies in fluorescence, colorimetric, and electrochemical signaling. Then we introduced novel portability-improving strategies based on lateral flow assays (LFAs), microfluidic chips, simplified instruments, and one-pot design. In the end, we also provide our outlook for the future development of CRISPR/Cas12a biosensors.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Wang Z, Yuan H, Yang L, et al (2023)

Decreasing predictable DNA off-target effects and narrowing editing windows of adenine base editors by fusing human Rad18 protein variant.

International journal of biological macromolecules, 253(Pt 7):127418.

Adenine base editors, enabling targeted A-to-G conversion in genomic DNA, have enormous potential in therapeutic applications. However, the currently used adenine base editors are limited by wide editing windows and off-target effects in genetic therapy. Here, we report human e18 protein, a RING type E3 ubiquitin ligase variant, fusing with adenine base editors can significantly improve the preciseness and narrow the editing windows compared with ABEmax and ABE8e by diminishing the abundance of base editor protein. As a proof of concept, ABEmax-e18 and ABE8e-e18 dramatically decrease Cas9-dependent and Cas9-independent off-target effects than traditional adenine base editors. Moreover, we utilized ABEmax-e18 to establish syndactyly mouse models and achieve accurate base conversion at human PCSK9 locus in HepG2 cells which exhibited its potential in genetic therapy. Furthermore, a truncated version of base editors-RING (ABEmax-RING or AncBE4max-RING), which fusing the 63 amino acids of e18 protein RING domain to the C terminal of ABEmax or AncBE4max, exhibited similar effect compared to ABEmax-e18 or AncBE4max-e18.In summary, the e18 or RING protein fused with base editors strengthens the precise toolbox in gene modification and maybe works well with various base editing tools with a more applicable to precise genetic therapies in the future.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Wang Z, Zhang Y, Kang Z, et al (2023)

Improvement of wheat drought tolerance through editing of TaATX4 by CRISPR/Cas9.

Journal of genetics and genomics = Yi chuan xue bao, 50(11):913-916.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Carvalho T (2023)

Discontinued CRISPR gene therapy for sickle-cell disease improves symptoms.

Nature medicine, 29(11):2669-2670.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Harisa GI, Faris TM, Sherif AY, et al (2023)

Gene-editing technology, from macromolecule therapeutics to organ transplantation: Applications, limitations, and prospective uses.

International journal of biological macromolecules, 253(Pt 5):127055.

Gene editing technologies (GETs) could induce gene knockdown or gene knockout for biomedical applications. The clinical success of gene silence by RNAi therapies pays attention to other GETs as therapeutic approaches. This review aims to highlight GETs, categories, mechanisms, challenges, current use, and prospective applications. The different academic search engines, electronic databases, and bibliographies of selected articles were used in the preparation of this review with a focus on the fundamental considerations. The present results revealed that, among GETs, CRISPR/Cas9 has higher editing efficiency and targeting specificity compared to other GETs to insert, delete, modify, or replace the gene at a specific location in the host genome. Therefore, CRISPR/Cas9 is talented in the production of molecular, tissue, cell, and organ therapies. Consequently, GETs could be used in the discovery of innovative therapeutics for genetic diseases, pandemics, cancer, hopeless diseases, and organ failure. Specifically, GETs have been used to produce gene-modified animals to spare human organ failure. Genetically modified pigs are used in clinical trials as a source of heart, liver, kidneys, and lungs for xenotransplantation (XT) in humans. Viral, non-viral, and hybrid vectors have been utilized for the delivery of GETs with some limitations. Therefore, extracellular vesicles (EVs) are proposed as intelligent and future cargoes for GETs delivery in clinical applications. This study concluded that GETs are promising for the production of molecular, cellular, and organ therapies. The use of GETs as XT is still in the early stage as well and they have ethical and biosafety issues.

RevDate: 2023-11-27
CmpDate: 2023-11-27

Kim SH, Shin S, Baek M, et al (2023)

Identification of hyperosmotic stress-responsive genes in Chinese hamster ovary cells via genome-wide virus-free CRISPR/Cas9 screening.

Metabolic engineering, 80:66-77.

Chinese hamster ovary (CHO) cells are the preferred mammalian host cells for therapeutic protein production that have been extensively engineered to possess the desired attributes for high-yield protein production. However, empirical approaches for identifying novel engineering targets are laborious and time-consuming. Here, we established a genome-wide CRISPR/Cas9 screening platform for CHO-K1 cells with 111,651 guide RNAs (gRNAs) targeting 21,585 genes using a virus-free recombinase-mediated cassette exchange-based gRNA integration method. Using this platform, we performed a positive selection screening under hyperosmotic stress conditions and identified 180 genes whose perturbations conferred resistance to hyperosmotic stress in CHO cells. Functional enrichment analysis identified hyperosmotic stress responsive gene clusters, such as tRNA wobble uridine modification and signaling pathways associated with cell cycle arrest. Furthermore, we validated 32 top-scoring candidates and observed a high rate of hit confirmation, demonstrating the potential of the screening platform. Knockout of the novel target genes, Zfr and Pnp, in monoclonal antibody (mAb)-producing recombinant CHO (rCHO) cells and bispecific antibody (bsAb)-producing rCHO cells enhanced their resistance to hyperosmotic stress, thereby improving mAb and bsAb production. Overall, the collective findings demonstrate the value of the screening platform as a powerful tool to investigate the functions of genes associated with hyperosmotic stress and to discover novel targets for rational cell engineering on a genome-wide scale in CHO cells.

RevDate: 2023-11-22

Theuerkauf SA, Herrera-Carrillo E, John F, et al (2023)

AAV vectors displaying bispecific DARPins enable dual-control targeted gene delivery.

Biomaterials, 303:122399 pii:S0142-9612(23)00407-6 [Epub ahead of print].

Precise delivery of genes to therapy-relevant cells is crucial for in vivo gene therapy. Receptor-targeting as prime strategy for this purpose is limited to cell types defined by a single cell-surface marker. Many target cells are characterized by combinations of more than one marker, such as the HIV reservoir cells. Here, we explored the tropism of adeno-associated viral vectors (AAV2) displaying designed ankyrin repeat proteins (DARPins) mono- and bispecific for CD4 and CD32a. Cryo-electron tomography revealed an unaltered capsid structure in the presence of DARPins. Surprisingly, bispecific AAVs transduced CD4/CD32a double-positive cells at much higher efficiencies than single-positive cells, even if present in low amounts in cell mixtures or human blood. This preference was confirmed when vector particles were systemically administered into mice. Cell trafficking studies revealed an increased cell entry rate for bispecific over monospecific AAVs. When equipped with an HIV genome-targeting CRISPR/Cas cassette, the vectors prevented HIV replication in T cell cultures. The data provide proof-of-concept for high-precision gene delivery through tandem-binding regions on AAV. Reminiscent of biological products following Boolean logic AND gating, the data suggest a new option for receptor-targeted vectors to improve the specificity and safety of in vivo gene therapy.

RevDate: 2023-11-22

Wu C, Yue Y, Huang B, et al (2023)

CRISPR-powered microfluidic biosensor for preamplification-free detection of ochratoxin A.

Talanta, 269:125414 pii:S0039-9140(23)01165-7 [Epub ahead of print].

The CRISPR technology, which does not require complex instruments, expensive reagents or professional operators, has attracted a lot of attention. When utilizing the CRISPR-Cas system for detection, the pre-amplification step is often necessary to enhance sensitivity. However, this approach tends to introduce complexity and prolong the time required. To address this issue, we employed Pd@PCN-222 nanozyme to label single-stranded DNA, referred to as Pd@PCN-222 CRISPR nanozyme, which serves as the reporter of the CRISPR system. Pd@PCN-222 nanozyme possess exceptional catalytic activity for the reduction of H2O2. Compared with traditional electrochemical probe ferrocene and methylene blue without catalytic activity, there is a significant amplification of the electrochemical signal. So the need for pre-amplification was eliminated. In this study, we constructed a CRISPR-Cas system for ochratoxin A, utilizing the Pd@PCN-222 CRISPR nanozyme to amplified signal avoiding pre-amplification with outstanding detection of 1.21 pg/mL. Furthermore, we developed a microfluidic electrochemical chip for the on-site detection of ochratoxin A. This achievement holds significant promise in establishing a practical on-site detection platform for identifying food safety hazards.

RevDate: 2023-11-22
CmpDate: 2023-11-22

Nagalakshmi U, Meier N, SP Dinesh-Kumar (2024)

Virus-Induced Heritable Gene Editing in Plants.

Methods in molecular biology (Clifton, N.J.), 2724:273-288.

Gene editing using clustered, regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) nuclease is an excellent tool for assessing gene function in plants. However, delivery of CRISPR/Cas-editing components into plant cells is still a major bottleneck and requires tissue culture-based approaches and regeneration of plants. To overcome this limitation, several plant viral vectors have recently been engineered to deliver single-guide RNA (sgRNA) targets into SpCas9-expressing plants. Here, we describe an optimized, step-by-step protocol based on the tobacco rattle virus (TRV)-based vector system to deliver sgRNAs fused to mobile tRNA sequences for efficient heritable editing in Nicotiana benthamiana and Arabidopsis thaliana model systems. The protocol described here could be adopted to study the function of any gene of interest.

RevDate: 2023-11-25
CmpDate: 2023-11-25

Wong C (2023)

UK first to approve CRISPR treatment for diseases: what you need to know.

Nature, 623(7988):676-677.

RevDate: 2023-11-21

Behrouzian Fard G, Ahmadi MH, Gholamin M, et al (2023)

CRISPR-Cas9 technology: As an efficient genome modification tool in the cancer diagnosis and treatment.

Biotechnology and bioengineering [Epub ahead of print].

Cancer is the second most common cause of death globally and is a major public health concern. Managing this disease is difficult due to its multiple stages and numerous genetic and epigenetic changes. Traditional cancer diagnosis and treatment methods have limitations, making it crucial to develop new modalities to combat the increasing burden of cancer. The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system has transformed genetic engineering due to its simplicity, specificity, low cytotoxicity, and cost-effectiveness. It has been proposed as an effective technology to enhance cancer diagnosis and treatment strategies. This article presents the most recent discoveries regarding the structure, mechanism, and delivery methods of the highly powerful genome editing tool, CRISPR-Cas9. In terms of diagnosis, the article examines the role of CRISPR-Cas9 in detecting microRNAs and DNA methylation, and discusses two popular gene detection techniques that utilize the CRISPR-Cas system: DNA endonuclease-targeted CRISPR trans reporter and specific high sensitivity enzymatic reporter unlocking. Regarding treatment, the article explores several genes that have been identified and modified by CRISPR-Cas9 for effective tumorigenesis of common cancers such as breast, lung, and colorectal cancer. The present review also addresses the challenges and ethical issues associated with using CRISPR-Cas9 as a diagnostic and therapeutic tool. Despite some limitations, CRISPR-Cas9-based cancer diagnosis has the potential to become the next generation of cancer diagnostic tools, and the continuous progress of CRISPR-Cas9 can greatly aid in cancer treatment.

RevDate: 2023-11-20

Ramos JN, Araújo MRB, Sant'Anna LO, et al (2023)

Molecular characterization and whole-genome sequencing of Corynebacterium diphtheriae causing skin lesion.

European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology [Epub ahead of print].

We present a case of skin lesion caused by nontoxigenic Corynebacterium diphtheriae. Genomic taxonomy analyses corroborated the preliminary identification provided by mass spectrometry. The strain showed a susceptible phenotype with increased exposure to penicillin, the first drug of choice for the treatment. An empty type 1 class integron carrying only the sul1 gene, which encodes sulfonamide resistance, was found flanked by transposases. Virulence factors involved in adherence and iron uptake, as well as the CRISPR-Cas system, were predicted. MLST analysis revealed the ST-681, previously reported in French Guiana, a European territory.

RevDate: 2023-11-23
CmpDate: 2023-11-22

Geng K, Merino LG, Veiga RG, et al (2024)

Intrinsic deletion at 10q23.31, including the PTEN gene locus, is aggravated upon CRISPR-Cas9-mediated genome engineering in HAP1 cells mimicking cancer profiles.

Life science alliance, 7(2):.

The CRISPR-Cas9 system is a powerful tool for studying gene functions and holds potential for disease treatment. However, precise genome editing requires thorough assessments to minimize unintended on- and off-target effects. Here, we report an unexpected 283-kb deletion on Chromosome 10 (10q23.31) in chronic myelogenous leukemia-derived HAP1 cells, which are frequently used in CRISPR screens. The deleted region encodes regulatory genes, including PAPSS2, ATAD1, KLLN, and PTEN We found that this deletion was not a direct consequence of CRISPR-Cas9 off-targeting but rather occurred frequently during the generation of CRISPR-Cas9-modified cells. The deletion was associated with global changes in histone acetylation and gene expression, affecting fundamental cellular processes such as cell cycle and DNA replication. We detected this deletion in cancer patient genomes. As in HAP1 cells, the deletion contributed to similar gene expression patterns among cancer patients despite interindividual differences. Our findings suggest that the unintended deletion of 10q23.31 can confound CRISPR-Cas9 studies and underscore the importance to assess unintended genomic changes in CRISPR-Cas9-modified cells, which could impact cancer research.

RevDate: 2023-11-20

Storz U (2023)

The CRISPR Cas patent files, part 1: Cas9 - where to we stand at the 10 year halftime ?.

Journal of biotechnology pii:S0168-1656(23)00199-2 [Epub ahead of print].

CRISPR Cas9 has turned out to be one of the most influential technologies in the life sciences. However, ferocious patent debates and an unclear licensing situation makes access to this technology difficult for Small and medium enterprises. This article gives an overview of the status quo 10 years after the seminal patents were filed.

RevDate: 2023-11-20

Puchta H, A Houben (2023)

Plant chromosome engineering - past, present and future.

The New phytologist [Epub ahead of print].

Spontaneous chromosomal rearrangements (CRs) play an essential role in speciation, genome evolution and crop domestication. To be able to use the potential of CRs for breeding, plant chromosome engineering was initiated by fragmenting chromosomes by X-ray irradiation. With the rise of the CRISPR/Cas system, it became possible to induce double-strand breaks (DSBs) in a highly efficient manner at will at any chromosomal position. This has enabled a completely new level of predesigned chromosome engineering. The genetic linkage between specific genes can be broken by inducing chromosomal translocations. Natural inversions, which suppress genetic exchange, can be reverted for breeding. In addition, various approaches for constructing minichromosomes by downsizing regular standard A or supernumerary B chromosomes, which could serve as future vectors in plant biotechnology, have been developed. Recently, a functional synthetic centromere could be constructed. Also, different ways of genome haploidization have been set up, some based on centromere manipulations. In the future, we expect to see even more complex rearrangements, which can be combined with previously developed engineering technologies such as recombinases. Chromosome engineering might help to redefine genetic linkage groups, change the number of chromosomes, stack beneficial genes on mini cargo chromosomes, or set up genetic isolation to avoid outcrossing.

RevDate: 2023-11-24

Altae-Tran H, Shmakov SA, Makarova KS, et al (2023)

Diversity, evolution, and classification of the RNA-guided nucleases TnpB and Cas12.

Proceedings of the National Academy of Sciences of the United States of America, 120(48):e2308224120.

The TnpB proteins are transposon-associated RNA-guided nucleases that are among the most abundant proteins encoded in bacterial and archaeal genomes, but whose functions in the transposon life cycle remain unknown. TnpB appears to be the evolutionary ancestor of Cas12, the effector nuclease of type V CRISPR-Cas systems. We performed a comprehensive census of TnpBs in archaeal and bacterial genomes and constructed a phylogenetic tree on which we mapped various features of these proteins. In multiple branches of the tree, the catalytic site of the TnpB nuclease is rearranged, demonstrating structural and probably biochemical malleability of this enzyme. We identified numerous cases of apparent recruitment of TnpB for other functions of which the most common is the evolution of type V CRISPR-Cas effectors on about 50 independent occasions. In many other cases of more radical exaptation, the catalytic site of the TnpB nuclease is apparently inactivated, suggesting a regulatory function, whereas in others, the activity appears to be retained, indicating that the recruited TnpB functions as a nuclease, for example, as a toxin. These findings demonstrate remarkable evolutionary malleability of the TnpB scaffold and provide extensive opportunities for further exploration of RNA-guided biological systems as well as multiple applications.

RevDate: 2023-11-24
CmpDate: 2023-11-24

Gong T, Liao L, Jiang B, et al (2024)

Ag[+]-stabilized DNA triplex coupled with catalytic hairpin assembly and CRISPR/Cas12a amplifications for sensitive metallothionein assay.

Talanta, 268(Pt 2):125392.

Metallothionein (MT) is a protein biomarker secreted by liver in response to the treatment for heavy metal toxicity and oncological diseases. On the basis of a new Ag[+]-stabilized DNA triplex probe (Ag[+]-SDTP), we establish a fluorescent biosensing system for high sensitivity detection of MT by combining catalytic hairpin assembly (CHA) and the CRISPR/Cas12a signal enhancements. The MT analyte complexes with Ag[+] in Ag[+]-SDTP to disrupt the triplex structure and to release the ssDNA strands, which trigger subsequent CHA formation of many protospacer adjacent motif (PAM)-containing dsDNAs from two hairpins. Cas12a/crRNA further recognizes these PAM sequences to activate its trans-catalytic activity to cyclically cleave the fluorescently quenched ssDNA reporters to recovery drastically amplified fluorescence for detecting MT down to 0.34 nM within the dynamic range of 1∼800 nM. Moreover, the sensing method is able to selectively discriminate MT from other non-specific molecules and can realize low level detection of MT in diluted human serums, manifesting its potentiality for monitoring the disease-specific MT biomarker at trace levels.

RevDate: 2023-11-24
CmpDate: 2023-11-24

Jin S, Yong H, Liu Y, et al (2023)

CRISPR/Cas9-mediated high-mobility group A2 knockout inhibits cell proliferation and invasion in papillary thyroid carcinoma cells.

Advances in medical sciences, 68(2):409-416.

PURPOSE: Metastasis and recurrence are the prognostic risk factor in patients with thyroid carcinoma. High-mobility group A2 (HMGA2) protein plays a crucial role in papillary thyroid carcinoma (PTC) metastasis. The aim of this study was to investigate the mechanisms underlying the HMGA2 effect on PTC cell proliferation and invasion.

MATERIALS AND METHODS: We used the CRISPR/Cas9 system to perform knockout of the HMGA2 gene in the human PTC cell line TPC-1. The knockout monoclonal cells were screened and verified by PCR analysis and genomic sequencing. Cell proliferation was examined after the knockout of the HMGA2 gene using cell counting kit-8 (CCK-8) assays. Furthermore, cell migration and invasion after the knockout were examined by cell scratch tests. Additionally, the changes in cell cycle and apoptosis after the knockout were detected by flow cytometry.

RESULTS: The results of the PCR analysis and the genomic sequencing confirmed that the human PTC TPC-1 ​cell line with knockout of HMGA2 gene was successfully established. The knockout of the HMGA2 gene significantly reduced the cell proliferation, growth, and invasion. Meanwhile, the knockout of the HMGA2 gene delayed the conversion of the G2/M phase and promoted cell necrosis.

CONCLUSION: The CRISPR/Cas9-mediated HMGA2 knockout in the TPC-1 ​cell line inhibited cell proliferation and invasion, which might be due to the blockage of the cell cycle in the G2/M phase and the promotion of cell necrosis.

RevDate: 2023-11-24
CmpDate: 2023-11-24

Xin Q, Jia H, Wang B, et al (2023)

CRISPR-dCpf1 mediated whole genome crRNA inhibition library for high-throughput screening of growth characteristic genes in Bacillus amyloliquefaciens LB1ba02.

International journal of biological macromolecules, 253(Pt 5):127179.

Bacillus amyloliquefaciens LB1ba02 is generally recognized as food safe (GRAS) microbial host and important enzyme-producing strain in the industry. However, autolysis affects the growth of bacteria, further affecting the yield of target products. Besides, the restriction-modification system, existed in B. amyloliquefaciens LB1ba02, results in a low transformation efficiency, which further leads to a lack of high-throughput screening tools. Here, we constructed a genome-wide crRNA inhibition library based on the CRISPR/dCpf1 system and high-throughput screening of related genes affecting the cell growth and autolysis using flow cytometry in B. amyloliquefaciens LB1ba02. The whole genome crRNA library was first validated for resistance to the toxic chemical 5-fluorouracil, and then used for validation of essential genes. In addition, seven gene loci (oppD, flil, tuaA, prmA, sigO, hslU, and GE03231) that affect the growth characteristics of LB1ba02 were screened. Among them, the Opp system had the greatest impact on growth. When the expression of operon oppA-oppB-oppC-oppD-oppF was inhibited, the cell growth difference was most significant. Inhibition of other sites could also promote rapid growth of bacteria to varying degrees; however, inhibition of GE03231 site accelerated cell autolysis. Therefore, the whole genome crRNA inhibition library is well suited for B. amyloliquefaciens LB1ba02 and can be further applied to high-throughput mining of other functional genes.

RevDate: 2023-11-24
CmpDate: 2023-11-24

Zhang S, Wang B, Li Q, et al (2023)

CRISPR/Cas9 mutated p-coumaroyl shikimate 3'-hydroxylase 3 gene in Populus tomentosa reveals lignin functioning on supporting tree upright.

International journal of biological macromolecules, 253(Pt 3):126762.

The lignin plays one of the most important roles in plant secondary metabolism. However, it is still unclear how lignin can contribute to the impressive height of wood growth. In this study, C3'H, a rate-limiting enzyme of the lignin pathway, was used as the target gene. C3'H3 was knocked out by CRISPR/Cas9 in Populus tomentosa. Compared with wild-type popular trees, c3'h3 mutants exhibited dwarf phenotypes, collapsed xylem vessels, weakened phloem thickening, decreased hydraulic conductivity and photosynthetic efficiency, and reduced auxin content, except for reduced total lignin content and significantly increased H-subunit lignin. In the c3'h3 mutant, the flavonoid biosynthesis genes CHS, CHI, F3H, DFR, ANR, and LAR were upregulated, and flavonoid metabolite accumulations were detected, indicating that decreasing the lignin biosynthesis pathway enhanced flavonoid metabolic flux. Furthermore, flavonoid metabolites, such as naringenin and hesperetin, were largely increased, while higher hesperetin content suppressed plant cell division. Thus, studying the c3'h3 mutant allows us to deduce that lignin deficiency suppresses tree growth and leads to the dwarf phenotype due to collapsed xylem and thickened phloem, limiting material exchanges and transport.

RevDate: 2023-11-20

Liu Y, Binda CS, Berkhout B, et al (2023)

CRISPR-Cas attack of HIV-1 proviral DNA can cause unintended deletion of surrounding cellular DNA.

Journal of virology [Epub ahead of print].

Although HIV replication can be effectively inhibited by antiretroviral therapy, this does not result in a cure as the available drugs do not inactivate the integrated HIV-1 DNA in infected cells. Consequently, HIV-infected individuals need lifelong therapy to prevent viral rebound. Several preclinical studies indicate that CRISPR-Cas gene-editing systems can be used to achieve permanent inactivation of the viral DNA. It was previously shown that this inactivation was due to small inactivating mutations at the targeted sites in the HIV genome and to excision or inversion of the viral DNA fragment between two target sites. We, here, demonstrate that CRISPR-Cas treatment also causes large unintended deletions, which can include surrounding chromosomal sequences. As the loss of chromosomal sequences may cause oncogenic transformation of the cell, such unintended large deletions form a potential safety risk in clinical application of this antiviral application and possibly all CRISPR-Cas gene-editing approaches.

RevDate: 2023-11-22
CmpDate: 2023-11-20

Moon J, C Liu (2023)

Asymmetric CRISPR enabling cascade signal amplification for nucleic acid detection by competitive crRNA.

Nature communications, 14(1):7504.

Nucleic acid detection powered by CRISPR technology provides a rapid, sensitive, and deployable approach to molecular diagnostics. While exciting, there remain challenges limiting its practical applications, such as the need for pre-amplification and the lack of quantitative ability. Here, we develop an asymmetric CRISPR assay for cascade signal amplification detection of nucleic acids by leveraging the asymmetric trans-cleavage behavior of competitive crRNA. We discover that the competitive reaction between a full-sized crRNA and split crRNA for CRISPR-Cas12a can induce cascade signal amplification, significantly improving the target detection signal. In addition, we find that CRISPR-Cas12a can recognize fragmented RNA/DNA targets, enabling direct RNA detection by Cas12a. Based on these findings, we apply our asymmetric CRISPR assay to quantitatively detect microRNA without the need for pre-amplification, achieving a detection sensitivity of 856 aM. Moreover, using this method, we analyze and quantify miR-19a biomarker in plasma samples from bladder cancer patients. This asymmetric CRISPR assay has the potential to be widely applied for simple and sensitive nucleic acid detection in various diagnostic settings.

RevDate: 2023-11-21
CmpDate: 2023-11-20

Chen Q, Chuai G, Zhang H, et al (2023)

Genome-wide CRISPR off-target prediction and optimization using RNA-DNA interaction fingerprints.

Nature communications, 14(1):7521.

The powerful CRISPR genome editing system is hindered by its off-target effects, and existing computational tools achieved limited performance in genome-wide off-target prediction due to the lack of deep understanding of the CRISPR molecular mechanism. In this study, we propose to incorporate molecular dynamics (MD) simulations in the computational analysis of CRISPR system, and present CRISOT, an integrated tool suite containing four related modules, i.e., CRISOT-FP, CRISOT-Score, CRISOT-Spec, CRISORT-Opti for RNA-DNA molecular interaction fingerprint generation, genome-wide CRISPR off-target prediction, sgRNA specificity evaluation and sgRNA optimization of Cas9 system respectively. Our comprehensive computational and experimental tests reveal that CRISOT outperforms existing tools with extensive in silico validations and proof-of-concept experimental validations. In addition, CRISOT shows potential in accurately predicting off-target effects of the base editors and prime editors, indicating that the derived RNA-DNA molecular interaction fingerprint captures the underlying mechanisms of RNA-DNA interaction among distinct CRISPR systems. Collectively, CRISOT provides an efficient and generalizable framework for genome-wide CRISPR off-target prediction, evaluation and sgRNA optimization for improved targeting specificity in CRISPR genome editing.

RevDate: 2023-11-23
CmpDate: 2023-11-23

Chen R, Zhao J, Han M, et al (2023)

DNA Extraction- and Amplification-Free Nucleic Acid Biosensor for the Detection of Foodborne Pathogens Based on CRISPR/Cas12a and Argonaute Protein-Mediated Cascade Signal Amplification.

Journal of agricultural and food chemistry, 71(46):18037-18045.

A novel method for detecting low levels of viable foodborne pathogens, specifically Salmonella typhimurium (S. typhimurium), has been developed. Traditional nucleic acid assay, such as polymerase chain reaction (PCR), often requires complex DNA extraction and amplification, making it challenging to differentiate between viable and nonviable pathogens. This assay employed a phage as the recognition element to precisely identify and lyse viable S. typhimurium that can undergo DNA extraction. It combined the efficient trans-cleavage activities of CRISPR/Cas12a with the specific cleavage advantages of Argonaute proteins, enabling ultrasensitive detection. This double-enzyme-mediated nucleic acid test can accurately distinguish viable and nonviable S. typhimurium with a detection limit of 23 CFU/mL without DNA amplification. The method was successfully applied to common food samples, producing results consistent with quantitative PCR tests. This work provides a promising platform for easily detecting viable foodborne pathogens with high sensitivity without the need for DNA extraction and amplification.

RevDate: 2023-11-23
CmpDate: 2023-11-23

Viswan A, Yoshikawa C, Yamagishi A, et al (2023)

Efficient genome editing by controlled release of Cas9 ribonucleoprotein in plant cytosol using polymer-modified microneedle array.

Biochemical and biophysical research communications, 686:149179.

Direct delivery of genome-editing proteins into plant tissues could be useful in obtaining DNA-free genome-edited crops obviating the need for backcrossing to remove vector-derived DNA from the host genome as in the case of genetically modified organisms generated using DNA vector. Previously, we successfully delivered Cas9 ribonucleoprotein (RNP) into plant tissue by inserting microneedle array (MNA) physisorbed with Cas9 RNPs. Here, to enhance protein delivery and improve genome-editing efficiency, we introduced a bioactive polymer DMA/HPA/NHS modification to the MNA, which allowed strong bonding between the proteins and MNA. Compared with other modifying agents, this MNA modification resulted in better release of immobilized protein in a plant cytosol-mimicking environment. The delivery of Cas9 RNPs in Arabidopsis thaliana reporter plants was improved from 4 out of 17 leaf tissues when using unmodified MNAs to 9 out of 17 when using the polymer-modified MNAs. Further improvements in delivery efficiency can be envisaged by optimizing the polymer modification conditions, which could have significant implications for the development of more effective plant genome editing techniques.

RevDate: 2023-11-23
CmpDate: 2023-11-23

Pradhan SK, Karuppannasamy A, Sujatha PM, et al (2023)

Embryonic microinjection of ribonucleoprotein complex (Cas9+sgRNA) of white gene in melon fly, Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae) produced white eye phenotype.

Archives of insect biochemistry and physiology, 114(4):e22059.

Melon fly, Zeugodacus cucurbitae (Coquillett) is a major pest of cucurbitaceous crops, and causes substantial yield losses and economic costs. CRISPR/Cas9 is a rapid and effective site-specific genome editing tool for the generation of genetic changes that are stable and heritable. The CRISPR/Cas9 tool uses synthetically designed single guide RNA (sgRNA) that is complementary to the target gene and guides the Cas9 enzyme to perform nuclease activity by making double-strand breaks in the target DNA sequences. This tool can be effectively exploited to improve traits critical for the management of insect pests by targeting specific genes encoding these traits without the need of extensive genetic information. The white gene is an important gene responsible for the transport of body pigment precursor molecules. In this study, we produced effective mutagenesis of the white gene of Z. cucurbitae using the CRISPR/Cas9 tool with double sgRNA to target multiple sites of white to increase the efficiency in the generation of frame-shift mutations resulting in the white eye phenotype in adults. This was achieved through embryonic microinjection of the ribonucleoprotein (RNP) complex in the pre-blastoderm embryo stage 1 h after embryo laying. Our success with the production of a white eye mutant fly by CRISPR/Cas9 mutagenesis is important for the research on gene function and protein-level modifications in melon fly and forms the basis for the development of new genetic control strategies such as precision guided sterile insect technique (pgSIT) for this pest of economic significance.

RevDate: 2023-11-23
CmpDate: 2023-11-23

Ashok K, Bhargava CN, Asokan R, et al (2023)

CRISPR/Cas9 mediated mutagenesis of the major sex pheromone gene, acyl-CoA delta-9 desaturase (DES9) in Fall armyworm Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae).

International journal of biological macromolecules, 253(Pt 2):126557.

The Fall armyworm, Spodoptera frugiperda is a significant global pest causing serious yield loss on several staple crops. In this regard, this pest defies several management approaches based on chemicals, Bt transgenics etc., requiring effective alternatives. Recently CRISPR/Cas9 mediated genome editing has opened up newer avenues to establish functions of various target genes before employing them for further application. The virgin female moths of S. frugiperda emit sex pheromones to draw conspecific males. Therefore, we have edited the key pheromone synthesis gene, fatty acyl-CoA Delta-9 desaturase (DES9) of the Indian population of S. frugiperda. In order to achieve a larger deletion of the DES9, we have designed two single guide RNA (sgRNA) in sense and antisense direction targeting the first exon instead of a single guide RNA. The sgRNA caused site-specific knockout with a larger deletion which impacted the mating. Crossing studies between wild male and mutant female resulted in no fecundity, while fecundity was normal when mutant male crossed with the wild female. This indicates that mating disruption is stronger in females where DES9 is mutated. The current work is the first of its kind to show that DES9 gene editing impacted the likelihood of mating in S. frugiperda.

RevDate: 2023-11-18

Li Y, Liu Y, Tang X, et al (2023)

CRISPR/Cas-Powered Amplification-Free Detection of Nucleic Acids: Current State of the Art, Challenges, and Futuristic Perspectives.

ACS sensors [Epub ahead of print].

CRISPR/Cas system is becoming an increasingly influential technology that has been repositioned in nucleic acid detection. A preamplification step is usually required to improve the sensitivity of CRISPR/Cas-based detection. The striking biological features of CRISPR/Cas, including programmability, high sensitivity and sequence specificity, and single-base resolution. More strikingly, the target-activated trans-cleavage could act as a biocatalytic signal transductor and amplifier, thereby empowering it to potentially perform nucleic acid detection without a preamplification step. The reports of such work are on the rise, which is not only scientifically significant but also promising for futuristic end-user applications. This review started with the introduction of the detection methods of nucleic acids and the CRISPR/Cas-based diagnostics (CRISPR-Dx). Next, we objectively discussed the pros and cons of preamplification steps for CRISPR-Dx. We then illustrated and highlighted the recently developed strategies for CRISPR/Cas-powered amplification-free detection that can be realized through the uses of ultralocalized reactors, cascade reactions, ultrasensitive detection systems, or others. Lastly, the challenges and futuristic perspectives were proposed. It can be expected that this work not only makes the researchers better understand the current strategies for this emerging field, but also provides insight for designing novel CRISPR-Dx without a preamplification step to win practicable use in the near future.

RevDate: 2023-11-20
CmpDate: 2023-11-20

Wang W, Geng L, Zhang Y, et al (2023)

Development of antibody-aptamer sandwich-like immunosensor based on RCA and Nicked-PAM CRISPR/Cas12a system for the ultra-sensitive detection of a biomarker.

Analytica chimica acta, 1283:341849.

Biomarkers are the most sensitive reactants and early indicators of many kinds of diseases. The development of highly sensitive and simple techniques to quantify them is challenging. In this study, based on rolling cycle amplification (RCA) and the Nicked PAM/CRISPR-Cas12a system (RNPC) as a signal reporter, a sandwich-type method was developed using antibody@magnetic beads and aptamer for the high-sensitive detection of the C-reactive protein (CRP). The antibody-antigen (target)-aptamer sandwich-like reaction was coupled to RCA, which can produce hundreds of similar binding sites and are discriminated by CRISPR/Cas12a for signal amplification. The ultrasensitivity is achieved based on the dual-signal enhancing strategy, which involves the special recognition of aptamers, RCA, and trans-cleavage of CRISPR/Cas12a. By incorporating the CRISPR/Cas12a system with cleaved PAM, the nonspecific amplification of the RCA reaction alone was greatly reduced, and the dual signal output of RCA and Cas12a improved the detection sensitivity. Our assay can be performed only in two steps. The first step takes only 20 min of target capture, followed by a one-pot reaction, where the target concentration can be obtained by fluorescence values as long as there are 37 °C reaction conditions. Under optimal conditions, this system detected CRP with high sensitivity. The fabricated biosensor showed detection limits of 0.40 pg/mL in phosphate-buffered saline and 0.73 pg/mL in diluted human serum and a broad linear dynamic range of 1.28 pg/mL to 100 ng/mL within a total readout time of 90 min. The method could be used to perform multi-step signal amplification, which can help in the ultrasensitive detection of other proteins. Overall, the proposed biosensor might be used as an immunosensor biosensor platform.

RevDate: 2023-11-17

Lammens E-M, Volke DC, Schroven K, et al (2023)

A SEVA-based, CRISPR-Cas3-assisted genome engineering approach for Pseudomonas with efficient vector curing.

Microbiology spectrum [Epub ahead of print].

The CRISPR-Cas3 editing system as presented here facilitates the creation of genomic alterations in Pseudomonas putida and Pseudomonas aeruginosa in a straightforward manner. By providing the Cas3 system as a vector set with Golden Gate compatibility and different antibiotic markers, as well as by employing the established Standard European Vector Architecture (SEVA) vector set to provide the homology repair template, this system is flexible and can readily be ported to a multitude of Gram-negative hosts. Besides genome editing, the Cas3 system can also be used as an effective and universal tool for vector curing. This is achieved by introducing a spacer that targets the origin-of-transfer, present on the majority of established (SEVA) vectors. Based on this, the Cas3 system efficiently removes up to three vectors in only a few days. As such, this curing approach may also benefit other genomic engineering methods or remove naturally occurring plasmids from bacteria.

RevDate: 2023-11-21
CmpDate: 2023-11-20

Ozyerli-Goknar E, Kala EY, Aksu AC, et al (2023)

Epigenetic-focused CRISPR/Cas9 screen identifies (absent, small, or homeotic)2-like protein (ASH2L) as a regulator of glioblastoma cell survival.

Cell communication and signaling : CCS, 21(1):328.

BACKGROUND: Glioblastoma is the most common and aggressive primary brain tumor with extremely poor prognosis, highlighting an urgent need for developing novel treatment options. Identifying epigenetic vulnerabilities of cancer cells can provide excellent therapeutic intervention points for various types of cancers.

METHOD: In this study, we investigated epigenetic regulators of glioblastoma cell survival through CRISPR/Cas9 based genetic ablation screens using a customized sgRNA library EpiDoKOL, which targets critical functional domains of chromatin modifiers.

RESULTS: Screens conducted in multiple cell lines revealed ASH2L, a histone lysine methyltransferase complex subunit, as a major regulator of glioblastoma cell viability. ASH2L depletion led to cell cycle arrest and apoptosis. RNA sequencing and greenCUT&RUN together identified a set of cell cycle regulatory genes, such as TRA2B, BARD1, KIF20B, ARID4A and SMARCC1 that were downregulated upon ASH2L depletion. Mass spectrometry analysis revealed the interaction partners of ASH2L in glioblastoma cell lines as SET1/MLL family members including SETD1A, SETD1B, MLL1 and MLL2. We further showed that glioblastoma cells had a differential dependency on expression of SET1/MLL family members for survival. The growth of ASH2L-depleted glioblastoma cells was markedly slower than controls in orthotopic in vivo models. TCGA analysis showed high ASH2L expression in glioblastoma compared to low grade gliomas and immunohistochemical analysis revealed significant ASH2L expression in glioblastoma tissues, attesting to its clinical relevance. Therefore, high throughput, robust and affordable screens with focused libraries, such as EpiDoKOL, holds great promise to enable rapid discovery of novel epigenetic regulators of cancer cell survival, such as ASH2L.

CONCLUSION: Together, we suggest that targeting ASH2L could serve as a new therapeutic opportunity for glioblastoma. Video Abstract.

RevDate: 2023-11-22
CmpDate: 2023-11-20

Dhingra Y, DG Sashital (2023)

A tool for more specific DNA integration.

Science (New York, N.Y.), 382(6672):768-769.

The efficiency of targeted DNA insertion by CRISPR transposons is improved.

RevDate: 2023-11-22
CmpDate: 2023-11-20

George JT, Acree C, Park JU, et al (2023)

Mechanism of target site selection by type V-K CRISPR-associated transposases.

Science (New York, N.Y.), 382(6672):eadj8543.

CRISPR-associated transposases (CASTs) repurpose nuclease-deficient CRISPR effectors to catalyze RNA-guided transposition of large genetic payloads. Type V-K CASTs offer potential technology advantages but lack accuracy, and the molecular basis for this drawback has remained elusive. Here, we reveal that type V-K CASTs maintain an RNA-independent, "untargeted" transposition pathway alongside RNA-dependent integration, driven by the local availability of TnsC filaments. Using cryo-electron microscopy, single-molecule experiments, and high-throughput sequencing, we found that a minimal, CRISPR-less transpososome preferentially directs untargeted integration at AT-rich sites, with additional local specificity imparted by TnsB. By exploiting this knowledge, we suppressed untargeted transposition and increased type V-K CAST specificity up to 98.1% in cells without compromising on-target integration efficiency. These findings will inform further engineering of CAST systems for accurate, kilobase-scale genome engineering applications.

RevDate: 2023-11-22
CmpDate: 2023-11-22

Li Y, Cai M, Zhang W, et al (2024)

Cas12a-based direct visualization of nanoparticle-stabilized fluorescence signal for multiplex detection of DNA methylation biomarkers.

Biosensors & bioelectronics, 244:115810.

The CRISPR-Cas12a RNA-guided complexes hold immense promise for nucleic acid detection. However, limitations arise from their specificity in detecting off-targets and the stability of the signal molecules. Here, we have developed a platform that integrates multiplex amplification and nanomolecular-reporting signals, allowing us to detect various clinically relevant nucleic acid targets with enhanced stability, sensitivity, and visual interpretation. Through the electrostatic co-assembly of the Oligo reporter with oppositely charged nanoparticles, we observed a significant enhancement in its stability in low-pollution environments, reaching up to a threefold increase compared to the original version. Additionally, the fluorescence efficiency was expanded by three orders of magnitude, broadening the detection range considerably. Utilizing a multiplex strategy, this assay can accomplish simultaneous detection of multiple targets and single-point indication detection of nine specific targets. This significant advancement heightened the sensitivity of disease screening and improved the accuracy of diagnosing disease-related changes. We tested this assay in a colorectal cancer model, demonstrating that it can identify DNA methylation features at the aM-level within 40-60 min. Validation using clinical samples yielded consistent results with qPCR and bisulfite sequencing, affirming the assay's reliability and potential for clinical applications.

RevDate: 2023-11-22
CmpDate: 2023-11-22

Lin Z, Chen L, Tang S, et al (2023)

Efficient CRISPR/Cas9-mediated genome editing in sheepgrass (Leymus chinensis).

Journal of integrative plant biology, 65(11):2416-2420.

The lack of genome editing platforms has hampered efforts to study and improve forage crops that can be grown on lands not suited to other crops. Here, we established efficient Agrobacterium-mediated clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) genome editing in a perennial, stress-tolerant forage grass, sheepgrass (Leymus chinensis). By screening for active single-guide RNAs (sgRNAs), accessions that regenerate well, suitable Agrobacterium strains, and optimal culture media, and co-expressing the morphogenic factor TaWOX5, we achieved 11% transformation and 5.83% editing efficiency in sheepgrass. Knocking out Teosinte Branched1 (TB1) significantly increased tiller number and biomass. This study opens avenues for studying gene function and breeding in sheepgrass.

RevDate: 2023-11-20
CmpDate: 2023-11-20

Kaiser J (2023)

Rewriting DNA in the body lowers cholesterol.

Science (New York, N.Y.), 382(6672):751.

Verve Therapeutics says its base-editing approach may help prevent heart disease in many people.

RevDate: 2023-11-16

Martínez-Alvarez L, Ramond J-B, Vikram S, et al (2023)

With a pinch of salt: metagenomic insights into Namib Desert salt pan microbial mats and halites reveal functionally adapted and competitive communities.

Applied and environmental microbiology [Epub ahead of print].

The hyperarid Namib Desert is one of the oldest deserts on Earth. It contains multiple clusters of playas which are saline-rich springs surrounded by halite evaporites. Playas are of great ecological importance, and their indigenous (poly)extremophilic microorganisms are potentially involved in the precipitation of minerals such as carbonates and sulfates and have been of great biotechnological importance. While there has been a considerable amount of microbial ecology research performed on various Namib Desert edaphic microbiomes, little is known about the microbial communities inhabiting its multiple playas. In this work, we provide a comprehensive taxonomic and functional potential characterization of the microbial, including viral, communities of sediment mats and halites from two distant salt pans of the Namib Desert, contributing toward a better understanding of the ecology of this biome.

RevDate: 2023-11-17
CmpDate: 2023-11-17

Cui C, TH Chen (2023)

CRISPR/Cas12a trans-cleavage triggered by cleavage ligation of dumbbell DNA for specific detection of human 8-oxoguanine DNA glycosylase activity.

Mikrochimica acta, 190(12):468.

Human 8-oxoguanine DNA glycosylase (hOGG1) is an essential enzyme that recognizes and removes 8-oxoguanine (8-oxoG), a common DNA oxidative damage caused by reactive oxygen species, to maintain genomic integrity of living organisms. Abnormal expression of hOGG1 has been proved to be associated with different diseases such as cancer and neurogenerative disorders, making it a potential biomarker and therapeutic target. In this study, we report the development of a novel strategy for detecting hOGG1 activity based on CRISPR/Cas12a trans-cleavage triggered by cleavage ligation of a dumbbell DNA probe (DBP) designed with a 3' overhang and an 8-oxoG modification. When hOGG1 is present, it cleaves the DBP at the 8-oxoG site, forming a 5' phosphate termini and exposing a single-strand region allowing complementary to the 3' overhang. After hybridization, the 3' and 5' termini in the juxtaposition are ligated by T4 DNA ligase, leading to a closed DBP for CRISPR/Cas12a-crRNA to recognize and initiate the trans-cleavage of the surrounding ssDNAs with fluorophore and quencher. The method achieves a limit of detection (LOD) with 370 μU/mL and high selectivity. Furthermore, it demonstrates a good compatibility for detecting hOGG1 activity in cell lysates, suggesting a good performance for further application in disease diagnosis and scientific research.

RevDate: 2023-11-15

Guo J, Zhu Y, P Miao (2023)

Nano-Impact Electrochemical Biosensing Based on a CRISPR-Responsive DNA Hydrogel.

Nano letters [Epub ahead of print].

Nano-impact electrochemistry (NIE) enables simple, rapid, and high-throughput biocoupling and biomolecular recognition. However, the low effective collision frequency limits the sensitivity. In this study, we propose a novel NIE sensing strategy amplified by the CRISPR-responsive DNA hydrogel and cascade DNA assembly. By controlling the phase transition of DNA hydrogel and the self-electrolysis of silver nanoparticles, we can obtain significant electrochemical responses. The whole process includes target miRNA-induced strand displacement amplification, catalytic hairpin assembly, and CRISPR/Cas trans-cutting. Thus, ultrahigh sensitivity is promised. This NIE biosensing strategy achieves a limit of detection as low as 4.21 aM for miR-141 and demonstrates a high specificity for practical applications. It may have wide applicability in nucleic acid sensing and shows great potential in disease diagnosis.

RevDate: 2023-11-16

Koller F, M Cieslak (2023)

A perspective from the EU: unintended genetic changes in plants caused by NGT-their relevance for a comprehensive molecular characterisation and risk assessment.

Frontiers in bioengineering and biotechnology, 11:1276226.

Several regions in the world are currently holding discussions in regard to the regulation of new genomic techniques (NGTs) and their application in agriculture. The European Commission, for instance, is proposing the introduction of specific regulation for NGT plants. Various questions need to be answered including e.g., the extent to which NGT-induced intended and unintended genetic modifications must be subjected to a mandatory risk assessment as part of an approval procedure. This review mostly focuses on findings in regard to unintended genetic changes that can be caused by the application of NGTs. More specifically, the review deals with the application of the nuclease CRISPR/Cas, which is currently the most important tool for developing NGT plants, and its potential to introduce double strand breaks (DSBs) at a targeted DNA sequence. For this purpose, we identified the differences in comparison to non-targeted mutagenesis methods used in conventional breeding. The review concludes that unintended genetic changes caused by NGT processes are relevant to risk assessment. Due to the technical characteristics of NGTs, the sites of the unintended changes, their genomic context and their frequency (in regard to specific sites) mean that the resulting gene combinations (intended or unintended) may be unlikely to occur with conventional methods. This, in turn, implies that the biological effects (phenotypes) can also be different and may cause risks to health and the environment. Therefore, we conclude that the assessment of intended as well as unintended genetic changes should be part of a mandatory comprehensive molecular characterisation and risk assessment of NGT plants that are meant for environmental releases or for market authorisation.

RevDate: 2023-11-16

Dhokane D, Shaikh A, Yadav A, et al (2023)

CRISPR-based bioengineering in microalgae for production of industrially important biomolecules.

Frontiers in bioengineering and biotechnology, 11:1267826.

Microalgae, as photosynthetic organisms, have the potential to produce biomolecules for use in food, feed, cosmetics, nutraceuticals, fuel, and other applications. Faster growth rates and higher protein and lipid content make microalgae a popular chassis for many industrial applications. However, challenges such as low productivity and high production costs have limited their commercialization. To overcome these challenges, bioengineering approaches such as genetic engineering, metabolic engineering, and synthetic biology have been employed to improve the productivity and quality of microalgae-based products. Genetic engineering employing genome editing tools like CRISPR/Cas allows precise and targeted genetic modifications. CRISPR/Cas systems are presently used to modify the genetic makeup of microalgae for enhanced production of specific biomolecules. However, these tools are yet to be explored explicitly in microalgae owing to some limitations. Despite the progress made in CRISPR-based bioengineering approaches, there is still a need for further research to optimize the production of microalgae-based products. This includes improving the efficiency of genome editing tools, understanding the regulatory mechanisms of microalgal metabolism, and optimizing growth conditions and cultivation strategies. Additionally, addressing the ethical, social, and environmental concerns associated with genetic modification of microalgae is crucial for the responsible development and commercialization of microalgae-based products. This review summarizes the advancements of CRISPR-based bioengineering for production of industrially important biomolecules and provides key considerations to use CRISPR/Cas systems in microalgae. The review will help researchers to understand the progress and to initiate genome editing experiments in microalgae.

RevDate: 2023-11-14

Labuschagne M (2023)

Biofortification to improve food security.

Emerging topics in life sciences pii:233741 [Epub ahead of print].

Crop biofortification has significantly progressed in the last few decades. The first biofortification success was quality protein maize, leading to double the amount of the essential amino acids lysine and tryptophan. This was followed by biofortification of staple crops such as maize, wheat, rice, legumes and cassava for nutrients such as Fe and Zn and provitamin A. These crops have reached millions of households, especially in the developing regions of the world. The development and release of these biofortified crops through conventional breeding generally took 8-10 years. To speed up the process, molecular markers, genome-wide association studies and genomic selection have been incorporated into breeding efforts. Genetic engineering has the potential to increase the efficiency of crop biofortification through multi-nutrient biofortification in a short timespan and to combine biofortification with climate resilience. Regulatory issues still prevent the dissemination of genetically modified crops in many countries. This could be overcome by CRISPR-Cas-mediated genome editing, as it seems that many countries will regulate products of genome editing less strictly than transgenic crops. Effective policies on national or regional level are needed for the sustainable production of biofortified crops. The availability of affordable quality biofortified seed and other inputs should be ensured through local seed systems, which will increase the production and adoption of biofortified crops. There is scope to expand the crops and the range of nutrients for biofortification. Genetic engineering should be combined with conventional breeding as a approach for future improvement of multi-nutrient crops.


RJR Experience and Expertise


Robbins holds BS, MS, and PhD degrees in the life sciences. He served as a tenured faculty member in the Zoology and Biological Science departments at Michigan State University. He is currently exploring the intersection between genomics, microbial ecology, and biodiversity — an area that promises to transform our understanding of the biosphere.


Robbins has extensive experience in college-level education: At MSU he taught introductory biology, genetics, and population genetics. At JHU, he was an instructor for a special course on biological database design. At FHCRC, he team-taught a graduate-level course on the history of genetics. At Bellevue College he taught medical informatics.


Robbins has been involved in science administration at both the federal and the institutional levels. At NSF he was a program officer for database activities in the life sciences, at DOE he was a program officer for information infrastructure in the human genome project. At the Fred Hutchinson Cancer Research Center, he served as a vice president for fifteen years.


Robbins has been involved with information technology since writing his first Fortran program as a college student. At NSF he was the first program officer for database activities in the life sciences. At JHU he held an appointment in the CS department and served as director of the informatics core for the Genome Data Base. At the FHCRC he was VP for Information Technology.


While still at Michigan State, Robbins started his first publishing venture, founding a small company that addressed the short-run publishing needs of instructors in very large undergraduate classes. For more than 20 years, Robbins has been operating The Electronic Scholarly Publishing Project, a web site dedicated to the digital publishing of critical works in science, especially classical genetics.


Robbins is well-known for his speaking abilities and is often called upon to provide keynote or plenary addresses at international meetings. For example, in July, 2012, he gave a well-received keynote address at the Global Biodiversity Informatics Congress, sponsored by GBIF and held in Copenhagen. The slides from that talk can be seen HERE.


Robbins is a skilled meeting facilitator. He prefers a participatory approach, with part of the meeting involving dynamic breakout groups, created by the participants in real time: (1) individuals propose breakout groups; (2) everyone signs up for one (or more) groups; (3) the groups with the most interested parties then meet, with reports from each group presented and discussed in a subsequent plenary session.


Robbins has been engaged with photography and design since the 1960s, when he worked for a professional photography laboratory. He now prefers digital photography and tools for their precision and reproducibility. He designed his first web site more than 20 years ago and he personally designed and implemented this web site. He engages in graphic design as a hobby.

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By delivering the Cas9 nuclease, complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be precisely cut at any desired location, allowing existing genes to be removed and/or new ones added. That is, the CRISPR-Cas system provides a tool for the cut-and-paste editing of genomes. Welcome to the brave new world of genome editing. R. Robbins

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Collection of publications by R J Robbins

Reprints and preprints of publications, slide presentations, instructional materials, and data compilations written or prepared by Robert Robbins. Most papers deal with computational biology, genome informatics, using information technology to support biomedical research, and related matters.

Research Gate page for R J Robbins

ResearchGate is a social networking site for scientists and researchers to share papers, ask and answer questions, and find collaborators. According to a study by Nature and an article in Times Higher Education , it is the largest academic social network in terms of active users.

Curriculum Vitae for R J Robbins

short personal version

Curriculum Vitae for R J Robbins

long standard version

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