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05 Jun 2023 at 01:39
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Bibliography on: CRISPR-Cas


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RJR: Recommended Bibliography 05 Jun 2023 at 01:39 Created: 


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: 2023-05-31

Garrett SC, Philippe C, Kim JG, et al (2023)

Investigation of CRISPR-Independent Phage Resistance Mechanisms Reveals a Role for FtsH in Phage Adsorption to Streptococcus thermophilus.

Journal of bacteriology [Epub ahead of print].

Prokaryotes are under constant pressure from phage infection and thus have evolved multiple means of defense or evasion. While CRISPR-Cas constitutes a robust immune system and appears to be the predominant means of survival for Streptococcus thermophilus when facing lytic phage infection, other forms of phage resistance coexist in this species. Here, we show that S. thermophilus strains with deleted CRISPR-Cas loci can still give rise to phage-resistant clones following lytic phage challenge. Notably, non-CRISPR phage-resistant survivors had multiple mutations which would truncate or recode a membrane-anchored host protease, FtsH. Phage adsorption was dramatically reduced in FtsH mutants, implicating this protein in phage attachment. Phages were isolated which could bypass FtsH-based resistance through mutations predicted to alter tape measure protein translation. Together, these results identify key components in phage propagation that are subject to mutation in the molecular arms race between phage and host cell. IMPORTANCE Streptococcus thermophilus is an important organism for production of cultured dairy foods, but it is susceptible to lytic phages which can lead to failed products. Consequently, mechanisms for phage resistance are an active area of research. One such mechanism is CRISPR-Cas, and S. thermophilus is a model organism for the study of this form of adaptive immunity. Here, we expand on known mechanisms with our finding that spontaneous mutations in ftsH, a gene encoding a membrane-anchored protease, protected against phage infection by disrupting phage adsorption. In turn, mutations in phage tail protein genes allowed phages to overcome ftsH-based resistance. Our results identified components in phage propagation that are subject to mutation in the molecular arms race between phage and host.

RevDate: 2023-05-31

Gehlert FO, Nickel L, Vakirlis N, et al (2023)

Active in vivo translocation of the Methanosarcina mazei Gö1 Casposon.

Nucleic acids research pii:7186994 [Epub ahead of print].

Casposons are transposable elements containing the CRISPR associated gene Cas1solo. Identified in many archaeal genomes, casposons are discussed as the origin of CRISPR-Cas systems due to their proposed Cas1solo-dependent translocation. However, apart from bioinformatic approaches and the demonstration of Cas1solo integrase and endonuclease activity in vitro, casposon transposition has not yet been shown in vivo. Here, we report on active casposon translocations in Methanosarcina mazei Gö1 using two independent experimental approaches. First, mini-casposons, consisting of a R6Kγ origin and two antibiotic resistance cassettes, flanked by target site duplications (TSDs) and terminal inverted repeats (TIRs), were generated, and shown to actively translocate from a suicide plasmid and integrate into the chromosomal MetMaz-C1 TSD IS1a. Second, casposon excision activity was confirmed in a long-term evolution experiment using a Cas1solo overexpression strain in comparison to an empty vector control under four different treatments (native, high temperature, high salt, mitomycin C) to study stress-induced translocation. Analysis of genomic DNA using a nested qPCR approach provided clear evidence of casposon activity in single cells and revealed significantly different casposon excision frequencies between treatments and strains. Our results, providing the first experimental evidence for in vivo casposon activity are summarized in a modified hypothetical translocation model.

RevDate: 2023-05-31

Casas-Mollano JA, Zinselmeier MH, Sychla A, et al (2023)

Efficient gene activation in plants by the MoonTag programmable transcriptional activator.

Nucleic acids research pii:7186991 [Epub ahead of print].

CRISPR/Cas-based transcriptional activators have been developed to induce gene expression in eukaryotic and prokaryotic organisms. The main advantages of CRISPR/Cas-based systems is that they can achieve high levels of transcriptional activation and are very easy to program via pairing between the guide RNA and the DNA target strand. SunTag is a second-generation system that activates transcription by recruiting multiple copies of an activation domain (AD) to its target promoters. SunTag is a strong activator; however, in some species it is difficult to stably express. To overcome this problem, we designed MoonTag, a new activator that works on the same basic principle as SunTag, but whose components are better tolerated when stably expressed in transgenic plants. We demonstrate that MoonTag is capable of inducing high levels of transcription in all plants tested. In Setaria, MoonTag is capable of inducing high levels of transcription of reporter genes as well as of endogenous genes. More important, MoonTag components are expressed in transgenic plants to high levels without any deleterious effects. MoonTag is also able to efficiently activate genes in eudicotyledonous species such as Arabidopsis and tomato. Finally, we show that MoonTag activation is functional across a range of temperatures, which is promising for potential field applications.

RevDate: 2023-06-02
CmpDate: 2023-06-01

Fu H, Shan C, Kang F, et al (2023)

CRISPR-GRANT: a cross-platform graphical analysis tool for high-throughput CRISPR-based genome editing evaluation.

BMC bioinformatics, 24(1):219.

BACKGROUD: CRISPR/Cas is an efficient genome editing system that has been widely used for functional genetic studies and exhibits high potential in biomedical translational applications. Indel analysis has thus become one of the most common practices in the lab to evaluate DNA editing events generated by CRISPR/Cas. Several indel analysis tools have been reported, however, it is often required that users have certain bioinformatics training and basic command-line processing capability.

RESULTS: Here, we developed CRISPR-GRANT, a stand-alone graphical CRISPR indel analysis tool, which could be easily installed for multi-platforms, including Linux, Windows, and macOS. CRISPR-GRANT offered a straightforward GUI by simple click-and-run for genome editing analysis of single or pooled amplicons and one-step analysis for whole-genome sequencing without the need of data pre-processing, making it ideal for novice lab scientists. Moreover, it also exhibited shorter run-time compared with tools currently available.

CONCLUSION: Therefore, CRISPR-GRANT is a valuable addition to the current CRISPR toolkits that significantly lower the barrier for wet-lab researchers to conduct indel analysis from large NGS datasets. CRISPR-GRANT binaries are freely available for Linux (above Ubuntu 16.04), macOS (above High Sierra 10.13) and Windows (above Windows 7) at https://github.com/fuhuancheng/CRISPR-GRANT . CRISPR-GRANT source code is licensed under the GPLv3 license and free to download and use.

RevDate: 2023-05-30

Lahm H, Dzilic E, Neb I, et al (2023)

Correction of a deleterious TBX5 mutation in an induced pluripotent stem cell line (DHMi004-A-1) using a completely plasmid-free CRISPR/Cas 9 approach.

Stem cell research, 70:103126 pii:S1873-5061(23)00112-5 [Epub ahead of print].

TBX5 is a transcription factor which plays an essential role at different checkpoints during cardiac differentiation. However, regulatory pathways affected by TBX5 still remain ill-defined. We have applied the CRISPR/Cas9 technology using a completely plasmid-free approach to correct a heterozygous causative "loss-of function" TBX5 mutation in an iPSC line (DHMi004-A), that has been established from a patient suffering from Holt-Oram syndrome (HOS). This isogenic iPSC line, DHMi004-A-1, represents a powerful in vitro tool to dissect the regulatory pathways affected by TBX5 in HOS.

RevDate: 2023-05-30

Li Q, Song ZL, Zhang Y, et al (2023)

Synergistic Incorporation of Two ssDNA Activators Enhances the Trans-Cleavage of CRISPR/Cas12a.

Analytical chemistry [Epub ahead of print].

CRISPR/Cas12a has been believed to be powerful in molecular detection and diagnostics due to its amplified trans-cleavage feature. However, the activating specificity and multiple activation mechanisms of the Cas12a system are yet to be elucidated fully. Herein, a "synergistic activator effect" is discovered, which supports an activation mechanism that a synergistic incorporation of two short ssDNA activators can promote the trans-cleavage of CRISPR/Cas12a, while either of them is too short to work independently. As a proof-of-concept example, the synergistic activator-triggered CRISPR/Cas12a system has been successfully harnessed in the AND logic operation and the discrimination of single-nucleotide variants, requiring no signal conversion elements or other amplified enzymes. Moreover, a single-nucleotide specificity has been achieved for the detection of single-nucleotide variants by pre-introducing a synthetic mismatch between crRNA and the "helper" activator. The finding of "synergistic activator effect" not only provides deeper insight into CRISPR/Cas12a but also may facilitate its expanded application and power the exploration of the undiscovered properties of other CRISPR/Cas systems.

RevDate: 2023-06-02
CmpDate: 2023-06-01

Zhang JX, Xu JH, Yuan B, et al (2023)

Detection of Burkholderia pseudomallei with CRISPR-Cas12a based on specific sequence tags.

Frontiers in public health, 11:1153352.

Melioidosis is a bacterial infection caused by Burkholderia pseudomallei (B. pseudomallei), posing a significant threat to public health. Rapid and accurate detection of B. pseudomallei is crucial for preventing and controlling melioidosis. However, identifying B. pseudomallei is challenging due to its high similarity to other species in the same genus. To address this issue, this study proposed a dual-target method that can specifically identify B. pseudomallei in less than 40 min. We analyzed 1722 B. pseudomallei genomes to construct large-scale pan-genomes and selected specific sequence tags in their core genomes that effectively distinguish B. pseudomallei from its closely related species. Specifically, we selected two specific tags, LC1 and LC2, which we combined with the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated proteins (Cas12a) system and recombinase polymerase amplification (RPA) pre-amplification. Our analysis showed that the dual-target RPA-CRISPR/Cas12a assay has a sensitivity of approximately 0.2 copies/reaction and 10 fg genomic DNA for LC1, and 2 copies/reaction and 20 fg genomic DNA for LC2. Additionally, our method can accurately and rapidly detect B. pseudomallei in human blood and moist soil samples using the specific sequence tags mentioned above. In conclusion, the dual-target RPA-CRISPR/Cas12a method is a valuable tool for the rapid and accurate identification of B. pseudomallei in clinical and environmental samples, aiding in the prevention and control of melioidosis.

RevDate: 2023-06-02
CmpDate: 2023-06-02

Lahm H, Stieglbauer S, Neb I, et al (2023)

Generation of three CRISPR/Cas9 edited human induced pluripotent stem cell lines (DHMi005-A-5, DHMi005-A-6 and DHMi005-A-7) carrying a Holt-Oram Syndrome patient-specific TBX5 mutation with known cardiac phenotype and a FLAG-tag after exon 9 of the TBX5 gene.

Stem cell research, 69:103123.

TBX5 is a transcription factor (TF) playing essential role during cardiogenesis. It is well known that TF mutations possibly result in non- or additional binding of the DNA due to conformational changes of the protein. We introduced a Holt-Oram Syndrome (HOS) patient-specific TBX5 mutation c.920_C > A heterozygously in a healthy induced pluripotent stell cell (iPSC) line. This TBX5 mutation results in conformational changes of the protein and displayed ventricular septal defects in the patient itself. Additionally we introduced a FLAG-tag on the TBX5 mutation-carrying allele. The resulting heterozygous TBX5-FLAG iPSC lines are a powerful tool to investigate altered TF activity bonding.

RevDate: 2023-06-02
CmpDate: 2023-06-02

Luo X, Yang K, Jiang M, et al (2023)

Generation of a homozygous GRIN2A gene knockout human embryonic stem cell line using CRISPR/Cas9 system.

Stem cell research, 69:103121.

Schizophrenia is a group of common psychosis of unknown etiology and GRIN2A gene has been a risk gene for schizophrenia. In order to understand the relationship between the GRIN2A and schizophrenia, we generated a GRIN2A-KO human embryonic stem cell line by CRISPR/Cas9 system, which could provide a valuable resource for investing pathogenic mechanisms underlying schizophrenia and facilitating the development of targeted medicine.

RevDate: 2023-06-02
CmpDate: 2023-06-02

Sun R, Cui Y, Liu Z, et al (2023)

A prime editor efficiently repaired human induced pluripotent stem cells with AR gene mutation (c.2710G > A; p. V904M).

Stem cell research, 69:103102.

Prime Editor (PE) is a precise genome manipulation technology based on the CRISPR-Cas9 system, while its application in human induced pluripotent stem cells (iPSCs) remains limited. Here, we established a repaired hiPS cell line (SKLRMi001-A-1) from hiPSCs with androgen receptor (AR) mutation (c.2710G > A; p.V904M). The repaired iPSC line expressed pluripotency markers, retained normal karyotype, showed the capability of differentiating into three germ layers and was absence of mycoplasma infection. The repaired iPSC line will help to elucidate the mechanism of androgen insensitivity syndrome (AIS) and benefit treatment for AIS in the future.

RevDate: 2023-06-02
CmpDate: 2023-06-02

Merkert S, Haase A, Dahlmann J, et al (2023)

Generation of two human NRF2 knockout iPSC clones using CRISPR/Cas9 editing.

Stem cell research, 69:103090.

The nuclear factor erythroid 2-related factor 2 (NFE2L2, known as NRF2) regulates the expression of antioxidative and anti-inflammatory proteins. In order to investigate its impact during viral infections and testing of antiviral compounds, we applied CRISPR/Cas9 editing to eliminate NRF2 in the human iPS cell line MHHi001-A and generated two NRF2 knockout iPSC clones MHHi001-A-6 and MHHi001-A-7. After differentiation into epithelia or endothelial cells, these cells are useful tools to examine the antiviral effects of activators of the NRF2 signaling pathway.

RevDate: 2023-06-02
CmpDate: 2023-06-02

Yılmaz VM, Ramnarine TJS, Königer A, et al (2023)

Tropical super flies: Integrating Cas9 into Drosophila ananassae and its phenotypic effects.

Journal of insect physiology, 147:104516.

Ectotherms such as insects are animals whose body temperature largely depends on ambient temperature and temperature variations provide a selection pressure affecting the geographical distribution of these species. However, over the course of evolution, some insect species managed to colonize environments characterized by various temperature ranges. Therefore, insects provide an excellent study system to investigate the basis of adaptation to temperature changes and extremes. We are generally using the vinegar fly Drosophila ananassae as a model system to investigate the genetic basis of cold tolerance. This species has expanded from its tropical ancestral range to more temperate regions resulting in a cosmopolitan, domestic distribution. Previously, we identified candidate genes significantly associated with cold tolerance in this species. We now established molecular genetic tools to assess the function of these genes. Using CRISPR/Cas9 methodology for genome editing and the PiggyBac system, the Cas9 enzyme was successfully integrated into the genome of three fly strains with different levels of cold tolerance. We further report on preliminary findings that the Cas9 integration itself did not have a consistent effect on tolerance to cold. In conclusion, we offer with our study the molecular tools that allow studying stress-related candidate genes in D. ananassae in the future. In addition, we point out and provide guidance on the challenges that come with genome editing in a non-model species.

RevDate: 2023-06-02
CmpDate: 2023-06-02

Rädecke K, Gore A, Burau K, et al (2023)

Generation of two homozygous SHOX2 knock-out human induced pluripotent stem cell lines using CRISPR/Cas9.

Stem cell research, 69:103089.

SHOX2 is a homeobox transcription factor associated with atrial fibrillation (AF) and sinus node dysfunction. Here, we generated two homozygous SHOX2 knock-out hiPSC lines from a healthy control line and a corrected AF patient line (disease-specific SHOX2 mutation corrected to WT) using CRISPR/Cas9. These cell lines maintained pluripotency, an ability to differentiate into all three germlayers and a normal karyotype, presenting a valuable tool to investigate the impact of a full SHOX2 knock-out with respect to arrhythmogenic diseases on a cellular level.

RevDate: 2023-06-02
CmpDate: 2023-06-02

Jo S, Kim JW, Noh H, et al (2023)

Generation of a PDGFRB-mCherry knock-in reporter human induced pluripotent stem cell line (KITi001-A-1), using CRISPR/Cas9 nuclease.

Stem cell research, 69:103081.

PDGFRB encodes platelet-derived growth factor receptor beta (PDGFR-β), a cell surface tyrosine kinase receptor for members of the platelet-derived growth factor family. It is required for the normal development of the vascular and nervous systems and rearrangement of the actin cytoskeleton. PDGFR-β plays an essential role in early liver diseases, including liver fibrosis. Here, we generated a human induced pluripotent stem cell (iPSC) line, KITi001-A-1, using CRISPR/Cas9. This reporter iPSC line and its derivatives are useful for tracing PDGFR-β-expressing cells and for screening for liver fibrosis-inducing compounds.

RevDate: 2023-06-02
CmpDate: 2023-06-02

Wang X, Sun J, Fu C, et al (2023)

Generation of a PPP1CA knockout human pluripotent stem cell line via CRISPR/Cas9.

Stem cell research, 69:103077.

The Protein phosphatase 1 catalytic subunit alpha (PPP1CA) is a alpha subunit of the PP1 complex, which known to be involved in the regulation of a variety of cellular processes, such as cell division, glycogen metabolism, muscle contractility, protein synthesis, and HIV-1 viral transcription. Increased PP1 activity has been observed in the end stage of heart failure. Here, a PPP1CA knockout human embryonic stem cell line, WAe009-A-B, was generated using the CRISPR/Cas9 system to further study the function of PPP1CA. The cell line confirmed with pluripotency, normal karyotype and differentiation potential.

RevDate: 2023-06-02
CmpDate: 2023-06-02

Wu X, X Wang (2023)

Generation of a TBX20-knockout human embryonic stem line by CRISPR/Cas9 system.

Stem cell research, 69:103082.

The TBX20 gene plays a crucial role in embryonic development and has been involved in various diseases, such as heart defects, intellectual disability, and cancer. Herein, we have established a TBX20-knockout human embryonic stem cell line (WAe009-A-84) that maintains stem cell-like features, pluripotency, a normal karyotype, and the ability to differentiate into all three germ layers in vivo. This cell line will be a valuable resource for exploring TBX20's role in human development and could have significant implications for regenerative medicine and disease modeling.

RevDate: 2023-06-02
CmpDate: 2023-06-02

Schmitz AS, Korneck M, Raju J, et al (2023)

Generation of a heterozygous and a homozygous CSF1R knockout line from iPSC using CRISPR/Cas9.

Stem cell research, 69:103066.

Mutations in Colony-stimulating factor 1 receptor (CSF1R) lead to CSF1R-related leukoencephalopathy, also known as Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP), a rapidly progressing neurodegenerative disease with severe cognitive and motor impairment. In this study, a homozygous and a heterozygous CSF1R knockout induced pluripotent stem cell (iPSC) line were generated by CRISPR/Cas9-based gene editing. These in vitro models will provide a helpful tool for investigating the still largely unknown pathophysiology of CSF1R-related leukoencephalopathy.

RevDate: 2023-06-02
CmpDate: 2023-06-02

Schuurmans IME, Wu KM, van Karnebeek CDM, et al (2023)

Generation of an induced pluripotent stem cell line carrying a biallelic deletion (SCTCi019-A) in GCDH using CRISPR/Cas9.

Stem cell research, 69:103069.

GCDH encodes for the enzyme catalyzing the sixth step of the lysine catabolism pathway. Biallelic pathogenic variants in GCDH have been associated with glutaric aciduria type 1 (GA1). In this study CRISPR/Cas9 technology was used to create an isogenic GCDH knock-out human iPSC line. One clone with a biallelic deletion (SCTCi019-A) in GCDH was obtained and fully characterized, revealing a normal karyotype, no off-targets detected and expression of pluripotency markers. This iPSC line can contribute to gain insights in the molecular mechanism of disease.

RevDate: 2023-06-02
CmpDate: 2023-06-02

Kim AH, Lee HM, Kim HS, et al (2023)

Establishment of TUBB3-mCherry knock-in human pluripotent stem cell line using CRISPR/Cas9 (SNUe003-A-4).

Stem cell research, 69:103064.

TUBB3 is a structural neuronal protein important for multiple neuronal functions including axonal guidance and maturation. This study aimed to generate a human pluripotent stem cell (hPSC) line with a TUBB3-mCherry reporter using CRISPR/SpCas9 nuclease. The stop codon in the last exon of TUBB3 was replaced with a T2A-mCherry cassette using CRISPR/SpCas9-mediated homologous recombination. The established TUBB3-mCherry knock-in cell line exhibited typical pluripotent characteristics. The mCherry reporter faithfully replicated the endogenous level of TUBB3 upon induction of neuronal differentiation. The reporter cell line could contribute to the investigation of neuronal differentiation, neuronal toxicity, and neuronal tracing.

RevDate: 2023-06-02
CmpDate: 2023-06-02

Shi H, Doench JG, H Chi (2023)

CRISPR screens for functional interrogation of immunity.

Nature reviews. Immunology, 23(6):363-380.

CRISPR-based technologies represent a major breakthrough in biomedical science as they offer a powerful platform for unbiased screening and functional genomics in various fields, including immunology. Pooled and arrayed CRISPR screens have uncovered previously unknown intracellular drivers in innate and adaptive immune cells for immune regulation as well as intercellular regulators mediating cell-cell interactions. Recent single-cell CRISPR screening platforms expand the readouts to the transcriptome and enable the inference of gene regulatory networks for better mechanistic insights. CRISPR screens also allow for mapping of genetic interactions to identify genes that synergize or alleviate complex immune phenotypes. Here, we review the progress in and emerging adaptation of CRISPR technologies to advance our fundamental immunological knowledge and identify novel disease targets for immunotherapy of infection, inflammation and cancer.

RevDate: 2023-06-02
CmpDate: 2023-06-02

Shao H, Jian J, Peng D, et al (2023)

Recombinase Polymerase Amplification Coupled with CRISPR-Cas12a Technology for Rapid and Highly Sensitive Detection of Heterodera avenae and Heterodera filipjevi.

Plant disease, 107(5):1365-1376.

The cereal cyst nematodes Heterodera avenae and Heterodera filipjevi are recognized as cyst nematodes that infect cereal crops and cause severe economic losses worldwide. Rapid, visual detection of cyst nematodes is essential for more effective control of this pest. In this study, recombinase polymerase amplification (RPA) combined with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a (formerly known as cpf1) was developed for the rapid detection of H. avenae and H. filipjevi from infested field samples. The RPA reaction was performed at a wide range of temperatures from 35 to 42°C within 15 min. There was no cross-reactivity between H. avenae, H. filipjevi, and the common closely related plant-parasitic nematodes, indicating the high specificity of this assay. The detection limit of RPA-Cas12a was as low as 10[-4] single second-stage juvenile (J2), 10[-5] single cyst, and 0.001 ng of genomic DNA, which is 10 times greater than that of RPA-lateral flow dipstick (LFD) detection. The RPA-Cas12a assay was able to detect 10[-1] single J2 of H. avenae and H. filipjevi in 10 g of soil. In addition, the RPA-LFD assay and RPA-Cas12a assays could both quickly detect H. avenae and H. filipjevi from naturally infested soil, and the entire detection process could be completed within 1 h. These results indicated that the RPA-Cas12a assay developed herein is a simple, rapid, specific, sensitive, and visual method that can be easily adapted for the quick detection of H. avenae and H. filipjevi in infested fields.

RevDate: 2023-06-01
CmpDate: 2023-06-01

Qi Q, Liu X, Fu F, et al (2023)

Utilizing Epigenetic Modification as a Reactive Handle To Regulate RNA Function and CRISPR-Based Gene Regulation.

Journal of the American Chemical Society, 145(21):11678-11689.

The current methods to control RNA functions in living conditions are limited. The new RNA-controlling strategy presented in this study involves utilizing 5-formylcytidine (f5C)-directed base manipulation. This study shows that malononitrile and pyridine boranes can effectively manipulate the folding, small molecule binding, and enzyme recognition of f5C-bearing RNAs. We further demonstrate the efficiency of f5C-directed reactions in controlling two different clustered regularly interspaced short palindromic repeat (CRISPR) systems. Although further studies are needed to optimize the efficiency of these reactions in vivo, this small molecule-based approach presents exciting new opportunities for regulating CRISPR-based gene expression and other applications.

RevDate: 2023-06-01
CmpDate: 2023-06-01

Chen X, Huang C, Hu Q, et al (2023)

Sensing platform for nucleic-acid detection based on a 2-aminopurine probe sheared by trans-cleavage activity of the CRISPR/Cas12a system.

The Analyst, 148(11):2482-2492.

Target double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) can activate the trans-cleavage activity of the CRISPR/Cas12a, cutting the surrounding non-target ssDNA arbitrarily. In a typical CRISPR/Cas12a system, this non-target ssDNA, with a fluorescent tag and its quencher incorporated at both ends (ssDNA-FQ), is usually used as the reporter. Here, a 2-aminopurine probe (T-pro 4), made by inserting four 2-APs in non-target ssDNA, was screened for using as a reporter in the CRISPR/Cas12a system. Compared with ssDNA-FQ, each 2-AP probe is cleaved by the activated CRISPR/Cas12a system, multi-unit signals are generated. Therefore, the CRISPR/Cas12a system using the 2-AP probe as a reporter may be more sensitive than the CRISPR/Cas12a system which uses ssDNA-FQ as the reporter. We achieved ssDNA detection at as little as 10[-11] M using the 2-AP probe as the reporter in the CRISPR/Cas12a system. Compared to the CRISPR/Cas12a system using ssDNA-FQ as the reporter, its sensitivity increased by an order of magnitude. Furthermore, the method that combines PCR and the 2-AP-probe-mediated CRISPR/Cas12a system can detect goat pox virus (GTPV) down to 8.35 × 10[-2] copies per μL, 10 times lower than the method that combines PCR and the ssDNA-FQ-mediated CRISPR/Cas12a system. These results indicate that the CRISPR/Cas12a system using the screened 2-AP probe as a reporter has potential in highly sensitive detection of viruses.

RevDate: 2023-06-01
CmpDate: 2023-06-01

Shen P, Si Z, Huang D, et al (2023)

CRISPR Cas12a-enabled biosensors coupled with commercial pregnancy test strips for the visible point-of-care testing of SARS-CoV-2.

The Analyst, 148(11):2573-2581.

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has promoted the development of nucleic acid diagnosis technology. Several platforms with isothermal amplification methods have achieved sensitive and specific detection of SARS-CoV-2. However, they still suffer from complicated operations, delicate instruments, and unintuitive signal output modes. Here, a system consisting of CRISPR Cas12a-based biosensors and commercial pregnancy test strips (CRISPR-PTS) was established for the point-of-care testing of SARS-CoV-2. The target viral nucleic acids were finally reflected on the test strips through four steps, namely sample pretreatment, RT-RAA amplification, CRISPR Cas12a reaction, and separation-free hCG detection. This CRISPR-PTS assay possessed an outstanding sensitivity of as low as 1 copy per μL for SARS-CoV-2 detection and showed an excellent specificity in distinguishing the SARS-CoV-2 pseudovirus as well as other SARS-like viral clinical samples. In addition, the CRISPR-PTS assay performed well in practical applications, with 96.3% agreement versus RT-qPCR in spiked samples. With the advantages of low reagent cost, simple operation procedure, and visible signal output, CRISPR-PTS assay was expected to provide a strong supplement in the prevention and early diagnosis of infectious diseases in resource-limited situations.

RevDate: 2023-06-01
CmpDate: 2023-06-01

Deng F, Pan J, Chen M, et al (2023)

Integrating CRISPR-Cas12a with catalytic hairpin assembly as a logic gate biosensing platform for the detection of polychlorinated biphenyls in water samples.

The Science of the total environment, 881:163465.

Polychlorinated biphenyls (PCBs) are ubiquitous persistent organic pollutants that cause harmful effects on environmental safety and human health. There is an urgent need to develop an intelligent method for PCBs sensing. In this work, we proposed a logic gate biosensing platform for simultaneous detection of multiple PCBs. 2,3',5,5'-tetrachlorobiphenyl (PCB72) and 3,3',4,4'-tetrachlorobiphenyl (PCB77) were used as the two inputs to construct biocomputing logic gates. We used 0 and 1 to encode the inputs and outputs. The aptamer was used to recognize the inputs and release the trigger DNA. A catalytic hairpin assembly (CHA) module is designed to convert and amplify each trigger DNA into multiple programmable DNA duplexes, which initiate the trans-cleavage activity of CRISPR/Cas12a for the signal output. The activated Cas12 cleaves the BHQ-Cy5 modified single-stranded DNA (ssDNA) to yield the fluorescence reporting signals. In the YES logic gate, PCB72 was used as the only input to carry out the logic operation. In the OR, AND, and INHIBIT logic gates, PCB72 and PCB77 were used as the two inputs. The output signals can be visualized by the naked eye under UV light transilluminators or quantified by a microplate reader. Our constructed biosensing platform possesses the merits of multiple combinations of inputs, intuitive digital output, and high flexibility and scalability, which holds great promise for the intelligent detection of different PCBs.

RevDate: 2023-05-31

Mipeshwaree Devi A, Khedashwori Devi K, Premi Devi P, et al (2023)

Metabolic engineering of plant secondary metabolites: prospects and its technological challenges.

Frontiers in plant science, 14:1171154.

Plants produce a wide range of secondary metabolites that play vital roles for their primary functions such as growth, defence, adaptations or reproduction. Some of the plant secondary metabolites are beneficial to mankind as nutraceuticals and pharmaceuticals. Metabolic pathways and their regulatory mechanism are crucial for targeting metabolite engineering. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated system has been widely applied in genome editing with high accuracy, efficiency, and multiplex targeting ability. Besides its vast application in genetic improvement, the technique also facilitates a comprehensive profiling approach to functional genomics related to gene discovery involved in various plant secondary metabolic pathways. Despite these wide applications, several challenges limit CRISPR/Cas system applicability in genome editing in plants. This review highlights updated applications of CRISPR/Cas system-mediated metabolic engineering of plants and its challenges.

RevDate: 2023-05-31

Marín-Sanz M, Barro F, S Sánchez-León (2023)

Unraveling the celiac disease-related immunogenic complexes in a set of wheat and tritordeum genotypes: implications for low-gluten precision breeding in cereal crops.

Frontiers in plant science, 14:1171882.

The development of low-gluten immunogenic cereal varieties is a suitable way to fight the increment of pathologies associated with the consumption of cereals. Although RNAi and CRISPR/Cas technologies were effective in providing low-gluten wheat, the regulatory framework, particularly in the European Union, is an obstacle to the short- or medium-term implementation of such lines. In the present work, we carried out a high throughput amplicon sequencing of two highly immunogenic complexes of wheat gliadins in a set of bread and durum wheat, and tritordeum genotypes. Bread wheat genotypes harboring the 1BL/1RS translocation were included in the analysis and their amplicons successfully identified. The number of CD epitopes and their abundances were determined in the alpha- and gamma-gliadin amplicons, including 40k-γ-secalin ones. Bread wheat genotypes not containing the 1BL/1RS translocation showed a higher average number of both alpha- and gamma-gliadin epitopes than those containing such translocation. Interestingly, alpha-gliadin amplicons not containing CD epitopes accounted for the highest abundance (around 53%), and the alpha- and gamma-gliadin amplicons with the highest number of epitopes were present in the D-subgenome. The durum wheat and tritordeum genotypes showed the lowest number of alpha- and gamma-gliadin CD epitopes. Our results allow progress in unraveling the immunogenic complexes of alpha- and gamma-gliadins and can contribute to the development of low-immunogenic varieties within precision breeding programs, by crossing or by CRISPR/Cas gene editing.

RevDate: 2023-05-31

Subedi U, Burton Hughes K, Chen G, et al (2023)

Eliciting Targeted Mutations in Medicago sativa Using CRISPR/Cas9-Mediated Genome Editing: A Potential Tool for the Improvement of Disease Resistance.

Methods in molecular biology (Clifton, N.J.), 2659:219-239.

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) has become a breeding tool of choice for eliciting targeted genetic alterations in crop species as a means of improving a wide range of agronomic traits, including disease resistance, in recent years. With the recent development of CRISPR/Cas9 technology in Medicago sativa (alfalfa), which is an important perennial forage legume grown worldwide, its use for the enhancement of pathogen resistance is almost certainly on the horizon. In this chapter, we present detailed procedures for the generation of a single nonhomologous end-joining-derived indel at a precise genomic locus of alfalfa via CRISPR/Cas9. This method encompasses crucial steps in this process, including guide RNA design, binary CRISPR vector construction, Agrobacterium-mediated transformation of alfalfa explants, and molecular assessments of transformed genotypes for transgene and edit identification.

RevDate: 2023-05-31
CmpDate: 2023-05-31

Cai B, Chang S, Tian Y, et al (2023)

CRISPR/Cas9 for hepatitis B virus infection treatment.

Immunity, inflammation and disease, 11(5):e866.

Hepatitis B virus (HBV) infection remains a global health challenge. Despite the availability of effective preventive vaccines, millions of people are at risk of cirrhosis and hepatocellular carcinoma. Current drug therapies inhibit viral replication, slow the progression of liver fibrosis and reduce infectivity, but they rarely remove the covalently sealed circular DNA (cccDNA) of the virus that causes HBV persistence. Alternative treatment strategies, including those based on CRISPR/cas9 knockout virus gene, can effectively inhibit HBV replication, so it has a good prospect. During chronic infection, some virus gene knockouts based on CRISPR/cas9 may even lead to cccDNA inactivation. This paper reviews the progress of different HBV CRISPR/cas9, vectors for delivering to the liver, and the current situation of preclinical and clinical research.

RevDate: 2023-05-31
CmpDate: 2023-05-31

Mabuchi A, Hata S, Genova M, et al (2023)

ssDNA is not superior to dsDNA as long HDR donors for CRISPR-mediated endogenous gene tagging in human diploid RPE1 and HCT116 cells.

BMC genomics, 24(1):289.

BACKGROUND: Recent advances in CRISPR technology have enabled us to perform gene knock-in in various species and cell lines. CRISPR-mediated knock-in requires donor DNA which serves as a template for homology-directed repair (HDR). For knock-in of short sequences or base substitutions, ssDNA donors are frequently used among various other forms of HDR donors, such as linear dsDNA. However, partly due to the complexity of long ssDNA preparation, it remains unclear whether ssDNA is the optimal type of HDR donors for insertion of long transgenes such as fluorescent reporters in human cells.

RESULTS: In this study, we established a nuclease-based simple method for the preparation of long ssDNA with high yield and purity, and comprehensively compared the performance of ssDNA and dsDNA donors with 90 bases of homology arms for endogenous gene tagging with long transgenes in human diploid RPE1 and HCT116 cells. Quantification using flow cytometry revealed lower efficiency of endogenous fluorescent tagging with ssDNA donors than with dsDNA. By analyzing knock-in outcomes using long-read amplicon sequencing and a classification framework, a variety of mis-integration events were detected regardless of the donor type. Importantly, the ratio of precise insertion was lower with ssDNA donors than with dsDNA. Moreover, in off-target integration analyses using donors without homology arms, ssDNA and dsDNA were comparably prone to non-homologous integration.

CONCLUSIONS: These results indicate that ssDNA is not superior to dsDNA as long HDR donors with relatively short homology arms for gene knock-in in human RPE1 and HCT116 cells.

RevDate: 2023-05-31
CmpDate: 2023-05-31

Iqbal Z, Rehman K, Xia J, et al (2023)

Biomaterial-assisted targeted and controlled delivery of CRISPR/Cas9 for precise gene editing.

Biomaterials science, 11(11):3762-3783.

RISPR-Cas9 has exhibited enormous potential in gene therapy. It can perform genome editing with single-nucleotide precision in various types of cell and tissue, providing a powerful breakthrough technology for genome editing in therapeutic development. But the limited delivery methods pose substantial challenges pertinent to safe and effective CRISPR/Cas9 delivery, thus hindering its application. These challenges should be tackled to develop next-generation genetic therapies. Biomaterial-based drug delivery systems can overcome these issues, for example using biomaterials as carriers for CRISPR/Cas9 targeted delivery, and conditional control of its function can improve precision, furnish on-demand and transient gene editing and reduce adverse consequences such as off-target events and immunogenicity, representing a promising direction for modern precision medicine. This review describes the application status and research progress of current CRISPR/Cas9 delivery approaches, including polymeric nanoparticles, liposomes, extracellular vesicles, inorganic nanoparticles and hydrogels. The unique properties of light-controlled and small-molecule drugs for spatially and temporally controlled genome editing are also illustrated. In addition, targetable delivery vehicles for the active delivery of CRISPR systems are also discussed. The perspectives to overcome the current limitations in the CRISPR/Cas9 delivery and their bench-to-bedside translation are also highlighted.

RevDate: 2023-05-30
CmpDate: 2023-05-29

Zein-Eddine R, Refrégier G, Cervantes J, et al (2023)

The future of CRISPR in Mycobacterium tuberculosis infection.

Journal of biomedical science, 30(1):34.

Clustered Regularly Interspaced Short Palindromic repeats (CRISPR)-Cas systems rapidly raised from a bacterial genetic curiosity to the most popular tool for genetic modifications which revolutionized the study of microbial physiology. Due to the highly conserved nature of the CRISPR locus in Mycobacterium tuberculosis, the etiological agent of one of the deadliest infectious diseases globally, initially, little attention was paid to its CRISPR locus, other than as a phylogenetic marker. Recent research shows that M. tuberculosis has a partially functional Type III CRISPR, which provides a defense mechanism against foreign genetic elements mediated by the ancillary RNAse Csm6. With the advent of CRISPR-Cas based gene edition technologies, our possibilities to explore the biology of M. tuberculosis and its interaction with the host immune system are boosted. CRISPR-based diagnostic methods can lower the detection threshold to femtomolar levels, which could contribute to the diagnosis of the still elusive paucibacillary and extrapulmonary tuberculosis cases. In addition, one-pot and point-of-care tests are under development, and future challenges are discussed. We present in this literature review the potential and actual impact of CRISPR-Cas research on human tuberculosis understanding and management. Altogether, the CRISPR-revolution will revitalize the fight against tuberculosis with more research and technological developments.

RevDate: 2023-05-29
CmpDate: 2023-05-29

Zhang F, Neik TX, Thomas WJW, et al (2023)

CRISPR-Based Genome Editing Tools: An Accelerator in Crop Breeding for a Changing Future.

International journal of molecular sciences, 24(10):.

Genome editing is an important strategy to maintain global food security and achieve sustainable agricultural development. Among all genome editing tools, CRISPR-Cas is currently the most prevalent and offers the most promise. In this review, we summarize the development of CRISPR-Cas systems, outline their classification and distinctive features, delineate their natural mechanisms in plant genome editing and exemplify the applications in plant research. Both classical and recently discovered CRISPR-Cas systems are included, detailing the class, type, structures and functions of each. We conclude by highlighting the challenges that come with CRISPR-Cas and offer suggestions on how to tackle them. We believe the gene editing toolbox will be greatly enriched, providing new avenues for a more efficient and precise breeding of climate-resilient crops.

RevDate: 2023-05-29
CmpDate: 2023-05-29

Jiang C, Geng L, Wang J, et al (2023)

Multiplexed Gene Engineering Based on dCas9 and gRNA-tRNA Array Encoded on Single Transcript.

International journal of molecular sciences, 24(10):.

Simultaneously, multiplexed genome engineering and targeting multiple genomic loci are valuable to elucidating gene interactions and characterizing genetic networks that affect phenotypes. Here, we developed a general CRISPR-based platform to perform four functions and target multiple genome loci encoded in a single transcript. To establish multiple functions for multiple loci targets, we fused four RNA hairpins, MS2, PP7, com and boxB, to stem-loops of gRNA (guide RNA) scaffolds, separately. The RNA-hairpin-binding domains MCP, PCP, Com and λN22 were fused with different functional effectors. These paired combinations of cognate-RNA hairpins and RNA-binding proteins generated the simultaneous, independent regulation of multiple target genes. To ensure that all proteins and RNAs are expressed in one transcript, multiple gRNAs were constructed in a tandemly arrayed tRNA (transfer RNA)-gRNA architecture, and the triplex sequence was cloned between the protein-coding sequences and the tRNA-gRNA array. By leveraging this system, we illustrate the transcriptional activation, transcriptional repression, DNA methylation and DNA demethylation of endogenous targets using up to 16 individual CRISPR gRNAs delivered on a single transcript. This system provides a powerful platform to investigate synthetic biology questions and engineer complex-phenotype medical applications.

RevDate: 2023-05-29
CmpDate: 2023-05-29

Feser CJ, Williams JM, Lammers DT, et al (2023)

Engineering Human Cells Expressing CRISPR/Cas9-Synergistic Activation Mediators for Recombinant Protein Production.

International journal of molecular sciences, 24(10):.

Recombinant engineering for protein production commonly employs plasmid-based gene templates for introduction and expression of genes in a candidate cell system in vitro. Challenges to this approach include identifying cell types that can facilitate proper post-translational modifications and difficulty expressing large multimeric proteins. We hypothesized that integration of the CRISPR/Cas9-synergistic activator mediator (SAM) system into the human genome would be a powerful tool capable of robust gene expression and protein production. SAMs are comprised of a "dead" Cas9 (dCas9) linked to transcriptional activators viral particle 64 (VP64), nuclear factor-kappa-B p65 subunit (p65), and heat shock factor 1 (HSF1) and are programmable to single or multiple gene targets. We integrated the components of the SAM system into human HEK293, HKB11, SK-HEP1, and HEP-g2 cells using coagulation factor X (FX) and fibrinogen (FBN) as proof of concept. We observed upregulation of mRNA in each cell type with concomitant protein expression. Our findings demonstrate the capability of human cells stably expressing SAM for user-defined singleplex and multiplex gene targeting and highlight their broad potential utility for recombinant engineering as well as transcriptional modulation across networks for basic, translational, and clinical modeling and applications.

RevDate: 2023-05-29
CmpDate: 2023-05-29

Huang S, Zhang H, Chen W, et al (2023)

CRISPR/Cas9-Mediated Knockout of tnfaip1 in Zebrafish Plays a Role in Early Development.

Genes, 14(5):.

TNF α-induced protein 1 (TNFAIP1) was first identified in human umbilical vein endothelial cells and can be induced by tumor necrosis factor α (TNFα). Early studies have found that TNFAIP1 is involved in the development of many tumors and is closely associated with the neurological disorder Alzheimer's disease. However, little is known about the expression pattern of TNFAIP1 under physiological conditions and its function during embryonic development. In this study, we used zebrafish as a model to illustrate the early developmental expression pattern of tnfaip1 and its role in early development. First, we examined the expression pattern of tnfaip1 during early zebrafish development using quantitative real-time PCR and whole mount in situ hybridization and found that tnfaip1 was highly expressed in early embryonic development and, subsequently, expression became localized to anterior embryonic structures. To investigate the function of tnfaip1 during early development, we constructed a model of a stably inherited tnfaip1 mutant using the CRISPR/Cas9 system. Tnfaip1 mutant embryos showed significant developmental delays as well as microcephaly and microphthalmia. At the same time, we found decreased expression of the neuronal marker genes tuba1b, neurod1, and ccnd1 in tnfaip1 mutants. Analysis of transcriptome sequencing data revealed altered expression of the embryonic development related genes dhx40, hspa13, tnfrsf19, nppa, lrp2b, hspb9, clul1, zbtb47a, cryba1a, and adgrg4a in the tnfaip1 mutants. These findings suggest an important role for tnfaip1 in the early development of zebrafish.

RevDate: 2023-05-30
CmpDate: 2023-05-29

Zhao X, Qu K, Curci B, et al (2023)

Comparison of In-Frame Deletion, Homology-Directed Repair, and Prime Editing-Based Correction of Duchenne Muscular Dystrophy Mutations.

Biomolecules, 13(5):.

Recent progress in CRISPR gene editing tools has substantially increased the opportunities for curing devastating genetic diseases. Here we compare in-frame deletion by CRISPR-based non-homologous blunt end joining (NHBEJ), homology-directed repair (HDR), and prime editing (PE, PE2, and PE3)-based correction of two Duchenne Muscular Dystrophy (DMD) loss-of-function mutations (c.5533G>T and c.7893delC). To enable accurate and rapid evaluation of editing efficiency, we generated a genomically integrated synthetic reporter system (VENUS) carrying the DMD mutations. The VENUS contains a modified enhanced green fluorescence protein (EGFP) gene, in which expression was restored upon the CRISPR-mediated correction of DMD loss-of-function mutations. We observed that the highest editing efficiency was achieved by NHBEJ (74-77%), followed by HDR (21-24%) and PE2 (1.5%) in HEK293T VENUS reporter cells. A similar HDR (23%) and PE2 (1.1%) correction efficiency is achieved in fibroblast VENUS cells. With PE3 (PE2 plus nicking gRNA), the c.7893delC correction efficiency was increased 3-fold. Furthermore, an approximately 31% correction efficiency of the endogenous DMD: c.7893delC is achieved in the FACS-enriched HDR-edited VENUS EGFP+ patient fibroblasts. We demonstrated that a highly efficient correction of DMD loss-of-function mutations in patient cells can be achieved by several means of CRISPR gene editing.

RevDate: 2023-05-30
CmpDate: 2023-05-30

Chen K, Dai L, Zhao J, et al (2023)

Temperature-boosted PAM-less activation of CRISPR-Cas12a combined with selective inhibitors enhances detection of SNVs with VAFs below 0.01.

Talanta, 261:124674.

The precise identification of rare single nucleotide variations (SNVs) concomitant with excess wild-type DNA is a valuable method for minimally invasive disease diagnosis and early prediction of drug responsiveness. Selective enrichment of mutant variants via strand displacement reaction offers an ideal approach of SNVs analysis but fails to differentiate wildtype from mutants with variant allele fraction (VAF) < 0.01%. Here, we demonstrate that integration of PAM-less CRISPR-Cas12a and adjacent mutation-enhanced inhibition of wild-type alleles enables highly sensitive measurement of SNVs well below the 0.01% VAF threshold. Raising the reaction temperature to the upper limit of LbaCas12a helps to boost PAM-less activation of collateral DNase activity, which can be further enhanced using PCR additives, leading to ideal discriminative performance for single point mutations. Along with selective inhibitors bearing additional adjacent mutation, it allowed detection of model EGFR L858R mutants down to 0.001% with high sensitivity and specificity. Preliminary investigation on adulterated genomic samples prepared in two different ways also suggests that it can accurately measure ultralow-abundance SNVs extracted directly from clinical samples. We believe that our design, which combines the superior SNV enrichment capability of strand displacement reaction and unparalleled programmability of CRISPR-Cas12a, has the potential to significantly advance current SNV profiling technologies.

RevDate: 2023-05-28

Gómez-Martínez J, Rocha-Gracia RDC, Bello-López E, et al (2023)

Comparative Genomics of Pseudomonas aeruginosa Strains Isolated from Different Ecological Niches.

Antibiotics (Basel, Switzerland), 12(5):.

The Pseudomonas aeruginosa genome can change to adapt to different ecological niches. We compared four genomes from a Mexican hospital and 59 genomes from GenBank from different niches, such as urine, sputum, and environmental. The ST analysis showed that high-risk STs (ST235, ST773, and ST27) were present in the genomes of the three niches from GenBank, and the STs of Mexican genomes (ST167, ST2731, and ST549) differed from the GenBank genomes. Phylogenetic analysis showed that the genomes were clustering according to their ST and not their niche. When analyzing the genomic content, we observed that environmental genomes had genes involved in adapting to the environment not found in the clinics and that their mechanisms of resistance were mutations in antibiotic resistance-related genes. In contrast, clinical genomes from GenBank had resistance genes, in mobile/mobilizable genetic elements in the chromosome, except for the Mexican genomes that carried them mostly in plasmids. This was related to the presence of CRISPR-Cas and anti-CRISPR; however, Mexican strains only had plasmids and CRISPR-Cas. blaOXA-488 (a variant of blaOXA50) with higher activity against carbapenems was more prevalent in sputum genomes. The virulome analysis showed that exoS was most prevalent in the genomes of urinary samples and exoU and pldA in sputum samples. This study provides evidence regarding the genetic variability among P. aeruginosa isolated from different niches.

RevDate: 2023-05-28

Jin Y, Li W, Zhang H, et al (2023)

The Post-Antibiotic Era: A New Dawn for Bacteriophages.

Biology, 12(5):.

Phages are the most biologically diverse entities in the biosphere, infecting specific bacteria. Lytic phages quickly kill bacteria, while lysogenic phages integrate their genomes into bacteria and reproduce within the bacteria, participating in the evolution of natural populations. Thus, lytic phages are used to treat bacterial infections. However, due to the huge virus invasion, bacteria have also evolved a special immune mechanism (CRISPR-Cas systems, discovered in 1987). Therefore, it is necessary to develop phage cocktails and synthetic biology methods to infect bacteria, especially against multidrug-resistant bacteria infections, which are a major global threat. This review outlines the discovery and classification of phages and the associated achievements in the past century. The main applications of phages, including synthetic biology and PT, are also discussed, in addition to the effects of PT on immunity, intestinal microbes, and potential safety concerns. In the future, combining bioinformatics, synthetic biology, and classic phage research will be the way to deepen our understanding of phages. Overall, whether phages are an important element of the ecosystem or a carrier that mediates synthetic biology, they will greatly promote the progress of human society.

RevDate: 2023-05-26

Awan MJA, Akram A, Amin I, et al (2023)

Viral vectors as carriers of genome-editing reagents.

Trends in plant science pii:S1360-1385(23)00168-1 [Epub ahead of print].

The presence of a transgene in the genome of plants is a regulatory challenge. Recently, Liu et al. reported an engineered tomato spotted wilt virus (TSWV) that can carry large clustered regularly interspaced palindromic repeats (CRISPR)/Cas reagents for targeted genome editing in various crops without the integration of the transgene into the genome.

RevDate: 2023-05-28

Jabran M, Ali MA, Zahoor A, et al (2023)

Intelligent reprogramming of wheat for enhancement of fungal and nematode disease resistance using advanced molecular techniques.

Frontiers in plant science, 14:1132699.

Wheat (Triticum aestivum L.) diseases are major factors responsible for substantial yield losses worldwide, which affect global food security. For a long time, plant breeders have been struggling to improve wheat resistance against major diseases by selection and conventional breeding techniques. Therefore, this review was conducted to shed light on various gaps in the available literature and to reveal the most promising criteria for disease resistance in wheat. However, novel techniques for molecular breeding in the past few decades have been very fruitful for developing broad-spectrum disease resistance and other important traits in wheat. Many types of molecular markers such as SCAR, RAPD, SSR, SSLP, RFLP, SNP, and DArT, etc., have been reported for resistance against wheat pathogens. This article summarizes various insightful molecular markers involved in wheat improvement for resistance to major diseases through diverse breeding programs. Moreover, this review highlights the applications of marker assisted selection (MAS), quantitative trait loci (QTL), genome wide association studies (GWAS) and the CRISPR/Cas-9 system for developing disease resistance against most important wheat diseases. We also reviewed all reported mapped QTLs for bunts, rusts, smuts, and nematode diseases of wheat. Furthermore, we have also proposed how the CRISPR/Cas-9 system and GWAS can assist breeders in the future for the genetic improvement of wheat. If these molecular approaches are used successfully in the future, they can be a significant step toward expanding food production in wheat crops.

RevDate: 2023-05-29
CmpDate: 2023-05-29

Yang X, B Zhang (2023)

A review on CRISPR/Cas: a versatile tool for cancer screening, diagnosis, and clinic treatment.

Functional & integrative genomics, 23(2):182.

Cancer is one of the leading causes of death worldwide and it has the trend of increase incidence. However, in the past decades, as quickly developed new technologies and modified old techniques for cancer screening, diagnosis, and treatment, the cancer-caused mortality rates dropped quickly, and the survival times of cancer patients are enhanced. However, the current death rate is still about 50% and the survival patients always suffer from the side effect of current cancer treatments. Recently developed Nobel Prize-winning CRISPR/Cas technology provides new hope on cancer screening, early diagnosis, and clinic treatment as well as new drug development. Currently, four major CRISPR/Cas9-derived genome editors, CRISPR/Cas9 nucleotide sequence editor, CRISPR/Cas base editor (BE), CRISPR prime editor (PE), and CRISPR interference (CRISPRi) (including both CRISPRa and CRISPRr), were well developed and used to various research and applications, including cancer biology study and cancer screening, diagnosis, and treatment. Additionally, CRISPR/Cas12 and CRISPR/Cas13 genome editors were also widely used in cancer-related basic and applied research as well as treatment. Cancer-associated SNPs and genetic mutations as well as both oncogenes and tumor suppressor genes are perfect targets for CRISPR/Cas-based gene therapy for cancer treatment. CRISPR/Cas is also employed to modify and generate new Chimeric antigen receptor (CAR) T-cells for improving its safety, efficiency, and longer-time last for treating various cancers. Currently, there are many clinic trails of CRISPR-based gene therapy for cancer treatments. Although all CRISPR/Cas-derived genome and epigenome tools are promising methods for cancer biology study and treatment, the efficiency and long term-safety are still the major concerns for CRISPR-based gene therapy. Developing new CRISPR/Cas delivery methods and reducing the potential side effects, including off-target impacts, will enhance CRISPR/Cas application in cancer-related research, diagnosis, and therapeutical treatment.

RevDate: 2023-05-29
CmpDate: 2023-05-29

Zhou S, Sun H, Dong J, et al (2023)

Highly sensitive and facile microRNA detection based on target triggered exponential rolling-circle amplification coupling with CRISPR/Cas12a.

Analytica chimica acta, 1265:341278.

MicroRNAs (miRNAs) play a crucial role in the regulation of gene expression and have been implicated in many diseases. Herein, we develop a target triggered exponential rolling-circle amplification coupling with CRISPR/Cas12a (T-ERCA/Cas12a) system, which can achieve the ultrasensitive detection with simple operation and no annealing procedure. In this assay, T-ERCA combines the exponential amplification with rolling-circle amplification by introducing a dumb-bell probe with two enzyme recognition sites. miRNA-155 targets are activators that trigger exponential rolling circle amplification to produce large amounts of ssDNA, which is then recognized by CRISPR/Cas12a for further amplification. Compared with single EXPAR or RCA combined with CRISPR/Cas12a, this assay shows higher amplification efficiency. Therefore, benefiting from the excellent amplification effect of T-ERCA and the high recognition specificity of CRISPR/Cas12a, the proposed strategy shows a wide detection range from 1 fM to 5 nM with a LOD (limit of detection) down to 0.31 fM. Moreover, it shows good application ability for assessing miRNA levels in different cells, indicating that the T-ERCA/Cas12a may provide a new guidance for molecular diagnosis and clinical practical application.

RevDate: 2023-05-29
CmpDate: 2023-05-29

Yang L, Zhang Y, Yi W, et al (2023)

A rapid and efficient platform for antiviral crRNA screening using CRISPR-Cas13a-based nucleic acid detection.

Frontiers in immunology, 14:1116230.

INTRODUCTION: Rapid and high-throughput screening of antiviral clustered regularly interspaced short palindromic repeat (CRISPR) RNAs (crRNAs) is urgently required for the CRISPR-Cas13a antiviral system. Based on the same principle, we established an efficient screening platform for antiviral crRNA through CRISPR-Cas13a nucleic acid detection.

METHOD: In this study, crRNAs targeting PA, PB1, NP, and PB2 of the influenza A virus (H1N1) were screened using CRISPR-Cas13a nucleic acid detection, and their antiviral effects were confirmed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The RNA secondary structures were predicted by bioinformatics methods.

RESULTS: The results showed that crRNAs screened by CRISPR-Cas13a nucleic acid detection could effectively inhibit viral RNA in mammalian cells. Besides, we found that this platform for antiviral crRNA screening was more accurate than RNA secondary structure prediction. In addition, we validated the feasibility of the platform by screening crRNAs targeting NS of the influenza A virus (H1N1).

DISCUSSION: This study provides a new approach for screening antiviral crRNAs and contributes to the rapid advancement of the CRISPR-Cas13a antiviral system.

RevDate: 2023-05-29
CmpDate: 2023-05-29

Migunova E, Rajamani S, Bonanni S, et al (2023)

Cardiac RNase Z edited via CRISPR-Cas9 drives heart hypertrophy in Drosophila.

PloS one, 18(5):e0286214.

Cardiomyopathy (CM) is a group of diseases distinguished by morphological and functional abnormalities in the myocardium. It is etiologically heterogeneous and may develop via cell autonomous and/or non-autonomous mechanisms. One of the most severe forms of CM has been linked to the deficiency of the ubiquitously expressed RNase Z endoribonuclease. RNase Z cleaves off the 3'-trailer of both nuclear and mitochondrial primary tRNA (pre-tRNA) transcripts. Cells mutant for RNase Z accumulate unprocessed pre-tRNA molecules. Patients carrying RNase Z variants with reduced enzymatic activity display a plethora of symptoms including muscular hypotonia, microcephaly and severe heart hypertrophy; still, they die primarily due to acute heart decompensation. Determining whether the underlying mechanism of heart malfunction is cell autonomous or not will provide an opportunity to develop novel strategies of more efficient treatments for these patients. In this study, we used CRISPR-TRiM technology to create Drosophila models that carry cardiomyopathy-linked alleles of RNase Z only in the cardiomyocytes. We found that this modification is sufficient for flies to develop heart hypertrophy and systolic dysfunction. These observations support the idea that the RNase Z linked CM is driven by cell autonomous mechanisms.

RevDate: 2023-05-29
CmpDate: 2023-05-29

Huang DQ, Wu Q, Yang JH, et al (2023)

Deciphering endogenous and exogenous regulations of anammox consortia in responding to lincomycin by multiomics: quorum sensing and CRISPR system.

Water research, 239:120061.

The widespread use of antibiotics has created an antibiotic resistance genes (ARGs)-enriched environment, which causes high risks on human and animal health. Although antibiotics can be partially adsorbed and degraded in wastewater treatment processes, striving for a complete understanding of the microbial adaptive mechanism to antibiotic stress remains urgent. Combined with metagenomics and metabolomics, this study revealed that anammox consortia could adapt to lincomycin by spontaneously changing the preference for metabolite utilization and establishing interactions with eukaryotes, such as Ascomycota and Basidiomycota. Specifically, quorum sensing (QS) based microbial regulation and the ARGs transfer mediated by clustered regularly interspaced short palindromic repeats (CRISPR) system and global regulatory genes were the principal adaptive strategies. Western blotting results validated that Cas9 and TrfA were mainly responsible for the alteration of ARGs transfer pathway. These findings highlight the potential adaptative mechanism of microbes to antibiotic stress and fill gaps in horizontal gene transfer pathways in the anammox process, further facilitating the ARGs control through molecular and synthetic biology techniques.

RevDate: 2023-05-29
CmpDate: 2023-05-29

Martin-Rufino JD, Castano N, Pang M, et al (2023)

Massively parallel base editing to map variant effects in human hematopoiesis.

Cell, 186(11):2456-2474.e24.

Systematic evaluation of the impact of genetic variants is critical for the study and treatment of human physiology and disease. While specific mutations can be introduced by genome engineering, we still lack scalable approaches that are applicable to the important setting of primary cells, such as blood and immune cells. Here, we describe the development of massively parallel base-editing screens in human hematopoietic stem and progenitor cells. Such approaches enable functional screens for variant effects across any hematopoietic differentiation state. Moreover, they allow for rich phenotyping through single-cell RNA sequencing readouts and separately for characterization of editing outcomes through pooled single-cell genotyping. We efficiently design improved leukemia immunotherapy approaches, comprehensively identify non-coding variants modulating fetal hemoglobin expression, define mechanisms regulating hematopoietic differentiation, and probe the pathogenicity of uncharacterized disease-associated variants. These strategies will advance effective and high-throughput variant-to-function mapping in human hematopoiesis to identify the causes of diverse diseases.

RevDate: 2023-05-29
CmpDate: 2023-05-29

Sudhakar S, Barkau CL, Chilamkurthy R, et al (2023)

Binding to the conserved and stably folded guide RNA pseudoknot induces Cas12a conformational changes during ribonucleoprotein assembly.

The Journal of biological chemistry, 299(5):104700.

Ribonucleoproteins (RNPs) comprise one or more RNA and protein molecules that interact to form a stable complex, which commonly involves conformational changes in the more flexible RNA components. Here, we propose that Cas12a RNP assembly with its cognate CRISPR RNA (crRNA) guide instead proceeds primarily through Cas12a conformational changes during binding to more stable, prefolded crRNA 5' pseudoknot handles. Phylogenetic reconstructions and sequence and structure alignments revealed that the Cas12a proteins are divergent in sequence and structure while the crRNA 5' repeat region, which folds into a pseudoknot and anchors binding to Cas12a, is highly conserved. Molecular dynamics simulations of three Cas12a proteins and their cognate guides revealed substantial flexibility for unbound apo-Cas12a. In contrast, crRNA 5' pseudoknots were predicted to be stable and independently folded. Limited trypsin hydrolysis, differential scanning fluorimetry, thermal denaturation, and CD analyses supported conformational changes of Cas12a during RNP assembly and an independently folded crRNA 5' pseudoknot. This RNP assembly mechanism may be rationalized by evolutionary pressure to conserve CRISPR loci repeat sequence, and therefore guide RNA structure, to maintain function across all phases of the CRISPR defense mechanism.

RevDate: 2023-05-29
CmpDate: 2023-05-29

Pantazica AM, van Eerde A, Dobrica MO, et al (2023)

The "humanized" N-glycosylation pathway in CRISPR/Cas9-edited Nicotiana benthamiana significantly enhances the immunogenicity of a S/preS1 Hepatitis B Virus antigen and the virus-neutralizing antibody response in vaccinated mice.

Plant biotechnology journal, 21(6):1176-1190.

The recent SARS-CoV-2 pandemic has taught the world a costly lesson about the devastating consequences of viral disease outbreaks but also, the remarkable impact of vaccination in limiting life and economic losses. Vaccination against human Hepatitis B Virus (HBV), a major human pathogen affecting 290 million people worldwide, remains a key action towards viral hepatitis elimination by 2030. To meet this goal, the development of improved HBV antigens is critical to overcome non-responsiveness to standard vaccines based on the yeast-produced, small (S) envelope protein. We have recently shown that combining relevant immunogenic determinants of S and large (L) HBV proteins in chimeric antigens markedly enhances the anti-HBV immune response. However, the demand for cost-efficient, high-quality antigens remains challenging. This issue could be addressed by using plants as versatile and rapidly scalable protein production platforms. Moreover, the recent generation of plants lacking β-1,2-xylosyltransferase and α-1,3-fucosyltransferase activities (FX-KO), by CRISPR/Cas9 genome editing, enables production of proteins with "humanized" N-glycosylation. In this study, we investigated the impact of plant N-glycosylation on the immunogenic properties of a chimeric HBV S/L vaccine candidate produced in wild-type and FX-KO Nicotiana benthamiana. Prevention of β-1,2-xylose and α-1,3-fucose attachment to the HBV antigen significantly increased the immune response in mice, as compared with the wild-type plant-produced counterpart. Notably, the antibodies triggered by the FX-KO-made antigen neutralized more efficiently both wild-type HBV and a clinically relevant vaccine escape mutant. Our study validates in premiere the glyco-engineered Nicotiana benthamiana as a substantially improved host for plant production of glycoprotein vaccines.

RevDate: 2023-05-29
CmpDate: 2023-05-29

Raffan S, Oddy J, Mead A, et al (2023)

Field assessment of genome-edited, low asparagine wheat: Europe's first CRISPR wheat field trial.

Plant biotechnology journal, 21(6):1097-1099.

RevDate: 2023-05-25

Li W, Wang Z, Jiang Z, et al (2023)

MiR-3960 inhibits bladder cancer progression via targeting of DEXI.

Biochemical and biophysical research communications, 668:8-18 pii:S0006-291X(23)00618-6 [Epub ahead of print].

PURPOSE: MicroRNAs (miRNAs) are dominant cargo in exosomes and act as master regulators of cell function, inhibiting mRNA translation and affecting gene silencing. Some aspects of tissue-specific miRNA transport in bladder cancer (BC) and its role in cancer progression are not fully understood.

MATERIALS AND METHODS: A microarray was used to identify miRNAs in mouse bladder carcinoma cell line MB49 exosomes. Real-time reverse transcription polymerase chain reaction was used to examine the expression of miRNAs in BC and healthy donor serum. Western blotting and immunohistochemical staining were used to examine the expression of dexamethasone-induced protein (DEXI) in patients with BC. CRISPR-Cas 9 was used to knock out Dexi in MB49, and flow cytometry was performed to test cell proliferation ability and apoptosis under chemotherapy. Human BC organoid culture, miR-3960 transfection, and 293T-exosome-loaded miR-3960 delivery were used to analyze the effect of miR-3960 on BC progression.

RESULTS: The results showed that miR-3960 levels in BC tissue were positively correlated with patient survival time. Dexi was a major target of miR-3960. Dexi knockout inhibited MB49 cell proliferation and promoted cisplatin- and gemcitabine-induced apoptosis. Transfection of miR-3960 mimic inhibited DEXI expression and organoid growth. In parallel, 293T-exosome-loaded miR-3960 delivery and Dexi knockout significantly inhibited subcutaneous growth of MB49 cells in vivo.

CONCLUSION: Our results demonstrate the potential role of miR-3960-mediated inhibition of DEXI as a therapeutic strategy against BC.

RevDate: 2023-05-27

Schmidt M, Kircheis W, Simons A, et al (2023)

A diachronic perspective on citation latency in Wikipedia articles on CRISPR/Cas-9: an exploratory case study.

Scientometrics, 128(6):3649-3673.

This paper analyzes Wikipedia's representation of the Nobel Prize winning CRISPR/Cas9 technology, a method for gene editing. We propose and evaluate different heuristics to match publications from several publication corpora against Wikipedia's central article on CRISPR and against the complete Wikipedia revision history in order to retrieve further Wikipedia articles relevant to the topic and to analyze Wikipedia's referencing patterns. We explore to what extent the selection of referenced literature of Wikipedia's central article on CRISPR adheres to scientific standards and inner-scientific perspectives by assessing its overlap with (1) the Web of Science (WoS) database, (2) a WoS-based field-delineated corpus, (3) highly-cited publications within this corpus, and (4) publications referenced by field-specific reviews. We develop a diachronic perspective on citation latency and compare the delays with which publications are cited in relevant Wikipedia articles to the citation dynamics of these publications over time. Our results confirm that a combination of verbatim searches by title, DOI, and PMID is sufficient and cannot be improved significantly by more elaborate search heuristics. We show that Wikipedia references a substantial amount of publications that are recognized by experts and highly cited, but that Wikipedia also cites less visible literature, and, to a certain degree, even not strictly scientific literature. Delays in occurrence on Wikipedia compared to the publication years show (most pronounced in case of the central CRISPR article) a dependence on the dynamics of both the field and the editor's reaction to it in terms of activity.

RevDate: 2023-05-25

Kayo D, Kimura S, Yamazaki T, et al (2023)

Spatio-temporal control of targeted gene expression in combination with CRISPR/Cas and Tet-On systems in Medaka.

Genesis (New York, N.Y. : 2000) [Epub ahead of print].

Spatial and temporal control of transgene expression is a powerful approach to understand gene functions in specific cells and tissues. The Tet-On system is a robust tool for controlling transgene expression spatially and temporally; however, few studies have examined whether this system can be applied to postembryonic stages of Medaka (Oryzias latipes) or other fishes. Here, we first improved a basal promoter sequence on the donor vector for a nonhomologous end joining (NHEJ)-based knock-in (KI) system. Next, using transgenic Medaka for establishing the Tet-On system by KI, we demonstrated that doxycycline administration for four or more days by feeding can be a stable and efficient method to achieve expression of the transduced reporter gene in adult fish. From these analyses, we propose an optimized approach for a spatio-temporal gene-expression system in the adult stage of Medaka and other small fishes.

RevDate: 2023-05-26
CmpDate: 2023-05-26

Walker-Sünderhauf D, Klümper U, Pursey E, et al (2023)

Removal of AMR plasmids using a mobile, broad host-range CRISPR-Cas9 delivery tool.

Microbiology (Reading, England), 169(5):.

Antimicrobial resistance (AMR) genes are widely disseminated on plasmids. Therefore, interventions aimed at blocking plasmid uptake and transfer may curb the spread of AMR. Previous studies have used CRISPR-Cas-based technology to remove plasmids encoding AMR genes from target bacteria, using either phage- or plasmid-based delivery vehicles that typically have narrow host ranges. To make this technology feasible for removal of AMR plasmids from multiple members of complex microbial communities, an efficient, broad host-range delivery vehicle is needed. We engineered the broad host-range IncP1-plasmid pKJK5 to encode cas9 programmed to target an AMR gene. We demonstrate that the resulting plasmid pKJK5::csg has the ability to block the uptake of AMR plasmids and to remove resident plasmids from Escherichia coli. Furthermore, due to its broad host range, pKJK5::csg successfully blocked AMR plasmid uptake in a range of environmental, pig- and human-associated coliform isolates, as well as in isolates of two species of Pseudomonas. This study firmly establishes pKJK5::csg as a promising broad host-range CRISPR-Cas9 delivery tool for AMR plasmid removal, which has the potential to be applied in complex microbial communities to remove AMR genes from a broad range of bacterial species.

RevDate: 2023-05-26
CmpDate: 2023-05-26

Yue Y, Wang S, Jin Q, et al (2023)

A triple amplification strategy using GR-5 DNAzyme as a signal medium for ultrasensitive detection of trace Pb[2+] based on CRISPR/Cas12a empowered electrochemical biosensor.

Analytica chimica acta, 1263:341241.

Lead ions (Pb[2+]) are a well-known toxic heavy metal that poses a significant threat to human health. Therefore, the development of a simple and ultrasensitive technique for detecting Pb[2+] is essential. With their trans-cleavage properties, the newly discovered CRISPR-V effectors have become a potential high-precision biometric tool. In this regard, a CRISPR/Cas12a-based electrochemical biosensor (E-CRISPR) has been developed, which is combined with the GR-5 DNAzyme that can specifically recognize Pb[2+]. In this strategy, the GR-5 DNAzyme acts as a signal-mediated intermediary, which can convert Pb[2+] into nucleic acid signals, thereby becoming single-stranded DNA that triggers strand displacement amplification (SDA) reaction. This is coupled with following activated CRISPR/Cas12a cleavage of the electrochemical signal probe, enabling cooperative signal amplification for ultrasensitive Pb[2+] detection. The proposed method has a detection limit as low as 0.02 pM. Therefore, we have developed an E-CRISPR detection platform with GR-5 DNAzyme as a signal medium (called SM-E-CRISPR biosensor). This provides a method for the CRISPR system to specifically detect non-nucleic substances by converting the signal using a medium.

RevDate: 2023-05-25

Miezner G, Turgeman-Grott I, Zatopek KM, et al (2023)

An archaeal Cas3 protein facilitates rapid recovery from DNA damage.

microLife, 4:uqad007.

CRISPR-Cas systems provide heritable acquired immunity against viruses to archaea and bacteria. Cas3 is a CRISPR-associated protein that is common to all Type I systems, possesses both nuclease and helicase activities, and is responsible for degradation of invading DNA. Involvement of Cas3 in DNA repair had been suggested in the past, but then set aside when the role of CRISPR-Cas as an adaptive immune system was realized. Here we show that in the model archaeon Haloferax volcanii a cas3 deletion mutant exhibits increased resistance to DNA damaging agents compared with the wild-type strain, but its ability to recover quickly from such damage is reduced. Analysis of cas3 point mutants revealed that the helicase domain of the protein is responsible for the DNA damage sensitivity phenotype. Epistasis analysis indicated that cas3 operates with mre11 and rad50 in restraining the homologous recombination pathway of DNA repair. Mutants deleted for Cas3 or deficient in its helicase activity showed higher rates of homologous recombination, as measured in pop-in assays using non-replicating plasmids. These results demonstrate that Cas proteins act in DNA repair, in addition to their role in defense against selfish elements and are an integral part of the cellular response to DNA damage.

RevDate: 2023-05-25

Alinejad T, Modarressi S, Sadri Z, et al (2023)

Diagnostic applications and therapeutic option of Cascade CRISPR/Cas in the modulation of miRNA in diverse cancers: promises and obstacles.

Journal of cancer research and clinical oncology [Epub ahead of print].

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas technology is a molecular tool specific to sequences for engineering genomes. Among diverse clusters of Cas proteins, the class 2/type II CRISPR/Cas9 system, despite several challenges, such as off-target effects, editing efficiency, and efficient delivery, has shown great promise for driver gene mutation discovery, high-throughput gene screening, epigenetic modulation, nucleic acid detection, disease modeling, and more importantly for therapeutic purposes. CRISPR-based clinical and experimental methods have applications across a wide range of areas, especially for cancer research and, possibly, anticancer therapy. On the other hand, given the influential role of microRNAs (miRNAs) in the regulations of cellular division, carcinogenicity, tumorigenesis, migration/invasion, and angiogenesis in diverse normal and pathogenic cellular processes, in different stages of cancer, miRNAs are either oncogenes or tumor suppressors, according to what type of cancer they are involved in. Hence, these noncoding RNA molecules are conceivable biomarkers for diagnosis and therapeutic targets. Moreover, they are suggested to be adequate predictors for cancer prediction. Conclusive evidence proves that CRISPR/Cas system can be applied to target small non-coding RNAs. However, the majority of studies have highlighted the application of the CRISPR/Cas system for targeting protein-coding regions. In this review, we specifically discuss diverse applications of CRISPR-based tools for probing miRNA gene function and miRNA-based therapeutic involvement in different types of cancers.

RevDate: 2023-05-26
CmpDate: 2023-05-26

Engstler M, T Beneke (2023)

Gene editing and scalable functional genomic screening in Leishmania species using the CRISPR/Cas9 cytosine base editor toolbox LeishBASEedit.

eLife, 12:.

CRISPR/Cas9 gene editing has revolutionised loss-of-function experiments in Leishmania, the causative agent of leishmaniasis. As Leishmania lack a functional non-homologous DNA end joining pathway however, obtaining null mutants typically requires additional donor DNA, selection of drug resistance-associated edits or time-consuming isolation of clones. Genome-wide loss-of-function screens across different conditions and across multiple Leishmania species are therefore unfeasible at present. Here, we report a CRISPR/Cas9 cytosine base editor (CBE) toolbox that overcomes these limitations. We employed CBEs in Leishmania to introduce STOP codons by converting cytosine into thymine and created http://www.leishbaseedit.net/ for CBE primer design in kinetoplastids. Through reporter assays and by targeting single- and multi-copy genes in L. mexicana, L. major, L. donovani, and L. infantum, we demonstrate how this tool can efficiently generate functional null mutants by expressing just one single-guide RNA, reaching up to 100% editing rate in non-clonal populations. We then generated a Leishmania-optimised CBE and successfully targeted an essential gene in a plasmid library delivered loss-of-function screen in L. mexicana. Since our method does not require DNA double-strand breaks, homologous recombination, donor DNA, or isolation of clones, we believe that this enables for the first time functional genetic screens in Leishmania via delivery of plasmid libraries.

RevDate: 2023-05-26
CmpDate: 2023-05-26

Lu J, S Wang (2023)

CRISPR/Cas9-Mediated Gene Knockout in Cells and Tissues Using Lentivirus.

Current protocols, 3(5):e772.

CRISPR-Cas9 has become a powerful and popular gene editing tool. However, successful application of this tool in the lab can still be quite daunting to many newcomers to molecular biology, mostly because it is a relatively lengthy process involving multiple steps with variations of each step. Here, we provide a reliable, stepwise, and newcomer-friendly protocol to knock out a target gene in wild-type human fibroblasts. This protocol involves sgRNA design using CRISPOR, construction of an "all-in-one" vector expressing both sgRNA and Cas9 using Golden Gate cloning, streamlined production of high-titer lentiviruses in 1 week after molecular cloning, and transduction of cells to generate a knockout cell pool. We further introduce a protocol for lentiviral transduction of ex vivo mouse embryonic salivary epithelial explants. In summary, our protocol is useful for new researchers to apply CRISPR-Cas9 to generate stable gene knockout cells and tissue explants using lentivirus. Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: sgRNA design Basic Protocol 2: Cloning sgRNA in plasmid vector containing Cas9 encoding sequence using golden gate cloning Basic Protocol 3: Lentivirus packaging Basic Protocol 4: Lentivirus transduction of cells Basic Protocol 5: Lentivirus transduction of salivary gland epithelial buds.

RevDate: 2023-05-26
CmpDate: 2023-05-26

Shi Y, Kopparapu N, Ohler L, et al (2023)

Efficient and rapid fluorescent protein knock-in with universal donors in mouse embryonic stem cells.

Development (Cambridge, England), 150(10):.

Fluorescent protein (FP) tagging is a key method for observing protein distribution, dynamics and interaction with other proteins in living cells. However, the typical approach using overexpression of tagged proteins can perturb cell behavior and introduce localization artifacts. To preserve native expression, fluorescent proteins can be inserted directly into endogenous genes. This approach has been widely used in yeast for decades, and more recently in invertebrate model organisms with the advent of CRISPR/Cas9. However, endogenous FP tagging has not been widely used in mammalian cells due to inefficient homology-directed repair. Recently, the CRISPaint system used non-homologous end joining for efficient integration of FP tags into native loci, but it only allows C-terminal knock-ins. Here, we have enhanced the CRISPaint system by introducing new universal donors for N-terminal insertion and for multi-color tagging with orthogonal selection markers. We adapted the procedure for mouse embryonic stem cells, which can be differentiated into diverse cell types. Our protocol is rapid and efficient, enabling live imaging in less than 2 weeks post-transfection. These improvements increase the versatility and applicability of FP knock-in in mammalian cells.

RevDate: 2023-05-26
CmpDate: 2023-05-26

Tanihara F, Hirata M, Namula Z, et al (2023)

GHR-mutant pig derived from domestic pig and microminipig hybrid zygotes using CRISPR/Cas9 system.

Molecular biology reports, 50(6):5049-5057.

BACKGROUND: Pigs are excellent large animal models with several similarities to humans. They provide valuable insights into biomedical research that are otherwise difficult to obtain from rodent models. However, even if miniature pig strains are used, their large stature compared with other experimental animals requires a specific maintenance facility which greatly limits their usage as animal models. Deficiency of growth hormone receptor (GHR) function causes small stature phenotypes. The establishment of miniature pig strains via GHR modification will enhance their usage as animal models. Microminipig is an incredibly small miniature pig strain developed in Japan. In this study, we generated a GHR mutant pig using electroporation-mediated introduction of the CRISPR/Cas9 system into porcine zygotes derived from domestic porcine oocytes and microminipig spermatozoa.

METHODS AND RESULTS: First, we optimized the efficiency of five guide RNAs (gRNAs) designed to target GHR in zygotes. Embryos that had been electroporated with the optimized gRNAs and Cas9 were then transferred into recipient gilts. After embryo transfer, 10 piglets were delivered, and one carried a biallelic mutation in the GHR target region. The GHR biallelic mutant showed a remarkable growth-retardation phenotype. Furthermore, we obtained F1 pigs derived from the mating of GHR biallelic mutant with wild-type microminipig, and GHR biallelic mutant F2 pigs through sib-mating of F1 pigs.

CONCLUSIONS: We have successfully demonstrated the generation of biallelic GHR-mutant small-stature pigs. Backcrossing of GHR-deficient pig with microminipig will establish the smallest pig strain which can contribute significantly to the field of biomedical research.

RevDate: 2023-05-26
CmpDate: 2023-05-26

Hu M, Liu R, Qiu Z, et al (2023)

Light-Start CRISPR-Cas12a Reaction with Caged crRNA Enables Rapid and Sensitive Nucleic Acid Detection.

Angewandte Chemie (International ed. in English), 62(23):e202300663.

The clustered regularly interspaced short palindromic repeats (CRISPR) system is a promising platform for nucleic acid detection. Regulating the CRISPR reaction would be extremely useful to improve the detection efficiency and speed of CRISPR diagnostic applications. Here, we have developed a light-start CRISPR-Cas12a reaction by employing caged CRISPR RNA (crRNA). When combined with recombinase polymerase amplification, a robust photocontrolled one-pot assay is achieved. The photocontrolled one-pot assay is simpler and is 50-fold more sensitive than the conventional assay. This improved detection efficiency also facilitates the development of a faster CRISPR diagnostic method. The detection of clinical samples demonstrated that 10-20 min is sufficient for effective detection, which is much faster than the current gold-standard technique PCR. We expect this advance in CRISPR diagnostics to promote its widespread detection applications in biomedicine, agriculture, and food safety.

RevDate: 2023-05-26
CmpDate: 2023-05-26

Davachi ND, Bartlewski PM, Masoudi R, et al (2023)

Suitability of a universal electroporation device for genome editing and production of transgenic rats.

Reproductive biology, 23(2):100755.

Mammalian genome editing has utilized expensive and highly specialized electroporator devices. The "Gene Pulser XCell," a modular electroporation system for transfecting all cell types, has not been used extensively in mammalian embryo genome editing. The present experiment was undertaken to determine the usefulness of the Gene Pulser XCell for inserting the CRISPR/Cas9 system into intact zygotes in order to obtain the enhanced green fluorescent protein reporter rats (eGFP-R). An electroporation pulse response test using mCherry mRNA was performed to optimize the settings of the electroporator. Forty-five combinations of five pulse voltages (15, 25, 30, 35 and 40 V), three pulse durations (5, 10 and 25 ms), and three pulse frequencies (2, 5 and 6 pulses) applied at a constant 100-ms pulse interval and temperature of 37.5 °C were evaluated. The test revealed that the 35 V was the only voltage suitable for insertion of mCherry mRNA into intact rat zygotes and the only one that resulted in the production of embryos attaining the blastocyst stage. The incorporation of mCherry mRNA increased but the survival of the electroporated embryos declined with an increment in the number of pulses. Subsequent transfer of 1112 surviving Sprague Dawley rat embryos (after 8 h of incubating 1800 zygotes electroporated with the CRISPR/Cas9) resulted in the production of 287 offspring (25.8%). Ensuing PCR and phenotypic evaluation confirmed that twenty animals (6.96%) expressed eGFP in all body organs/tissues except for blood and blood vessels. The mortality of males and females before the attainment of puberty was 2 and 3 pups, respectively, and the final number/ratio of male to female of offspring was 9:11. All the surviving rats mated naturally and successfully transmitted the GFP transgene to their progeny. The Gene Pulser XCell total system with the settings predetermined in the present experiment can effectively be used to produce transgenic rats through the CRISPR/Cas9-mediated genome editing of zygotes.

RevDate: 2023-05-26
CmpDate: 2023-05-26

Li X, Liu X, Wei J, et al (2023)

Ultrasensitive detection of microRNAs based on click chemistry-terminal deoxynucleotidyl transferase combined with CRISPR/Cas12a.

Biochimie, 208:38-45.

The specificity and sensitivity of microRNA (miRNA) detection play a vital role in the early diagnosis of cancer and the treatment of various diseases. Here, we constructed a fluorescent biosensor based on click chemistry-terminal deoxynucleotidyl transferase (ccTdT) combined with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas)12a cascade amplification system to achieve ultrasensitive miRNA-21 detection. Target miRNA-21 was employed as a template for click chemistry ligation of two nucleic acid probes, the product of which can be combined with magnetic microbeads (MBs). Then the 3'-end of the ligated nucleic acid and complementary strand miRNA-21 was extended by TdT. The extended poly-T tails activated the trans-cleavage ability of CRISPR/Cas12a, cleaving the reporter gene to generate the fluorescent signal. The proposed biosensor has a wide linear detection range, from 1 pM to 10[5] pM, with detection limits as low as 88 fM under optimal experimental conditions. Hence, this fluorescent biosensor enables simple, sensitive detection of miRNAs and offers a promising analytical platform for clinical diagnostics and biomedical research.

RevDate: 2023-05-23

Anonymous (2023)

Correction to: Clonally Selected Lines After CRISPR-Cas Editing Are Not Isogenic by Panda et al. The CRISPR Journal, 2023;6(2):176-182; DOI: 10.1089/crispr.2022.0050.

RevDate: 2023-05-23

Chen D, Huang W, Zhang Y, et al (2023)

CRISPR-Mediated Profiling of Viral RNA at Single Nucleotide Resolution.

Angewandte Chemie (International ed. in English) [Epub ahead of print].

Mass pathogen screening is critical to preventing the outbreaks and spread of infectious diseases. The large-scale epidemic of COVID-19 and the rapid mutation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus have put forward new requirements for virus detection and identification techniques. Here, we report a CRISPR-based Amplification-free Viral RNA Electrical Detection platform (CAVRED) for the rapid detection and identification of SARS-CoV-2 variants. A series of CRISPR RNA assays were designed to amplify the CRISPR-Cas system's ability to discriminate between mutant and wild RNA genomes with a single-nucleotide difference. The identified viral RNA information was converted into readable electrical signals through field-effect transistor biosensors for the achievement of highly sensitive detection of single-base mutations. CAVRED can detect the SARS-CoV-2 virus genome as low as 1 cp/µL within 20 mins without amplification, and this value is comparable to the detection limit of real-time quantitative polymerase chain reaction. Based on the excellent RNA mutation detection ability, an 8-in-1 CAVRED array was constructed and realized the rapid identification of 40 simulated throat swab samples of SARS-CoV-2 variants with a 95.0% accuracy. The advantages of accuracy, sensitivity, and fast speed of CAVRED promise its application in rapid and large-scale epidemic screening.

RevDate: 2023-05-24
CmpDate: 2023-05-24

Takebayashi K, Wittayarat M, Lin Q, et al (2023)

Efficiency of genetic modification in gene-knockout sperm-derived zygotes followed by electroporation of guide RNA targeting the same gene.

Animal science journal = Nihon chikusan Gakkaiho, 94(1):e13842.

Genetic mosaicism is considered one of the main limitations of the electroporation method used to transfer CRISPR-Cas9/guide RNA (gRNA) into porcine zygotes. We hypothesized that fertilization of oocytes with sperm from gene-deficient boars, in combination with electroporation (EP) to target the same region of the gene in subsequent zygotes, would increase the gene modification efficiency. As myostatin (MSTN) and α1,3-galactosyltransferase (GGTA1) have beneficial effects on agricultural production and xenotransplantation, respectively, we used these two genes to test our hypothesis. Spermatozoa from gene-knockout boars were used for oocyte fertilization in combination with EP to transfer gRNAs targeting the same gene region to zygotes. No significant differences in the rates of cleavage and blastocyst formation as well as in the mutation rates of blastocysts were observed between the wild-type and gene-deficient spermatozoa groups, irrespective of the targeted gene. In conclusion, the combination of fertilization with gene-deficient spermatozoa and gene editing of the same targeted gene region using EP had no beneficial effects on embryo genetic modification, indicating that EP alone is a sufficient tool for genome modification.

RevDate: 2023-05-22

Lyu M, Sun Y, Yan N, et al (2023)

Efficient CRISPR/Cas9-mediated gene editing in mammalian cells by the novel selectable traffic light reporters.

International journal of biological macromolecules pii:S0141-8130(23)01820-2 [Epub ahead of print].

CRISPR/Cas9 is a powerful tool for gene editing in various cell types and organisms. However, it is still challenging to screen genetically modified cells from an excess of unmodified cells. Our previous studies demonstrated that surrogate reporters can be used for efficient screening of genetically modified cells. Here, we developed two novel traffic light screening reporters, puromycin-mCherry-EGFP (PMG) based on single-strand annealing (SSA) and homology-directed repair (HDR), respectively, to measure the nuclease cleavage activity within transfected cells and to select genetically modified cells. We found that the two reporters could be self-repaired coupling the genome editing events driven by different CRISPR/Cas nucleases, resulting in a functional puromycin-resistance and EGFP selection cassette that can be afforded to screen genetically modified cells by puromycin selection or FACS enrichment. We further compared the novel reporters with different traditional reporters at several endogenous loci in different cell lines, for the enrichment efficiencies of genetically modified cells. The results indicated that the SSA-PMG reporter exhibited improvements in enriching gene knockout cells, while the HDR-PMG system was very useful in enriching knock-in cells. These results provide robust and efficient surrogate reporters for the enrichment of CRISPR/Cas9-mediated editing in mammalian cells, thereby advancing basic and applied research.

RevDate: 2023-05-22

Hamdi I, Boni F, Shen Q, et al (2023)

Characteristics of subtype III-A CRISPR-Cas system in Mycobacterium tuberculosis: An overview.

Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases pii:S1567-1348(23)00043-6 [Epub ahead of print].

CRISPR-Cas systems are the only RNA- guided adaptive immunity pathways that trigger the detection and destruction of invasive phages and plasmids in bacteria and archaea. Due to its prevalence and mystery, the Class 1 CRISPR-Cas system has lately been the subject of several studies. This review highlights the specificity of CRISPR-Cas system III-A in Mycobacterium tuberculosis, the tuberculosis-causing pathogen, for over twenty years. We discuss the difference between the several subtypes of Type III and their defence mechanisms. The anti-CRISPRs (Acrs) recently described, the critical role of Reverse transcriptase (RT) and housekeeping nuclease for type III CRISPR-Cas systems, and the use of this cutting-edge technology, its impact on the search for novel anti-tuberculosis drugs.

RevDate: 2023-05-25
CmpDate: 2023-05-25

Fatehi S, Marks RM, Rok MJ, et al (2023)

Advances in CRISPR/Cas9 Genome Editing for the Treatment of Muscular Dystrophies.

Human gene therapy, 34(9-10):388-403.

Muscular dystrophies (MDs) comprise a diverse group of inherited disorders characterized by progressive muscle loss and weakness. Given the genetic etiology underlying MDs, researchers have explored the potential of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing as a therapeutic intervention, resulting in significant advances. Here, we review recent progress on the use of CRISPR/Cas9 genome editing as a potential therapy for MDs. Significant strides have been made in this realm, made possible through innovative techniques such as precision genetic editing by modified forms of CRISPR/Cas9. These approaches have shown varying degrees of success in animal models of MD, including Duchenne MD, congenital muscular dystrophy type 1A, and myotonic dystrophy type 1. Even so, there are several challenges facing the development of CRISPR/Cas9-based MD therapies, including the targeting of satellite cells, improved editing efficiency in skeletal and cardiac muscle tissue, delivery vehicle enhancements, and the host immunogenic response. Although more work is needed to advance CRISPR/Cas9 genome editing past the preclinical stages, its therapeutic potential for MD is extremely promising and justifies concentrated efforts to move into clinical trials.

RevDate: 2023-05-25
CmpDate: 2023-05-25

Chemello F, Olson EN, R Bassel-Duby (2023)

CRISPR-Editing Therapy for Duchenne Muscular Dystrophy.

Human gene therapy, 34(9-10):379-387.

Duchenne muscular dystrophy (DMD) is a debilitating genetic disorder that results in progressive muscle degeneration and premature death. DMD is caused by mutations in the gene encoding dystrophin protein, a membrane-associated protein required for maintenance of muscle structure and function. Although the genetic mutations causing the disease are well known, no curative therapies have been developed to date. The advent of genome-editing technologies provides new opportunities to correct the underlying mutations responsible for DMD. These mutations have been successfully corrected in human cells, mice, and large animal models through different strategies based on CRISPR-Cas9 gene editing. Ideally, CRISPR-editing could offer a one-time treatment for DMD by correcting the genetic mutations and enabling normal expression of the repaired gene. However, numerous challenges remain to be addressed, including optimization of gene editing, delivery of gene-editing components to all the muscles of the body, and the suppression of possible immune responses to the CRISPR-editing therapy. This review provides an overview of the recent advances toward CRISPR-editing therapy for DMD and discusses the opportunities and the remaining challenges in the path to clinical translation.

RevDate: 2023-05-25
CmpDate: 2023-05-25

Iyer DN, Schimmer AD, H Chang (2023)

Applying CRISPR-Cas9 screens to dissect hematological malignancies.

Blood advances, 7(10):2252-2270.

Bit by bit, over the last few decades, functional genomic tools have been piecing together the molecular puzzle driving tumorigenesis in human patients. Nevertheless, our understanding of the role of several genes and regulatory elements that drive critical cancer-associated physiological processes from disease development to progression to spread is very limited, which significantly affects our ability of applying these insights in the context of improved disease management. The recent advent of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-based technology and its application in cancer genomics has, however, allowed the generation of a wealth of knowledge that has helped decipher several critical questions associated with translational cancer research. Precisely, the high-throughput capability coupled with a high level of technological plasticity associated with the CRISPR-Cas9 screens have expanded our horizons from a mere struggle to appreciate cancer as a genetic disease to observing the integrated genomic/epigenomic network of numerous malignancies and correlating it with our present knowledge of drugging strategies to develop innovative approaches for next-generation precision cancer medicine. Specifically, within blood cancers, current CRISPR screens have specifically focused on improving our understanding of drug resistance mechanisms, disease biology, the development of novel therapeutic approaches, and identifying the molecular mechanisms of current therapies, with an underlying aim of improving disease outcomes. Here, we review the development of the CRISPR-Cas9 genome-editing strategy, explicitly focusing on the recent advances in the CRISPR-Cas9-based screening approaches, its current capabilities, limitations, and future applications in the context of hematological malignancies.

RevDate: 2023-05-22

Zhou F, Yu X, Gan R, et al (2023)

CRISPRimmunity: an interactive web server for CRISPR-associated Important Molecular events and Modulators Used in geNome edIting Tool identifYing.

Nucleic acids research pii:7175359 [Epub ahead of print].

The CRISPR-Cas system is a highly adaptive and RNA-guided immune system found in bacteria and archaea, which has applications as a genome editing tool and is a valuable system for studying the co-evolutionary dynamics of bacteriophage interactions. Here introduces CRISPRimmunity, a new web server designed for Acr prediction, identification of novel class 2 CRISPR-Cas loci, and dissection of key CRISPR-associated molecular events. CRISPRimmunity is built on a suite of CRISPR-oriented databases providing a comprehensive co-evolutionary perspective of the CRISPR-Cas and anti-CRISPR systems. The platform achieved a high prediction accuracy of 0.997 for Acr prediction when tested on a dataset of 99 experimentally validated Acrs and 676 non-Acrs, outperforming other existing prediction tools. Some of the newly identified class 2 CRISPR-Cas loci using CRISPRimmunity have been experimentally validated for cleavage activity in vitro. CRISPRimmunity offers the catalogues of pre-identified CRISPR systems to browse and query, the collected resources or databases to download, a well-designed graphical interface, a detailed tutorial, multi-faceted information, and exportable results in machine-readable formats, making it easy to use and facilitating future experimental design and further data mining. The platform is available at http://www.microbiome-bigdata.com/CRISPRimmunity. Moreover, the source code for batch analysis are published on Github (https://github.com/HIT-ImmunologyLab/CRISPRimmunity).

RevDate: 2023-05-24
CmpDate: 2023-05-24

Han W, Li Z, Guo Y, et al (2023)

Efficient precise integration of large DNA sequences with 3'-overhang dsDNA donors using CRISPR/Cas9.

Proceedings of the National Academy of Sciences of the United States of America, 120(22):e2221127120.

CRISPR/Cas9 genome-editing tools have tremendously boosted our capability of manipulating the eukaryotic genomes in biomedical research and innovative biotechnologies. However, the current approaches that allow precise integration of gene-sized large DNA fragments generally suffer from low efficiency and high cost. Herein, we developed a versatile and efficient approach, termed LOCK (Long dsDNA with 3'-Overhangs mediated CRISPR Knock-in), by utilizing specially designed 3'-overhang double-stranded DNA (odsDNA) donors harboring 50-nt homology arm. The length of the 3'-overhangs of odsDNA is specified by the five consecutive phosphorothioate modifications. Compared with existing methods, LOCK allows highly efficient targeted insertion of kilobase-sized DNA fragments into the mammalian genomes with low cost and low off-target effects, yielding >fivefold higher knock-in frequencies than conventional homologous recombination-based approaches. This newly designed LOCK approach based on homology-directed repair is a powerful tool suitable for gene-sized fragment integration that is urgently needed for genetic engineering, gene therapies, and synthetic biology.

RevDate: 2023-05-23

Srivastava P, D Prasad (2023)

Isothermal nucleic acid amplification and its uses in modern diagnostic technologies.

3 Biotech, 13(6):200.

Nucleic acids are prominent biomarkers for diagnosing infectious pathogens using nucleic acid amplification techniques (NAATs). PCR, a gold standard technique for amplifying nucleic acids, is widely used in scientific research and diagnosis. Efficient pathogen detection is a key to adequate food safety and hygiene. However, using bulky thermal cyclers and costly laboratory setup limits its uses in developing countries, including India. The isothermal amplification methods are exploited to develop miniaturized sensors against viruses, bacteria, fungi and other pathogenic organisms and have been applied for in situ diagnosis. Isothermal amplification techniques have been found suitable for POC techniques and follow WHO's ASSURED criteria. LAMP, NASBA, SDA, RCA and RPA are some of the isothermal amplification techniques which are preferable for POC diagnostics. Furthermore, methods such as WGA, CPA, HDA, EXPAR, SMART, SPIA and DAMP were introduced for even more accuracy and robustness. Using recombinant polymerases and other nucleic acid-modifying enzymes has dramatically broadened the detection range of target pathogens under the scanner. The coupling of isothermal amplification methods with advanced technologies such as CRISPR/Cas systems, fluorescence-based chemistries, microfluidics and paper-based sensors has significantly influenced the biosensing and diagnosis field. This review comprehensively analyzed isothermal nucleic acid amplification methods, emphasizing their advantages, disadvantages and limitations.

RevDate: 2023-05-23

Fichter KM, Setayesh T, P Malik (2023)

Strategies for precise gene edits in mammalian cells.

Molecular therapy. Nucleic acids, 32:536-552.

CRISPR-Cas technologies have the potential to revolutionize genetic medicine. However, work is still needed to make this technology clinically efficient for gene correction. A barrier to making precise genetic edits in the human genome is controlling how CRISPR-Cas-induced DNA breaks are repaired by the cell. Since error-prone non-homologous end-joining is often the preferred cellular repair pathway, CRISPR-Cas-induced breaks often result in gene disruption. Homology-directed repair (HDR) makes precise genetic changes and is the clinically desired pathway, but this repair pathway requires a homology donor template and cycling cells. Newer editing strategies, such as base and prime editing, can affect precise repair for relatively small edits without requiring HDR and circumvent cell cycle dependence. However, these technologies have limitations in the extent of genetic editing and require the delivery of bulky cargo. Here, we discuss the pros and cons of precise gene correction using CRISPR-Cas-induced HDR, as well as base and prime editing for repairing small mutations. Finally, we consider emerging new technologies, such as recombination and transposases, which can circumvent both cell cycle and cellular DNA repair dependence for editing the genome.

RevDate: 2023-05-24
CmpDate: 2023-05-24

Migliara A, Cappelluti MA, Giannese F, et al (2023)

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing.

Journal of visualized experiments : JoVE.

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among traditional technologies, RNA interference suffers from partial target abrogation and the requirement for life-long treatments. In contrast, artificial nucleases can impose stable gene inactivation through induction of a DNA double strand break (DSB), but recent studies are questioning the safety of this approach. Targeted epigenetic editing via engineered transcriptional repressors (ETRs) may represent a solution, as a single administration of specific ETR combinations can lead to durable silencing without inducing DNA breaks. ETRs are proteins containing a programmable DNA-binding domain (DBD) and effectors from naturally occurring transcriptional repressors. Specifically, a combination of three ETRs equipped with the KRAB domain of human ZNF10, the catalytic domain of human DNMT3A and human DNMT3L, was shown to induce heritable repressive epigenetic states on the ETR-target gene. The hit-and-run nature of this platform, the lack of impact on the DNA sequence of the target, and the possibility to revert to the repressive state by DNA demethylation on demand, make epigenetic silencing a game-changing tool. A critical step is the identification of the proper ETRs' position on the target gene to maximize on-target and minimize off-target silencing. Performing this step in the final ex vivo or in vivo preclinical setting can be cumbersome. Taking the CRISPR/catalytically dead Cas9 system as a paradigmatic DBD for ETRs, this paper describes a protocol consisting of the in vitro screen of guide RNAs (gRNAs) coupled to the triple-ETR combination for efficient on-target silencing, followed by evaluation of the genome-wide specificity profile of top hits. This allows for reduction of the initial repertoire of candidate gRNAs to a short list of promising ones, whose complexity is suitable for their final evaluation in the therapeutically relevant setting of interest.

RevDate: 2023-05-24
CmpDate: 2023-05-24

Mao G, Luo X, Ye S, et al (2023)

Fluorescence and Colorimetric Analysis of African Swine Fever Virus Based on the RPA-Assisted CRISPR/Cas12a Strategy.

Analytical chemistry, 95(20):8063-8069.

It is well-established that different detection modes are necessary for corresponding applications, which can effectively reduce matrix interference and improve the detection accuracy. Here, we reported a magnetic separation method based on recombinase polymerase amplification (RPA)-assisted clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a for dual-mode analysis of African swine fever virus (ASFV) genes, including colorimetry and fluorescence. The ASFV gene was selected as the initial RPA template to generate the amplicon. The RPA amplicon was then recognized by CRISPR-associated RNA (crRNA), activating the trans-cleavage activity of Cas12a and leading to the nonspecific cleavage of ssDNA as well as a significant release of alkaline phosphatase (ALP) in the ALP-ssDNA modified magnetic bead. The released ALP can catalyze para-nitrophenyl phosphate to generate para-nitrophenol, resulting in substantial changes in absorbance and fluorescence, both of which can be used for detection with the naked eye. This strategy allows the sensitive detection of ASFV DNA, with a 20 copies/mL detection limit; no cross-reactivity with other viruses was observed. A good linear relationship was obtained in serum. In addition, this sensor displayed 100% specificity and sensitivity for clinical sample analysis. This method integrates the high sensitivity of fluorescence with easy readout of colorimetry and enables a simple, low-cost, and highly sensitive dual-mode detection of viral nucleic acid, thereby providing a broad prospect for the practical application in the diagnosis of virus infection.

RevDate: 2023-05-24
CmpDate: 2023-05-24

Xiang X, Song M, Xu X, et al (2023)

Microfluidic Biosensor Integrated with Signal Transduction and Enhancement Mechanism for Ultrasensitive Noncompetitive Assay of Multiple Mycotoxins.

Analytical chemistry, 95(20):7993-8001.

To achieve high-throughput ultrasensitive detection of mycotoxins in food, a functional DNA-guided transition-state CRISPR/Cas12a microfluidic biosensor (named FTMB) was successfully constructed. The signal transduction CRISPR/Cas12a strategy in FTMB has utilized DNA sequences with a specific recognition function and activators to form trigger switches. Meanwhile, the transition-state CRISPR/Cas12a system was constructed by adjusting the composition ratio of crRNA and activator to achieve a high response for low concentrations of target mycotoxins. On the other hand, the signal enhancement of FTMB has efficiently integrated the signal output of quantum dots (QDs) with the fluorescence enhancement effect of photonic crystals (PCs). The construction of universal QDs for the CRISPR/Cas12a system and PC films matching the photonic bandgap produced a significant signal enhancement by a factor of 45.6. Overall, FTMB exhibited a wide analytic range (10[-5]-10[1] ng·mL[-1]), low detection of limit (fg·mL[-1]), short detection period (∼40 min), high specificity, good precision (coefficients of variation <5%), and satisfactory practical sample analysis capacity (the consistency with HPLC at 88.76%-109.99%). It would provide a new and reliable solution for the rapid detection of multiple small molecules in the fields of clinical diagnosis and food safety.

RevDate: 2023-05-24
CmpDate: 2023-05-24

Gu J, Sun J, Tian K, et al (2023)

Reversal of hepatic fibrosis by the co-delivery of drug and ribonucleoprotein-based genome editor.

Biomaterials, 298:122133.

Liver fibrosis is a chronic disease without effective treatment in the clinic. Gene editing systems such as the well-known CRISPR/Cas9 have shown great potential in the biomedical field. However, the delivery of the ribonucleoprotein is challenging due to the unstable RNA probe and the requirement for the entrance to the nucleus. Recently, a structure-guided endonuclease (SGN) has been reported as an effective gene-editing system composed of a nuclease and stable DNA probes, which can regulate the protein expression by targeting specific mRNA outside the nucleus. Here, we conjugated the SGN to a nanomicelle as the delivery system. In the resulting material, the chance of the collision between the endonuclease and the probe was raised due to the confinement of the two components within the 40-nm nanomicelle, thus the mRNA can be cleaved immediately after being captured by the probe, resulting in a space-induced nucleotide identification-cleavage acceleration effect. The delivery system was used to treat liver fibrosis via the co-delivery of SGN and a drug rosiglitazone to the hepatic stellate cells, which separately downregulated the expression of tissue inhibitor of metalloprotease-1 and inactivated the hepatic stellate cells. The system successfully reversed the liver fibrosis in mice through the bidirectional regulatory that simultaneously promoted the degradation and inhibited the production of the collagen, demonstrating the great potency of the SGN system as gene medicine.

RevDate: 2023-05-23

Fang L, Yang L, Han M, et al (2023)

CRISPR-cas technology: A key approach for SARS-CoV-2 detection.

Frontiers in bioengineering and biotechnology, 11:1158672.

The CRISPR (Clustered Regularly Spaced Short Palindromic Repeats) system was first discovered in prokaryotes as a unique immune mechanism to clear foreign nucleic acids. It has been rapidly and extensively used in basic and applied research owing to its strong ability of gene editing, regulation and detection in eukaryotes. Hererin in this article, we reviewed the biology, mechanisms and relevance of CRISPR-Cas technology and its applications in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnosis. CRISPR-Cas nucleic acid detection tools include CRISPR-Cas9, CRISPR-Cas12, CRISPR-Cas13, CRISPR-Cas14, CRISPR nucleic acid amplification detection technology, and CRISPR colorimetric readout detection system. The above CRISPR technologies have been applied to the nucleic acid detection, including SARS-CoV-2 detection. Common nucleic acid detection based on CRISPR derivation technology include SHERLOCK, DETECTR, and STOPCovid. CRISPR-Cas biosensing technology has been widely applied to point-of-care testing (POCT) by targeting recognition of both DNA molecules and RNA Molecules.

RevDate: 2023-05-23
CmpDate: 2023-05-23

Yang X, Zeng X, Chen X, et al (2023)

Development of a CRISPR/Cas12a-recombinase polymerase amplification assay for visual and highly specific identification of the Congo Basin and West African strains of mpox virus.

Journal of medical virology, 95(5):e28757.

Human mpox is a zoonotic disease, similar to smallpox, caused by the mpox virus, which is further subdivided into Congo Basin and West African clades with different pathogenicity. In this study, a novel diagnostic protocol utilizing clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 12a nuclease (CRISPR/Cas12a)-mediated recombinase polymerase amplification (RPA) was developed to identify mpox in the Congo Basin and West Africa (CRISPR-RPA). Specific RPA primers targeting D14L and ATI were designed. CRISPR-RPA assay was performed using various target templates. In the designed CRISPR-RPA reaction system, the exponentially amplified RPA amplification products with a protospacer adjacent motif (PAM) site can locate the Cas12a/crRNA complex to its target regions, which successfully activates the CRISPR/Cas12a effector and achieves ultrafast trans-cleavage of a single-stranded DNA probe. The limit of detection for the CRISPR-RPA assay was 10 copies per reaction for D14L- and ATI-plasmids. No cross-reactivity was observed with non-mpox strains, confirming the high specificity of the CRISPR-RPA assay for distinguishing between the Congo Basin and West African mpox. The CRISPR-RPA assay can be completed within 45 min using real-time fluorescence readout. Moreover, the cleavage results were visualized under UV light or an imaging system, eliminating the need for a specialized apparatus. In summary, the developed CRISPR/RPA assay is a visual, rapid, sensitive, and highly specific detection technique that can be used as an attractive potential identification tool for Congo Basin and West African mpox in resource-limited laboratories.

RevDate: 2023-05-21

Chirco KR, Martinez C, DA Lamba (2023)

Advancements in pre-clinical development of gene editing-based therapies to treat inherited retinal diseases.

Vision research, 209:108257 pii:S0042-6989(23)00081-0 [Epub ahead of print].

One of the major goals in the inherited retinal disease (IRD) field is to develop an effective therapy that can be applied to as many patients as possible. Significant progress has already been made toward this end, with gene editing at the forefront. The advancement of gene editing-based tools has been a recent focus of many research groups around the world. Here, we provide an update on the status of CRISPR/Cas-derived gene editors, promising options for delivery of these editing systems to the retina, and animal models that aid in pre-clinical testing of new IRD therapeutics.

RevDate: 2023-05-23
CmpDate: 2023-05-23

Keppeke GD, Diogenes L, Gomes K, et al (2023)

"Untargeting" autoantibodies using genome editing, a proof-of-concept study.

Clinical immunology (Orlando, Fla.), 251:109343.

Autoantibodies (AAbs) are useful biomarkers and many have direct pathogenic role. Current standard therapies for elimination of specific B/plasma-cell clones are not fully efficient. We apply CRISPR/Cas9 genome-editing to knockout V(D)J rearrangements that produce pathogenic AAbs in vitro. HEK293T cell-lines were established stably expressing a humanized anti-dsDNA Ab (clone 3H9) and a human-derived anti-nAChR-α1 Ab (clone B12L). For each clone, five CRISPR/Cas9 heavy-chain's CDR2/3-targeting guided-RNAs (T-gRNAs) were designed. Non-Target-gRNA (NT-gRNA) was control. After editing, levels of secreted Abs were evaluated, as well as 3H9 anti-dsDNA and B12L anti-AChR reactivities. T-gRNAs editing decreased expression of heavy-chain genes to ∼50-60%, compared to >90% in NT-gRNA, although secreted Abs levels and reactivity to their respective antigens in T-gRNAs decreased ∼90% and ∼ 95% compared with NT-gRNA for 3H9 and B12L, respectively. Sequencing indicated indels at Cas9 cut-site, which could lead to codon jam, and consequently, knockout. Additionally, remaining secreted 3H9-Abs presented variable dsDNA reactivity among the five T-gRNA, suggesting the exact Cas9 cut-site and indels further interfere with antibody-antigen interaction. CRISPR/Cas9 genome-editing was very effective to knockout the Heavy-Chain-IgG genes, considerably affecting AAbs secretion and binding capacity, fostering application of this concept to in vivo models as a potential novel therapeutic approach for AAb-mediated diseases.

RevDate: 2023-05-23
CmpDate: 2023-05-23

Carusillo A, Haider S, Schäfer R, et al (2023)

A novel Cas9 fusion protein promotes targeted genome editing with reduced mutational burden in primary human cells.

Nucleic acids research, 51(9):4660-4673.

Precise genome editing requires the resolution of nuclease-induced DNA double strand breaks (DSBs) via the homology-directed repair (HDR) pathway. In mammals, this is typically outcompeted by non-homologous end-joining (NHEJ) that can generate potentially genotoxic insertion/deletion mutations at DSB sites. Because of higher efficacy, clinical genome editing has been restricted to imperfect but efficient NHEJ-based approaches. Hence, strategies that promote DSB resolution via HDR are essential to facilitate clinical transition of HDR-based editing strategies and increase safety. Here we describe a novel platform that consists of a Cas9 fused to DNA repair factors to synergistically inhibit NHEJ and favor HDR for precise repairing of Cas-induced DSBs. Compared to canonical CRISPR/Cas9, the increase in error-free editing ranges from 1.5-fold to 7-fold in multiple cell lines and in primary human cells. This novel CRISPR/Cas9 platform accepts clinically relevant repair templates, such as oligodeoxynucleotides (ODNs) and adeno-associated virus (AAV)-based vectors, and has a lower propensity to induce chromosomal translocations as compared to benchmark CRISPR/Cas9. The observed reduced mutational burden, resulting from diminished indel formation at on- and off-target sites, provides a remarkable gain in safety and advocates this novel CRISPR system as an attractive tool for therapeutic applications depending on precision genome editing.

RevDate: 2023-05-23
CmpDate: 2023-05-23

Byun G, Yang J, SW Seo (2023)

CRISPRi-mediated tunable control of gene expression level with engineered single-guide RNA in Escherichia coli.

Nucleic acids research, 51(9):4650-4659.

Precise control of gene expression is essential for flux redistribution in metabolic pathways. Although the CRISPR interference (CRISPRi) system can effectively repress gene expression at the transcriptional level, it has still been difficult to precisely control the level without loss of specificity or an increase in cell toxicity. In this study, we developed a tunable CRISPRi system that performs transcriptional regulation at various levels. We constructed a single-guide RNA (sgRNA) library targeting repeat, tetraloop, and anti-repeat regions to modulate the binding affinity against dCas9. Each screened sgRNA could regulate the gene expression at a certain level between fully-repressing and non-repressing states (>45-fold). These sgRNAs also enabled modular regulation with various target DNA sequences. We applied this system to redistribute the metabolic flux to produce violacein derivatives in a predictable ratio and optimize lycopene production. This system would help accelerate the flux optimization processes in metabolic engineering and synthetic biology.

RevDate: 2023-05-23
CmpDate: 2023-05-23

Pan C, Li R, Shui L, et al (2023)

A new method to synthesize multiple gRNA libraries and functional mapping of mammalian H3K4me3 regions.

Nucleic acids research, 51(9):e50.

Genetic screening based on the clustered regularly interspaced palindromic repeat (CRISPR) system has been indicated to be a powerful tool for identifying regulatory genes or cis-elements. However, when applying CRISPR screens to pinpoint functional elements at particular loci, a large number of guide RNA (gRNA) spacers may be required to achieve saturated coverage. Here, we present a controlled template-dependent elongation (CTDE) method relying on reversible terminators to synthesize gRNA libraries with genomic regions of interest. By applying this approach to H3K4me3 chromatin immunoprecipitation (ChIP)-derived DNA of mammalian cells, mega-sized gRNA libraries were synthesized in a tissue-specific manner, with which we conducted screening experiments to annotate essential sites for cell proliferation. Additionally, we confirmed that an essential site within the intron of LINC00339 regulates its own mRNA and that LINC00339 is a novel regulator of the cell cycle that maintains HepG2 proliferation. The CTDE method has the potential to be automated with high efficiency at low cost, and will be widely used to identify functional elements in mammalian genomes.

RevDate: 2023-05-23
CmpDate: 2023-05-23

Rahman K, Jamal M, Chen X, et al (2022)

Reprogramming Mycobacterium tuberculosis CRISPR System for Gene Editing and Genome-wide RNA Interference Screening.

Genomics, proteomics & bioinformatics, 20(6):1180-1196.

Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), which is still the leading cause of mortality from a single infectious disease worldwide. The development of novel anti-TB drugs and vaccines is severely hampered by the complicated and time-consuming genetic manipulation techniques for M. tuberculosis. Here, we harnessed an endogenous type III-A CRISPR/Cas10 system of M. tuberculosis for efficient gene editing and RNA interference (RNAi). This simple and easy method only needs to transform a single mini-CRISPR array plasmid, thus avoiding the introduction of exogenous protein and minimizing proteotoxicity. We demonstrated that M. tuberculosis genes can be efficiently and specifically knocked in/out by this system as confirmed by DNA high-throughput sequencing. This system was further applied to single- and multiple-gene RNAi. Moreover, we successfully performed genome-wide RNAi screening to identify M. tuberculosis genes regulating in vitro and intracellular growth. This system can be extensively used for exploring the functional genomics of M. tuberculosis and facilitate the development of novel anti-TB drugs and vaccines.

RevDate: 2023-05-21

Wang X, Jing S, Wang W, et al (2023)

Direct and noninvasive fluorescence analysis of an RNA-protein interaction based on a CRISPR/Cas12a-powered assay.

Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy, 299:122884 pii:S1386-1425(23)00569-3 [Epub ahead of print].

RNA-protein interactions (RPIs) play critical roles in gene transcription and protein expression, but current analytical methods for RPIs are mainly performed in an invasive manner, involving special RNA/protein labeling, hampering access to intact and precise information on RPIs. In this work, we present the first CRISPR/Cas12a-based fluorescence assay for the direct analysis of RPIs without RNA/protein labeling steps. Select vascular endothelial growth factor 165 (VEGF165)/its RNA aptamer interaction as a model, the RNA sequence simultaneously serves as both the aptamer of VEGF165 and crRNA of CRISPR/Cas12a system, and the presence of VEGF165 facilitates VEGF165/its RNA aptamer interaction, thus prohibiting the formation of Cas12a-crRNA-DNA ternary complex along with low fluorescence signal. The assay showed a detection limit of 0.23 pg mL[-1], and good performance in serum-spiked samples with an RSD of 0.4 %-13.1 %. This simple and selective strategy opens the door for establishing CRISPR/Cas-based biosensors for gaining intact information on RPIs, and shows widespread potential for other RPIs analysis.

RevDate: 2023-05-20

Wu J, Tao Y, Deng D, et al (2023)

The applications of CRISPR/Cas-mediated genome editing in genetic hearing loss.

Cell & bioscience, 13(1):93.

Hearing loss (HL) can be caused by a number of different genetic factors. Non-syndromic HL refers that HL occurs as an isolated symptom in an individual, whereas syndromic HL refers that HL is associated with other symptoms or abnormalities. To date, more than 140 genes have been identified as being associated with non-syndromic HL, and approximately 400 genetic syndromes can include HL as one of the clinical symptoms. However, no gene therapeutic approaches are currently available to restore or improve hearing. Therefore, there is an urgent necessity to elucidate the possible pathogenesis of specific mutations in HL-associated genes and to investigate the promising therapeutic strategies for genetic HL. The development of the CRISPR/Cas system has revolutionized the field of genome engineering, which has become an efficacious and cost-effective tool to foster genetic HL research. Moreover, several in vivo studies have demonstrated the therapeutic efficacy of the CRISPR/Cas-mediated treatments for specific genetic HL. In this review, we briefly introduce the progress in CRISPR/Cas technique as well as the understanding of genetic HL, and then we detail the recent achievements of CRISPR/Cas technique in disease modeling and therapeutic strategies for genetic HL. Furthermore, we discuss the challenges for the application of CRISPR/Cas technique in future clinical treatments.

RevDate: 2023-05-20

Even-Zohar N, Metin-Armagan D, Ben-Shlomo A, et al (2023)

Generation of isogenic and homozygous MEN1 mutant cell lines from patient-derived iPSCs using CRISPR/Cas9.

Stem cell research, 69:103124 pii:S1873-5061(23)00110-1 [Epub ahead of print].

MEN1, an autosomal dominant disorder caused by mutations in the tumor suppressor gene MEN1, manifests with co-occurrence of multiple endocrine/neuroendocrine neoplasms. An iPSC line derived from an index patient carrying the mutation c.1273C>T (p.Arg465*) was edited using a single multiplex CRISPR/Cas approach to create an isogenic control non-mutated line and a homozygous double mutant line. These cell lines will be useful for elucidating subcellular MEN1 pathophysiology and for screening to identify potential MEN1 therapeutic targets.

RevDate: 2023-05-22
CmpDate: 2023-05-22

Lee K, C Lee (2023)

Generation of CRISPR-Cas9-mediated knockin mutant models in mice and MEFs for studies of polymorphism in clock genes.

Scientific reports, 13(1):8109.

The creation of mutant mice has been invaluable for advancing biomedical science, but is too time- and resource-intensive for investigating the full range of mutations and polymorphisms. Cell culture models are therefore an invaluable complement to mouse models, especially for cell-autonomous pathways like the circadian clock. In this study, we quantitatively assessed the use of CRISPR to create cell models in mouse embryonic fibroblasts (MEFs) as compared to mouse models. We generated two point mutations in the clock genes Per1 and Per2 in mice and in MEFs using the same sgRNAs and repair templates for HDR and quantified the frequency of the mutations by digital PCR. The frequency was about an order of magnitude higher in mouse zygotes compared to that in MEFs. However, the mutation frequency in MEFs was still high enough for clonal isolation by simple screening of a few dozen individual cells. The Per mutant cells that we generated provide important new insights into the role of the PAS domain in regulating PER phosphorylation, a key aspect of the circadian clock mechanism. Quantification of the mutation frequency in bulk MEF populations provides a valuable basis for optimizing CRISPR protocols and time/resource planning for generating cell models for further studies.

RevDate: 2023-05-22

Wu J, Xu H, Hu F, et al (2023)

CRISPR-Cas and catalytic hairpin assembly technology for target-initiated amplification detection of pancreatic cancer specific tsRNAs.

Frontiers in bioengineering and biotechnology, 11:1169424.

Transfer RNA-derived small RNAs (tsRNAs) tRF-LeuCAG-002 (ts3011a RNA) is a novel class of non-coding RNAs biomarker for pancreatic cancer (PC). Reverse transcription polymerase chain reaction (RT-qPCR) has been unfit for community hospitals that are short of specialized equipment or laboratory setups. It has not been reported whether isothermal technology can be used for detection, because the tsRNAs have rich modifications and secondary structures compared with other non-coding RNAs. Herein, we have employed a catalytic hairpin assembly (CHA) circuit and clustered regularly interspaced short palindromic repeats (CRISPR) to develop an isothermal and target-initiated amplification method for detecting ts3011a RNA. In the proposed assay, the presence of target tsRNA triggers the CHA circuit that transforms new DNA duplexes to activate collateral cleavage activity of CRISPR-associated proteins (CRISPR-Cas) 12a, achieving cascade signal amplification. This method showed a low detection limit of 88 aM at 37 °C within 2 h. Moreover, it was demonstrated for the first time that, this method is less likely to produce aerosol contamination than RT-qPCR by simulating aerosol leakage experiments. This method has good consistency with RT-qPCR in the detection of serum samples and showed great potential for PC-specific tsRNAs point-of-care testing (POCT).

RevDate: 2023-05-19

Shi L, Su J, Cho MJ, et al (2023)

Promoter editing for the genetic improvement of crops.

Journal of experimental botany pii:7173264 [Epub ahead of print].

The proper gene expression plays a fundamental role in the regulation of agronomically important traits in crop plants. The genetic manipulation of plant promoters through genome editing has emerged as an effective strategy to create favorable traits in crops by altering the expression pattern of the pertinent genes. Promoter editing can be applied in a directed manner, where nucleotide sequences associated with favorable traits are precisely generated. Alternatively, promoter editing can also be exploited as a random mutagenic approach to generate novel genetic variations within a designated promoter, from which elite alleles are selected based on their phenotypic effects. Pioneering studies have demonstrated the potential of promoter editing in engineering agronomically important traits as well as in mining novel promoter alleles valuable for plant breeding. In this review article, we update the progress in the application of promoter editing in crops for increased yield, enhanced tolerance to biotic and abiotic stresses, and improved quality. We also discuss several remaining technical bottlenecks and how this strategy may be better employed for the genetic improvement of crops in the future.

RevDate: 2023-05-22
CmpDate: 2023-05-22

Chaudhry A, Hassan AU, Khan SH, et al (2023)

The changing landscape of agriculture: role of precision breeding in developing smart crops.

Functional & integrative genomics, 23(2):167.

Food plants play a crucial role in human survival, providing them essential nutrients. However, traditional breeding methods have not been able to keep up with the demands of the growing population. The improvement of food plants aims to increase yield, quality, and resistance to biotic and abiotic stresses. With CRISPR/Cas9, researchers can identify and edit key genes conferring desirable qualities in agricultural plants, including increased yield, enhanced product quality attributes, and increased tolerance to biotic and abiotic challenges. These modifications have enabled the creation of "smart crops" that exhibit rapid climatic adaptation, resistance to extreme weather conditions and high yield and quality. The use of CRISPR/Cas9 combined with viral vectors or growth regulators has made it possible to produce more efficient modified plants with certain conventional breeding methods. However, ethical and regulatory aspects of this technology must be carefully considered. Proper regulation and application of genome editing technology can bring immense benefits to agriculture and food security. This article provides an overview of genetically modified genes and conventional as well as emerging tools, including CRISPR/Cas9, that have been utilized to enhance the quality of plants/fruits and their products. The review also discusses the challenges and prospects associated with these techniques.

RevDate: 2023-05-19

Zhang J, Zhu A, Mei M, et al (2023)

Repurposing CRISPR/Cas to Discover SARS-CoV-2 Detecting and Neutralizing Aptamers.

Advanced science (Weinheim, Baden-Wurttemberg, Germany) [Epub ahead of print].

RNA aptamers provide useful biological probes and therapeutic agents. New methodologies to screen RNA aptamers will be valuable by complementing the traditional Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Meanwhile, repurposing clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated systems (Cas) has expanded their utility far beyond their native nuclease function. Here, CRISmers, a CRISPR/Cas-based novel screening system for RNA aptamers based on binding to a chosen protein of interest in a cellular context, is presented. Using CRISmers, aptamers are identified specifically targeting the receptor binding domain (RBD) of the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Two aptamer leads enable sensitive detection and potent neutralization of SARS-CoV-2 Delta and Omicron variants in vitro. Intranasal administration of one aptamer, further modified with 2'-fluoro pyrimidines (2'-F), 2'-O-methyl purines (2'-O), and conjugation with both cholesterol and polyethylene glycol of 40 kDa (PEG40K), achieves effective prophylactic and therapeutic antiviral activity against live Omicron BA.2 variants in vivo. The study concludes by demonstrating the robustness, consistency, and potential broad utility of CRISmers using two newly identified aptamers but switching CRISPR, selection marker, and host species.

RevDate: 2023-05-22
CmpDate: 2023-05-22

Ma Y, Zou L, Liang Y, et al (2023)

[Rapid detection and genotyping of SARS-CoV-2 Omicron BA.4/5 variants using a RT-PCR and CRISPR-Cas12a-based assay].

Nan fang yi ke da xue xue bao = Journal of Southern Medical University, 43(4):516-526.

OBJECTIVE: To establish a rapid detection and genotyping method for SARS-CoV-2 Omicron BA.4/5 variants using CRISPPR-Cas12a gene editing technology.

METHODS: We combined reverse transcription-polymerase chain reaction (RT-PCR) and CRISPR gene editing technology and designed a specific CRISPPR RNA (crRNA) with suboptimal protospacer adjacent motifs (PAM) for rapid detection and genotyping of SARS- CoV-2 Omicron BA.4/5 variants. The performance of this RT- PCR/ CRISPPR-Cas12a assay was evaluated using 43 clinical samples of patients infected by wild-type SARS-CoV-2 and the Alpha, Beta, Delta, Omicron BA. 1 and BA. 4/5 variants and 20 SARS- CoV- 2-negative clinical samples infected with 11 respiratory pathogens. With Sanger sequencing method as the gold standard, the specificity, sensitivity, concordance (Kappa) and area under the ROC curve (AUC) of RT-PCR/CRISPPR-Cas12a assay were calculated.

RESULTS: This assay was capable of rapid and specific detection of SARS- CoV-2 Omicron BA.4/5 variant within 30 min with the lowest detection limit of 10 copies/μL, and no cross-reaction was observed in SARS-CoV-2-negative clinical samples infected with 11 common respiratory pathogens. The two Omicron BA.4/5 specific crRNAs (crRNA-1 and crRNA-2) allowed the assay to accurately distinguish Omicron BA.4/5 from BA.1 sublineage and other major SARS-CoV-2 variants of concern. For detection of SARS-CoV-2 Omicron BA.4/5 variants, the sensitivity of the established assay using crRNA-1 and crRNA-2 was 97.83% and 100% with specificity of 100% and AUC of 0.998 and 1.000, respectively, and their concordance rate with Sanger sequencing method was 92.83% and 96.41%, respectively.

CONCLUSION: By combining RT-PCR and CRISPPR-Cas12a gene editing technology, we successfully developed a new method for rapid detection and identification of SARS-CoV-2 Omicron BA.4/5 variants with a high sensitivity, specificity and reproducibility, which allows rapid detection and genotyping of SARS- CoV-2 variants and monitoring of the emerging variants and their dissemination.

RevDate: 2023-05-22
CmpDate: 2023-05-22

Liu C, Yao X, Liu C, et al (2023)

Development and evaluation of RPA-NFO-LFT and RPA-Cas12a-LFT systems for the detection of Candida albicans.

Analytical methods : advancing methods and applications, 15(19):2355-2365.

Recently, the growing number of medical interventions has led to the risk of invasive candidiasis. Among them, Candida albicans (C. albicans) infection has the highest incidence, which has led to great demand for developing early diagnosis methods. In this study, two lateral flow device based molecular assay systems, RPA-NFO-LFT and RPA-Cas12a-LFT, were established and optimized to achieve the detection of C. albicans. Firstly, efficient and specific primers for C. albicans detection were designed and screened, and the purification of amplification products was also explored. Then, many important conditions and issues for each system were investigated and discussed to improve the performances of the test strip devices in C. albicans detection. An evaluation study revealed that both systems showed favorable specificity and sensitivity in the detection of C. albicans samples with a lower detection limit of 10[3] CFU ml[-1], while RPA-Cas12a-LFT is more accurate for visual interpretation and more stable toward samples that exhibit serum nucleic acid interference. Finally, the performances of RPA-NFO-LFT and RPA-Cas12a-LFT were compared with that of the conventional qPCR method. This work might provide a reference for the development of molecular assay devices in practical candidiasis diagnosis.

RevDate: 2023-05-22
CmpDate: 2023-05-22

Foss DV, Muldoon JJ, Nguyen DN, et al (2023)

Peptide-mediated delivery of CRISPR enzymes for the efficient editing of primary human lymphocytes.

Nature biomedical engineering, 7(5):647-660.

CRISPR-mediated genome editing of primary human lymphocytes is typically carried out via electroporation, which can be cytotoxic, cumbersome and costly. Here we show that the yields of edited primary human lymphocytes can be increased substantially by delivering a CRISPR ribonucleoprotein mixed with an amphiphilic peptide identified through screening. We evaluated the performance of this simple delivery method by knocking out genes in T cells, B cells and natural killer cells via the delivery of Cas9 or Cas12a ribonucleoproteins or an adenine base editor. We also show that peptide-mediated ribonucleoprotein delivery paired with an adeno-associated-virus-mediated homology-directed repair template can introduce a chimaeric antigen receptor gene at the T-cell receptor α constant locus, and that the engineered cells display antitumour potency in mice. The method is minimally perturbative, does not require dedicated hardware, and is compatible with multiplexed editing via sequential delivery, which minimizes the risk of genotoxicity. The peptide-mediated intracellular delivery of ribonucleoproteins may facilitate the manufacturing of engineered T cells.


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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E-mail: RJR8222@gmail.com

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

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Curriculum Vitae for R J Robbins

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