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30 Mar 2023 at 01:43
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Bibliography on: CRISPR-Cas


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RJR: Recommended Bibliography 30 Mar 2023 at 01:43 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: 2023-03-28

Ordon J, Kiel N, Becker D, et al (2023)

Targeted gene deletion with SpCas9 and multiple guide RNAs in Arabidopsis thaliana: four are better than two.

Plant methods, 19(1):30.

BACKGROUND: In plant genome editing, RNA-guided nucleases such as Cas9 from Streptococcus pyogenes (SpCas9) predominantly induce small insertions or deletions at target sites. This can be used for inactivation of protein-coding genes by frame shift mutations. However, in some cases, it may be advantageous to delete larger chromosomal segments. This is achieved by simultaneously inducing double strand breaks upstream and downstream of the segment to be deleted. Experimental approaches for the deletion of larger chromosomal segments have not been systematically evaluated.

RESULTS: We designed three pairs of guide RNAs for deletion of a ~ 2.2 kb chromosomal segment containing the Arabidopsis WRKY30 locus. We tested how the combination of guide RNA pairs and co-expression of the exonuclease TREX2 affect the frequency of wrky30 deletions in editing experiments. Our data demonstrate that compared to one pair of guide RNAs, two pairs increase the frequency of chromosomal deletions. The exonuclease TREX2 enhanced mutation frequency at individual target sites and shifted the mutation profile towards larger deletions. However, TREX2 did not elevate the frequency of chromosomal segment deletions.

CONCLUSIONS: Multiplex editing with at least two pairs of guide RNAs (four guide RNAs in total) elevates the frequency of chromosomal segment deletions at least at the AtWRKY30 locus, and thus simplifies the selection of corresponding mutants. Co-expression of the TREX2 exonuclease can be used as a general strategy to increase editing efficiency in Arabidopsis without obvious negative effects.

RevDate: 2023-03-28

Kolvenbach CM, Dworschak GC, Rieke JM, et al (2023)

Modelling human lower urinary tract malformations in zebrafish.

Molecular and cellular pediatrics, 10(1):2.

Advances in molecular biology are improving our understanding of the genetic causes underlying human congenital lower urinary tract (i.e., bladder and urethral) malformations. This has recently led to the identification of the first disease-causing variants in the gene BNC2 for isolated lower urinary tract anatomical obstruction (LUTO), and of WNT3 and SLC20A1 as genes implicated in the pathogenesis of the group of conditions called bladder-exstrophy-epispadias complex (BEEC). Implicating candidate genes from human genetic data requires evidence of their influence on lower urinary tract development and evidence of the found genetic variants' pathogenicity. The zebrafish (Danio rerio) has many advantages for use as a vertebrate model organism for the lower urinary tract. Rapid reproduction with numerous offspring, comparable anatomical kidney and lower urinary tract homology, and easy genetic manipulability by Morpholino®-based knockdown or CRISPR/Cas editing are among its advantages. In addition, established marker staining for well-known molecules involved in urinary tract development using whole-mount in situ hybridization (WISH) and the usage of transgenic lines expressing fluorescent protein under a tissue-specific promoter allow easy visualization of phenotypic abnormalities of genetically modified zebrafish. Assays to examine the functionality of the excretory organs can also be modeled in vivo with the zebrafish. The approach of using these multiple techniques in zebrafish not only enables rapid and efficient investigation of candidate genes for lower urinary tract malformations derived from human data, but also cautiously allows transferability of causality from a non-mammalian vertebrate to humans.

RevDate: 2023-03-28

Wang C, Sun Z, Hu Y, et al (2023)

A Novel Anti-CRISPR AcrIE9.2 Is Associated with Dissemination of blaKPC Plasmids in Klebsiella pneumoniae Sequence Type 15.

Antimicrobial agents and chemotherapy [Epub ahead of print].

Sequence type (ST) 15 has become an emerging clone of carbapenem-resistant Klebsiella pneumoniae in which type I-E* CRISPR-Cas usually exists, indicating that the CRISPR-Cas system may not be able to block the transfer of blaKPC plasmids. The purpose of this study was to explore the mechanisms underlying dissemination of blaKPC plasmids in K. pneumoniae ST15. The type I-E* CRISPR-Cas system was present in 98.0% of 612 nonduplicate K. pneumoniae ST15 strains (88 clinical isolates and 524 from the NCBI database). Twelve ST15 clinical isolates were completely sequenced, and self-targeted protospacers were found on blaKPC plasmids flanked by a protospacer adjacent motif (PAM) of AAT in 11 isolates. The type I-E* CRISPR-Cas system was cloned from a clinical isolate and expressed in Escherichia coli BL21(DE3). In BL21(DE3) harboring the CRISPR system, the transformation efficiency of protospacer-bearing plasmids with a PAM of AAT was reduced by 96.2% compared to the empty vector, indicating that the type I-E* CRISPR-Cas system impeded blaKPC plasmid transfer. BLAST for known anti-CRISPR (Acr) amino acid sequences uncovered a novel AcrIE9-like protein with 40.5% to 44.6% sequence identity with AcrIE9 designated AcrIE9.2, which was present in 90.1% (146 of 162) of ST15 strains carrying both blaKPC and the CRISPR-Cas system. When AcrIE9.2 was cloned and expressed in a ST15 clinical isolate, the conjugation frequency of a CRISPR-targeted blaKPC plasmid was increased from 3.96 × 10[-6] to 2.01 × 10[-4] compared to the AcrIE9.2 absent strain. In conclusion, AcrIE9.2 may be associated with the dissemination of blaKPC in ST15 by repressing CRISPR-Cas activity.

RevDate: 2023-03-28

Li X, Bao N, Yan Z, et al (2023)

Degradation of Antibiotic Resistance Genes by VADER with CRISPR-Cas Immunity.

Applied and environmental microbiology [Epub ahead of print].

The evolution and dissemination of antibiotic resistance genes (ARGs) are prompting severe health and environmental issues. While environmental processes, e.g., biological wastewater treatment, are key barriers to prevent the spread of ARGs, they are often sources of ARGs at the same time, requiring upgraded biotechnology. Here, we present VADER, a synthetic biology system for the degradation of ARGs based on CRISPR-Cas immunity, an archaeal and bacterial immune system for eliminating invading foreign DNAs, to be implemented for wastewater treatment processes. Navigated by programmable guide RNAs, VADER targets and degrades ARGs depending on their DNA sequences, and by employing an artificial conjugation machinery, IncP, it can be delivered via conjugation. The system was evaluated by degrading plasmid-borne ARGs in Escherichia coli and further demonstrated via the elimination of ARGs on the environmentally relevant RP4 plasmid in Pseudomonas aeruginosa. Next, a prototype conjugation reactor at a 10-mL scale was devised, and 100% of the target ARG was eliminated in the transconjugants receiving VADER, giving a proof of principle for the implementation of VADER in bioprocesses. By generating a nexus of synthetic biology and environmental biotechnology, we believe that our work is not only an enterprise for tackling ARG problems but also a potential solution for managing undesired genetic materials in general in the future. IMPORTANCE Antibiotic resistance has been causing severe health problems and has led to millions of deaths in recent years. Environmental processes, especially those of the wastewater treatment sector, are an important barrier to the spread of antibiotic resistance from the pharmaceutical industry, hospitals, or civil sewage. However, they have been identified as a nonnegligible source of antibiotic resistance at the same time, as antibiotic resistance with its main cause, antibiotic resistance genes (ARGs), may accumulate in biological treatment units. Here, we transplanted the CRISPR-Cas system, an immune system via programmable DNA cleavage, to tackle the antibiotic resistance problem raised in wastewater treatment processes, and we propose a new sector specialized in ARG removal with a conjugation reactor to implement the CRISPR-Cas system. Our study provides a new angle for resolving public health issues via the implementation of synthetic biology in environmental contexts at the process level.

RevDate: 2023-03-28

Song D, Wang F, Ju Y, et al (2023)

Application and development of noninvasive biomarkers for colorectal cancer screening: a systematic review.

International journal of surgery (London, England) pii:01279778-990000000-00206 [Epub ahead of print].

BACKGROUND: Colorectal cancer (CRC) is the second most common cause of cancer-related death (9.4% of the 9.9 million cancer deaths). However, CRC develops slowly, and early detection and intervention can effectively improve the survival rate and quality of life. Although colonoscopy can detect and diagnose CRC, it is unsuitable for CRC screening in average-risk populations. Some commercial kits based on DNA mutation or methylation are approved for screening, but the low sensitivity for advanced adenoma or early-stage CRC would limit the applications.

MAIN RESULTS: Recently, researchers have focused on developing noninvasive or minimally invasive, easily accessible biomarkers with higher sensitivity and accuracy for CRC screening. Numerous reports describe advances in biomarkers, including DNA mutations and methylation, mRNA and miRNA, gut microbes, and metabolites, as well as low-throughput multiomics panels. In small cohorts, the specificity and sensitivity improved when fecal immunochemical testing combined with other biomarkers; further verification in large cohorts is expected. In addition, the continuous improvement of laboratory technology has also improved the sensitivity of detection technology, such as PCR, and the application of CRISPR/Cas technology. Besides, artificial intelligence has extensively promoted the mining of biomarkers. Machine learning was performed to construct a diagnosis model for CRC screening based on the cfDNA fragment features from whole-genome sequencing data. In another study, multiomics markers, including cfDNA, epigenetic, and protein signals, were also discovered by machine learning. Finally, advancements in sensor technology promote the applicability of volatile organic compounds in CRC early detection.

CONCLUSION: Here, the authors review advances in early detection and screening of CRC based on different biomarker types. Most studies reported optimistic findings based on preliminary research, and prospective clinical studies are ongoing. These promising biomarkers are expected to more accurately identify early-stage patients with CRC and be applied in the future.

RevDate: 2023-03-29
CmpDate: 2023-03-29

Huang W, Wei H, Wang C, et al (2023)

[Establishment and preliminary evaluation of a fluorescent recombinase-aided amplification/CRISPR-Cas12a system for rapid detection of Plasmodium falciparum].

Zhongguo xue xi chong bing fang zhi za zhi = Chinese journal of schistosomiasis control, 35(1):38-43.

OBJECTIVE: To establish a fluorescent assay for rapid detection of Plasmodium falciparum based on recombinaseaided amplification (RAA) and CRISPR-Cas12a system,and to preliminarily evaluate the diagnostic efficiency of this system.

METHODS: The 18S ribosomal RNA (rRNA) gene of P. falciparum was selected as the target sequence, and three pairs of RAA primers and CRISPR-derived RNA (crRNA) were designed and synthesized. The optimal combination of RAA primers and crRNA was screened and the reaction conditions of the system were optimized to create a fluorescent RAA/CRISPR-Cas12a system. The plasmid containing 18S rRNA gene of the P. falciparum strain 3D7 was generated, and diluted into concentrations of 1 000, 100, 10, 1 copy/μL for the fluorescent RAA/CRISPR-Cas12a assay, and its sensitivity was evaluated. The genomic DNA from P. vivax, P. malariae, P. ovum, hepatitis B virus, human immunodeficiency virus and Treponema pallidum was employed as templates for the fluorescent RAA/CRISPR-Cas12a assay, and its specificity was evaluated. Fifty malaria clinical samples were subjected to the fluorescent RAA/CRISPR-Cas12a assay and nested PCR assay, and the consistency between two assays was compared. In addition, P. falciparum strain 3D7 was cultured in vitro. Then, the culture was diluted into blood samples with parasite densities of 1 000, 500, 200, 50, 10 parasites/μL with healthy volunteers' O-positive red blood cells for the RAA/CRISPR-Cas12a assay, and the detection efficiency was tested.

RESULTS: The Pf-F3/Pf-R3/crRNA2 combination, 2.5 μL as the addition amount of B buffer, 40 min as the RAA reaction time, 37 °C as the reaction temperature of the CRISPR-Cas12a system were employed to establish the fluorescent RAA/CRISPR-Cas12a system. Such a system was effective to detect the plasmid containing 18S rRNA gene of the P. falciparum strain 3D7 at a concentration of 1 copy/μL, and presented fluorescent signals for detection of P. falciparum, but failed to detect P. ovum, P. malariae, P. vivax, T. pallidum, hepatitis B virus or human immunodeficiency virus. The fluorescent RAA/CRISPR-Cas12a system and nested PCR assay showed completely consistent results for detection of 50 malaria clinical samples (kappa = 1.0, P < 0.001). Following 6-day in vitro culture of the P. falciparum strain 3D7, 10 mL cultures were generated and the fluorescent RAA/CRISPR-Cas12a system showed the minimal detection limit of 50 parasites/μL.

CONCLUSIONS: The fluorescent RAA/CRISPR-Cas12a system is rapid, sensitive and specific for detection of P. falciparum, which shows promising value for rapid detection and risk monitoring of P. falciparum.

RevDate: 2023-03-29
CmpDate: 2023-03-29

Sconocchia T, Foßelteder J, Köhnke T, et al (2023)

Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human Hematopoietic Stem and Progenitor Cells.

Journal of visualized experiments : JoVE.

Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional properties such as proliferation and differentiation, thereby promoting the development of hematologic malignancies. Efficient and precise genetic manipulation of HSPCs is required to model, characterize, and better understand the functional consequences of recurrent somatic mutations. Mutations can have a deleterious effect on a gene and result in loss-of-function (LOF) or, in stark contrast, may enhance function or even lead to novel characteristics of a particular gene, termed gain-of-function (GOF). In contrast to LOF mutations, GOF mutations almost exclusively occur in a heterozygous fashion. Current genome-editing protocols do not allow for the selective targeting of individual alleles, hampering the ability to model heterozygous GOF mutations. Here, we provide a detailed protocol on how to engineer heterozygous GOF hotspot mutations in human HSPCs by combining CRISPR/Cas9-mediated homology-directed repair and recombinant AAV6 technology for efficient DNA donor template transfer. Importantly, this strategy makes use of a dual fluorescent reporter system to allow for the tracking and purification of successfully heterozygously edited HSPCs. This strategy can be employed to precisely investigate how GOF mutations affect HSPC function and their progression toward hematological malignancies.

RevDate: 2023-03-29
CmpDate: 2023-03-29

Abdulmalek HW, A Yazgan-Karataş (2023)

Improvement of Bacilysin Production in Bacillus subtilis by CRISPR/Cas9-Mediated Editing of the 5'-Untranslated Region of the bac Operon.

Journal of microbiology and biotechnology, 33(3):410-418.

Bacilysin is a dipeptide antibiotic composed of L-alanine and L-anticapsin produced by certain strains of Bacillus subtilis. Bacilysin is gaining increasing attention in industrial agriculture and pharmaceutical industries due to its potent antagonistic effects on various bacterial, fungal, and algal pathogens. However, its use in industrial applications is hindered by its low production in the native producer. The biosynthesis of bacilysin is mainly based on the bacABCDEF operon. Examination of the sequence surrounding the upstream of the bac operon did not reveal a clear, strong ribosome binding site (RBS). Therefore, in this study, we aimed to investigate the impact of RBS as a potential route to improve bacilysin production. For this, the 5' untranslated region (5'UTR) of the bac operon was edited using the CRISPR/Cas9 approach by introducing a strong ribosome binding sequence carrying the canonical Shine-Dalgarno sequence (TAAGGAGG) with an 8 nt spacing from the AUG start codon. Strong RBS substitution resulted in a 2.87-fold increase in bacilysin production without affecting growth. Strong RBS substitution also improved the mRNA stability of the bac operon. All these data revealed that extensive RBS engineering is a promising key option for enhancing bacilysin production in its native producers.

RevDate: 2023-03-29
CmpDate: 2023-03-29

Mathew SM (2023)

Strategies for generation of mice via CRISPR/HDR-mediated knock-in.

Molecular biology reports, 50(4):3189-3204.

CRISPR/Cas9 framework is generally used to generate genetically modified mouse models. The clustered regularly interspaced short palindromic repeat gene editing technique, can efficiently generate knock-outs using the non-homologous end-joining repair pathway. Small knock-ins also work precisely using a repair template with help of homology-directed-repair (HDR) mechanism. However, when the fragment size is larger than 4-5 kb, the knock-in tends to be error prone and the efficiency decreases. Certain types of modifications, in particular insertions of very large DNA fragments (10-100 kb) or entire gene replacements, are still difficult. The HDR process needs further streamlining and improvement. Here in this review, we describe methods to enhance the efficiency of the knock-in through checking each step from the guide design to the microinjection and choice of the oocyte donors. This helps in understanding the parameters that can be modified to get improved knock-in efficiency via CRISPR targeting.

RevDate: 2023-03-29
CmpDate: 2023-03-29

Beracochea V, Stritzler M, Radonic L, et al (2023)

CRISPR/Cas9-mediated knockout of SPL13 radically increases lettuce yield.

Plant cell reports, 42(3):645-647.

RevDate: 2023-03-28

Li M, Qin Z, Yin K, et al (2023)

[Application of CRISPR/Cas systems in the nucleic acid detection of pathogens: a review].

Zhongguo xue xi chong bing fang zhi za zhi = Chinese journal of schistosomiasis control, 35(1):98-103.

Rapid, sensitive and specific detection tools are critical for the prevention and control of infectious diseases. The in vitro nucleic acid amplification assays, including polymerase chain reaction and isothermal amplification technology, have been widely used for the detection of pathogens. Recently, nucleic acid detection-based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) have been developed, which are rapid, highly sensitive, highly specific, and portable. This review describes the classification and principle of CRISPR/Cas systems and their applications in pathogen detection, and discusses the prospects of CRISPR/Cas systems.

RevDate: 2023-03-28

Hu Y, Jiang J, Wang D, et al (2023)

Coexistence of bla KPC-IncFII plasmids and type I-E[*] CRISPR-Cas systems in ST15 Klebsiella pneumoniae.

Frontiers in microbiology, 14:1125531.

The CRISPR-Cas system in Klebsiella pneumoniae can prevent the entry of bla KPC-IncF plasmids. However, some clinical isolates bear the KPC-2 plasmids despite carrying the CRISPR-Cas system. The purpose of this study was to characterize the molecular features of these isolates. A total of 697 clinical K. pneumoniae isolates were collected from 11 hospitals in China, and tested for the presence of CRISPR-Cas systems using polymerase chain reaction. Overall, 164 (23.5%) of 697 K. pneumoniae isolates had type I-E[*] (15.9%) or type I-E (7.7%) CRISPR-Cas systems. The most prevalent sequence type among isolates carrying type I-E[*] CRISPR was ST23 (45.9%), followed by ST15 (18.9%). Isolates with CRISPR-Cas system were more susceptible to ten antimicrobials tested, including carbapenems, compared with the CRISPR-negative isolates. However, there were still 21 CRISPR-Cas-carrying isolates that showed resistance to carbapenems, and these isolates were subjected to whole-genome sequencing. Thirteen of these 21 isolates carried bla KPC-2-bearing plasmids, of which nine had a new plasmid type, IncFIIK34, and two had IncFII(PHN7A8) plasmids. In addition, 12 of these 13 isolates belonged to ST15, while only eight (5.6%, 8/143) isolates belonged to ST15 in carbapenem-susceptible K. pneumoniae carrying CRISPR-Cas systems. In conclusion, we found that bla KPC-2-bearing IncFII plasmids could co-exist with the type I-E[*] CRISPR-Cas systems in ST15 K. pneumoniae.

RevDate: 2023-03-28

Papaioannou NY, Patsali P, Naiisseh B, et al (2023)

High-efficiency editing in hematopoietic stem cells and the HUDEP-2 cell line based on in vitro mRNA synthesis.

Frontiers in genome editing, 5:1141618.

Introduction: Genome editing tools, such as CRISPR/Cas, TALE nucleases and, more recently, double-strand-break-independent editors, have been successfully used for gene therapy and reverse genetics. Among various challenges in the field, tolerable and efficient delivery of editors to target cells and sites, as well as independence from commercially available tools for flexibility and fast adoption of new editing technology are the most pressing. For many hematopoietic research applications, primary CD34[+] cells and the human umbilical cord-derived progenitor erythroid 2 (HUDEP-2) cell line are highly informative substrates and readily accessible for in vitro manipulation. Moreover, ex vivo editing of CD34[+] cells has immediate therapeutic relevance. Both cell types are sensitive to standard transfection procedures and reagents, such as lipofection with plasmid DNA, calling for more suitable methodology in order to achieve high efficiency and tolerability of editing with editors of choice. These challenges can be addressed by RNA delivery, either as a mixture of guide RNA and mRNA for CRISRP/Cas-based systems or as a mixture of mRNAs for TALENs. Compared to ribonucleoproteins or proteins, RNA as vector creates flexibility by removing dependence on commercial availability or laborious in-house preparations of novel editor proteins. Compared to DNA, RNA is less toxic and by obviating nuclear transcription and export of mRNA offers faster kinetics and higher editing efficiencies. Methods: Here, we detail an in vitro transcription protocol based on plasmid DNA templates with the addition of Anti-Reverse Cap Analog (ARCA) using T7 RNA polymerase, and poly (A) tailing using poly (A) polymerase, combined with nucleofection of HUDEP-2 and patient-derived CD34[+] cells. Our protocol for RNA-based delivery employs widely available reagents and equipment and can easily be adopted for universal in vitro delivery of genome editing tools. Results and Discussion: Drawing on a common use case, we employ the protocol to target a β-globin mutation and to reactivate γ-globin expression as two potential therapies for β-hemoglobinopathies, followed by erythroid differentiation and functional analyses. Our protocol allows high editing efficiencies and unimpaired cell viability and differentiation, with scalability, suitability for functional assessment of editing outcomes and high flexibility in the application to different editors.

RevDate: 2023-03-28

Kumari A, Sharma D, Sharma P, et al (2023)

Meta-QTL and haplo-pheno analysis reveal superior haplotype combinations associated with low grain chalkiness under high temperature in rice.

Frontiers in plant science, 14:1133115.

Chalk, an undesirable grain quality trait in rice, is primarily formed due to high temperatures during the grain-filling process. Owing to the disordered starch granule structure, air spaces and low amylose content, chalky grains are easily breakable during milling thereby lowering head rice recovery and its market price. Availability of multiple QTLs associated with grain chalkiness and associated attributes, provided us an opportunity to perform a meta-analysis and identify candidate genes and their alleles contributing to enhanced grain quality. From the 403 previously reported QTLs, 64 Meta-QTLs encompassing 5262 non-redundant genes were identified. MQTL analysis reduced the genetic and physical intervals and nearly 73% meta-QTLs were narrower than 5cM and 2Mb, revealing the hotspot genomic regions. By investigating expression profiles of 5262 genes in previously published datasets, 49 candidate genes were shortlisted on the basis of their differential regulation in at least two of the datasets. We identified non-synonymous allelic variations and haplotypes in 39 candidate genes across the 3K rice genome panel. Further, we phenotyped a subset panel of 60 rice accessions by exposing them to high temperature stress under natural field conditions over two Rabi cropping seasons. Haplo-pheno analysis uncovered haplotype combinations of two starch synthesis genes, GBSSI and SSIIa, significantly contributing towards the formation of grain chalk in rice. We, therefore, report not only markers and pre-breeding material, but also propose superior haplotype combinations which can be introduced using either marker-assisted breeding or CRISPR-Cas based prime editing to generate elite rice varieties with low grain chalkiness and high HRY traits.

RevDate: 2023-03-27

Nieves-Cordones M, Amo J, Hurtado-Navarro L, et al (2023)

Inhibition of SlSKOR by SlCIPK23-SlCBL1/9 uncovers CIPK-CBL-target network rewiring in land plants.

The New phytologist [Epub ahead of print].

Transport of K[+] to the xylem is a key process in the mineral nutrition of the shoots. Although CIPK-CBL complexes have been widely shown to regulate K[+] uptake transport systems, no information is available about the xylem ones. Here, we studied the physiological roles of the voltage-gated K[+] channel SlSKOR and its regulation by the SlCIPK23-SlCBL1/9 complexes in tomato plants. We phenotyped gene-edited slskor and slcipk23 tomato knock-out mutants and carried out two-electrode voltage-clamp (TEVC) and BiFC assays in Xenopus oocytes as key approaches. SlSKOR was preferentially expressed in the root stele and was important not only for K[+] transport to shoots but also, indirectly, for that of Ca[2+] , Mg[2+] , Na[+] , NO3 [-] and Cl[-] . Surprisingly, the SlCIPK23-SlCBL1/9 complexes turned out to be negative regulators of SlSKOR. Inhibition of SlSKOR by SlCIPK23-SlCBL1/9 was observed in Xenopus oocytes and tomato plants. Regulation of SKOR-like channels by CIPK23-CBL1 complexes was also present in Medicago, grapevine and lettuce but not in Arabidopsis and saltwater cress. Our results provide a molecular framework for coordinating root K[+] uptake and its translocation to the shoot by SlCIPK23-SlCBL1/9 in tomato plants. Moreover, they evidenced that CIPK-CBL-target networks have evolved differently in land plants.

RevDate: 2023-03-28
CmpDate: 2023-03-28

Hosoba K, Morita T, Zhang Y, et al (2023)

High-efficient CRISPR/Cas9-mediated gene targeting to establish cell models of ciliopathies.

Methods in cell biology, 175:85-95.

Primary cilia are antenna-like structures developed on the cell surface of mammalian cells during the quiescent G0 phase. Primary cilia in mammalian cells receive extracellular signals for early development and cell tissue homeostasis. Ciliopathies characterized with congenital anomalies such as cerebellar hypoplasia, polycystic kidney and polydactyly are caused by germline mutations of ciliary structure- and function-related genes. Gene knock-out techniques in ciliated cultured cells with the uniformed genetic background are useful to evaluate the pathophysiological roles of ciliopathy-related gene products. Genome editing technology has been applied into the gene knock-out in many types of cultured cell lines. However, the frequency of genome editing varies according to cell species and cycle because of dependency on error-free homology-directed repair (HDR) activity. The human telomerase reverse transcriptase-immortalized retinal pigmented epithelial cell line (hTERT-RPE1) is well known for its suitability in cilia research. However, the efficacy of the HDR-mediated knock-out clone isolation was low. Here, we introduce the clustered regularly interspaced short palindromic repeats-obligate ligation-gated recombination (CRISPR-ObLiGaRe) system, which is a nonhomologous end-joining (NHEJ)-mediated gene targeting method, to generate the knock-out clones effectively even in the lower-HDR activity cell lines including hTERT-RPE1 cell. This CRISPR-ObLiGaRe system is a powerful tool for establishing ciliopathy model cell libraries and identifying each gene function in cilia-related phenotypes.

RevDate: 2023-03-28
CmpDate: 2023-03-28

June CH (2023)

The Unlikely Development of CAR T Cells: a Brief History and Prospects for the Future.

The Keio journal of medicine, 72(1):26.

The quest to cure cancer has been one of the Holy Grails of medicine. The discovery I am going to share with you, CAR T cells, is a promising new form of therapy of cancer that offers the prospect of curing cancer using the immune system. CAR T cells are the first form of synthetic biology to enter the practice of medicine. The notion of using the immune system to fight cancer is an old idea. Over a century ago, bacteria were ground up and injected into patients with late-stage cancers, and occasionally the cancer would disappear. However, we now have precise tools for genetic editing and gene insertion like CRISPR/Cas9 to rewrite the DNA code, offering the possibility to improve the immune system over what has evolved in a Darwinian fashion. In 2017 for the first time, CAR T cells were approved for the treatment of cancer. Today they are used worldwide in more than 15,000 patients and they offer the promise to move beyond cancer to other fields of medicine such as autoimmune disease and heart disease. Here I will discuss the promises and challenges faced by the evolving CAR T cell industry.

RevDate: 2023-03-25

Bendixen L, Jensen TI, RO Bak (2023)

CRISPR/Cas-mediated transcriptional modulation: The therapeutic promises of CRISPRa and CRISPRi.

Molecular therapy : the journal of the American Society of Gene Therapy pii:S1525-0016(23)00145-4 [Epub ahead of print].

The CRISPR/Cas system is commonly known for its ability to cleave DNA in a programmable manner, which has democratized gene editing and facilitated recent breakthroughs in gene therapy. However, newer iterations of the technology using nuclease-disabled Cas enzymes have spurred a variety of different types of genetic engineering platforms such as transcriptional modulation using the CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) systems. This review introduces the creation of these programmable transcriptional modulators, various methods of delivery utilized for these systems, and recent technological developments. CRISPRa and CRISPRi have also been implemented in genetic screens for interrogating gene function and discovering genes involved in various biological pathways. We describe recent compelling examples of how these tools have become powerful means to unravel genetic networks and uncovering important information about devastating diseases. Finally, we provide an overview of preclinical studies in which transcriptional modulation has been used therapeutically, and we discuss potential future directions of these novel modalities.

RevDate: 2023-03-28
CmpDate: 2023-03-28

Li L, Tian H, Wang G, et al (2023)

Ready-to-use interactive dual-readout differential lateral flow biosensor for two genotypes of human papillomavirus.

Biosensors & bioelectronics, 228:115224.

Ready-to-use in vitro diagnosis of multiple genotypes is vital for the prevention and treatment of cervical cancer. Herein, a paper-film-based interactive dual readout differential lateral flow biosensor is proposed to simultaneously assay two high-risk types of human papillomavirus (HPV) within the body-fluid. The CuCo2S4/ZnIn2S4 heterostructure is fabricated on the paper-film compound chip with high thermostability, and surface sulfur vacancy is introduced by mild annealing treatment to endow unexceptionable photoexcitation activity, such structure can be served as an initial energy harvester and converter. With the assistance of differential channels, the dual-target-propelled self-assembly of annular DNA and the cleavage activity of CRISPR-Cas12a are stepwise activated by sequential solution transfer. Accordingly, the input and release of polydopamine-coated gold nanoparticles with photothermal/photoelectric characteristic were implemented. The fabricated biosensor not only realized intelligent thermal-response without large instruments, but also actuated dynamic interfacial charge separation and transfer kinetics to further transmit photoelectric-signal, resulting in desirable interactive dual-signal with low limit-of-detection (0.21 pM for HPV-18 and 42.92 pM for HPV-16). Thanks to the sophisticated design of differential lateral flow paper-film compound chip and interactive dual-signal amplification strategy, sensitive detection of two HPV genotypes is realized, which provides a promising candidate for home medical intelligent diagnosis.

RevDate: 2023-03-28
CmpDate: 2023-03-28

Zhu Z, Guo Y, Wang C, et al (2023)

An ultra-sensitive one-pot RNA-templated DNA ligation rolling circle amplification-assisted CRISPR/Cas12a detector assay for rapid detection of SARS-CoV-2.

Biosensors & bioelectronics, 228:115179.

Rapid, sensitive, and one-pot diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays an extremely important role in point-of-care testing (POCT). Herein, we report an ultra-sensitive and rapid one-pot enzyme-catalyzed rolling circle amplification-assisted CRISPR/FnCas12a assay, termed OPERATOR. OPERATOR employs a single well-designed single-strand padlock DNA, containing a protospacer adjacent motif (PAM) site and a sequence complementary to the target RNA which procedure converts and amplifies genomic RNA to DNA by RNA-templated DNA ligation and multiply-primed rolling circle amplification (MRCA). The MRCA amplicon of single-stranded DNA is cleaved by the FnCas12a/crRNA complex and detected via a fluorescence reader or lateral flow strip. OPERATOR presents outstanding advantages including ultra-sensitivity (1.625 copies per reaction), high specificity (100%), rapid reaction speed (∼30 min), easy operation, low cost, and on-spot visualization. Furthermore, we established a POCT platform by combining OPERATOR with rapid RNA release and a lateral flow strip without professional equipment. The high performance of OPERATOR in SARS-CoV-2 tests was confirmed using both reference materials and clinical samples, and the results suggest that is readily adaptable for point-of-care testing of other RNA viruses.

RevDate: 2023-03-28
CmpDate: 2023-03-28

Boontawon T, Nakazawa T, Choi YJ, et al (2023)

Double-gene targeting with preassembled Cas9 ribonucleoprotein for safe genome editing in the edible mushroom Pleurotus ostreatus.

FEMS microbiology letters, 370:.

CRISPR/Cas9 has potential for efficient molecular breeding. Recently, a foreign-DNA-free gene-targeting technology was established by introducing a preassembled Cas9 ribonucleoprotein (RNP) complex into the oyster mushroom Pleurotus ostreatus. However, the target gene was restricted to such a gene like pyrG, since screening of a genome-edited strain was indispensable and could be performed via examination of 5-fluoroorotic acid (5-FOA) resistance caused by the disruption of the target gene. In this study, we simultaneously introduced the Cas9 RNP complex targeting fcy1, a mutation that conferred P. ostreatus resistance to 5-fluorocytosine (5-FC), together with that targeting pyrG. A total of 76 5-FOA resistant strains were isolated during the first screening. Subsequently, a 5-FC resistance examination was conducted, and three strains exhibited resistance. Genomic PCR experiments followed by DNA sequencing revealed that mutations were successfully introduced into fcy1 and pyrG in the three strains. The results indicated that double gene-edited mutants could be obtained in one experiment employing 5-FOA resistance screening for strains with Cas9 RNP incorporation. This work may pave the way for safe CRISPR/Cas9 technology to isolate mutant strains in any gene of interest without an ectopic marker gene.

RevDate: 2023-03-28
CmpDate: 2023-03-28

Sheikh Beig Goharrizi MA, Ghodsi S, MR Memarjafari (2023)

Implications of CRISPR-Cas9 Genome Editing Methods in Atherosclerotic Cardiovascular Diseases.

Current problems in cardiology, 48(5):101603.

Today, new methods have been developed to treat or modify the natural course of cardiovascular diseases (CVDs), including atherosclerosis, by the clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) system. Genome-editing tools are CRISPR-related palindromic short iteration systems such as CRISPR-Cas9, a valuable technology for achieving somatic and germinal genomic manipulation in model cells and organisms for various applications, including the creation of deletion alleles. Mutations in genomic deoxyribonucleic acid and new genes' placement have emerged. Based on World Health Organization fact sheets, 17.9 million people die from CVDs each year, an estimated 32% of all deaths worldwide. 85% of all CVD deaths are due to acute coronary events and strokes. This review discusses the applications of CRISPR-Cas9 technology throughout atherosclerotic disease research and the prospects for future in vivo genome editing therapies. We also describe several limitations that must be considered to achieve the full scientific and therapeutic potential of cardiovascular genome editing in the treatment of atherosclerosis.

RevDate: 2023-03-28
CmpDate: 2023-03-28

Liu S, Duan X, Peng F, et al (2023)

A tunable genome editing system of the prime editor mediated by dihydrofolate reductase.

Journal of genetics and genomics = Yi chuan xue bao, 50(3):204-207.

RevDate: 2023-03-26

Ma L, Zhang W, Yin L, et al (2023)

A SERS-signalled, CRISPR/Cas-powered bioassay for amplification-free and anti-interference detection of SARS-CoV-2 in foods and environmental samples using a single tube-in-tube vessel.

Journal of hazardous materials, 452:131195 [Epub ahead of print].

The pandemic of COVID-19 creates an imperative need for sensitive and portable detection of SARS-CoV-2. We devised a SERS-read, CRISPR/Cas-powered nanobioassay, termed as OVER-SARS-CoV-2 (One-Vessel Enhanced RNA test on SARS-CoV-2), which enabled supersensitive, ultrafast, accurate and portable detection of SARS-CoV-2 in a single vessel in an amplification-free and anti-interference manner. The SERS nanoprobes were constructed by conjugating gold nanoparticles with Raman reporting molecular and single-stranded DNA (ssDNA) probes, whose aggregation-to-dispersion changes can be finely tuned by target-activated Cas12a though trans-cleavage of linker ssDNA. As such, the nucleic acid signals could be dexterously converted and amplified to SERS signals. By customizing an ingenious vessel, the steps of RNA reverse transcription, Cas12a trans-cleavage and SERS nanoprobes crosslinking can be integrated into a single and disposal vessel. It was proved that our proposed nanobioassay was able to detect SARS-CoV-2 as low as 200 copies/mL without any pre-amplification within 45 min. In addition, the proposed nanobioassay was confirmed by clinical swab samples and challenged for SARS-CoV-2 detection in simulated complex environmental and food samples. This work enriches the arsenal of CRISPR-based diagnostics (CRISPR-Dx) and provides a novel and robust platform for SARS-CoV-2 decentralized detection, which can be put into practice in the near future.

RevDate: 2023-03-27

Rubio A, Sprang M, Garzón A, et al (2023)

Analysis of bacterial pangenomes reduces CRISPR dark matter and reveals strong association between membranome and CRISPR-Cas systems.

Science advances, 9(12):eadd8911.

CRISPR-Cas systems are prokaryotic acquired immunity mechanisms, which are found in 40% of bacterial genomes. They prevent viral infections through small DNA fragments called spacers. However, the vast majority of these spacers have not yet been associated with the virus they recognize, and it has been named CRISPR dark matter. By analyzing the spacers of tens of thousands of genomes from six bacterial species, we have been able to reduce the CRISPR dark matter from 80% to as low as 15% in some of the species. In addition, we have observed that, when a genome presents CRISPR-Cas systems, this is accompanied by particular sets of membrane proteins. Our results suggest that when bacteria present membrane proteins that make it compete better in its environment and these proteins are, in turn, receptors for specific phages, they would be forced to acquire CRISPR-Cas.

RevDate: 2023-03-27
CmpDate: 2023-03-27

Jeon W, Jung SY, Lee CY, et al (2023)

Evaluation of Radiation Sensitivity Differences in Mouse Liver Tumor Organoids Using CRISPR/Cas9-Mediated Gene Mutation.

Technology in cancer research & treatment, 22:15330338231165125.

BACKGROUND: To assess the radiosensitivity of liver tumors harboring different genetic mutations, mouse liver tumors were generated in vivo through the hydrodynamic injection of clustered regularly interspaced short palindromic repeat/caspase 9 (CRISPR/Cas9) constructs encoding single-guide RNAs (sgRNAs) targeting Tp53, Pten, Nf1, Nf2, Tsc2, Cdkn2a, or Rb1.

METHODS: The plasmid vectors were delivered to the liver of adult C57BL/6 mice via hydrodynamic tail vein injection. The vectors were injected into 10 mice in each group. Organoids were generated from mouse liver tumors. The radiation response of the organoids was assessed using an ATP cell viability assay.

RESULTS: The mean survival period of mice injected with vectors targeting Nf2 (4.8 months) was lower than that of other mice. Hematoxylin and eosin staining, immunohistochemical (IHC) staining, and target sequencing analyses revealed that mouse liver tumors harbored the expected mutations. Tumor organoids were established from mouse liver tumors. Histological evaluation revealed marked morphological similarities between the mouse liver tumors and the generated tumor organoids. Moreover, IHC staining indicated that the parental tumor protein expression pattern was maintained in the organoids. The results of the ATP cell viability assay revealed that the tumor organoids with mutated Nf2 were more resistant to high-dose radiation than those with other gene mutations.

CONCLUSIONS: This study developed a radiation response assessment system for mouse tumors with mutant target genes using CRISPR/Cas9 and organoids. The Tp53 and Pten double mutation in combination with the Nf2 mutation increased the radiation resistance of tumors. The system used in this study can aid in elucidating the mechanism underlying differential intrinsic radiation sensitivity of individual tumors.

RevDate: 2023-03-23

Botelho J, Tüffers L, Fuss J, et al (2023)

Phylogroup-specific variation shapes the clustering of antimicrobial resistance genes and defence systems across regions of genome plasticity in Pseudomonas aeruginosa.

EBioMedicine, 90:104532 pii:S2352-3964(23)00097-X [Epub ahead of print].

BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen consisting of three phylogroups (hereafter named A, B, and C). Here, we assessed phylogroup-specific evolutionary dynamics across available and also new P. aeruginosa genomes.

METHODS: In this genomic analysis, we first generated new genome assemblies for 18 strains of the major P. aeruginosa clone type (mPact) panel, comprising a phylogenetically diverse collection of clinical and environmental isolates for this species. Thereafter, we combined these new genomes with 1991 publicly available P. aeruginosa genomes for a phylogenomic and comparative analysis. We specifically explored to what extent antimicrobial resistance (AMR) genes, defence systems, and virulence genes vary in their distribution across regions of genome plasticity (RGPs) and "masked" (RGP-free) genomes, and to what extent this variation differs among the phylogroups.

FINDINGS: We found that members of phylogroup B possess larger genomes, contribute a comparatively larger number of pangenome families, and show lower abundance of CRISPR-Cas systems. Furthermore, AMR and defence systems are pervasive in RGPs and integrative and conjugative/mobilizable elements (ICEs/IMEs) from phylogroups A and B, and the abundance of these cargo genes is often significantly correlated. Moreover, inter- and intra-phylogroup interactions occur at the accessory genome level, suggesting frequent recombination events. Finally, we provide here the mPact panel of diverse P. aeruginosa strains that may serve as a valuable reference for functional analyses.

INTERPRETATION: Altogether, our results highlight distinct pangenome characteristics of the P. aeruginosa phylogroups, which are possibly influenced by variation in the abundance of CRISPR-Cas systems and are shaped by the differential distribution of other defence systems and AMR genes.

FUNDING: German Science Foundation, Max-Planck Society, Leibniz ScienceCampus Evolutionary Medicine of the Lung, BMBF program Medical Infection Genomics, Kiel Life Science Postdoc Award.

RevDate: 2023-03-27
CmpDate: 2023-03-27

Chornyi S, Koster J, HR Waterham (2023)

Applying CRISPR-Cas9 Genome Editing to Study Genes Involved in Peroxisome Biogenesis or Peroxisomal Functions.

Methods in molecular biology (Clifton, N.J.), 2643:233-245.

The development and application of the CRISPR-Cas9 technology for genome editing of mammalian cells have opened up a wealth of possibilities for genetically modifying and manipulating human cells, and use in functional studies or therapeutic approaches.Here we describe the approach that we have been using successfully to generate multiple human cell lines with targeted (partial) gene deletions, i.e., knockout cells, or human cells with modified genomic nucleotide sequences, i.e., knock-in cells, in genes encoding known or putative proteins involved in peroxisome biogenesis or peroxisomal functions.

RevDate: 2023-03-27
CmpDate: 2023-03-27

Zhao J, Zuo S, Huang L, et al (2023)

CRISPR-Cas12a-based genome editing and transcriptional repression for biotin synthesis in Pseudomonas mutabilis.

Journal of applied microbiology, 134(3):.

AIMS: To establish a dual-function clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system combined genome editing and transcriptional repression for multiplex metabolic engineering of Pseudomonas mutabilis.

MATERIALS AND RESULTS: This CRISPR-Cas12a system consisted of two plasmids that enabled single gene deletion, replacement, and inactivation with efficiency >90% for most targets within 5 days. With the guidance of truncated crRNA containing 16 bp spacer sequences, a catalytically active Cas12a could be employed to repress the expression of the reporter gene eGFP up to 66.6%. When bdhA deletion and eGFP repression were tested simultaneously by transforming a single crRNA plasmid and Cas12a plasmid, the knockout efficiency reached 77.8% and the expression of eGFP was decreased by >50%. Finally, the dual-functional system was demonstrated to increase the production of biotin by 3.84-fold, with yigM deletion and birA repression achieved simultaneously.

CONCLUSIONS: This CRISPR-Cas12a system is an efficient genome editing and regulation tool to facilitate the construction of P. mutabilis cell factories.

RevDate: 2023-03-27
CmpDate: 2023-03-27

Peri KVR, Faria-Oliveira F, Larsson A, et al (2023)

Split-marker-mediated genome editing improves homologous recombination frequency in the CTG clade yeast Candida intermedia.

FEMS yeast research, 23:.

Genome-editing toolboxes are essential for the exploration and exploitation of nonconventional yeast species as cell factories, as they facilitate both genome studies and metabolic engineering. The nonconventional yeast Candida intermedia is a biotechnologically interesting species due to its capacity to convert a wide range of carbon sources, including xylose and lactose found in forestry and dairy industry waste and side-streams, into added-value products. However, possibilities of genetic manipulation have so far been limited due to lack of molecular tools for this species. We describe here the development of a genome editing method for C. intermedia, based on electroporation and gene deletion cassettes containing the Candida albicans NAT1 dominant selection marker flanked by 1000 base pair sequences homologous to the target loci. Linear deletion cassettes targeting the ADE2 gene originally resulted in <1% targeting efficiencies, suggesting that C. intermedia mainly uses nonhomologous end joining for integration of foreign DNA fragments. By developing a split-marker based deletion technique for C. intermedia, we successfully improved the homologous recombination rates, achieving targeting efficiencies up to 70%. For marker-less deletions, we also employed the split-marker cassette in combination with a recombinase system, which enabled the construction of double deletion mutants via marker recycling. Overall, the split-marker technique proved to be a quick and reliable method for generating gene deletions in C. intermedia, which opens the possibility to uncover and enhance its cell factory potential.

RevDate: 2023-03-27
CmpDate: 2023-03-27

Yang W, Zhao H, Dou Y, et al (2023)

CYP3A4 and CYP3A5 Expression is Regulated by CYP3A4*1G in CRISPR/Cas9-Edited HepG2 Cells.

Drug metabolism and disposition: the biological fate of chemicals, 51(4):492-498.

Functional CYP3A4*1G (G>A, rs2242480) in cytochrome P450 3A4 (CYP3A4) regulates the drug-metabolizing enzyme CYP3A4 expression. The objective of this study was to investigate whether CYP3A4*1G regulates both basal and rifampicin (RIF)-induced expression and enzyme activity of CYP3A4 and CYP3A5 in gene-edited human HepG2 cells. CYP3A4*1G GG and AA genotype HepG2 cells were established using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) single nucleotide polymorphism technology and homology-directed repair in the CYP3A4*1G GA HepG2 cell line. In CYP3A4*1G GG, GA, and AA HepG2 cells, CYP3A4*1G regulated expression of CYP3A4 and CYP3A5 mRNA and protein in an allele-dependent manner. Of note, significantly decreased expression level of CYP3A4 and CYP3A5 was observed in CYP3A4*1G AA HepG2 cells. Moreover, the results after RIF treatment showed that CYP3A4*1G decreased the induction level of CYP3A4 and CYP3A5 mRNA expression in CYP3A4*1G AA HepG2 cells. At the same time, CYP3A4*1G decreased CYP3A4 enzyme activity and tacrolimus metabolism, especially in CYP3A4*1G GA HepG2 cells. In summary, we successfully constructed CYP3A4*1G GG and AA homozygous HepG2 cell models and found that CYP3A4*1G regulates both basal and RIF-induced expression and enzyme activity of CYP3A4 and CYP3A5 in CRISPR/Cas9 CYP3A4*1G HepG2 cells. SIGNIFICANCE STATEMENT: Cytochrome P450 (CYP) 3A4*1G regulates both basal and rifampicin (RIF)-induced expression and enzyme activity of CYP3A4 and CYP3A5. This study successfully established CYP3A4*1G (G>A, rs2242480), GG, and AA HepG2 cell models using CRISPR/Cas9, thus providing a powerful tool for studying the mechanism by which CYP3A4*1G regulates the basal and RIF-induced expression of CYP3A4 and CYP3A5.

RevDate: 2023-03-27
CmpDate: 2023-03-27

Zhang R, Zhang S, Li J, et al (2023)

CRISPR/Cas9-targeted mutagenesis of TaDCL4, TaDCL5 and TaRDR6 induces male sterility in common wheat.

Plant biotechnology journal, 21(4):839-853.

Phased, small interfering RNAs (phasiRNAs) are important for plant anther development, especially for male sterility. PhasiRNA biogenesis is dependent on genes like RNA polymerase 6 (RDR6), DICER-LIKE 4 (DCL4), or DCL5 to produce 21- or 24 nucleotide (nt) double-strand small RNAs. Here, we generated mutants of DCL4, DCL5 and RDR6 using CRISPR/Cas9 system and studied their effects on plant reproductive development and phasiRNA production in wheat. We found that RDR6 mutation caused sever consequence throughout plant development starting from seed germination and the dcl4 mutants grew weaker with thorough male sterility, while dcl5 plants developed normally but exhibited male sterility. Correspondingly, DCL4 and DCL5, respectively, specified 21- and 24-nt phasiRNA biogenesis, while RDR6 contributed to both. Also, the three key genes evolved differently in wheat, with TaDCL5-A/B becoming non-functioning and TaRDR6-A being lost after polyploidization. Furthermore, we found that PHAS genes (phasiRNA precursors) identified via phasiRNAs diverged rapidly among sub-genomes of polyploid wheat. Despite no similarity being found among phasiRNAs of grasses, their targets were enriched for similar biological functions. In light of the important roles of phasiRNA pathways in gametophyte development, genetic dissection of the function of key genes may help generate male sterile lines suitable for hybrid wheat breeding.

RevDate: 2023-03-27
CmpDate: 2023-03-27

Cheng J, Hill C, Han Y, et al (2023)

New semi-dwarfing alleles with increased coleoptile length by gene editing of gibberellin 3-oxidase 1 using CRISPR-Cas9 in barley (Hordeum vulgare L.).

Plant biotechnology journal, 21(4):806-818.

The green revolution was based on genetic modification of the gibberellin (GA) hormone system with "dwarfing" gene mutations that reduces GA signals, conferring shorter stature, thus enabling plant adaptation to modern farming conditions. Strong GA-related mutants with shorter stature often have reduced coleoptile length, discounting yield gain due to their unsatisfactory seedling emergence under drought conditions. Here we present gibberellin (GA) 3-oxidase1 (GA3ox1) as an alternative semi-dwarfing gene in barley that combines an optimal reduction in plant height without restricting coleoptile and seedling growth. Using large-scale field trials with an extensive collection of barley accessions, we showed that a natural GA3ox1 haplotype moderately reduced plant height by 5-10 cm. We used CRISPR/Cas9 technology, generated several novel GA3ox1 mutants and validated the function of GA3ox1. We showed that altered GA3ox1 activities changed the level of active GA isoforms and consequently increased coleoptile length by an average of 8.2 mm, which could provide essential adaptation to maintain yield under climate change. We revealed that CRISPR/Cas9-induced GA3ox1 mutations increased seed dormancy to an ideal level that could benefit the malting industry. We conclude that selecting HvGA3ox1 alleles offers a new opportunity for developing barley varieties with optimal stature, longer coleoptile and additional agronomic traits.

RevDate: 2023-03-27
CmpDate: 2023-03-27

Luo W, Suzuki R, R Imai (2023)

Precise in planta genome editing via homology-directed repair in wheat.

Plant biotechnology journal, 21(4):668-670.

RevDate: 2023-03-27
CmpDate: 2023-03-27

Zhu Y, Lin Y, Fan Y, et al (2023)

CRISPR/Cas9-mediated restoration of Tamyb10 to create pre-harvest sprouting-resistant red wheat.

Plant biotechnology journal, 21(4):665-667.

RevDate: 2023-03-27
CmpDate: 2023-03-27

Zhang Z, Wu X, Yang J, et al (2023)

Highly efficient base editing in rabbit by using near-PAMless engineered CRISPR/Cas9 variants.

Science China. Life sciences, 66(3):635-638.

RevDate: 2023-03-25

Huo G, Shepherd J, X Pan (2023)

Craspase: A novel CRISPR/Cas dual gene editor.

Functional & integrative genomics, 23(2):98.

RevDate: 2023-03-25

Daròs JA, Pasin F, F Merwaiss (2023)

CRISPR-Cas-based plant genome engineering goes viral.

Molecular plant pii:S1674-2052(23)00074-6 [Epub ahead of print].

RevDate: 2023-03-24

Didara Z, Reithofer F, Zöttl K, et al (2023)

Inhibition of adenovirus replication by CRISPR-Cas9-mediated targeting of the viral E1A gene.

Molecular therapy. Nucleic acids, 32:48-60.

DNA-targeting CRISPR-Cas systems are able to cleave dsDNA in mammalian cells. Accordingly, they have been employed to target the genomes of dsDNA viruses, mostly when present in cells in a non-replicative state with low copy numbers. However, the sheer amount of viral DNA produced within a very short time by certain lytically replicating viruses potentially brings the capacities of CRISPR-Cas systems to their limits. The accessibility of viral DNA replication sites, short time of accessibility of the DNA before encapsidation, or its complexation with shielding proteins are further potential hurdles. Adenoviruses are fast-replicating dsDNA viruses for which no approved antiviral therapy currently exists. We evaluated the potency of CRISPR-Cas9 in inhibiting the replication of human adenovirus 5 in vitro by targeting its master regulator E1A with a set of guide RNAs and observed a decrease in infectious virus particles by up to three orders of magnitude. Target DNA cleavage also negatively impacted the amount of viral DNA accumulated during the infection cycle. This outcome was mainly caused by specific deletions, inversions, and duplications occurring between target sites, which abolished most E1A functions in most cases. Additionally, we compared two strategies for multiplex gRNA expression and obtained comparable results.

RevDate: 2023-03-24
CmpDate: 2023-03-24

Ravikiran KT, Thribhuvan R, Sheoran S, et al (2023)

Tailoring crops with superior product quality through genome editing: an update.

Planta, 257(5):86.

In this review, using genome editing, the quality trait alterations in important crops have been discussed, along with the challenges encountered to maintain the crop products' quality. The delivery of economic produce with superior quality is as important as high yield since it dictates consumer's acceptance and end use. Improving product quality of various agricultural and horticultural crops is one of the important targets of plant breeders across the globe. Significant achievements have been made in various crops using conventional plant breeding approaches, albeit, at a slower rate. To keep pace with ever-changing consumer tastes and preferences and industry demands, such efforts must be supplemented with biotechnological tools. Fortunately, many of the quality attributes are resultant of well-understood biochemical pathways with characterized genes encoding enzymes at each step. Targeted mutagenesis and transgene transfer have been instrumental in bringing out desired qualitative changes in crops but have suffered from various pitfalls. Genome editing, a technique for methodical and site-specific modification of genes, has revolutionized trait manipulation. With the evolution of versatile and cost effective CRISPR/Cas9 system, genome editing has gained significant traction and is being applied in several crops. The availability of whole genome sequences with the advent of next generation sequencing (NGS) technologies further enhanced the precision of these techniques. CRISPR/Cas9 system has also been utilized for desirable modifications in quality attributes of various crops such as rice, wheat, maize, barley, potato, tomato, etc. The present review summarizes salient findings and achievements of application of genome editing for improving product quality in various crops coupled with pointers for future research endeavors.

RevDate: 2023-03-22

Zhan X, Zhou J, Jiang Y, et al (2023)

DNA tetrahedron-based CRISPR bioassay for treble-self-amplified and multiplex HPV-DNA detection with elemental tagging.

Biosensors & bioelectronics, 229:115229 pii:S0956-5663(23)00171-9 [Epub ahead of print].

Sensitive quantification of multiple analytes of interest is of great significance for clinical diagnosis. CRISPR Cas platforms offer a strategy for improving the specificity, sensitivity, and speed of nucleic acid-based diagnostics, while their multiplex analysis capability is still limited and challenging. Herein, we develop a novel DNA Tetrahedron (DTN)-supported biosensor based on the spatially separated CRISPR Cas self-amplification strategy and multiple-metal-nanoparticle tagging coupled with inductively coupled plasma mass spectrometry (ICP-MS) detection to improve the sensitivity and feasibility of the platform for multiplex detection of HPV-DNA (HPV-16, HPV-18 and HPV-52). Given target DNA induces robust trans-cleavage activity of the Cas12a/crRNA duplex, and the surrounding corresponding single-stranded DNA (ssDNA) linker are cleaved into short fragments that are unable to bond metal-nanoparticle probes ([197]Au, [107]Ag, [195]Pt) onto DTN modified magnetic beads probe (MBs-DTN), resulting in obvious ICP-MS signal change. Of note, compared with ssDNA functionalized MBs, a higher Signal-to-Noise Ratio was obtained by using MBs-DTN in our system, further amplifying the signal by regulating probes on the surface of MBs. As expected, the HPV-DNA could be detected with detection limits as low as 218 fM and be multiplexed assayed at one test with high accuracy and specificity by this proposed strategy. Furthermore, we demonstrated that the HPV-DNA in cervical swab samples could be detected, showing high consistency with DNA sequencing results. We believe that this work provides a promising option in designing CRISPR based multiplex detection system for high sensitivity, good specificity, and clinical molecular diagnostics.

RevDate: 2023-03-24
CmpDate: 2023-03-24

Huang H, Zhang W, Zhang J, et al (2023)

Epigenome editing based on CRISPR/dCas9[p300] facilitates transdifferentiation of human fibroblasts into Leydig-like cells.

Experimental cell research, 425(2):113551.

Recently, Leydig cell (LCs) transplantation has a promising potential to treat male hypogonadism. However, the scarcity of seed cells is the actual barrier impeding the application of LCs transplantation. Utilizing the cutting-edge CRISPR/dCas9[VP64] technology, human foreskin fibroblasts (HFFs) were transdifferentiated into Leydig-like cells(iLCs) in previous study, but the efficiency of transdifferentiation is not very satisfactory. Therefore, this study was conducted to further optimize the CRISPR/dCas9 system for obtaining sufficient iLCs. First, the stable CYP11A1-Promoter-GFP-HFFs cell line was established by infecting HFFs with CYP11A1-Promoter-GFP lentiviral vectors, and then co-infected with dCas9[p300] and the combination of sgRNAs targeted to NR5A1, GATA4 and DMRT1. Next, this study adopted quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blot, and immunofluorescence to determine the efficiency of transdifferentiation, the generation of testosterone, the expression levels of steroidogenic biomarkers. Moreover, we utilized chromatin immuno-precipitation (ChIP) followed by quantitative polymerase chain reaction (ChIP-qPCR) to measure the levels of acetylation of targeted H3K27. The results revealed that advanced dCas9[p300] facilitated generation of iLCs. Moreover, the dCas9[p300]-mediated iLCs significantly expressed the steroidogenic biomarkers and produced more testosterone with or without LH treatment than the dCas9[VP64]-mediated. Additionally, preferred enrichment in H3K27ac at the promoters was detected only with dCas9[p300] treatment. The data provided here imply that the improved version of dCas9 can aid in the harvesting of iLCs, and will provide sufficient seed cells for cell transplantation treatment of androgen deficiency in the future.

RevDate: 2023-03-23
CmpDate: 2023-03-23

Liang F, Dong Z, Ye J, et al (2023)

In vivo DNA methylation editing in zebrafish.

Epigenetics, 18(1):2192326.

The CRISPR/dCas9-based epigenome editing technique has driven much attention. Fused with a catalytic domain from Dnmt or Tet protein, the CRISPR/dCas9-DnmtCD or -TetCD systems possess the targeted DNA methylation editing ability and have established a series of in vitro and in vivo disease models. However, no publication has been reported on zebrafish (Danio rerio), an important animal model in biomedicine. The present study demonstrated that CRISPR/dCas9-Dnmt7 and -Tet2 catalytic domain fusions could site-specifically edit genomic DNA methylation in vivo in zebrafish and may serve as an efficient toolkit for DNA methylation editing in the zebrafish model.

RevDate: 2023-03-22

Chen D, Liang Y, Wang H, et al (2023)

CRISPR-Cas-Driven Single Micromotor (Cas-DSM) Enables Direct Detection of Nucleic Acid Biomarkers at the Single-Molecule Level.

Analytical chemistry [Epub ahead of print].

The target-dependent endonuclease activity (also known as the trans-cleavage activity) of CRISPR-Cas systems has stimulated great interest in the development of nascent sensing strategies for nucleic acid diagnostics. Despite many attempts, the majority of the sensitive CRISPR-Cas diagnostics strategies mainly rely on nucleic acid preamplification, which generally needs complex probes/primers designs, multiple experimental steps, and a longer testing time, as well as introducing the risk of false-positive results. In this work, we propose the CRISPR-Cas-Driven Single Micromotor (Cas-DSM), which can directly detect the nucleic acid targets at a single-molecule level with high specificity. We have demonstrated that the Cas-DSM is a reliable and practical method for the quantitative detection of DNA/RNA in various complex clinical samples as well as in individual cells without any preamplification processes. Due to the excellent features of the CRISPR/Cas system, including constant temperature, simple design, high specificity, and flexible programmability, the Cas-DSM could serve as a simple and universal platform for nucleic acid detection. More importantly, this work will provide a breakthrough for the development of next-generation amplification-free CRISPR/Cas sensing toolboxes.

RevDate: 2023-03-20

Hussain MS, Anand V, M Kumar (2023)

Functional PAM sequence for DNA interference by CRISPR-Cas I-B system of Leptospira interrogans and the role of LinCas11b encoded within lincas8b.

International journal of biological macromolecules pii:S0141-8130(23)00980-7 [Epub ahead of print].

Pathogenic species of Leptospira are recalcitrant for genetic manipulation using conventional tools, and therefore there is a need to explore techniques of higher efficiency. Application of endogenous CRISPR-Cas tool is emerging and efficient; nevertheless, it is limited by a poor understanding of interference machinery in the bacterial genome and its associated protospacer adjacent motif (PAM). In this study, interference machinery of CRISPR-Cas subtype I-B (Lin_I-B) from L. interrogans was experimentally validated in E. coli using the various identified PAM (TGA, ATG, ATA). The overexpression of the Lin_I-B interference machinery in E. coli demonstrated that LinCas5, LinCas6, LinCas7, and LinCas8b can self-assemble on cognate CRISPR RNA to form an interference complex (LinCascade). Moreover, a robust interference of target plasmids containing a protospacer with a PAM suggested a functional LinCascade. We also recognized a small open reading frame within lincas8b that independently co-translates LinCas11b. A mutant variant of LinCascade[-Cas11b] that lacks LinCas11b co-expression erred to mount target plasmid interference. At the same time, LinCas11b complementation in LinCascade[-Cas11b] rescued target plasmid interference. Thus, the present study establishes Leptospira subtype I-B interference machinery to be functional and, soon, may pave the way for scientists to harness it as a programmable endogenous genetic manipulation tool.

RevDate: 2023-03-20

Suliman Maashi M (2023)

CRISPR/Cas-based Aptasensor as an Innovative Sensing Approaches for Food Safety Analysis: Recent Progresses and New Horizons.

Critical reviews in analytical chemistry [Epub ahead of print].

Food safety is one of the greatest public problems occurring around the world. Chemical, physical, and microbiological hazards could lead to food safety problems, which might occur at all stages of the supply chain. To tackle food safety problems and protect consumer health, specific, accurate, and rapid diagnosis techniques meeting various requirements are the imperative measures to ensure food safety. CRISPR-Cas system, a novel emerging technology, is effectively repurposed in (bio)sensing and has shown a tremendous capability to develop on-site and portable diagnostic methods with high specificity and sensitivity. Among numerous existing CRISPR/Cas systems, CRISPR/Cas13a and CRISPR/Cas12a are extensively employed in the design of biosensors, owing to their ability to cleave both non-target and target sequences. However, the specificity limitation in CRISPR/Cas has hindered its progress. Nowadays, nucleic acid aptamers recognized for their specificity and high-affinity characteristics for their analytes are incorporated into CRISPR/Cas systems. With the benefits of reproducibility, high durability, portability, facile operation, and cost-effectiveness, CRISPR/Cas-based aptasensing approaches are an ideal choice for fabricating highly specific point-of-need analytical tools with enhanced response signals. In the current study, we explore some of the most recent progress in the CRISPR/Cas-mediated aptasensors for detecting food risk factors including veterinary drugs, pesticide residues, pathogens, mycotoxins, heavy metals, illegal additives, food additives, and other contaminants. The nanomaterial engineering support with CRISPR/Cas aptasensors is also signified to achieve a hopeful perspective to provide new straightforward test kits toward trace amounts of different contaminants encountered in food samples.

RevDate: 2023-03-20

Kedlaya MN, Puzhankara L, Prasad R, et al (2023)

Periodontal Disease Pathogens, Pathogenesis, and Therapeutics: The CRISPR-Cas Effect.

The CRISPR journal [Epub ahead of print].

Periodontal disease (PD) is an immune-inflammatory disease affecting the supporting structures of the teeth, which results in progressive destruction of the hard and soft tissues surrounding teeth, ultimately resulting in tooth loss. The primary etiological factor for this disease is the presence of pathogenic microorganisms. Pathogenic bacteria face antagonistic conditions and foreign DNA components during the infection stage and depend on defense mechanisms such as clustered regularly interspaced short palindromic repeats (CRISPR)-Cas to counter them. Virulence genes regulated by the CRISPR-Cas system are often expressed by bacteria as part of the stress response to the presence of stress conditions and foreign elements. There is ever-growing evidence regarding the role of CRISPR-Cas in virulence of periodontal pathogens. The same CRISPR-Cas system may also be targeted to reduce bacterial virulence and it may also be utilized to develop diagnostic and therapeutic strategies for prevention and control of PD progression. This review article describes the CRISPR-Cas systems in the periodontal dysbiotic microbial communities, their role in the virulence of periodontal pathogens, and their potential role in understanding the pathogenesis of periodontitis and treatment of PD.

RevDate: 2023-03-23
CmpDate: 2023-03-23

Ledford H (2023)

Why CRISPR babies are still too risky - embryo studies highlight challenges.

Nature, 615(7953):568-569.

RevDate: 2023-03-23
CmpDate: 2023-03-23

Liu Y, Ma L, Liu W, et al (2023)

RPA-CRISPR/Cas12a Combined with Rolling Circle Amplification-Enriched DNAzyme: A Homogeneous Photothermal Sensing Strategy for Plant Pathogens.

Journal of agricultural and food chemistry, 71(11):4736-4744.

Alternaria is an endemic fungus associated with brown spot disease, which is one of the most serious citrus diseases. In addition, the mycotoxins metabolized by Alternaria threaten human health seriously. Herein, a novel homogeneous and portable qualitative photothermal method based on recombinase polymerase amplification (RPA), CRISPR/Cas12a, and rolling circle amplification (RCA) for the detection of Alternaria is described. Using RCA primers as substrates for CRISPR/Cas12a trans-cleavage, the two systems, RPA-CRISPR/Cas12a and RCA-enriched G-quadruplex/hemin DNAzyme, are intelligently combined. Target DNA at fg/μL levels can be detected with high specificity. Additionally, the practicability of the proposed method is demonstrated by analyzing cultured Alternaria from different fruit and vegetable samples, as well as citrus fruit samples collected in the field. Furthermore, the implementation of this method does not require any sophisticated equipment and complicated washing steps. Therefore, it has great potential to screen Alternaria in poor laboratories.

RevDate: 2023-03-23
CmpDate: 2023-03-23

Papadopoulos C, MM Albà (2023)

Newly evolved genes in the human lineage are functional.

Trends in genetics : TIG, 39(4):235-236.

Genes restricted to a given species or lineage are mysterious. Many emerged de novo from ancestral noncoding genomic regions rather than from pre-existing genes. A new study by Vakirlis and colleagues shows that, in humans, many of these are associated with phenotypic effects, accelerating our understanding of their functional importance.

RevDate: 2023-03-23
CmpDate: 2023-03-23

Bachtarzi H (2023)

Genome Editing: Moving Toward a New Era of Innovation, Development, and Approval.

Human gene therapy, 34(5-6):171-176.

Therapeutic genome editing is currently reshaping and transforming the development of advanced therapies as more ex vivo and in vivo gene editing-based technologies are used to treat a broad range of debilitating and complex disorders. With first-generation gene editing modalities (notably those based on ZFNs, TALENs and CRISPR/Cas9), comes a new second-generation of gene editing-based therapeutics including base editing, prime editing and other nuclease-free genome editing modalities. Such ground-breaking innovative products warrant careful considerations from a product development and regulatory perspective, that take into account not only the common development considerations that apply to standard gene and cell therapy products, but also other specific considerations linked with the technology being used. This article sheds light into specific considerations for developing safe and effective in vivo and ex vivo genome editing medicines that will continue to push barriers even further for the cell and gene therapy field.

RevDate: 2023-03-23
CmpDate: 2023-03-23

Nishimura A, Tanahashi R, Oi T, et al (2023)

Plasmid-free CRISPR/Cas9 genome editing in Saccharomyces cerevisiae.

Bioscience, biotechnology, and biochemistry, 87(4):458-462.

The current CRISPR/Cas9 systems in the yeast Saccharomyces cerevisiae cannot be considered a non-genetic modification technology because it requires the introduction of Cas9 and sgRNA into yeast cells using plasmid expression systems. Our present study showed that the yeast genome can be edited without plasmid expression systems by using a commercially available protein transfection reagent and chemically modified sgRNAs.

RevDate: 2023-03-21

Hu M, Chen S, Ni Y, et al (2023)

CRISPR/Cas9-mediated genome editing in vancomycin-producing strain Amycolatopsis keratiniphila.

Frontiers in bioengineering and biotechnology, 11:1141176.

Amycolatopsis is an important source of diverse valuable bioactive natural products. The CRISPR/Cas-mediated gene editing tool has been established in some Amycolatopsis species and has accomplished the deletion of single gene or two genes. The goal of this study was to develop a high-efficient CRISPR/Cas9-mediated genome editing system in vancomycin-producing strain A. keratiniphila HCCB10007 and enhance the production of vancomycin by deleting the large fragments of ECO-0501 BGC. By adopting the promoters of gapdhp and ermE*p which drove the expressions of scocas9 and sgRNA, respectively, the all-in-one editing plasmid by homology-directed repair (HDR) precisely deleted the single gene gtfD and inserted the gene eGFP with the efficiency of 100%. Furthermore, The CRISPR/Cas9-mediated editing system successfully deleted the large fragments of cds13-17 (7.7 kb), cds23 (12.7 kb) and cds22-23 (21.2 kb) in ECO-0501 biosynthetic gene cluster (BGC) with high efficiencies of 81%-97% by selecting the sgRNAs with a suitable PAM sequence. Finally, a larger fragment of cds4-27 (87.5 kb) in ECO-0501 BGC was deleted by a dual-sgRNA strategy. The deletion of the ECO-0501 BGCs revealed a noticeable improvement of vancomycin production, and the mutants, which were deleted the ECO-0501 BGCs of cds13-17, cds22-23 and cds4-27, all achieved a 30%-40% increase in vancomycin yield. Therefore, the successful construction of the CRISPR/Cas9-mediated genome editing system and its application in large fragment deletion in A. keratiniphila HCCB10007 might provide a powerful tool for other Amycolatopsis species.

RevDate: 2023-03-21

Tang Y, Wang F, Wang Y, et al (2023)

In vitro characterization of a pAgo nuclease TtdAgo from Thermococcus thioreducens and evaluation of its effect in vivo.

Frontiers in bioengineering and biotechnology, 11:1142637.

In spite of the development of genome-editing tools using CRISPR-Cas systems, highly efficient and effective genome-editing tools are still needed that use novel programmable nucleases such as Argonaute (Ago) proteins to accelerate the construction of microbial cell factories. In this study, a prokaryotic Ago (pAgo) from a hyperthermophilic archaeon Thermococcus thioreducens (TtdAgo) was characterized in vitro. Our results showed that TtdAgo has a typical DNA-guided DNA endonuclease activity, and the efficiency and accuracy of cleavage are modulated by temperature, divalent ions, and the phosphorylation and length of gDNAs and their complementarity to the DNA targets. TtdAgo can utilize 5'-phosphorylated (5'-P) or 5'- hydroxylated (5'-OH) DNA guides to cleave single-stranded DNA (ssDNA) at temperatures ranging from 30°C to 95°C in the presence of Mn[2+] or Mg[2+] and displayed no obvious preference for the 5'-end-nucleotide of the guide. In addition, single-nucleotide mismatches had little effects on cleavage efficiency, except for mismatches at position 4 or 8 that dramatically reduced target cleavage. Moreover, TtdAgo performed programmable cleavage of double-stranded DNA at 75°C. We further introduced TtdAgo into an industrial ethanologenic bacterium Zymomonas mobilis to evaluate its effect in vivo. Our preliminary results indicated that TtdAgo showed cell toxicity toward Z. mobilis, resulting in a reduced growth rate and final biomass. In conclusion, we characterized TtdAgo in vitro and investigated its effect on Z. mobilis in this study, which lays a foundation to develop Ago-based genome-editing tools for recalcitrant industrial microorganisms in the future.

RevDate: 2023-03-21
CmpDate: 2023-03-21

Deng L, He X, Liu K, et al (2023)

One-pot RPA-Cas12a assay for instant and visual detection of Burkholderia pseudomallei.

Analytica chimica acta, 1252:341059.

Burkholderia pseudomallei is the causative agent of melioidosis, a potentially life-threatening infectious disease, and poses public health risks in endemic areas. Due to the high mortality, intrinsic antibiotic resistance, and atypical manifestations, establishing a rapid, accurate, and sensitive identification of B. pseudomallei enables earlier diagnosis, proper treatments, and better outcomes of melioidosis. Herein, we present a One-Pot CRISPR-integrated assay for Instant and Visual Detection (termed OPC-IVD) of B. pseudomallei. The integration of recombinase polymerase amplification and CRISPR-Cas12a recognition-activated trans-cleavage, achieved a true all-in-one single-tube reaction system, initiating the amplification and cleavage simultaneously, which realized a facile sample-to-answer assay. This approach could be performed with simplified DNA extraction and completed around 30 min by holding the reaction tube in the hand. The detection limit of our OPC-IVD was determined to be 2.19 copy/uL of plasmid DNA, 12.5 CFU/mL of B. pseudomallei, and 61.5 CFU/mL of bacteria in spiked blood samples, respectively. Furthermore, the introduction of internal amplification control effectively reduced the occurrence of false negatives, which was incorporated in the reaction system, and amplified simultaneously with the target and read by naked eyes. The assay exhibited 100% accuracy when evaluated in clinical isolates and samples. The streamlined workflow of our OPC-IVD of B. pseudomallei enables a field-deployable, instrument-free, and ultra-fast approach that can be utilized by non-expert personnel in the field of molecular diagnosis of melioidosis especially in under-resourced setting.

RevDate: 2023-03-21
CmpDate: 2023-03-21

Schary Y, Rotem I, Caller T, et al (2023)

CRISPR-Cas9 editing of TLR4 to improve the outcome of cardiac cell therapy.

Scientific reports, 13(1):4481.

Inflammation and fibrosis limit the reparative properties of human mesenchymal stromal cells (hMSCs). We hypothesized that disrupting the toll-like receptor 4 (TLR4) gene would switch hMSCs toward a reparative phenotype and improve the outcome of cell therapy for infarct repair. We developed and optimized an improved electroporation protocol for CRISPR-Cas9 gene editing. This protocol achieved a 68% success rate when applied to isolated hMSCs from the heart and epicardial fat of patients with ischemic heart disease. While cell editing lowered TLR4 expression in hMSCs, it did not affect classical markers of hMSCs, proliferation, and migration rate. Protein mass spectrometry analysis revealed that edited cells secreted fewer proteins involved in inflammation. Analysis of biological processes revealed that TLR4 editing reduced processes linked to inflammation and extracellular organization. Furthermore, edited cells expressed less NF-ƙB and secreted lower amounts of extracellular vesicles and pro-inflammatory and pro-fibrotic cytokines than unedited hMSCs. Cell therapy with both edited and unedited hMSCs improved survival, left ventricular remodeling, and cardiac function after myocardial infarction (MI) in mice. Postmortem histologic analysis revealed clusters of edited cells that survived in the scar tissue 28 days after MI. Morphometric analysis showed that implantation of edited cells increased the area of myocardial islands in the scar tissue, reduced the occurrence of transmural scar, increased scar thickness, and decreased expansion index. We show, for the first time, that CRISPR-Cas9-based disruption of the TLR4-gene reduces pro-inflammatory polarization of hMSCs and improves infarct healing and remodeling in mice. Our results provide a new approach to improving the outcomes of cell therapy for cardiovascular diseases.

RevDate: 2023-03-21
CmpDate: 2023-03-21

Karlikow M, Amalfitano E, Yang X, et al (2023)

CRISPR-induced DNA reorganization for multiplexed nucleic acid detection.

Nature communications, 14(1):1505.

Nucleic acid sensing powered by the sequence recognition of CRIPSR technologies has enabled major advancement toward rapid, accurate and deployable diagnostics. While exciting, there are still many challenges facing their practical implementation, such as the widespread need for a PAM sequence in the targeted nucleic acid, labile RNA inputs, and limited multiplexing. Here we report FACT (Functionalized Amplification CRISPR Tracing), a CRISPR-based nucleic acid barcoding technology compatible with Cas12a and Cas13a, enabling diagnostic outputs based on cis- and trans-cleavage from any sequence. Furthermore, we link the activation of CRISPR-Cas12a to the expression of proteins through a Reprogrammable PAIRing system (RePAIR). We then combine FACT and RePAIR to create FACTOR (FACT on RePAIR), a CRISPR-based diagnostic, that we use to detect infectious disease in an agricultural use case: honey bee viral infection. With high specificity and accuracy, we demonstrate the potential of FACTOR to be applied to the sensing of any nucleic acid of interest.

RevDate: 2023-03-21
CmpDate: 2023-03-21

Thakur N, Nigam M, Awasthi G, et al (2023)

Synergistic soil-less medium for enhanced yield of crops: a step towards incorporating genomic tools for attaining net zero hunger.

Functional & integrative genomics, 23(2):86.

Globally, industrial farming endangers crucial ecological mechanisms upon which food production relies, while 815 million people are undernourished and a significant number are malnourished. Zero Hunger aims to concurrently solve global ecological sustainability and food security concerns. Recent breakthroughs in molecular tools and approaches have allowed scientists to detect and comprehend the nature and structure of agro-biodiversity at the molecular and genetic levels, providing us an advantage over traditional methods of crop breeding. These bioinformatics techniques let us optimize our target plants for our soil-less medium and vice versa. Most of the soil-borne and seed-borne diseases are the outcomes of non-treated seed and growth media, which are important factors in low productivity. The farmers do not consider these issues, thereby facing problems growing healthy crops and suffering economic losses. This study is going to help the farmers increase their eco-friendly, chemical residue-free, quality yield of crops and their economic returns. The present invention discloses a synergistic soil-less medium that consists of only four ingredients mixed in optimal ratios by weight: vermicompost (70-80%), vermiculite (10-15%), coco peat (10-15%), and Rhizobium (0-1%). The medium exhibits better physical and chemical characteristics than existing conventional media. The vermiculite to coco peat ratio is reduced, while the vermicompost ratio is increased, with the goals of lowering toxicity, increasing plant and water holding capacity, avoiding drying of the media, and conserving water. The medium provides balanced nutrition and proper ventilation for seed germination and the growth of seedlings. Rhizobium is also used to treat the plastic bags and seeds. The results clearly show that the current synergistic soil-less environment is best for complete plant growth. Securing genetic advantages via sexual recombination, induced random mutations, and transgenic techniques have been essential for the development of improved agricultural varieties. The recent availability of targeted genome-editing technology provides a new path for integrating beneficial genetic modifications into the most significant agricultural species on the planet. Clustered regularly interspaced short palindromic repeats and associated protein 9 (CRISPR/Cas9) has evolved into a potent genome-editing tool for imparting genetic modifications to crop species. In addition, the integration of analytical methods like population genomics, phylogenomics, and metagenomics addresses conservation problems, while whole genome sequencing has opened up a new dimension for explaining the genome architecture and its interactions with other species. The in silico genomic and proteomic investigation was also conducted to forecast future investigations for the growth of French beans on a synergistic soil-less medium with the purpose of studying how a blend of vermicompost, vermiculite, cocopeat, and Rhizobium secrete metal ions, and other chemical compounds into the soil-less medium and affect the development of our target plant as well as several other plants. This interaction was studied using functional and conserved region analysis, phylogenetic analysis, and docking tools.

RevDate: 2023-03-21
CmpDate: 2023-03-21

Du SW, K Palczewski (2023)

Eye on genome editing.

The Journal of experimental medicine, 220(5):.

CRISPR/Cas9 genome editing techniques have the potential to treat previously untreatable inherited genetic disorders of vision by correcting mutations that cause these afflictions. Using a prime editor, Qin et al. (2023. J. Exp. Med.https://doi.org/10.1084/jem.20220776) restored visual functions in a mouse model (rd10) of retinitis pigmentosa.

RevDate: 2023-03-21
CmpDate: 2023-03-21

Huang M, Coral D, Ardalani H, et al (2023)

Identification of a weight loss-associated causal eQTL in MTIF3 and the effects of MTIF3 deficiency on human adipocyte function.

eLife, 12:.

Genetic variation at the MTIF3 (Mitochondrial Translational Initiation Factor 3) locus has been robustly associated with obesity in humans, but the functional basis behind this association is not known. Here, we applied luciferase reporter assay to map potential functional variants in the haplotype block tagged by rs1885988 and used CRISPR-Cas9 to edit the potential functional variants to confirm the regulatory effects on MTIF3 expression. We further conducted functional studies on MTIF3-deficient differentiated human white adipocyte cell line (hWAs-iCas9), generated through inducible expression of CRISPR-Cas9 combined with delivery of synthetic MTIF3-targeting guide RNA. We demonstrate that rs67785913-centered DNA fragment (in LD with rs1885988, r[2] > 0.8) enhances transcription in a luciferase reporter assay, and CRISPR-Cas9-edited rs67785913 CTCT cells show significantly higher MTIF3 expression than rs67785913 CT cells. Perturbed MTIF3 expression led to reduced mitochondrial respiration and endogenous fatty acid oxidation, as well as altered expression of mitochondrial DNA-encoded genes and proteins, and disturbed mitochondrial OXPHOS complex assembly. Furthermore, after glucose restriction, the MTIF3 knockout cells retained more triglycerides than control cells. This study demonstrates an adipocyte function-specific role of MTIF3, which originates in the maintenance of mitochondrial function, providing potential explanations for why MTIF3 genetic variation at rs67785913 is associated with body corpulence and response to weight loss interventions.

RevDate: 2023-03-21
CmpDate: 2023-03-21

Chen Z, Zheng S, C Fu (2023)

Shotgun knockdown of RNA by CRISPR-Cas13d in fission yeast.

Journal of cell science, 136(6):.

The CRISPR-Cas13d system has a single small effector protein that targets RNA and does not require the presence of a protospacer flanking site in the targeted transcript. These features make CRISPR-Cas13d an attractive system for RNA manipulation. Here, we report the successful implementation of the CRISPR-Cas13d system in fission yeast for RNA knockdown. A high effectiveness of the CRISPR-Cas13d system was ensured by using an array of CRISPR RNAs (crRNAs) that are flanked by two self-cleaving ribozymes and are expressed from an RNA polymerase II promoter. Given the repressible nature of the promoter, RNA knockdown by the CRISPR-Cas13d system is reversible. Moreover, using the CRISPR-Cas13d system, we identified an effective crRNA array targeting the transcript of gfp and the effectiveness was demonstrated by successful knockdown of the transcripts of noc4-gfp, bub1-gfp and ade6-gfp. In principle, the effective GFP crRNA array allows knockdown of any transcript carrying the GFP sequences. This new CRISPR-Cas13d-based toolkit is expected to have a wide range of applications in many aspects of biology, including dissection of gene function and visualization of RNA.

RevDate: 2023-03-21
CmpDate: 2023-03-21

Dubois M (2023)

Phishing alert! A Cas9-based method reveals the identity of promoter-bound transcription factors.

Plant physiology, 191(3):1462-1464.

RevDate: 2023-03-21
CmpDate: 2023-03-21

Wang Z, He Z, Liu Z, et al (2023)

A reverse chromatin immunoprecipitation technique based on the CRISPR-dCas9 system.

Plant physiology, 191(3):1505-1519.

DNA-protein interaction is one of the most crucial interactions in biological processes. However, the technologies available to study DNA-protein interactions are all based on DNA hybridization; however, DNA hybridization is not highly specific and is relatively low in efficiency. RNA-guided DNA recognition is highly specific and efficient. To overcome the limitations of technologies based on DNA hybridization, we built a DNA-binding protein capture technology based on the clustered regularly interspaced palindromic repeats (CRISPR)-dead Cas9 (dCas9) system and transient genetic transformation, termed reverse chromatin immunoprecipitation based on CRISPR-dCas9 system (R-ChIP-dCas9). In this system, dCas9 was fused with Strep-Tag II to form a fusion protein for StrepTactin affinity purification. Transient transformation was performed for the expression of dCas9 and guide RNA (gRNA) to form the dCas9-gRNA complex in birch (Betula platyphylla) plants, which binds to the target genomic DNA region. The dCas9-gRNA-DNA complex was crosslinked, then the chromatin was sonicated into fragments, and purified using StrepTactin beads. The proteins binding to the target genomic DNA region were identified using mass spectrometry. Using this method, we determined the upstream regulators of a NAM, ATAF, and CUC (NAC) transcription factor (TF), BpNAC090, and 32 TFs potentially regulating BpNAC090 were identified. The reliability of R-ChIP-dCas9 was further confirmed by chromatin immunoprecipitation, electrophoretic mobility shift assays, and yeast one-hybrid. This technology can be adapted to various plant species and does not depend on the availability of a stable transformation system; therefore, it has wide application in identifying proteins bound to genomic DNA.

RevDate: 2023-03-21
CmpDate: 2023-03-21

Musunuru K (2023)

CRISPR and cardiovascular diseases.

Cardiovascular research, 119(1):79-93.

CRISPR technologies have progressed by leaps and bounds over the past decade, not only having a transformative effect on biomedical research but also yielding new therapies that are poised to enter the clinic. In this review, I give an overview of (i) the various CRISPR DNA-editing technologies, including standard nuclease gene editing, base editing, prime editing, and epigenome editing, (ii) their impact on cardiovascular basic science research, including animal models, human pluripotent stem cell models, and functional screens, and (iii) emerging therapeutic applications for patients with cardiovascular diseases, focusing on the examples of hypercholesterolaemia, transthyretin amyloidosis, and Duchenne muscular dystrophy.

RevDate: 2023-03-20

Gu B, Zhuo C, Xu X, et al (2023)

Editorial: Molecular diagnostics for infectious diseases: Novel approaches, clinical applications and future challenges.

Frontiers in microbiology, 14:1153827.

RevDate: 2023-03-20

Xiao H, Hu J, Huang C, et al (2023)

CRISPR techniques and potential for the detection and discrimination of SARS-CoV-2 variants of concern.

Trends in analytical chemistry : TRAC, 161:117000.

The continuing evolution of the SARS-CoV-2 virus has led to the emergence of many variants, including variants of concern (VOCs). CRISPR-Cas systems have been used to develop techniques for the detection of variants. These techniques have focused on the detection of variant-specific mutations in the spike protein gene of SARS-CoV-2. These sequences mostly carry single-nucleotide mutations and are difficult to differentiate using a single CRISPR-based assay. Here we discuss the specificity of the Cas9, Cas12, and Cas13 systems, important considerations of mutation sites, design of guide RNA, and recent progress in CRISPR-based assays for SARS-CoV-2 variants. Strategies for discriminating single-nucleotide mutations include optimizing the position of mismatches, modifying nucleotides in the guide RNA, and using two guide RNAs to recognize the specific mutation sequence and a conservative sequence. Further research is needed to confront challenges in the detection and differentiation of variants and sublineages of SARS-CoV-2 in clinical diagnostic and point-of-care applications.

RevDate: 2023-03-19

Marino ND (2023)

Phage against the machine: discovery and mechanism of type V anti-CRISPRs.

Journal of molecular biology pii:S0022-2836(23)00110-9 [Epub ahead of print].

The discovery of diverse bacterial CRISPR-Cas systems has reignited interest in understanding bacterial defense pathways while yielding exciting new tools for genome editing. CRISPR-Cas systems are widely distributed in prokaryotes, found in 40% of bacteria and 90% of archaea, where they function as adaptive immune systems against bacterial viruses (phage) and other mobile genetic elements. In turn, phage have evolved inhibitors, called anti-CRISPR proteins, to prevent targeting. Type V CRISPR-Cas12 systems have emerged as a particularly exciting arena in this co-evolutionary arms race. Type V anti-CRISPRs have highly diverse and novel mechanisms of action, some of which appear to be unusually potent or widespread. In this review, we discuss the discovery and mechanism of these anti-CRISPRs as well as future areas for exploration.

RevDate: 2023-03-18

Bhatia S, Pooja , SK Yadav (2023)

CRISPR-Cas for genome editing: Classification, mechanism, designing and applications.

International journal of biological macromolecules pii:S0141-8130(23)00948-0 [Epub ahead of print].

Clustered regularly interspersed short pallindromic repeats (CRISPR) and CRISPR associated proteins (Cas) system (CRISPR-Cas) came into light as prokaryotic defence mechanism for adaptive immune response. CRISPR-Cas works by integrating short sequences of the target genome (spacers) into the CRISPR locus. The locus containing spacers interspersed repeats is further expressed into small guide CRISPR RNA (crRNA) which is then deployed by the Cas proteins to evade the target genome. Based on the Cas proteins CRISPR-Cas is classified according to polythetic system of classification. The characteristic of the CRISPR-Cas9 system to target DNA sequences using programmable RNAs has opened new arenas due to which today CRISPR-Cas has evolved as cutting end technique in the field of genome editing. Here, we discuss about the evolution of CRISPR, its classification and various Cas systems including the designing and molecular mechanism of CRISPR-Cas. Applications of CRISPR-Cas as a genome editing tools are also highlighted in the areas such as agriculture, and anticancer therapy. Briefly discuss the role of CRISPR and its Cas systems in the diagnosis of COVID-19 and its possible preventive measures. The challenges in existing CRISP-Cas technologies and their potential solutions are also discussed briefly.

RevDate: 2023-03-17

Rostain W, Grebert T, Vyhovskyi D, et al (2023)

Cas9 off-target binding to the promoter of bacterial genes leads to silencing and toxicity.

Nucleic acids research pii:7079636 [Epub ahead of print].

Genetic tools derived from the Cas9 RNA-guided nuclease are providing essential capabilities to study and engineer bacteria. While the importance of off-target effects was noted early in Cas9's application to mammalian cells, off-target cleavage by Cas9 in bacterial genomes is easily avoided due to their smaller size. Despite this, several studies have reported experimental setups in which Cas9 expression was toxic, even when using the catalytic dead variant of Cas9 (dCas9). Specifically, dCas9 was shown to be toxic when in complex with guide RNAs sharing specific PAM (protospacer adjacent motif)-proximal sequence motifs. Here, we demonstrate that this toxicity is caused by off-target binding of Cas9 to the promoter of essential genes, with silencing of off-target genes occurring with as little as 4 nt of identity in the PAM-proximal sequence. Screens performed in various strains of Escherichia coli and other enterobacteria show that the nature of toxic guide RNAs changes together with the evolution of sequences at off-target positions. These results highlight the potential for Cas9 to bind to hundreds of off-target positions in bacterial genomes, leading to undesired effects. This phenomenon must be considered in the design and interpretation of CRISPR-Cas experiments in bacteria.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Wu ZS, Gao Y, Du YT, et al (2023)

The protocol of tagging endogenous proteins with fluorescent tags using CRISPR-Cas9 genome editing.

Yi chuan = Hereditas, 45(2):165-175.

The currently widely used CRISPR-Cas9 genome editing technology enables the editing of target genes (knock-out or knock-in) with high accuracy and efficiency. Guided by the small guide RNA, the Cas9 nuclease induces a DNA double-strand break at the targeted genomic locus. The DNA double-strand break can be repaired by the homology-directed repair pathway in the presence of a repair template. With the repair template containing the coding sequence of a fluorescent tag, the targeted gene can be inserted with the sequence of a fluorescent tag at the designed position. The genome editing mediated labeling of endogenous proteins with fluorescent tags avoids the potential artifacts caused by gene overexpression and substantially improves the reproductivity of imaging experiments. This protocol focuses on creating mammalian cell lines with endogenous proteins tagged with fluorescent proteins or self-labeling protein tags using CRISPR-Cas9 genome editing.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Liu MZ, Wang LR, Li YM, et al (2023)

Generation of genetically modified rat models via the CRISPR/Cas9 technology.

Yi chuan = Hereditas, 45(1):78-87.

The RNA-guided CRISPR/Cas9 genomic editing system consists of a single guide RNA (sgRNA) and a Cas9 nuclease. The two components form a complex in cells and target the genomic loci complementary to the sgRNA. The Cas9 nuclease cleaves the target site creating a double stranded DNA break (DSB). In mammalian cells, DSBs are often repaired via error prone non-homologous end joining (NHEJ) or via homology directed repair (HDR) with the presence of donor DNA templates. Micro-injection of the CRISPR/Cas9 system into the rat embryos enables generation of genetically modified rat models. Here, we describe a detailed protocol for creating gene knockout or knockin rat models via the CRISPR/Cas9 technology.

RevDate: 2023-03-20
CmpDate: 2023-03-20

D'Antonio L, Fieni C, Ciummo SL, et al (2023)

Inactivation of interleukin-30 in colon cancer stem cells via CRISPR/Cas9 genome editing inhibits their oncogenicity and improves host survival.

Journal for immunotherapy of cancer, 11(3):.

BACKGROUND: Progression of colorectal cancer (CRC), a leading cause of cancer-related death worldwide, is driven by colorectal cancer stem cells (CR-CSCs), which are regulated by endogenous and microenvironmental signals. Interleukin (IL)-30 has proven to be crucial for CSC viability and tumor progression. Whether it is involved in CRC tumorigenesis and impacts clinical behavior is unknown.

METHODS: IL30 production and functions, in stem and non-stem CRC cells, were determined by western blot, immunoelectron microscopy, flow cytometry, cell viability and sphere formation assays. CRISPR/Cas9-mediated deletion of the IL30 gene, RNA-Seq and implantation of IL30 gene transfected or deleted CR-CSCs in NSG mice allowed to investigate IL30's role in CRC oncogenesis. Bioinformatics and immunopathology of CRC samples highlighted the clinical implications.

RESULTS: We demonstrated that both CR-CSCs and CRC cells express membrane-anchored IL30 that regulates their self-renewal, via WNT5A and RAB33A, and/or proliferation and migration, primarily by upregulating CXCR4 via STAT3, which are suppressed by IL30 gene deletion, along with WNT and RAS pathways. Deletion of IL30 gene downregulates the expression of proteases, such as MMP2 and MMP13, chemokine receptors, mostly CCR7, CCR3 and CXCR4, and growth and inflammatory mediators, including ANGPT2, CXCL10, EPO, IGF1 and EGF. These factors contribute to IL30-driven CR-CSC and CRC cell expansion, which is abrogated by their selective blockade. IL30 gene deleted CR-CSCs displayed reduced tumorigenicity and gave rise to slow-growing and low metastatic tumors in 80% of mice, which survived much longer than controls. Bioinformatics and CIBERSORTx of the 'Colorectal Adenocarcinoma TCGA Nature 2012' collection, and morphometric assessment of IL30 expression in clinical CRC samples revealed that the lack of IL30 in CRC and infiltrating leucocytes correlates with prolonged overall survival.

CONCLUSIONS: IL30 is a new CRC driver, since its inactivation, which disables oncogenic pathways and multiple autocrine loops, inhibits CR-CSC tumorigenicity and metastatic ability. The development of CRISPR/Cas9-mediated targeting of IL30 could improve the current therapeutic landscape of CRC.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Wang R, Zhang R, Khodaverdian A, et al (2023)

Theoretical guarantees for phylogeny inference from single-cell lineage tracing.

Proceedings of the National Academy of Sciences of the United States of America, 120(12):e2203352120.

Lineage-tracing technologies based on Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9 (CRISPR-Cas9) genome editing have emerged as a powerful tool for investigating development in single-cell contexts, but exact reconstruction of the underlying clonal relationships in experiment is complicated by features of the data. These complications are functions of the experimental parameters in these systems, such as the Cas9 cutting rate, the diversity of indel outcomes, and the rate of missing data. In this paper, we develop two theoretically grounded algorithms for the reconstruction of the underlying single-cell phylogenetic tree as well as asymptotic bounds for the number of recording sites necessary for exact recapitulation of the ground truth phylogeny at high probability. In doing so, we explore the relationship between the problem difficulty and the experimental parameters, with implications for experimental design. Lastly, we provide simulations showing the empirical performance of these algorithms and showing that the trends in the asymptotic bounds hold empirically. Overall, this work provides a theoretical analysis of phylogenetic reconstruction in single-cell CRISPR-Cas9 lineage-tracing technologies.

RevDate: 2023-03-18

Meng X, Wu TG, Lou QY, et al (2023)

Optimization of CRISPR-Cas system for clinical cancer therapy.

Bioengineering & translational medicine, 8(2):e10474.

Cancer is a genetic disease caused by alterations in genome and epigenome and is one of the leading causes for death worldwide. The exploration of disease development and therapeutic strategies at the genetic level have become the key to the treatment of cancer and other genetic diseases. The functional analysis of genes and mutations has been slow and laborious. Therefore, there is an urgent need for alternative approaches to improve the current status of cancer research. Gene editing technologies provide technical support for efficient gene disruption and modification in vivo and in vitro, in particular the use of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems. Currently, the applications of CRISPR-Cas systems in cancer rely on different Cas effector proteins and the design of guide RNAs. Furthermore, effective vector delivery must be met for the CRISPR-Cas systems to enter human clinical trials. In this review article, we describe the mechanism of the CRISPR-Cas systems and highlight the applications of class II Cas effector proteins. We also propose a synthetic biology approach to modify the CRISPR-Cas systems, and summarize various delivery approaches facilitating the clinical application of the CRISPR-Cas systems. By modifying the CRISPR-Cas system and optimizing its in vivo delivery, promising and effective treatments for cancers using the CRISPR-Cas system are emerging.

RevDate: 2023-03-18

Khambhati K, Bhattacharjee G, Gohil N, et al (2023)

Phage engineering and phage-assisted CRISPR-Cas delivery to combat multidrug-resistant pathogens.

Bioengineering & translational medicine, 8(2):e10381.

Antibiotic resistance ranks among the top threats to humanity. Due to the frequent use of antibiotics, society is facing a high prevalence of multidrug resistant pathogens, which have managed to evolve mechanisms that help them evade the last line of therapeutics. An alternative to antibiotics could involve the use of bacteriophages (phages), which are the natural predators of bacterial cells. In earlier times, phages were implemented as therapeutic agents for a century but were mainly replaced with antibiotics, and considering the menace of antimicrobial resistance, it might again become of interest due to the increasing threat of antibiotic resistance among pathogens. The current understanding of phage biology and clustered regularly interspaced short palindromic repeats (CRISPR) assisted phage genome engineering techniques have facilitated to generate phage variants with unique therapeutic values. In this review, we briefly explain strategies to engineer bacteriophages. Next, we highlight the literature supporting CRISPR-Cas9-assisted phage engineering for effective and more specific targeting of bacterial pathogens. Lastly, we discuss techniques that either help to increase the fitness, specificity, or lytic ability of bacteriophages to control an infection.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Chen H, Li B, Shi S, et al (2023)

Au-Fe3O4 nanozyme coupled with CRISPR-Cas12a for sensitive and visual antibiotic resistance diagnosing.

Analytica chimica acta, 1251:341014.

The accumulation and spread of antibiotic resistance bacteria (ARB) in the environment may accelerate the formation of superbugs and seriously threaten the health of all living beings. The timeliness and accurate diagnosing of antibiotic resistance is essential to controlling the propagation of superbugs in the environment and formulating effective public health management programs. Herein, we developed a speedy, sensitive, accurate, and user-friendly colorimetric assay for antibiotic resistance, via a synergistic combination of the peroxidase-like property of the Au-Fe3O4 nanozyme and the specific gene identification capability of the CRISPR-Cas12a. Once the CRISPR-Cas12a system recognizes a target resistance gene, it activates its trans-cleavage activity and subsequently releases the Au-Fe3O4 nanozymes, which oxidizes the 3,3,5,5-tetramethylbenzidine (TMB) with color change from transparent to blue. The diagnosing signals could be captured and analyzed by a smartphone. This method detected kanamycin-resistance genes, ampicillin-resistance genes, and chloramphenicol-resistance genes by simple operation steps with high sensitivity (<0.1 CFU μL[-1]) and speediness (<1 h). This approach may prove easy for the accurate and sensitive diagnosis of the ARGs or ARB in the field, thus surveilling and controlling the microbial water quality flexibly and efficiently.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Niu C, Liu J, Xing X, et al (2023)

CRISPR-Cas12a-assisted elimination of the non-specific signal from non-specific amplification in the Exponential Amplification Reaction.

Analytica chimica acta, 1251:340998.

Non-specific amplification is a major problem in nucleic acid amplification resulting in false-positive results, especially for exponential amplification reactions (EXPAR). Although efforts were made to suppress the influence of non-specific amplification, such as chemical blocking of the template's 3'-ends and sequence-independent weakening of template-template interactions, it is still a common problem in many conventional EXPAR reactions. In this study, we propose a novel strategy to eliminate the non-specific signal from non-specific amplification by integrating the CRISPR-Cas12a system into two-templates EXPAR. An EXPAR-Cas12a strategy named EXPCas was developed, where the Cas12a system acted as a filter to filter out non-specific amplificons in EXPAR, suppressing and eliminating the influence of non-specific amplification. As a result, the signal-to-background ratio was improved from 1.3 to 15.4 using this method. With microRNA-21 (miRNA-21) as a target, the detection can be finished in 40 min with a LOD of 103 fM and no non-specific amplification was observed.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Yin Z, Su R, Ge L, et al (2023)

Single-cell resolution reveals RalA GTPase expanding hematopoietic stem cells and facilitating of BCR-ABL1-driven leukemogenesis in a CRISPR/Cas9 gene editing mouse model.

International journal of biological sciences, 19(4):1211-1227.

BCR-ABL oncogene-mediated Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML) is suggested to originate from leukemic stem cells (LSCs); however, factors regulating self-renewal of LSC and normal hematopoietic stem cells (HSCs) are largely unclear. Here, we show that RalA, a small GTPase in the Ras downstream signaling pathway, has a critical effect on regulating the self-renewal of LSCs and HSCs. A RalA knock-in mouse model (RalA[Rosa26-Tg/+]) was initially constructed on the basis of the Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR/Cas9) assay to analyze normal hematopoietic differentiation frequency using single-cell resolution and flow cytometry. RalA overexpression promoted cell cycle progression and increased the frequency of granulocyte-monocyte progenitors (GMPs), HSCs and multipotent progenitors (MPPs). The uniform manifold approximation and projection (UMAP) plot revealed heterogeneities in HSCs and progenitor cells (HSPCs) and identified the subclusters of HSCs and GMPs with a distinct molecular signature. RalA also promoted BCR-ABL-induced leukemogenesis and self-renewal of primary LSCs and shortened the survival of leukemic mice. RalA knockdown prolonged survival and promoted sensitivity to imatinib in a patient-derived tumor xenograft model. Immunoprecipitation plus single-cell RNA sequencing of the GMP population confirmed that RalA induced this effect by interacting with RAC1. RAC1 inhibition by azathioprine effectively reduced the self-renewal, colony formation ability of LSCs and prolonged the survival in BCR-ABL1-driven RalA overexpression CML mice. Collectively, RalA was detected to be a vital factor that regulates the abilities of HSCs and LSCs, thus facilitating BCR-ABL-triggered leukemia in mice. RalA inhibition serves as the therapeutic approach to eradicate LSCs in CML.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Fudge JB (2023)

Cardiac defect corrected in vivo with CRISPR.

Nature biotechnology, 41(3):323.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Dyke E, Bijnagte-Schoenmaker C, Wu KM, et al (2023)

Generation of induced pluripotent stem cell line carrying frameshift variants in NPHP1 (UCSFi001-A-68) using CRISPR/Cas9.

Stem cell research, 68:103053.

NPHP1 (Nephrocystin 1) is a protein that localizes to the transition zone of the cilium, a small organelle that projects from the plasma membrane of most cells and allows for integration and coordination of signalling pathways during development and homeostasis. Loss of NPHP1 function due to biallelic NPHP1 gene mutations can lead to the development of ciliopathies - a heterogeneous spectra of disorders characterized by ciliary dysfunction. Here we report the generation of an NPHP1-null hiPSC line (UCSFi001-A-68) via CRISPR/Cas9-mediated non-homologous end joining in the UCSFi001-A background, for study of the role that this protein plays in different tissues.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Zhu Y, J Champer (2023)

Simulations Reveal High Efficiency and Confinement of a Population Suppression CRISPR Toxin-Antidote Gene Drive.

ACS synthetic biology, 12(3):809-819.

Though engineered gene drives hold great promise for spreading through and suppressing populations of disease vectors or invasive species, complications such as resistance alleles and spatial population structure can prevent their success. Additionally, most forms of suppression drives, such as homing drives or driving Y chromosomes, will generally spread uncontrollably between populations with even small levels of migration. The previously proposed CRISPR-based toxin-antidote system called toxin-antidote dominant embryo (TADE) suppression drive could potentially address the issues of confinement and resistance. However, it is a relatively weak form of drive compared to homing drives, which might make it particularly vulnerable to spatial population structure. In this study, we investigate TADE suppression drive using individual-based simulations in a continuous spatial landscape. We find that the drive is actually more confined than in simple models without space, even in its most efficient form with low cleavage rate in embryos from maternally deposited Cas9. Furthermore, the drive performed well in continuous space scenarios if the initial release requirements were met, suppressing the population in a timely manner without being severely affected by chasing, a phenomenon in which wild-type individuals avoid the drive by recolonizing empty areas. At higher embryo cut rates, the drive loses its ability to spread, but a single, widespread release can often still induce rapid population collapse. Thus, if TADE suppression gene drives can be successfully constructed, they may play an important role in control of disease vectors and invasive species when stringent confinement to target populations is desired.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Du L, Zhang D, Luo Z, et al (2023)

Molecular basis of stepwise cyclic tetra-adenylate cleavage by the type III CRISPR ring nuclease Crn1/Sso2081.

Nucleic acids research, 51(5):2485-2495.

The cyclic oligoadenylates (cOAs) act as second messengers of the type III CRISPR immunity system through activating the auxiliary nucleases for indiscriminate RNA degradation. The cOA-degrading nucleases (ring nucleases) provide an 'off-switch' regulation of the signaling, thereby preventing cell dormancy or cell death. Here, we describe the crystal structures of the founding member of CRISPR-associated ring nuclease 1 (Crn1) Sso2081 from Saccharolobus solfataricus, alone, bound to phosphate ions or cA4 in both pre-cleavage and cleavage intermediate states. These structures together with biochemical characterizations establish the molecular basis of cA4 recognition and catalysis by Sso2081. The conformational changes in the C-terminal helical insert upon the binding of phosphate ions or cA4 reveal a gate-locking mechanism for ligand binding. The critical residues and motifs identified in this study provide a new insight to distinguish between cOA-degrading and -nondegrading CARF domain-containing proteins.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Ham O, Kim S, Lee Y, et al (2023)

Generation of telomeric repeat binding factor 1 (TRF1)-knockout human embryonic stem cell lines, KRIBBe010-A-95, KRIBBe010-A-96, and KRIBBe010-A-97, using CRISPR/Cas9 technology.

Stem cell research, 68:103045.

Telomeric repeat binding factor 1 (TRF1) plays an essential role in maintaining telomere length. Here, we established TRF1-knockout human pluripotent stem cells (hPSCs; hTRF1-KO) using the CRISPR/Cas9 technology. The hTRF1-KO cell lines expressed pluripotency markers and demonstrated a normal karyotype (46, XX) and DNA profile. In addition, hTRF1-KOcells spontaneously differentiated into all three germ layers in vitro. Thus, these cell lines could be useful models in various research fields.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Huan YW, Torraca V, Brown R, et al (2023)

P1 Bacteriophage-Enabled Delivery of CRISPR-Cas9 Antimicrobial Activity Against Shigella flexneri.

ACS synthetic biology, 12(3):709-721.

The discovery of clustered, regularly interspaced, short palindromic repeats (CRISPR) and the Cas9 RNA-guided nuclease provides unprecedented opportunities to selectively kill specific populations or species of bacteria. However, the use of CRISPR-Cas9 to clear bacterial infections in vivo is hampered by the inefficient delivery of cas9 genetic constructs into bacterial cells. Here, we use a broad-host-range P1-derived phagemid to deliver the CRISPR-Cas9 chromosomal-targeting system into Escherichia coli and the dysentery-causing Shigella flexneri to achieve DNA sequence-specific killing of targeted bacterial cells. We show that genetic modification of the helper P1 phage DNA packaging site (pac) significantly enhances the purity of packaged phagemid and improves the Cas9-mediated killing of S. flexneri cells. We further demonstrate that P1 phage particles can deliver chromosomal-targeting cas9 phagemids into S. flexneri in vivo using a zebrafish larvae infection model, where they significantly reduce the bacterial load and promote host survival. Our study highlights the potential of combining P1 bacteriophage-based delivery with the CRISPR chromosomal-targeting system to achieve DNA sequence-specific cell lethality and efficient clearance of bacterial infection.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Stępniewski J, Jeż M, J Dulak (2023)

Generation of miR-15a/16-1 cluster-deficient human induced pluripotent stem cell line (DMBi001-A-2) using CRISPR/Cas9 gene editing.

Stem cell research, 68:103046.

miR-15a/16-1 cluster, composed of MIR15A and MIR16-1 genes located in close proximity on chromosome 13 was described to regulate post-natal cell cycle withdrawal of cardiomyocytes in mice. In humans, on the other hand, the level of miR-15a-5p and miR-16-p was negatively associated with the severity of cardiac hypertrophy. Therefore, to better understand the role of these microRNAs in human cardiomyocytes in regard to their proliferative potential and hypertrophic growth, we generated hiPSC line with complete deletion of miR-15a/16-1 cluster using CRISPR/Cas9 gene editing. Obtained cells demonstrate expression of pluripotency markers, differentiation capacity into all three germ layers and normal karyotype.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Jeong SH, Kim HJ, SJ Lee (2023)

New Target Gene Screening Using Shortened and Random sgRNA Libraries in Microbial CRISPR Interference.

ACS synthetic biology, 12(3):800-808.

CRISPR interference (CRISPRi) screening has been used for identification of target genes related to specific phenotypes using single-molecular guide RNA (sgRNA) libraries. In CRISPRi screening, the sizes of random sgRNA libraries contained with the original target recognition sequences are large (∼10[12]). Here, we demonstrated that the length of the target recognition sequence (TRS) can be shortened in sgRNAs from the original 20 nucleotides (N20) to 9 nucleotides (N9) that is still sufficient for dCas9 to repress target genes in the xylose operon of Escherichia coli, regardless of binding to a promoter or open reading frame region. Based on the results, we constructed random sgRNA plasmid libraries with 5'-shortened TRS lengths, and identified xylose metabolic target genes by Sanger sequencing of sgRNA plasmids purified from Xyl[-] phenotypic cells. Next, the random sgRNA libraries were harnessed to screen for target genes to enhance violacein pigment production in synthetic E. coli cells. Seventeen target genes were selected by analyzing the redundancy of the TRS in sgRNA plasmids in dark purple colonies. Among them, seven genes (tyrR, pykF, cra, ptsG, pykA, sdaA, and tnaA) have been known to increase the intracellular l-tryptophan pool, the precursor of a violacein. Seventeen cells with a single deletion of each target gene exhibited a significant increase in violacein production. These results indicate that using shortened random TRS libraries for CRISPRi can be simple and cost-effective for phenotype-based target gene screening.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Nguyen TTT, Tanaka Y, Sanada M, et al (2023)

CRISPR/Cas9-Mediated Induction of Relapse-Specific NT5C2 and PRPS1 Mutations Confers Thiopurine Resistance as a Relapsed Lymphoid Leukemia Model.

Molecular pharmacology, 103(4):199-210.

6-Mercaptopurine (6-MP) is a key component in maintenance therapy for childhood acute lymphoblastic leukemia (ALL). Recent next-generation sequencing analysis of childhood ALL clarified the emergence of the relapse-specific mutations of the NT5C2 and PRPS1 genes, which are involved in thiopurine metabolism. In this scenario, minor clones of leukemia cells could acquire the 6-MP-resistant phenotype as a result of the NT5C2 or PRPS1 mutation during chemotherapy (including 6-MP treatment) and confer disease relapse after selective expansion. Thus, to establish new therapeutic modalities overcoming 6-MP resistance in relapsed ALL, human leukemia models with NT5C2 and PRPS1 mutations in the intrinsic genes are urgently required. Here, mimicking the initiation process of the above clinical course, we sought to induce two relapse-specific hotspot mutations (R39Q mutation of the NT5C2 gene and S103N mutation of the PRPS1 gene) into a human lymphoid leukemia cell line by homologous recombination (HR) using the CRISPR/Cas9 system. After 6-MP selection of the cells transfected with Cas9 combined with single-guide RNA and donor DNA templates specific for either of those two mutations, we obtained the sublines with the intended NT5C2-R39Q and PRPS1-S103N mutation as a result of HR. Moreover, diverse in-frame small insertion/deletions were also confirmed in the 6-MP-resistant sublines at the target sites of the NT5C2 and PRPS1 genes as a result of nonhomologous end joining. These sublines are useful for molecular pharmacological evaluation of the NT5C2 and PRPS1 gene mutations in the 6-MP sensitivity and development of therapy overcoming the thiopurine resistance of leukemia cells. SIGNIFICANCE STATEMENT: Mimicking the initiation process of relapse-specific mutations of the NT5C2 and PRPS1 genes in childhood acute lymphoblastic leukemia treated with 6-mercaptopurine (6-MP), this study sought to introduce NT5C2-R39Q and PRPS1-S103N mutations into a human lymphoid leukemia cell line by homologous recombination using the CRISPR/Cas9 system. In the resultant 6-MP-resistant sublines, the intended mutations and diverse in-frame small insertions/deletions were confirmed, indicating that the obtained sublines are useful for molecular pharmacological evaluation of the NT5C2 and PRPS1 gene mutations.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Lackner M, Helmbrecht N, Pääbo S, et al (2023)

Detection of unintended on-target effects in CRISPR genome editing by DNA donors carrying diagnostic substitutions.

Nucleic acids research, 51(5):e26.

CRISPR nucleases can introduce double-stranded DNA breaks in genomes at positions specified by guide RNAs. When repaired by the cell, this may result in the introduction of insertions and deletions or nucleotide substitutions provided by exogenous DNA donors. However, cellular repair can also result in unintended on-target effects, primarily larger deletions and loss of heterozygosity due to gene conversion. Here we present a strategy that allows easy and reliable detection of unintended on-target effects as well as the generation of control cells that carry wild-type alleles but have demonstratively undergone genome editing at the target site. Our 'sequence-ascertained favorable editing' (SAFE) donor approach relies on the use of DNA donor mixtures containing the desired nucleotide substitutions or the wild-type alleles together with combinations of additional 'diagnostic' substitutions unlikely to have any effects. Sequencing of the target sites then results in that two different sequences are seen when both chromosomes are edited with 'SAFE' donors containing different sets of substitutions, while a single sequence indicates unintended effects such as deletions or gene conversion. We analyzed more than 850 human embryonic stem cell clones edited with 'SAFE' donors and detect all copy number changes and almost all clones with gene conversion.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Zulhussnain M, Zahoor MK, Ranian K, et al (2023)

CRISPR Cas9 mediated knockout of sex determination pathway genes in Aedes aegypti.

Bulletin of entomological research, 113(2):243-252.

The vector role of Aedes aegypti for viral diseases including dengue and dengue hemorrhagic fever makes it imperative for its proper control. Despite various adopted control strategies, genetic control measures have been recently focused against this vector. CRISPR Cas9 system is a recent and most efficient gene editing tool to target the sex determination pathway genes in Ae. aegypti. In the present study, CRISPR Cas9 system was used to knockout Ae. aegypti doublesex (Aaedsx) and Ae. aegypti sexlethal (AaeSxl) genes in Ae. aegypti embryos. The injection mixes with Cas9 protein (333 ng ul[-1]) and gRNAs (each at 100 ng ul[-1]) were injected into eggs. Injected eggs were allowed to hatch at 26 ± 1°C, 60 ± 10% RH. The survival and mortality rate was recorded in knockout Aaedsx and AaeSxl. The results revealed that knockout produced low survival and high mortality. A significant percentage of eggs (38.33%) did not hatch as compared to control groups (P value 0.00). Highest larval mortality (11.66%) was found in the knockout of Aaedsx female isoform, whereas, the emergence of only male adults also showed that the knockout of Aaedsx (female isoform) does not produce male lethality. The survival (3.33%) of knockout for AaeSxl eggs to the normal adults suggested further study to investigate AaeSxl as an efficient upstream of Aaedsx to target for sex transformation in Ae. aegypti mosquitoes.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Lee S, Lee H, Baek G, et al (2023)

Precision mitochondrial DNA editing with high-fidelity DddA-derived base editors.

Nature biotechnology, 41(3):378-386.

Bacterial toxin DddA-derived cytosine base editors (DdCBEs)-composed of split DddAtox (a cytosine deaminase specific to double-stranded DNA), custom-designed TALE (transcription activator-like effector) DNA-binding proteins, and a uracil glycosylase inhibitor-enable mitochondrial DNA (mtDNA) editing in human cells, which may pave the way for therapeutic correction of pathogenic mtDNA mutations in patients. The utility of DdCBEs has been limited by off-target activity, which is probably caused by spontaneous assembly of the split DddAtox deaminase enzyme, independent of DNA-binding interactions. We engineered high-fidelity DddA-derived cytosine base editors (HiFi-DdCBEs) with minimal off-target activity by substituting alanine for amino acid residues at the interface between the split DddAtox halves. The resulting domains cannot form a functional deaminase without binding of their linked TALE proteins at adjacent sites on DNA. Whole mitochondrial genome sequencing shows that, unlike conventional DdCBEs, which induce hundreds of unwanted off-target C-to-T conversions in human mtDNA, HiFi-DdCBEs are highly efficient and precise, avoiding collateral off-target mutations, and as such, they will probably be desirable for therapeutic applications.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Christie KA, Guo JA, Silverstein RA, et al (2023)

Precise DNA cleavage using CRISPR-SpRYgests.

Nature biotechnology, 41(3):409-416.

Methods for in vitro DNA cleavage and molecular cloning remain unable to precisely cleave DNA directly adjacent to bases of interest. Restriction enzymes (REs) must bind specific motifs, whereas wild-type CRISPR-Cas9 or CRISPR-Cas12 nucleases require protospacer adjacent motifs (PAMs). Here we explore the utility of our previously reported near-PAMless SpCas9 variant, named SpRY, to serve as a universal DNA cleavage tool for various cloning applications. By performing SpRY DNA digests (SpRYgests) using more than 130 guide RNAs (gRNAs) sampling a wide diversity of PAMs, we discovered that SpRY is PAMless in vitro and can cleave DNA at practically any sequence, including sites refractory to cleavage with wild-type SpCas9. We illustrate the versatility and effectiveness of SpRYgests to improve the precision of several cloning workflows, including those not possible with REs or canonical CRISPR nucleases. We also optimize a rapid and simple one-pot gRNA synthesis protocol to streamline SpRYgest implementation. Together, SpRYgests can improve various DNA engineering applications that benefit from precise DNA breaks.

RevDate: 2023-03-20
CmpDate: 2023-03-20

Grünewald J, Miller BR, Szalay RN, et al (2023)

Engineered CRISPR prime editors with compact, untethered reverse transcriptases.

Nature biotechnology, 41(3):337-343.

The CRISPR prime editor PE2 consists of a Streptococcus pyogenes Cas9 nickase (nSpCas9) fused at its C-terminus to a Moloney murine leukemia virus reverse transcriptase (MMLV-RT). Here we show that separated nSpCas9 and MMLV-RT proteins function as efficiently as intact PE2 in human cells. We use this Split-PE system to rapidly identify and engineer more compact prime editor architectures that also broaden the types of RTs used for prime editing.

RevDate: 2023-03-18

Zakrzewska M, M Burmistrz (2023)

Mechanisms regulating the CRISPR-Cas systems.

Frontiers in microbiology, 14:1060337.

The CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats- CRISPR associated proteins) is a prokaryotic system that enables sequence specific recognition and cleavage of nucleic acids. This is possible due to cooperation between CRISPR array which contains short fragments of DNA called spacers that are complimentary to the targeted nucleic acid and Cas proteins, which take part in processes of: acquisition of new spacers, processing them into their functional form as well as recognition and cleavage of targeted nucleic acids. The primary role of CRISPR-Cas systems is to provide their host with an adaptive and hereditary immunity against exogenous nucleic acids. This system is present in many variants in both Bacteria and Archea. Due to its modular structure, and programmability CRISPR-Cas system become attractive tool for modern molecular biology. Since their discovery and implementation, the CRISPR-Cas systems revolutionized areas of gene editing and regulation of gene expression. Although our knowledge on how CRISPR-Cas systems work has increased rapidly in recent years, there is still little information on how these systems are controlled and how they interact with other cellular mechanisms. Such regulation can be the result of both auto-regulatory mechanisms as well as exogenous proteins of phage origin. Better understanding of these interaction networks would be beneficial for optimization of current and development of new CRISPR-Cas-based tools. In this review we summarize current knowledge on the various molecular mechanisms that affect activity of CRISPR-Cas systems.

RevDate: 2023-03-18

Cerbino GN, Traglia GM, Ayala Nuñez T, et al (2023)

Comparative genome analysis of the genus Shewanella unravels the association of key genetic traits with known and potential pathogenic lineages.

Frontiers in microbiology, 14:1124225.

Shewanella spp. are Gram-negative rods widely disseminated in aquatic niches that can also be found in human-associated environments. In recent years, reports of infections caused by these bacteria have increased significantly. Mobilome and resistome analysis of a few species showed that they are versatile; however, comprehensive comparative studies in the genus are lacking. Here, we analyzed the genetic traits of 144 genomes from Shewanella spp. isolates focusing on the mobilome, resistome, and virulome to establish their evolutionary relationship and detect unique features based on their genome content and habitat. Shewanella spp. showed a great diversity of mobile genetic elements (MGEs), most of them associated with monophyletic lineages of clinical isolates. Furthermore, 79/144 genomes encoded at least one antimicrobial resistant gene with their highest occurrence in clinical-related lineages. CRISPR-Cas systems, which confer immunity against MGEs, were found in 41 genomes being I-E and I-F the more frequent ones. Virulome analysis showed that all Shewanella spp. encoded different virulence genes (motility, quorum sensing, biofilm, adherence, etc.) that may confer adaptive advantages for survival against hosts. Our data revealed that key accessory genes are frequently found in two major clinical-related groups, which encompass the opportunistic pathogens Shewanella algae and Shewanella xiamenensis together with several other species. This work highlights the evolutionary nature of Shewanella spp. genomes, capable of acquiring different key genetic traits that contribute to their adaptation to different niches and facilitate the emergence of more resistant and virulent isolates that impact directly on human and animal health.

RevDate: 2023-03-18

Pedrazzoli E, Bianchi A, Umbach A, et al (2023)

An optimized SpCas9 high-fidelity variant for direct protein delivery.

Molecular therapy : the journal of the American Society of Gene Therapy pii:S1525-0016(23)00128-4 [Epub ahead of print].

Electroporation of the Cas9 ribonucleoprotein (RNP) complex offers the advantage of preventing off-target cleavages and potential immune responses produced by long-term expression of the nuclease. Nevertheless, the majority of engineered high-fidelity Streptococcus pyogenes Cas9 (SpCas9) variants are less active than the wild-type enzyme and are not compatible with RNP delivery. Building on our previous studies on evoCas9, we developed a high-fidelity SpCas9 variant suitable for RNP delivery. The editing efficacy and precision of the recombinant high-fidelity Cas9 (rCas9HF), characterized by the K526D substitution, was compared with the R691A mutant (HiFi Cas9), which is currently the only available high-fidelity Cas9 that can be used as an RNP. The comparative analysis was extended to gene substitution experiments where the two high fidelities were used in combination with a DNA donor template, generating different ratios of non-homologous end joining (NHEJ) versus homology-directed repair (HDR) for precise editing. The analyses revealed a heterogeneous efficacy and precision indicating different targeting capabilities between the two variants throughout the genome. The development of rCas9HF, characterized by an editing profile diverse from the currently used HiFi Cas9 in RNP electroporation, increases the genome editing solutions for the highest precision and efficient applications.

RevDate: 2023-03-16

Wang X, Mu X, Li J, et al (2023)

A novel nanoparticle surface-constrained CRISPR-Cas12a 3D DNA walker-like nanomachines for sensitive and stable miRNAs detection.

Analytica chimica acta, 1251:340950.

The CRISPR-Cas system has broad prospects as a new type of nucleic acid signal amplification technology based on the trans-cleavage activity of Cas12a to single-stranded DNA, but the trans-cleavage reaction efficiency is relatively low in solution. In order to overcome this negative factor, a new 3D DNA nanomachine whose CRISPR-Cas12a is limited to the surface of nanoparticles is used for sensitive and stable detection of miRNA. By loading Cas12a activator onto spherical nucleic acid (SNA), the CRISPR-Cas12a activator system on the surface of Au nanoparticles (AuNPs) acts as a walker to carry out continuous recognition-walking-cutting reaction on the surface of AuNPs, which enhances the trans-cleavage activity of Cas12a to SNAs. Benefiting from the confinement effect of spherical nucleic acids surface, a 3D DNA nanomachine has been developed for the detection of miRNA-21, which has achieved high sensitivity and accuracy, and the detection limit is able to reach 8.0 pM. This new 3D DNA walker-like nanomachine provided another insight for future bioanalysis and early clinical diagnoses of disease and liquid biopsy.

RevDate: 2023-03-16

Mikkelsen NS, Hernandez SS, Jensen TI, et al (2023)

Enrichment of transgene integrations by transient CRISPR activation of a silent reporter gene.

Molecular therapy. Methods & clinical development, 29:1-16.

CRISPR-Cas-mediated site-specific integration of transgenes by homology-directed repair (HDR) is challenging, especially in primary cells, where inferior editing efficiency may impede the development of gene- and cellular therapies. Various strategies for enrichment of cells with transgene integrations have been developed, but most strategies either generate unwanted genomic scars or rely on permanent integration and expression of a reporter gene used for selection. However, stable expression of a reporter gene may perturb cell homeostasis and function. Here we develop a broadly applicable and versatile enrichment strategy by harnessing the capability of CRISPR activation (CRISPRa) to transiently induce expression of a therapeutically relevant reporter gene used for immunomagnetic enrichment. This strategy is readily adaptable to primary human T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs), where enrichment of 1.8- to 3.3-fold and 3.2- to 3.6-fold was achieved, respectively. Furthermore, chimeric antigen receptor (CAR) T cells were enriched 2.5-fold and demonstrated improved cytotoxicity over non-enriched CAR T cells. Analysis of HDR integrations showed a proportion of cells harboring deletions of the transgene cassette arising either from impartial HDR or truncated adeno-associated virus (AAV) vector genomes. Nonetheless, this novel enrichment strategy expands the possibility to enrich for transgene integrations in research settings and in gene and cellular therapies.

RevDate: 2023-03-17
CmpDate: 2023-03-16

Jianwei L, Jobichen C, Machida S, et al (2023)

Structures of apo Cas12a and its complex with crRNA and DNA reveal the dynamics of ternary complex formation and target DNA cleavage.

PLoS biology, 21(3):e3002023.

Cas12a is a programmable nuclease for adaptive immunity against invading nucleic acids in CRISPR-Cas systems. Here, we report the crystal structures of apo Cas12a from Lachnospiraceae bacterium MA2020 (Lb2) and the Lb2Cas12a+crRNA complex, as well as the cryo-EM structure and functional studies of the Lb2Cas12a+crRNA+DNA complex. We demonstrate that apo Lb2Cas12a assumes a unique, elongated conformation, whereas the Lb2Cas12a+crRNA binary complex exhibits a compact conformation that subsequently rearranges to a semi-open conformation in the Lb2Cas12a+crRNA+DNA ternary complex. Notably, in solution, apo Lb2Cas12a is dynamic and can exist in both elongated and compact forms. Residues from Met493 to Leu523 of the WED domain undergo major conformational changes to facilitate the required structural rearrangements. The REC lobe of Lb2Cas12a rotates 103° concomitant with rearrangement of the hinge region close to the WED and RuvC II domains to position the RNA-DNA duplex near the catalytic site. Our findings provide insight into crRNA recognition and the mechanism of target DNA cleavage.

RevDate: 2023-03-14

Spasskaya DS, Davletshin AI, Bachurin SS, et al (2023)

Improving the on-target activity of high-fidelity Cas9 editors by combining rational design and random mutagenesis.

Applied microbiology and biotechnology [Epub ahead of print].

Genomic and post-genomic editors based on CRISPR/Cas systems are widely used in basic research and applied sciences, including human gene therapy. Most genome editing tools are based on the CRISPR/Cas9 type IIA system from Streptococcus pyogenes. Unfortunately, a number of drawbacks have hindered its application in therapeutic approaches, the most serious of which is the relatively high level of off-targets. To overcome this obstacle, various high-fidelity Cas9 variants have been created. However, they show reduced on-target activity compared to wild-type Cas9 possibly due to increased sensitivity to eukaryotic chromatin. Here, we combined a rational approach with random mutagenesis to create a set of new Cas9 variants showing high specificity and increased activity in Saccharomyces cerevisiae yeast. Moreover, a novel mutation in the PAM (protospacer adjacent motif)-interacting Cas9 domain was found, which increases the on-target activity of high-fidelity Cas9 variants while retaining their high specificity. The obtained data suggest that this mutation acts by weakening the eukaryotic chromatin barrier for Cas9 and rearranging the RuvC active center. Improved Cas9 variants should further advance genome and post-genome editing technologies. KEY POINTS: • D147Y and P411T mutations increase the activity of high-fidelity Cas9 variants. • The new L1206P mutation further increases the activity of high-fidelity Cas9 variants. • The L1206P mutation weakens the chromatin barrier for Cas9 editors.

RevDate: 2023-03-15
CmpDate: 2023-03-15

Wang D, Fletcher GC, Gagic D, et al (2023)

Comparative genome identification of accessory genes associated with strong biofilm formation in Vibrio parahaemolyticus.

Food research international (Ottawa, Ont.), 166:112605.

Vibrio parahaemolyticus biofilms on the seafood processing plant surfaces are a potential source of seafood contamination and subsequent food poisoning. Strains differ in their ability to form biofilm, but little is known about the genetic characteristics responsible for biofilm development. In this study, pangenome and comparative genome analysis of V. parahaemolyticus strains reveals genetic attributes and gene repertoire that contribute to robust biofilm formation. The study identified 136 accessory genes that were exclusively present in strong biofilm forming strains and these were functionally assigned to the Gene Ontology (GO) pathways of cellulose biosynthesis, rhamnose metabolic and catabolic processes, UDP-glucose processes and O antigen biosynthesis (p < 0.05). Strategies of CRISPR-Cas defence and MSHA pilus-led attachment were implicated via Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation. Higher levels of horizontal gene transfer (HGT) were inferred to confer more putatively novel properties on biofilm-forming V. parahaemolyticus. Furthermore, cellulose biosynthesis, a neglected potential virulence factor, was identified as being acquired from within the order Vibrionales. The cellulose synthase operons in V. parahaemolyticus were examined for their prevalence (22/138, 15.94 %) and were found to consist of the genes bcsG, bcsE, bcsQ, bcsA, bcsB, bcsZ, bcsC. This study provides insights into robust biofilm formation of V. parahaemolyticus at the genomic level and facilitates: identification of key attributes for robust biofilm formation, elucidation of biofilm formation mechanisms and development of potential targets for novel control strategies of persistent V. parahaemolyticus.


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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