CRISPR-associated Protein Research Articles

Convergence of Surface-Enhanced Raman Scattering with Molecular Diagnostics: A Perspective on Future Directions.

Molecular diagnostics (MD) is widely employed in multiple scientific disciplines, such as oncology, pathogen detection, forensic investigations, and the pharmaceutical industry. Techniques such as polymerase chain reaction (PCR) revolutionized the rapid and accurate identification of nucleic acids (DNA, RNA). More recently, CRISPR and its CRISPR-associated protein (Cas) have been a ground-breaking discovery that is the latest revolution in molecular biology, including MD. Surface-enhanced Raman scattering (SERS) is a very attractive alternative to fluorescence as the currently most widely used optical readout in MD. In this Perspective, milestones in the development of MD, SERS-PCR, and next-generation approaches to MD, such as Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) and DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR), are briefly summarized. Our perspective on the future convergence of SERS with MD is focused on SERS-based CRISPR/Cas (SERS-CRISPR) since we anticipate many promising applications in this rapidly emerging field. We predict that major future developments will exploit the advantages of real-time monitoring with the superior brightness, photostability, and spectral multiplexing potential of SERS nanotags in an automated workflow for rapid assays under isothermal, amplification-free conditions.

OsLRR-RLP2 Gene Regulates Immunity to Magnaporthe oryzae in Japonica Rice.

Rice is an important cereal crop worldwide, the growth of which is affected by rice blast disease, caused by the fungal pathogen Magnaporthe oryzae. As climate change increases the diversity of pathogens, the disease resistance genes (R genes) in plants must be identified. The major blast-resistance genes have been identified in indica rice varieties; therefore, japonica rice varieties with R genes now need to be identified. Because leucine-rich repeat (LRR) domain proteins possess R-gene properties, we used bioinformatics analysis to identify the rice candidate LRR domain receptor-like proteins (OsLRR-RLPs). OsLRR-RLP2, which contains six LRR domains, showed differences in the DNA sequence, containing 43 single-nucleotide polymorphisms (SNPs) in indica and japonica subpopulations. The results of the M. oryzae inoculation analysis indicated that indica varieties with partial deletion of OsLRR-RLP2 showed susceptibility, whereas japonica varieties with intact OsLRR-RLP2 showed resistance. The oslrr-rlp2 mutant, generated using clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), showed increased pathogen susceptibility, whereas plants overexpressing this gene showed pathogen resistance. These results indicate that OsLRR-RLP2 confers resistance to rice, and OsLRR-RLP2 may be useful for breeding resistant cultivars.

CRISPR-Cas9 In Vivo Gene Editing of KLKB1 for Hereditary Angioedema

BackgroundHereditary angioedema is a rare genetic disease that leads to severe and unpredictable swelling attacks. NTLA-2002 is an in vivo gene-editing therapy based on clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9. NTLA-2002 targets the gene encoding kallikrein B1 (KLKB1), with the goal of lifelong control of angioedema attacks after a single dose.MethodsIn this phase 1 dose-escalation portion of a combined phase 1–2 trial of NTLA-2002 in adults with hereditary angioedema, we administered NTLA-2002 at a single dose of 25 mg, 50 mg, or 75 mg. The primary end points were the safety and side-effect profile of NTLA-2002 therapy. Secondary and exploratory end points included pharmacokinetics, pharmacodynamics, and clinical efficacy determined on the basis of investigator-confirmed angioedema attacks.ResultsThree patients received 25 mg of NTLA-2002, four received 50 mg, and three received 75 mg. At all dose levels, the most common adverse events were infusion-related reactions and fatigue. No dose-limiting toxic effects, serious adverse events, grade 3 or higher adverse events, or clinically important laboratory findings were observed after the administration of NTLA-2002. Dose-dependent reductions in the total plasma kallikrein protein level were observed between baseline and the latest assessment, with a mean percentage change of −67% in the 25-mg group, −84% in the 50-mg group, and −95% in the 75-mg group. The mean percentage change in the number of angioedema attacks per month between baseline and weeks 1 through 16 (primary observation period) was −91% in the 25-mg group, −97% in the 50-mg group, and −80% in the 75-mg group. Among all the patients, the mean percentage change in the number of angioedema attacks per month from baseline through the latest assessment was −95%.ConclusionsIn this small study, a single dose of NTLA-2002 led to robust, dose-dependent, and durable reductions in total plasma kallikrein levels, and no severe adverse events were observed. In exploratory analyses, reductions in the number of angioedema attacks per month were observed at all dose levels. (Funded by Intellia Therapeutics; ClinicalTrials.gov number, NCT05120830.)

LncRNA CHKB-DT Downregulation Enhances Dilated Cardiomyopathy Through ALDH2.

Human cardiac long noncoding RNA (lncRNA) profiles in patients with dilated cardiomyopathy (DCM) were previously analyzed, and the long noncoding RNA CHKB (choline kinase beta) divergent transcript (CHKB-DT) levels were found to be mostly downregulated in the heart. In this study, the function of CHKB-DT in DCM was determined. Long noncoding RNA expression levels in the human heart tissues were measured via quantitative reverse transcription-polymerase chain reaction and in situ hybridization assays. A CHKB-DT heterozygous or homozygous knockout mouse model was generated using the clustered regularly interspaced palindromic repeat (CRISPR)/CRISPR-associated protein 9 system, and the adeno-associated virus with a cardiac-specific promoter was used to deliver the RNA in vivo. Sarcomere shortening was performed to assess the primary cardiomyocyte contractility. The Seahorse XF cell mitochondrial stress test was performed to determine the energy metabolism and ATP production. Furthermore, the underlying mechanisms were explored using quantitative proteomics, ribosome profiling, RNA antisense purification assays, mass spectrometry, RNA pull-down, luciferase assay, RNA-fluorescence in situ hybridization, and Western blotting. CHKB-DT levels were remarkably decreased in patients with DCM and mice with transverse aortic constriction-induced heart failure. Heterozygous knockout of CHKB-DT in cardiomyocytes caused cardiac dilation and dysfunction and reduced the contractility of primary cardiomyocytes. Moreover, CHKB-DT heterozygous knockout impaired mitochondrial function and decreased ATP production as well as cardiac energy metabolism. Mechanistically, ALDH2 (aldehyde dehydrogenase 2) was a direct target of CHKB-DT. CHKB-DT physically interacted with the mRNA of ALDH2 and fused in sarcoma (FUS) through the GGUG motif. CHKB-DT knockdown aggravated ALDH2 mRNA degradation and 4-HNE (4-hydroxy-2-nonenal) production, whereas overexpression of CHKB-DT reversed these molecular changes. Furthermore, restoring ALDH2 expression in CHKB-DT+/- mice alleviated cardiac dilation and dysfunction. CHKB-DT is significantly downregulated in DCM. CHKB-DT acts as an energy metabolism-associated long noncoding RNA and represents a promising therapeutic target against DCM.

Nanomaterials for intelligent CRISPR-Cas tools: improving environment sustainability.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) is a desirable gene modification tool covering a wide area in various sectors of medicine, agriculture, and microbial biotechnology. The role of this incredible genetic engineering technology has been extensively investigated; however, it remains formidable with cargo choices, nonspecific delivery, and insertional mutagenesis. Various nanomaterials including lipid, polymeric, and inorganic are being used to deliver the CRISPR-Cas system. Progress in nanomaterials could potentially address these challenges by accelerating precision targeting, cost-effectiveness, and one-step delivery. In this review, we highlighted the advances in nanotechnology and nanomaterials as smart delivery systems for CRISPR-Cas so as to ameliorate applications for environmental remediation including biomedical research and healthcare, strategies for mitigating antimicrobial resistance, and to be used as nanofertilizers for enhancing crop growth, and reducing the environmental impact of traditional fertilizers. The timely co-evolution of nanotechnology and CRISPR technologies has contributed to smart novel nanostructure hybrids for improving the onerous tasks of environmental remediation and biological sustainability.

ChemSpaceAL: An Efficient Active Learning Methodology Applied to Protein-Specific Molecular Generation.

The incredible capabilities of generative artificial intelligence models have inevitably led to their application in the domain of drug discovery. Within this domain, the vastness of chemical space motivates the development of more efficient methods for identifying regions with molecules that exhibit desired characteristics. In this work, we present a computationally efficient active learning methodology and demonstrate its applicability to targeted molecular generation. When applied to c-Abl kinase, a protein with FDA-approved small-molecule inhibitors, the model learns to generate molecules similar to the inhibitors without prior knowledge of their existence and even reproduces two of them exactly. We also show that the methodology is effective for a protein without any commercially available small-molecule inhibitors, the HNH domain of the CRISPR-associated protein 9 (Cas9) enzyme. To facilitate implementation and reproducibility, we made all of our software available through the open-source ChemSpaceAL Python package.

Fine mapping and identification of two NtTOM2A homeologs responsible for tobacco mosaic virus replication in tobacco (Nicotiana tabacum L.)

BackgroundTobacco mosaic virus (TMV) is a widely distributed viral disease that threatens many vegetables and horticultural species. Using the resistance gene N which induces a hypersensitivity reaction, is a common strategy for controlling this disease in tobacco (Nicotiana tabacum L.). However, N gene-mediated resistance has its limitations, consequently, identifying resistance genes from resistant germplasms and developing resistant cultivars is an ideal strategy for controlling the damage caused by TMV.ResultsHere, we identified highly TMV-resistant tobacco germplasm, JT88, with markedly reduced viral accumulation following TMV infection. We mapped and cloned two tobamovirus multiplication protein 2A (TOM2A) homeologs responsible for TMV replication using an F2 population derived from a cross between the TMV-susceptible cultivar K326 and the TMV-resistant cultivar JT88. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated loss-of-function mutations of two NtTOM2A homeologs almost completely suppressed TMV replication; however, the single gene mutants showed symptoms similar to those of the wild type. Moreover, NtTOM2A natural mutations were rarely detected in 577 tobacco germplasms, and CRISPR/Cas9-mediated variation of NtTOM2A led to shortened plant height, these results indicating that the natural variations in NtTOM2A were rarely applied in tobacco breeding and the NtTOM2A maybe has an impact on growth and development.ConclusionsThe two NtTOM2A homeologs are functionally redundant and negatively regulate TMV resistance. These results deepen our understanding of the molecular mechanisms underlying TMV resistance in tobacco and provide important information for the potential application of NtTOM2A in TMV resistance breeding.

Highly sensitive detection of aflatoxin B1 byCRISPR/Cas12a-assisted single nanoparticle counting

CRISPR (clustered regularly interspaced short palindromic repeats) and CRISPR-associated protein (Cas) have gained extensive applications in bioassays. However, CRISPR-based detection platforms are often hampered by limited analytical sensitivity, while nucleic acid-based amplification strategies are usually indispensable for additional signal enhancement with potential risks of amplification leakages. To address these challenges, an amplification-free CRISPR-based bioassay of aflatoxin B1 (AFB1) was proposed by applying single nanoparticle counting. Single-particle mode inductively coupled plasma mass spectrometry (Sp-ICPMS) has been regarded as a sensitive tool for nanoparticle counting since one nanoparticle can generate considerable signals above backgrounds. With AFB1, activator strands were introduced to initiate the trans-cleavage of CRISPR/Cas12a for cutting the nanoparticles-tagged-magnetic beads, which were transduced to nanoparticle count signals after separation. Finally, a pico-mole level limit-of-detections (LODs) with moderate selectivity was achieved. Certified reference materials (CRMs) analysis and recovery tests were conducted with promising results. To our best knowledge, this is the first report of the single particle counting-based CRISPR/Cas12a biosensing study.

CRISPR/Cas StNRL1 gene knockout increases resistance to late blight and susceptibility to early blight in potato.

With the development of genome editing technologies, editing susceptible genes is a promising method to modify plants for resistance to stress. NPH3/RPT2-LIKE1 protein (NRL1) interacts with effector Pi02860 of Phytophthora infestans and creates a protein complex, promoting the proteasome-mediated degradation of the guanine nucleotide exchange factor SWAP70. SWAP70, as a positive regulator, enhances cell death triggered by the perception of the P. infestans pathogen-associated molecular pattern (PAMP) INF1. Using a clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, a construct was made to introduce four guide RNAs into the potato cultivar Agria. A total of 60 putative transgenic lines were regenerated, in which 10 transgenic lines with deletions were selected and analyzed. A mutant line with a four-allelic knockdown of StNRL1 gene was obtained, showing an ~90% reduction in StNRL1 expression level, resulting in enhanced resistance to P. infestans. Surprisingly, mutant lines were susceptible to Alternaria alternata, suggesting that StNRL1 may play a role as a resistance gene; hence, silencing StNRL1 enhances resistance to P. infestans.

ANGPTL3 deficiency impairs lipoprotein production and produces adaptive changes in hepatic lipid metabolism

Angiopoietin-like protein 3 (ANGPTL3) is a hepatically secreted protein and therapeutic target for reducing plasma triglyceride-rich lipoproteins and low-density lipoprotein (LDL) cholesterol. Although ANGPTL3 modulates the metabolism of circulating lipoproteins, its role in triglyceride-rich lipoprotein assembly and secretion remains unknown. CRISPR-associated protein 9 (CRISPR/Cas9) was used to target ANGPTL3 in HepG2 cells (ANGPTL3−/−) whereupon we observed ∼50% reduction of apolipoprotein B100 (ApoB100) secretion, accompanied by an increase in ApoB100 early presecretory degradation via a predominantly lysosomal mechanism. Despite defective particle secretion in ANGPTL3−/− cells, targeted lipidomic analysis did not reveal neutral lipid accumulation in ANGPTL3−/− cells; rather ANGPTL3−/− cells demonstrated decreased secretion of newly synthesized triglycerides and increased fatty acid oxidation. Furthermore, RNA sequencing demonstrated significantly altered expression of key lipid metabolism genes, including targets of peroxisome proliferator-activated receptor α, consistent with decreased lipid anabolism and increased lipid catabolism. In contrast, CRISPR/Cas9 LDL receptor (LDLR) deletion in ANGPTL3−/− cells did not result in a secretion defect at baseline, but proteasomal inhibition strongly induced compensatory late presecretory degradation of ApoB100 and impaired its secretion. Additionally, these ANGPTL3−/−;LDLR−/− cells rescued the deficient LDL clearance of LDLR−/− cells. In summary, ANGPTL3 deficiency in the presence of functional LDLR leads to the production of fewer lipoprotein particles due to early presecretory defects in particle assembly that are associated with adaptive changes in intrahepatic lipid metabolism. In contrast, when LDLR is absent, ANGPTL3 deficiency is associated with late presecretory regulation of ApoB100 degradation without impaired secretion. Our findings therefore suggest an unanticipated intrahepatic role for ANGPTL3, whose function varies with LDLR status.

Development of Cas13a-based therapy for cancer treatment.

Gene therapy has become a major focus of current biomedical research. CRISPR (Clustered Regularly Inter spaced Short Palindromic Repeats) systems have been extensively researched for disease treatment applications through genome editing specificity. Compared with Cas9 (CRISPR-associated proteins, Cas), a commonly used tool enzyme for genome editing, Cas13a exhibits RNA-dependent endonuclease activity, including collateral cleavage without obvious potential genetic risks. With its high specificity, Cas13a has significantly improved the sensitivity of viral diagnosis and shown potential to eliminate viruses. However, its efficacy in tumor therapy has not been determined. This review introduces the mechanism and research developments associated with the CRISPR-Cas13a system in tumor treatments and its potential to be used as a new tool for gene therapy. We hope more research would apply Cas13a-based therapy in cancer treatment in the future.

Identification and functional verification of Y-chromosome-specific gene typo-gyf in Bactrocera dorsalis.

Genes on the Y chromosome play important roles in male sex determination and development. The identification of Y-chromosome-specific genes not only provides a theoretical basis for the study of male reproductive development, but also offers genetic control targets for agricultural pests. However, Y-chromosome genes are rarely characterized due to their high repeatability and high heterochromatinization, especially in the oriental fruit fly. In this study, 1011 Y-chromosome-specific candidate sequences were screened from 2 to 4h Bactrocera dorsalis embryo datasets with the chromosome quotient method, 6 of which were identified as Y-chromosome-specific sequences by polymerase chain reaction, including typo-gyf, a 19126-bp DNA sequence containing a 575-amino acid open reading frame. Testicular deformation and a significant reduction in sperm number were observed after typo-gyf knockdown with RNA interference in embryos. After typo-gyf knockout with clustered regularly interspaced palindromic repeats (CRISPR) / CRISPR-associated protein 9 in the embryonic stage, the sex ratio of the emergent adults was unbalanced, with far more females than males. A genotype analysis of these females with the Y-chromosome gene MoY revealed no sex reversal. Typo-gyf knockout led to the death of XY individuals in the embryonic stage. We conclude that typo-gyf is an essential gene for male survival, and is also involved in testicular development and spermatogenesis. The identification of typo-gyf and its functional verification provide insight into the roles of Y-chromosome genes in male development.

The pAblo·pCasso self-curing vector toolset for unconstrained cytidine and adenine base-editing in Gram-negative bacteria.

A synthetic biology toolkit, exploiting clustered regularly interspaced short palindromic repeats (CRISPR) and modified CRISPR-associated protein (Cas) base-editors, was developed for genome engineering in Gram-negative bacteria. Both a cytidine base-editor (CBE) and an adenine base-editor (ABE) have been optimized for precise single-nucleotide modification of plasmid and genome targets. CBE comprises a cytidine deaminase conjugated to a Cas9 nickase from Streptococcus pyogenes (SpnCas9), resulting in C→T (or G→A) substitutions. Conversely, ABE consists of an adenine deaminase fused to SpnCas9 for A→G (or T→C) editing. Several nucleotide substitutions were achieved using these plasmid-borne base-editing systems and a novel protospacer adjacent motif (PAM)-relaxed SpnCas9 (SpRY) variant. Base-editing was validated in Pseudomonas putida and other Gram-negative bacteria by inserting premature STOP codons into target genes, thereby inactivating both fluorescent proteins and metabolic (antibiotic-resistance) functions. The functional knockouts obtained by engineering STOP codons via CBE were reverted to the wild-type genotype using ABE. Additionally, a series of induction-responsive vectors was developed to facilitate the curing of the base-editing platform in a single cultivation step, simplifying complex strain engineering programs without relying on homologous recombination and yielding plasmid-free, modified bacterial cells.

In vivo liver targeted genome editing as therapeutic approach: progresses and challenges.

The liver is an essential organ of the body that performs several vital functions, including the metabolism of biomolecules, foreign substances, and toxins, and the production of plasma proteins, such as coagulation factors. There are hundreds of genetic disorders affecting liver functions and, for many of them, the only curative option is orthotopic liver transplantation, which nevertheless entails many risks and long-term complications. Some peculiar features of the liver, such as its large blood flow supply and the tolerogenic immune environment, make it an attractive target for in vivo gene therapy approaches. In recent years, several genome-editing tools mainly based on the clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR-Cas9) system have been successfully exploited in the context of liver-directed preclinical or clinical therapeutic applications. These include gene knock-out, knock-in, activation, interference, or base and prime editing approaches. Despite many achievements, important challenges still need to be addressed to broaden clinical applications, such as the optimization of the delivery methods, the improvement of the editing efficiency, and the risk of on-target or off-target unwanted effects and chromosomal rearrangements. In this review, we highlight the latest progress in the development of in vivo liver-targeted genome editing approaches for the treatment of genetic disorders. We describe the technological advancements that are currently under investigation, the challenges to overcome for clinical applicability, and the future perspectives of this technology.

CRISPR/Cas detection with nanodevices: moving deeper into liquid biopsy.

The emerging field of liquid biopsy has garnered significant interest in precision diagnostics, offering a non-invasive and repetitive method for analyzing bodily fluids to procure real-time diagnostic data. The precision and accuracy offered by the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (CRISPR/Cas) technology have advanced and broadened the applications of liquid biopsy. Significantly, when combined with swiftly advancing nanotechnology, CRISPR/Cas-mediated nanodevices show vast potential in precise liquid biopsy applications. However, persistent challenges are still associated with off-target effects, and the current platforms also constrain the performance of the assays. In this review, we highlight the merits of CRISPR/Cas systems in liquid biopsy, tracing the development of CRISPR/Cas systems and their current applications in disease diagnosis particularly in liquid biopsies. We also outline ongoing efforts to design nanoscale devices with improved sensing and readout capabilities, aiming to enhance the performance of CRISPR/Cas detectors in liquid biopsy. Finally, we identify the critical obstacles hindering the widespread adoption of CRISPR/Cas liquid biopsy and explore potential solutions. This feature article presents a comprehensive overview of CRISPR/Cas-mediated liquid biopsies, emphasizing the progress in integrating nanodevices to improve specificity and sensitivity. It also sheds light on future research directions in employing nanodevices for CRISPR/Cas-based liquid biopsies in the realm of precision medicine.

Targeted Modification of Epigenetic Marks Using CRISPR/dCas9-SunTag-Based Modular Epigenetic Toolkit.

The epigenome, consisting of chemical modifications to DNA and histone proteins, can alter gene expression. Clustered regularly interspaced short palindromic repeats/dead CRISPR-associated protein 9 (CRISPR/dCas9) systems enable precise target gene-specific gene modulation by attaching different "effector" domains to the dCas9 protein to activate or repress specific genes. CRISPR/dCas9-SunTag is an improved system version, allowing more efficient and precise gene activation or repression by recruiting multiple copies of the protein of interest. A CRISPR/dCas9-SunTag-based modular epigenetic toolkit was developed, enabling gene-specific epigenetic architecture modulation. This protocol generated a stable SH-SY5Y cell line expressing the CRISPR/dCas9-SunTag-JARID1A system to study H3K4Me3-mediated promoter regulation at a 200-400bp of fine resolution. The procedure involved designing sgRNAs, subcloning dCas9-5XGCN4 into pLvx-DsRed, validating epigenetic mark changes with ChIP, and validating gene expression changes with RT-qPCR. This epigenetic toolkit is valuable for researchers to understand the relationship between gene-specific epigenetic modifications and gene expression.

Application of the CRISPR/Cas9 Gene Editing Method for Modulating Antibody Fucosylation in CHO Cells.

Genetic engineering plays an essential role in the development of cell lines for biopharmaceutical manufacturing. Advanced gene editing tools can improve both the productivity of recombinant cell lines as well as the quality of therapeutic antibodies. Antibody glycosylation is a critical quality attribute for therapeutic biologics because the glycan patterns on the antibody fragment crystallizable (Fc) region can alter its clinical efficacy and safety as a therapeutic drug. As an example, recombinant antibodies derived from Chinese hamster ovary (CHO) cells are generally highly fucosylated; the absence of α1,6-fucose significantly enhances antibody-dependent cell-mediated cytotoxicity (ADCC) against cancer cells. This chapter describes a protocol applying clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) approach with different formats to disrupt the α-1,6-fucosyltransferase (FUT8) gene and subsequently inhibit α-1,6 fucosylation on antibodies expressed in CHO cells.

Modifying peptide/lipid-associated nucleic acids (PLANAs) for CRISPR/Cas9 ribonucleoprotein delivery

With the first reports on the possibility of genome editing by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas)9 surfacing in 2005, the enthusiasm for protein silencing via nucleic acid delivery experienced a resurgence following a period of diminished enthusiasm due to challenges in delivering small interfering RNAs (siRNA), especially in vivo. However, delivering the components necessary for this approach into the nucleus is challenging, maybe even more than the cytoplasmic delivery of siRNA. We previously reported the birth of peptide/lipid-associated nucleic acids (PLANAs) for siRNA delivery. This project was designed to investigate the efficiency of these nanoparticles for in vitro delivery of CRISPR/Cas9 ribonucleoproteins. Our initial experiments indicated higher toxicity for PLANAs with the more efficient reverse transfection method. Therefore, polyethylene glycol (PEG) was added to the composition for PEGylation of the nanoparticles by partially replacing two of the lipid components with the PEG-conjugated counterparts. The results indicated a more significant reduction in the toxicity of the nanoparticle, less compromise in encapsulation efficiency and more PEGylation of the surface of the nanoparticles using DOPE-PEG2000 at 50 % replacement of the naïve lipid. The cell internalization and transfection efficiency showed a comparable efficiency for the PEGylated and non-PEGylated PLANAs and the commercially available Lipofectamine™ CRISPRMAX™. Next Generation Sequencing of the cloned cells showed a variety of indels in the transfected cell population. Overall, our results indicate the efficiency and safety of PEGylated PLANAs for in vitro transfection with CRISPR/Cas9 ribonucleoproteins. PEGylation has been studied extensively for in vivo delivery, and PEGylated PLANAs will be candidates for future in vivo studies.

Role of RNA interference in drought stress management: physiological, biochemical and molecular approach

Plants frequently encounter unfavourable growth conditions due to various biotic and abiotic stress factors. Drought is a significant abiotic stress factor that negatively impacts plant growth and development resulting in reduced crop productivity. Through evolution, plants have developed dynamic mechanisms that involve complex cross-talk between different regulatory levels, providing flexibility in their response to environmental stressors. Small non-coding RNAs (ncRNAs), particularly microRNAs (miRNAs) and small interfering RNAs (siRNAs), have been discovered to play a crucial role in regulating molecular response to stress, and to be the key players in the RNA interference (RNAi) process. RNAi is a reliable strategy for precise regulation of gene expression and has emerged as a game-changer in mitigating plant responses to abiotic stress such as drought. This review summarises the role of RNAi in drought mitigation and its mechanism of action, compiling the current understanding of drought-responsive miRNAs functional at physiological, biochemical and molecular levels in major cereals. Moreover, we discuss the fine-tuning of miRNAs using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9) system, a genome editing tool which can enhance the precision of gene expression regulation and increase plant tolerance to drought stress. These technologies have significant potential in ensuring global food security and sustainable agriculture by increasing crop yield and resilience to environmental stress.

Employing CRISPR-Cas9 to Enhance T Cell Effector Function.

As part of the adaptive immune system, T cells are critical to maintain immune homeostasis. T cells provide protective immunity by killing infected cells and combatting cancerous cells. To do so, T cells produce and secrete effector molecules, such as granzymes, perforin, and cytokines such as tumor necrosis factor α and interferon γ. However, in immune suppressive environments, such as tumors, T cells gradually lose the capacity to perform their effector function. One way T cell effector function can be enhanced is through genetic engineering with tools such as clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9). This protocol explains in a step-by-step fashion how to perform a controlled electroporation-based CRISPR experiment to enhance human T cell effector function. Of note, these steps are suitable for CRISPR-mediated genome editing in T cells in general and can thus also be used to study proteins of interest that do not influence T cell effector function.