Editing Methods Research Articles

Establishment of CRISPR-Cpf1-assisted gene editing tool and engineering of 4-hydroxyisoleucine biosynthesis in Corynebacterium glutamicum.

Corynebacterium glutamicum, an important industrial producer, is a model microorganism. However, the limited gene editing methods and their defects limit the efficient genome editing of C. glutamicum. To improve the screening efficiency of second-cross-over strains of traditional SacB editing system, a universal pCS plasmid which harbors CRISPR-Cpf1 system targeting kan gene of SacB system was designed and established to kill the false positive single-cross-over strains remained abundantly after the second-cross-over events. The lethality of pCS plasmid to C. glutamicum carrying kan gene on its genome was as high as 98.6%. In the example of PodhA::PilvBNC replacement, pCS plasmid improved the screening efficiency of second-cross-over bacteria from 5% to over 95%. Then this pCS-assisted gene editing system was applied to improve the supply of precursors and reduce the generation of by-products in the production of 4-hydroxyisoleucine (4-HIL). The 4-HIL titer of one edited strain SC01-TD5IM reached 137.0 ± 33.9 mM, while the weakening of lysE by promoter engineering reduced Lys content by 19.0-47.7% and 4-HIL titer by 16.4-64.5%. These editing demonstrates again the efficiency of this novel CRISPR-Cpf1-assisted gene editing tool, suggesting it as a useful tool for improving the genome editing and metabolic engineering in C. glutamicum.

OrthoAligner: Image-based Teeth Alignment Prediction via Latent Style Manipulation.

In this paper, we present OrthoAligner, a novel method to predict the visual outcome of orthodontic treatment in a portrait image. Unlike the state-of-the-art method, which relies on a 3D teeth model obtained from dental scanning, our method generates realistic alignment effects in images without requiring additional 3D information as input and thus making our system readily available to average users. The key of our approach is to employ the 3D geometric information encoded in an unsupervised generative model, i.e., StyleGAN in this paper. Instead of directly conducting translation in the image space, we embed the teeth region extracted from a given portrait to the latent space of the StyleGAN generator and propose a novel {latent} editing method to discover a geometrically meaningful editing path that yields the alignment process in the image space. To blend the edited mouth region with the original portrait image, we further introduce a BlendingNet to remove boundary artifacts and correct color inconsistency. We also extend our method to short video clips by propagating the alignment effects across neighboring frames. We evaluate our method in various orthodontic cases, compare it to the state-of-the-art and competitive baselines, and validate the effectiveness of each component.

SketchFaceNeRF: Sketch-based Facial Generation and Editing in Neural Radiance Fields

Realistic 3D facial generation based on Neural Radiance Fields (NeRFs) from 2D sketches benefits various applications. Despite the high realism of free-view rendering results of NeRFs, it is tedious and difficult for artists to achieve detailed 3D control and manipulation. Meanwhile, due to its conciseness and expressiveness, sketching has been widely used for 2D facial image generation and editing. Applying sketching to NeRFs is challenging due to the inherent uncertainty for 3D generation with 2D constraints, a significant gap in content richness when generating faces from sparse sketches, and potential inconsistencies for sequential multi-view editing given only 2D sketch inputs. To address these challenges, we present SketchFaceNeRF, a novel sketch-based 3D facial NeRF generation and editing method, to produce free-view photo-realistic images. To solve the challenge of sketch sparsity, we introduce a Sketch Tri-plane Prediction net to first inject the appearance into sketches, thus generating features given reference images to allow color and texture control. Such features are then lifted into compact 3D tri-planes to supplement the absent 3D information, which is important for improving robustness and faithfulness. However, during editing, consistency for unseen or unedited 3D regions is difficult to maintain due to limited spatial hints in sketches. We thus adopt a Mask Fusion module to transform free-view 2D masks (inferred from sketch editing operations) into the tri-plane space as 3D masks, which guide the fusion of the original and sketch-based generated faces to synthesize edited faces. We further design an optimization approach with a novel space loss to improve identity retention and editing faithfulness. Our pipeline enables users to flexibly manipulate faces from different viewpoints in 3D space, easily designing desirable facial models. Extensive experiments validate that our approach is superior to the state-of-the-art 2D sketch-based image generation and editing approaches in realism and faithfulness.

Guided Linear Upsampling

Guided upsampling is an effective approach for accelerating high-resolution image processing. In this paper, we propose a simple yet effective guided upsampling method. Each pixel in the high-resolution image is represented as a linear interpolation of two low-resolution pixels, whose indices and weights are optimized to minimize the upsampling error. The downsampling can be jointly optimized in order to prevent missing small isolated regions. Our method can be derived from the color line model and local color transformations. Compared to previous methods, our method can better preserve detail effects while suppressing artifacts such as bleeding and blurring. It is efficient, easy to implement, and free of sensitive parameters. We evaluate the proposed method with a wide range of image operators, and show its advantages through quantitative and qualitative analysis. We demonstrate the advantages of our method for both interactive image editing and real-time high-resolution video processing. In particular, for interactive editing, the joint optimization can be precomputed, thus allowing for instant feedback without hardware acceleration.

UniTune: Text-Driven Image Editing by Fine Tuning a Diffusion Model on a Single Image

Text-driven image generation methods have shown impressive results recently, allowing casual users to generate high quality images by providing textual descriptions. However, similar capabilities for editing existing images are still out of reach. Text-driven image editing methods usually need edit masks, struggle with edits that require significant visual changes and cannot easily keep specific details of the edited portion. In this paper we make the observation that image-generation models can be converted to image-editing models simply by fine-tuning them on a single image. We also show that initializing the stochastic sampler with a noised version of the base image before the sampling and interpolating relevant details from the base image after sampling further increase the quality of the edit operation. Combining these observations, we propose UniTune, a novel image editing method. UniTune gets as input an arbitrary image and a textual edit description, and carries out the edit while maintaining high fidelity to the input image. UniTune does not require additional inputs, like masks or sketches, and can perform multiple edits on the same image without retraining. We test our method using the Imagen model in a range of different use cases. We demonstrate that it is broadly applicable and can perform a surprisingly wide range of expressive editing operations, including those requiring significant visual changes that were previously impossible.

Accelerated Editing Method for Vehicle Durability Fatigue Load Spectrum Based on Wigner-Ville Transform.

A Wigner-Ville transform-based (WVT-based) load spectrum fast editing method for vehicle parts is proposed to improve the efficiency of durability tests. In this method, the instantaneous energy spectrum (IES) of the original time-domain signal is obtained via the Wigner-Ville transform, which is used as a criterion to identify time-domain points of ineffective damage contribution. A genetic algorithm (GA) based threshold optimization model is also proposed to automatically set the threshold of the IES under consideration of the relative damage requirements and statistical parameters of the signal. The effectiveness of the above proposed editing method is demonstrated by compiling an SUV's suspension coil spring signal obtained from physical sensor-based measurements. Meanwhile, the same spectrum is also processed using time-domain editing, Short-time Fourier-transform, and S-transform methods for comparison. The results show that the WVT-based edited spectrum has a time-duration retention ratio of about 76.30%, which is significantly superior to other methods, with the same pseudo-damage retention and statistical parameter error constraints. Moreover, in combination with the fatigue simulation analysis, it verifies that the load effect of the edited spectrum matches well with that of the original. Thus, the proposed method is considered more effective for compiling component load signals in vehicle acceleration durability tests.

Detection of TALEN-mediated genome cleavage during the early embryonic stage of the starfish Patiria pectinifera.

Echinoderms have long been utilized as experimental materials to study the genetic control of developmental processes and their evolution. Among echinoderms, the molecular study of starfish embryos has received considerable attention across research topics such as gene regulatory network evolution and larval regeneration. Recently, experimental techniques to manipulate gene functions have been gradually established in starfish as the feasibility of genome editing methods was reported. However, it is still unclear when these techniques cause genome cleavage during the development of starfish, which is critical to understand the timeframe and applicability of the experiment during early development of starfish. We herein reported that gene functions can be analyzed by the genome editing method TALEN in early embryos, such as the blastula of the starfish Patiria pectinifera. We injected the mRNA of TALEN targeting rar, which was previously constructed, into eggs of P. pectinifera and examined the efficiency of genome cleavage through developmental stages from 6 to 48 hours post fertilization. The results will be key knowledge not only when designing TALEN-based experiments but also when assessing the results.

On matching and mismatching in protein chemistry

Post-translational protein editing methods that rely upon precise chemical alteration of sequence and hence function are of increasing utility. Whilst the role of proteins as chiral environments is functionally ubiquitous in their interaction with substrates or ligands, the influence of stereochemical ‘matching/mismatching’ effects upon the reactivity of chemical reagents designed for such editing is essentially unexplored. The reagent dibromohexanediamide (DBHDA) is a useful reagent for the chemical transformation of cysteine to dehydroalanine (Dha) in proteins and peptides. Differences in reactivities of the stereoisomers of DBHDA, if any, in different protein substrates are unknown. Here, we report synthetic access to these isomers and reveal apparent stereochemically ‘matched vs mismatched’ differences in reactivity, that, in principle, in the future might be exploited to expand the methods available for residue- and site-selective editing.

Cystathionine β-synthase affects organization of cytoskeleton and modulates carcinogenesis in colorectal carcinoma cells.

Cystathionine β-synthase (CBS), one of three enzymes that endogenously produce hydrogen sulfide, is extensively studied for its relevance in the cells of various tumors. In our previous work, we observed that the immunofluorescence pattern of CBS is very similar to that of tubulin and actin. Therefore, we focused on the potential interaction of CBS with cytoskeletal proteins β-actin and β-tubulin and the functional relevance of the potential interaction of these proteins in colorectal carcinoma cell lines. To study the potential interaction of CBS with cytoskeletal proteins and its functional consequences, a CBS-knockout DLD1 (DLDx) cell line was established by using the CRISPR/Cas9 gene editing method. The interaction of the selected cytoskeletal protein with CBS was studied by immunoprecipitation, Western blot analysis, immunofluorescence, and proximity ligation assay. The functional consequences were studied by proliferation and migration assays and by generation of xenografts in SCID/bg mice. We have found that CBS, an enzyme that endogenously produces H2S, binds to cytoskeletal β-tubulin and, to a lesser extent, also to β-actin in colorectal carcinoma-derived cells. When CBS was knocked out by the CRISPR/Cas9 technique (DLDx), we observed a de-arranged cytoskeleton compared to the unmodified DLD1 cell line. Treatment of these cells with a slow sulfide donor GYY4137 resulted in normal organization of the cytoskeleton, thus pointing to the role of CBS in microtubule dynamics. To evaluate the physiological importance of this observation, both DLD1 and DLDx cells were injected into SCID/bg mice, and the size and mass of the developed xenografts were evaluated. Significantly larger tumors developed from DLDx compared to the DLD1 cells, which correlated with the increased proliferation of these cells. Taken together, in colorectal cancer DLD1 cells, CBS binds to the cytoskeleton, modulates microtubule dynamics, and thus affects the proliferation and migration in the colorectal carcinoma stable cell line.

CRISPR/Cas9 as a therapeutic tool for triple negative breast cancer: from bench to clinics.

Clustered regularly interspaced short palindromic repeats (CRISPR) is a third-generation genome editing method that has revolutionized the world with its high throughput results. It has been used in the treatment of various biological diseases and infections. Various bacteria and other prokaryotes such as archaea also have CRISPR/Cas9 systems to guard themselves against bacteriophage. Reportedly, CRISPR/Cas9-based strategy may inhibit the growth and development of triple-negative breast cancer (TNBC) via targeting the potentially altered resistance genes, transcription, and epigenetic regulation. These therapeutic activities could help with the complex issues such as drug resistance which is observed even in TNBC. Currently, various methods have been utilized for the delivery of CRISPR/Cas9 into the targeted cell such as physical (microinjection, electroporation, and hydrodynamic mode), viral (adeno-associated virus and lentivirus), and non-viral (liposomes and lipid nano-particles). Although different models have been developed to investigate the molecular causes of TNBC, but the lack of sensitive and targeted delivery methods for in-vivo genome editing tools limits their clinical application. Therefore, based on the available evidences, this review comprehensively highlighted the advancement, challenges limitations, and prospects of CRISPR/Cas9 for the treatment of TNBC. We also underscored how integrating artificial intelligence and machine learning could improve CRISPR/Cas9 strategies in TNBC therapy.

Сравнение методик расчета инспекционного интервала при реализации риск- ориентированного подхода к инспекции технологического оборудования

The article deals with the problems of conducting periodic inspections of equipment. This is due to the high frequency of equipment shutdown for its technical diagnostics and examination in accordance with the requirements of regulatory and technical documentation, in particular the Federal norms and rules «Rules for the safe operation of process pipelines». It is noted that the inspection is an important tool for ensuring the integrity of the equipment and, as a result, the industrial safety of the facility. At the same time, it is indicated that the inspection can be implemented on the basis of one of two approaches: regulated and risk-oriented. A general description of the inspection methodology, taking into account risk factors, is presented. It is pointed out that a risk-based approach to inspections is widespread in a number of countries. Various methods of implementing this approach are given, taking into account a large number of standards. The basics of API 581 3rd Edition and S-RBI techniques used for inspections are described. The technological process of the gas condensate stabilization system of a plant for the production of liquefied natural gas is described. A process pipeline is considered as an object of the study, for which an inspection interval is determined using the API 581 3rd Edition methodology. At the first stage of determining the inspection interval according to the S-RBI methodology, the process of assessing the criticality of the failure is described. The second step describes the process of determining the confidence factor, where the focus is on the estimation of the uncertainty in predicting the corrosion rate. At the third stage, the coefficient of the inspection interval is determined based on the data on the criticality of the failure and the reliability factor. Then an example of calculating the inspection interval for the object under study is given. Based on the results obtained, a conclusion was made about the comparability of API 581 3rd Edition and S-RBI methods. Both methods have their advantages and disadvantages, but at the same time they provide optimization of the scope of inspection work while maintaining a high level of industrial safety of the facility. In order to improve production efficiency, reliability and safety of pressure equipment, and optimize inspection intervals (which will allow to reduce the financial costs of enterprises), it is advisable to apply the above practices in the Russian Federation, providing the required legal and regulatory framework.

Counterfactual Representation Augmentation for Cross-Domain Sentiment Analysis

Cross-domain sentiment analysis aims to adapt sentiment classification models trained on one or more source domains to the target domain, which can effectively alleviate the problem of insufficient labelled data in specific domains. Unlike most previous methods of adjusting models based on observed data, we propose a novel counterfactual representation augmentation (CRA) method, which aims to improve target-domain generalization by constructing new counterfactual representations for training. Specifically, we train a domain discriminator to learn the domain discrepancy between the source and target domains on unlabeled data, and use a gradient editing method to directly construct counterfactual representations, which reduces the inductive bias of the source domain and augments the training data. Moreover, we further leverage an ensemble-based training method to indirectly encourage the target-domain classifier to rely more on robust features for prediction. Extensive experiments on a widely-used cross-domain sentiment classification benchmark dataset show that our method consistently surpasses different baseline methods on different tasks, demonstrating the strong ability to improve domain generalization. We also find that our model can effectively adjust the decision boundary to make the classifier more robust and generalized, through extensive qualitative and quantitative analysis.

Development and validation of a natural dynamic facial expression stimulus set.

Emotion research commonly uses either controlled and standardised pictures or natural video stimuli to measure participants' reactions to emotional content. Natural stimulus materials can be beneficial; however, certain measures such as neuroscientific methods, require temporally and visually controlled stimulus material. The current study aimed to create and validate video stimuli in which a model displays positive, neutral and negative expressions. These stimuli were kept as natural as possible while editing timing and visual features to make them suitable for neuroscientific research (e.g. EEG). The stimuli were successfully controlled regarding their features and the validation studies show that participants reliably classify the displayed expression correctly and perceive it as genuine. In conclusion, we present a motion stimulus set that is perceived as natural and that is suitable for neuroscientific research, as well as a pipeline describing successful editing methods for controlling natural stimuli.

CRISPR-Based Gene Editing in Acinetobacter baumannii to Combat Antimicrobial Resistance.

Antimicrobial resistance (AMR) poses a significant threat to the health, social, environment, and economic sectors on a global scale and requires serious attention to addressing this issue. Acinetobacter baumannii was given top priority among infectious bacteria because of its extensive resistance to nearly all antibiotic classes and treatment options. Carbapenem-resistant A. baumannii is classified as one of the critical-priority pathogens on the World Health Organization (WHO) priority list of antibiotic-resistant bacteria for effective drug development. Although available genetic manipulation approaches are successful in A. baumannii laboratory strains, they are limited when employed on newly acquired clinical strains since such strains have higher levels of AMR than those used to select them for genetic manipulation. Recently, the CRISPR-Cas (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) system has emerged as one of the most effective, efficient, and precise methods of genome editing and offers target-specific gene editing of AMR genes in a specific bacterial strain. CRISPR-based genome editing has been successfully applied in various bacterial strains to combat AMR; however, this strategy has not yet been extensively explored in A. baumannii. This review provides detailed insight into the progress, current scenario, and future potential of CRISPR-Cas usage for AMR-related gene manipulation in A. baumannii.

Wood cellulose microfibrils have a 24-chain core-shell nanostructure in seed plants.

Wood cellulose microfibril (CMF) is the most abundant organic substance on Earth but its nanostructure remains poorly understood. There are controversies regarding the glucan chain number (N) of CMFs during initial synthesis and whether they become fused afterward. Here, we combined small-angle X-ray scattering, solid-state nuclear magnetic resonance and X-ray diffraction analyses to resolve CMF nanostructures in native wood. We developed small-angle X-ray scattering measurement methods for the cross-section aspect ratio and area of the crystalline-ordered CMF core, which has a higher scattering length density than the semidisordered shell zone. The 1:1 aspect ratio suggested that CMFs remain mostly segregated, not fused. The area measurement reflected the chain number in the core zone (Ncore). To measure the ratio of ordered cellulose over total cellulose (Roc) by solid-state nuclear magnetic resonance, we developed a method termed global iterative fitting of T1ρ-edited decay (GIFTED), in addition to the conventional proton spin relaxation editing method. Using the formula N = Ncore/Roc, most wood CMFs were found to contain 24 glucan chains, conserved between gymnosperm and angiosperm trees. The average CMF has a crystalline-ordered core of ~2.2 nm diameter and a semidisordered shell of ~0.5 nm thickness. In naturally and artificially aged wood, we observed only CMF aggregation (contact without crystalline continuity) but not fusion (forming a conjoined crystalline unit). This further argued against the existence of partially fused CMFs in new wood, overturning the recently proposed 18-chain fusion hypothesis. Our findings are important for advancing wood structural knowledge and more efficient use of wood resources in sustainable bio-economies.

O-223 Sperm Genome Editing by CRISPR-Cas9 through an Ooplasm-Mediated Approach

Abstract Study question Can we edit the sperm genome by CRISPR-Cas9 through an ooplasmic-mediated approach? Summary answer The ooplasm-mediated technique induced the decondensation of the male gamete nucleus and allowed CRISPR-Cas9 to access the sperm DNA to carry out successful edits. What is known already Previous studies on genome editing using CRISPR-Cas9 have been performed at the S-phase or zygote stage, but challenges were represented by mosaicism and possible off-target edits. To address these issues, gene editing at the gamete level would be ideal. While this may be feasible in oocytes, it appears ambitious in the spermatozoon due to the DNA hypercoiling around the protamine core and the chromatin compaction. In preliminary experiments, permeabilization of sperm membrane allowed penetration of CRISPR-Cas9 to enter the cell, however, genomic editing was proved unsuccessful. Study design, size, duration In the past 5 months, a total of 128 oocytes were divided into 2 groups. To edit exclusively the male genome through oocyte-mediated sperm decondensation (OMSD), a single spermatozoon was injected into an enucleated oocyte to produce a haploid androgenetic embryo. The control cohort consisted of embryos generated through standard ICSI with the established heritable genome editing (HGE) approach. Both groups were treated with CRISPR-Cas9 aiming to knockout Tyr gene to create an albino phenotype. Participants/materials, setting, methods B6D2F1 mice were used to retrieve oocytes and spermatozoa. A cohort of oocytes used for OSMD approach were enucleated. While the intact oocytes were used for HGE control. All oocytes were injected with a single spermatozoon together with CRISPR-Cas9 solution, containing Tyr gRNA. All embryos were cultured up to the 8-cell stage. Individual blastomeres were isolated and sequenced to proof editing. DNA was extracted and amplified for T7E1 analysis to validate genome editing efficiency. Main results and the role of chance Of the 128 oocytes used for the study, 51 were enucleated for the OMSD experiments, while 77 were used for HGE control. After undergoing ICSI with CRISPR-Cas9 solution, 84.3% (43/51) of the OMSD cohort displayed a single male pronucleus and 79.2% (61/77) control HGE fertilized. In a time-lapse microscopy, 95.3% (41/43) of the OMSD experimental group developed to the 2-cell stage, comparable to the HGE control development at 96.7% (59/61). After 48 hours in culture, the HGE control reached 8-cell development at 88.5% (54/61), while the OMSD cohort cleaved at the lower rate at 60.4% (26/43, P<0.001). A 423-bp region around the CRISPR target site was amplified on DNA extracted by isolated blastomere. Gene modification at the target site was confirmed in 33.7% (70/208) of OMSD cohort and 29.7% (88/296) of the HGE control embryos. Limitations, reasons for caution While the experimental observations are still limited, it is important to note that these results may be underestimated since embryos with completely uniform modifications may go undetected by T7E1 assay. The technique needs further refinement to optimize targeting efficiency and support embryo development. Wider implications of the findings Once the genetic editing method is optimized, individual pseudo-blastomeres generated through this OMSD approach can be utilized as male gametes to fertilize oocyte. The generation of live offspring with the corrected haplotype will serve to prove the safety of genome editing through this technique. Trial registration number N/A

CRISPR/Cas9-mediated multiple guide RNA-targeted mutagenesis in the potato.

CRISPR/Cas9 technology has become the most efficient method for genome editing in many plant species, including important industrial crops such as potatoes. This study used three target regions (T1, T2, and T3) in gbss exon I, whose sequences were first inserted into the BbsI sites in the appropriate guide RNA (gRNA) vector (pEn-Chimera, pMR203, pMR204, and pMR205), and then localized between the AtU6 promoter and the gRNA scaffold sequence. Expression vectors were constructed by introducing gRNA genes into the pMR287 (pYUCas9Plus) plasmids using the MultiSite Gateway system by attR and attL sites. The three target regions of mutant potato lines were analyzed. The use of CRISPR/Cas9-mediated multiple guide RNA-targeted mutagenesis allowed tri- or tetra-allelic mutant potato lines to be generated. Multiple nucleotide substitutions and indels within and around the three target sites caused a frameshift mutation that led to a premature stop codon, resulting in the production of gbss-knockout plants. Mutation frequencies and analysis of mutation patterns suggested that the stably transformed Cas9/multiple guide RNA expression constructs used in this study can induce targeted mutations efficiently in the potato genome. Full knockout of the gbss gene was analyzed by CAPS, Sanger sequencing and iodine staining. The present study demonstrated successful CRISPR/Cas9-mediated multiple guide RNA-targeted mutagenesis in the potato gbss gene by Agrobacterium-mediated transformation, resulting in an amylose-free phenotype.

Acetylation-Mimic Mutation of TRIM28-Lys304 to Gln Attenuates the Interaction with KRAB-Zinc-Finger Proteins and Affects Gene Expression in Leukemic K562 Cells.

TRIM28/KAP1/TIF1β is a crucial epigenetic modifier. Genetic ablation of trim28 is embryonic lethal, although RNAi-mediated knockdown in somatic cells yields viable cells. Reduction in TRIM28 abundance at the cellular or organismal level results in polyphenism. Posttranslational modifications such as phosphorylation and sumoylation have been shown to regulate TRIM28 activity. Moreover, several lysine residues of TRIM28 are subject to acetylation, but how acetylation of TRIM28 affects its functions remains poorly understood. Here, we report that, compared with wild-type TRIM28, the acetylation-mimic mutant TRIM28-K304Q has an altered interaction with Krüppel-associated box zinc-finger proteins (KRAB-ZNFs). The TRIM28-K304Q knock-in cells were created in K562 erythroleukemia cells by CRISPR-Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein nuclease 9) gene editing method. Transcriptome analysis revealed that TRIM28-K304Q and TRIM28 knockout K562 cells had similar global gene expression profiles, yet the profiles differed considerably from wild-type K562 cells. The expression levels of embryonic-related globin gene and a platelet cell marker integrin-beta 3 were increased in TRIM28-K304Q mutant cells, indicating the induction of differentiation. In addition to the differentiation-related genes, many zinc-finger-proteins genes and imprinting genes were activated in TRIM28-K304Q cells; they were inhibited by wild-type TRIM28 via binding with KRAB-ZNFs. These results suggest that acetylation/deacetylation of K304 in TRIM28 constitutes a switch for regulating its interaction with KRAB-ZNFs and alters the gene regulation as demonstrated by the acetylation mimic TRIM28-K304Q.

ADVICES TO LIFE: A PHILOLOGICAL STUDY OF THE NAZAM TEXT LIFE ADVICES

Nazam is a Malay Islamic literary work which is an important means of delivering religious dakwah in Minangkabau. This research uses th uses the Nazam Life Advice Manuscript from Surau Parak Laweh, West Sumatra as the object and primary source of the research. This research raises the issue of the advice that must be done in living the life contained in the Nazam Naskah Advice on Life. In this regard, this study aims to describe the manuscript in terms of the condition of the manuscript, the presentation of the text edition and the disclosure of the contents of the text related to life advice that is relevant to this day. Through this research, the researcher discusses and explores the issue of life advice that is relevant to the current conditions based on a study of the main source of the Nazam Nazam Naskah Life Advice. Source reading is done using philological and hermeneutical approaches. The philological approach is used to describe the manuscript, presenting the edition of the text which is carried out using the critical edition method to produce a text that can be read by researchers and the general public. After getting the text edition, contextualization was carried out using a hermeneutic approach. Based on the reading results, it shows that there are three main pieces of advice contained in this Nazam Life Advice text. First, advice in protecting property and children. Second, advice to be sincere and stay away from riya. Third, advice to stay away from arrogant.

Improved prime editing allows for routine predictable gene editing in Physcomitrium patens.

Efficient and precise gene editing is the gold standard of any reverse genetic study. The recently developed prime editing approach, a modified CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein] editing method, has reached the precision goal but its editing rate can be improved. We present an improved methodology that allows for routine prime editing in the model plant Physcomitrium patens, whilst exploring potential new prime editing improvements. Using a standardized protoplast transfection procedure, multiple prime editing guide RNA (pegRNA) structural and prime editor variants were evaluated targeting the APT reporter gene through direct plant selection. Together, enhancements of expression of the prime editor, modifications of the 3' extension of the pegRNA, and the addition of synonymous mutation in the reverse transcriptase template sequence of the pegRNA dramatically improve the editing rate without affecting the quality of the edits. Furthermore, we show that prime editing is amenable to edit a gene of interest through indirect selection, as demonstrated by the generation of a Ppdek10 mutant. Additionally, we determine that a plant retrotransposon reverse transcriptase enables prime editing. Finally, we show for the first time the possibility of performing prime editing with two independently coded peptides.