Target Site Selection Research Articles

Transposition with Tn3-family elements occurs through interaction with the host β-sliding clamp processivity factor.

Tn3 family transposons are a widespread group of replicative transposons, notorious for contributing to the dissemination of antibiotic resistance, particularly the global prevalence of carbapenem resistance. The transposase (TnpA) of these elements catalyzes DNA breakage and rejoining reactions required for transposition. However, the molecular mechanism for target site selection with these elements remains unclear. Here, we identify a QLxxLR motif in N-terminal of Tn3 TnpAs and demonstrate that this motif allows interaction between TnpA of Tn3 family transposon Tn1721 and the host β-sliding clamp (DnaN), the major processivity factor of the DNA replication machinery. The TnpA-DnaN interaction is essential for Tn1721 transposition. Our work unveils a mechanism whereby Tn3 family transposons can bias transposition into certain replisomes through an interaction with the host replication machinery. This study further expands the diversity of mobile elements that use interaction with the host replication machinery to bias integration.

Natural and Engineered Guide RNA-Directed Transposition with CRISPR-Associated Tn7-Like Transposons.

CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated nuclease) defense systems have been naturally coopted for guide RNA-directed transposition on multiple occasions. In all cases, cooption occurred with diverse elements related to the bacterial transposon Tn7. Tn7 tightly controls transposition; the transposase is activated only when special targets are recognized by dedicated target-site selection proteins. Tn7 and the Tn7-like elements that coopted CRISPR-Cas systems evolved complementary targeting pathways: one that recognizes a highly conserved site in the chromosome and a second pathway that targets mobile plasmids capable of cell-to-cell transfer. Tn7 and Tn7-like elements deliver a single integration into the site they recognize and also control the orientation of the integration event, providing future potential for use as programmable gene-integration tools. Early work has shown that guide RNA-directed transposition systems can be adapted to diverse hosts, even within microbial communities, suggesting great potential for engineering these systems as powerful gene-editing tools.

AB067. New drug development for the use of PARP1-E2F1 transcriptional inhibitors in the treatment of glioblastoma.

Glioblastoma (GBM) is the most malignant brain tumor and ranks among the most lethal of all human cancers, without improvements in survival over the last 30 years. Data obtained in our group suggest that PARP1, a well-known DNA-repairing protein, could also play a key role in the regulation of cell cycle through its interaction with the transcription factor E2F1. Therefore, considering that most oncogenic processes are associated with cell cycle deregulation, we hypothesized that disruption of PARP1-E2F1 interaction would provide a novel therapeutic approach to different types of cancer. The identification of novel compounds disrupting PARP1-E2F1 interaction was carried out by combining in silico and in vitro screening, using a rational drug design. The virtual screen was performed using a molecular library of several million compounds at the selected target site, using AtomNet® (Atomwise, San Francisco, CA, USA), the first deep learning neural network for structure-based drug design and discovery. Since there is no complete structural information of the PARP1-E2F1 protein-protein interaction, a homologous structure of the BRCT domain of BRCA1 complex with the phospho-peptide (PDBID: 1T2V) was used to identify the potential binding interface of BRCT domain of PARP-1 (PDBID: 2COK) and the E2F1 protein. Top scoring compounds were clustered and filtered to obtain a final subset of 83 compounds that were incorporated to our in vitro screening, which included both transcriptional E2F1 activity and survival studies. Complete culture medium supplemented with the compounds selected in the in silico screening (10 μM) were added and incubated for 24 hours. E2F1 activity was observed by measuring luminescence. For the viability assay, the fluorescence reading was performed (excitation 544 nm and emission 590 nm). The in silico and in vitro screening resulted in 12 compounds that inhibited E2F1 transcriptional activity and significantly reduced cell number. The highest inhibition of both E2F1 transcriptional activity and cell growth was observed with compound 3797, which was selected for further studies. Both in silico and in vitro results indicate that inhibition of PARP1-E2F1 transcriptional activity may provide a new rationale for designing novel therapeutic approaches for the treatment of GBM.

A programmable seekRNA guides target selection by IS1111 and IS110 type insertion sequences

IS1111 and IS110 insertion sequence (IS) family members encode an unusual DEDD transposase type and exhibit specific target site selection. The IS1111 group include identifiable subterminal inverted repeats (sTIR) not found in the IS110 type1. IS in both families include a noncoding region (NCR) of significant length and, as each individual IS or group of closely related IS selects a different site, we had previously proposed that an NCR-derived RNA was involved in target selection2. Here, we find that the NCR is usually downstream of the transposase gene in IS1111 family IS and upstream in the IS110 type. Four IS1111 and one IS110 family members that target different sequences are used to demonstrate that the NCR determines a short seeker RNA (seekRNA) that co-purified with the transposase. The seekRNA is essential for transposition of the IS or a cargo flanked by IS ends from and to the preferred target. Short sequences matching both top and bottom strands of the target are present in the seekRNA but their order in IS1111 and IS110 family IS is reversed. Reprogramming the seekRNA and donor flank to target a different site is demonstrated, indicating future biotechnological potential for these systems.

Building a carbon dioxide storage portfolio for the Barrow-Dampier sub-basins through regional screening – an integrated geoscience approach

Safe geological carbon dioxide (CO2) storage (GCS) requires rocks with suitable injectivity, capacity and sealing properties to ensure secure long-term containment of injected CO2. A regional understanding of the subsurface is essential to determine the potential for GCS of a basin and to select target sites. This is best addressed by integrating the basin’s tectono-stratigraphic evolution, its gross depositional environment, and its hydrodynamic, thermal and stress regimes. A basin-scale GCS assessment for the Barrow-Dampier sub-basins was conducted by Geoscience Australia and SLB. The objective of the study is to high-grade geological intervals and sites for potential GCS and to understand potential storage capacity and key risk factors. Stratigraphic and structural mapping of key storage intervals was performed using the reprocessed seismic volume and well database associated with the project. Analysis of critically stressed faults was used to estimate the likelihood of reactivation based on the far-field regional stress field and fault mechanical properties. Pressure, temperature, porosity, permeability, and water geochemistry data have been screened for >500 wells for assessing the storage unit intervals and predicting the hydrodynamic regime. Calibrated 2D basin models provide information on the regional pressure-temperature regime, porosity/permeability distribution, and sealing effectiveness. Potentiometric surface maps for the aquifer systems inform the distribution of potential CO2 plume migration. Results of this integrated regional basin study are used to quantify the risk of identified storage containers and to map the chance of success for GCS at a regional scale. The project results are publicly available via NOPIMS.

Effects of PNA Sequence and Target Site Selection on Function of a 4.5S Non-Coding RNA.

Peptide nucleic acid (PNA) based antisense strategy is a promising therapeutic approach to specifically inhibit target gene expression. However, unlike protein coding genes, identification of an ideal PNA binding site for non-coding RNA is not straightforward. Here, we compare the inhibitory activities of PNA molecules that bind a non-coding 4.5S RNA called SRP RNA, a key component of the bacterial signal recognition particle (SRP). A 9-mer PNA (PNA9) complementary to the tetraloop region of the RNA was more potent in inhibiting its interaction with the SRP protein, compared to an 8-mer PNA (PNA8) targeting a stem-loop. PNA9, which contained a homo-pyrimidine sequence could form a triplex with the complementary stretch of RNA in vitro as confirmed using a fluorescent derivative of PNA9 (F-PNA13). The RNA-PNA complex formation resulted in inhibition of SRP function with PNA9 and F-PNA13, but not PNA8 highlighting the importance of target site selection. Surprisingly, F-PNA13 which was more potent in inhibiting SRP function in vitro, showed weaker antibacterial activity compared to PNA9 likely due to poor cell penetration of the longer PNA. Our results underscore the importance of suitable target site selection and optimum PNA length to develop better antisense molecules against non-coding RNA.

How to use CRISPR/Cas9 in plants: from target site selection to DNA repair.

A tool for precise, target-specific, efficient, and affordable genome editing is a dream for many researchers, from those who conduct basic research to those who use it for applied research. Since 2012, we have tool that almost fulfils such requirements; it is based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems. However, even CRISPR/Cas has limitations and obstacles that might surprise its users. In this review, we focus on the most frequently used variant, CRISPR/Cas9 from Streptococcus pyogenes, and highlight key factors affecting its mutagenesis outcomes: (i) factors affecting the CRISPR/Cas9 activity, such as the effect of the target sequence, chromatin state, or Cas9 variant, and how long it remains in place after cleavage; and (ii) factors affecting the follow-up DNA repair mechanisms including mostly the cell type and cell cycle phase, but also, for example, the type of DNA ends produced by Cas9 cleavage (blunt/staggered). Moreover, we note some differences between using CRISPR/Cas9 in plants, yeasts, and animals, as knowledge from individual kingdoms is not fully transferable. Awareness of these factors can increase the likelihood of achieving the expected results of plant genome editing, for which we provide detailed guidelines.

Abstract 4483: New drug development opportunities for PARP1

Abstract Personalized therapies allow, not only the increase of its therapeutic efficiency, but also the reduction of the secondary effects associated to the treatment of cancer. Data obtained in our group suggest that PARP1 could play a key role in the cell cycle regulation through its interaction with E2F1 transcription factor. Considering that most oncogenic processes is associated with cell cycle deregulation, the disruption of this interaction would provide a new therapeutic target of great interest and wide spectrum of indications. Therefore, our goal was to find molecules that interfere its role in cell-cycle regulation without affecting its function on DNA repair. The identification of novel compounds disrupting the PARP1/E2F1 interaction was carried out by combining in silico and in vitro screening, using a rational drug design. The virtual screen was performed using a molecular library of several million compounds at the selected target site, using AtomNet® (Atomwise), the first deep learning neural network for structure-based drug design and discovery. Since there is no complete structural information of the PARP-1/E2F1 protein-protein interaction, a homologous structure of BRCT domain of BRCA1 complex with the phospho-peptide (PDBID: 1T2V) was utilized to identify the potential binding interface of BRCT domain of PARP-1 (PDBID: 2COK) and the E2F1 protein. Top scoring compounds were clustered and filtered to arrive at a final subset of 83 compounds, library that were incorporated to our in vitro screening, which included both transcriptional (E2F1 activity) and survival studies. Twelve compounds were finally selected, based upon their higher capacity of inhibiting E2F1 activity.This work was supported by Agencia Estatal de Investigación (AEl/10.13039/501100011033), Xunta de Galicia (GPC GI-1862, ED431B 2020/26; ED431G 2019/02) and European Regional Development Fund-ERDF. AIMS Awards Program - Project A19-513, Atomwise Inc. Citation Format: Pablo Iglesias, David Moreiras, Laura Porres-Ventin, Lara Gonzalez-Rendo, Jose A. Zumalave, Victor M. Arce, Jose A. Costoya. New drug development opportunities for PARP1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4483.

Combining CRISPR-Cas-mediated terminal resolution with a novel genetic workflow to achieve high-diversity adenoviral libraries

While recombinant adenoviruses (rAds) are widely used in both laboratory and medical gene transfer, library-based applications using this vector platform are not readily available. Recently, we developed a new method, the CRISPR-Cas9 mediated invivo terminal resolution aiding high-efficiency rescue of rAds from recombinant DNA. Here we report on a genetic workflow that allows construction of bacterial artificial chromosome-based rAd libraries reconstituted using highly efficient terminal resolution. We utilized frequent, pre-existing genomic sequences to allow the insertion of a selection marker, complementing two selected target sites into novel endonuclease recognition sites. In the second step, this selection marker is replaced with a transgene or mutation of interest via Gibson assembly. Our approach does not cause unwanted genomic off-target mutations while providing substantial flexibility for the site and nature of the genetic modification. This new genetic workflow, which we termed half site-directed fragment replacement (HFR) allows the introduction of more than 106 unique modifications into rAd encoding BACs using laboratory scale methodology. To demonstrate the power of HFR, we rescued barcoded viral vector libraries yielding a diversity of approximately 2.5×104 unique rAds per cm2 of transfected cell culture.

Streamlined Agrobacterium rhizogenes-mediated hairy root transformation for efficient CRISPR/Cas9-based gene editing evaluation in diverse Citrullus varieties

The CRISPR/Cas9 genome-editing system serves as a pivotal tool for enhancing crop genetics. Within this system, single guide RNAs (sgRNAs) are instrumental in the precise cleavage of DNA double strands. However, the efficiency of gene editing varies among sgRNAs, emphasizing the need to meticulously select target sites, especially in the context of Citrullus lanatus, a species notorious for its challenging and inefficient generation of transgenic plants through stable transformation. This study employed an Agrobacterium rhizogenes-mediated hairy root method to assess effective target sites for gene editing of ClCIPK17 across various Citrullus species. Hairy roots were successfully induced in different plant tissues at diverse growth stages of Citrullus lanatus, Citrullus mucosospermus, and Citrullus amarus. Employing a vector with two sgRNAs (sgRNA1 and sgRNA5) positioned within conserved regions of exon 1 and exon 5 of ClCIPK17 in the CRISPR/Cas9 system, targeted mutations were detected in 90.9% of accessions across the four Citrullus species. Notably, 73.94% of all examined hairy roots exhibited mutations at the sgRNA1 site, while the sgRNA5 site showed no mutations. Among the 31 different mutation types identified at the sgRNA1 site, base deletion was the most prevalent. Using the sgRNA1 site of ClCIPK17, stable transgenic watermelon buds were obtained from explants, and the targeted mutations of the sgRNA1 site were confirmed. These findings underscore the viability of the hairy root transformation system in assessing the editing efficiency of sgRNA targets in diverse Citrullus species.

Application Of The CRISPR-Cas9 System to Drug Discovery in Breast Cancer

To elucidate the functioning of the CRISPR-Cas9 system in the realm of drug discovery, a series of experiments were undertaken. Firstly, CRISPR-Cas9 was employed to facilitate both positive and negative selection screening within human cells. Subsequently, an exploration of genes vital for pluripotent stem cell and cancer cell viability was conducted. Following this, in a melanoma model, a screen was performed to identify genes whose loss contributes to resistance against vemurafenib, a therapeutic agent targeting the mutant protein kinase BRAF. Notably, the top-ranking candidate genes included previously verified genes such as NF1 and MED12, alongside novel discoveries in the form of CUL3, NF2, TADA1, and TADA2B. This investigation revealed a remarkable consistency among distinct guide RNAs directed at the same gene, with an elevated rate of successful hit confirmation. These findings underscore the potential of genome-scale screening employing Cas9, thus substantiating CRISPR-Cas9 as a potent methodology for large-scale sequencing and the selection of target sites.

Relationship between land tenure and land use in unplanned settlements in Lusaka, Zambia

How do land tenure and land use relate to informal settlements? Many studies have examined the effects of land regularisation on communities. Land regularisation is considered a highly effective solution for improving the residential environment in informal settlements and has been implemented in numerous developing countries. However, few studies have explored how land regularisation impacts land use in informal settlements. In this study, we investigated how residents secure land tenure and use their land in unplanned settlements in Lusaka, the capital city of Zambia, focusing on people's practices in detail. We focussed on two unplanned settlements in Lusaka: Chaisa Ward, an improvement area where land regularisation has occurred, and Chazanga Ward, a customary area where land is not being regularised. As the investigation method, we conducted interviews with community management organisations and 31 dwelling owners and an observational survey about land use in selected target sites in Chaisa and Chazanga in 2009, 2011, and 2014. The study findings showed that in informal settlements, where land tenure is secured by authorities within the community, communal regulation supported by community-based social bonds is an important element of land use management. However, official recognition of land rights through land regularisation could undermine communal relationships and communal land use regulations, resulting in a disorganised and congested living environment. Therefore, we point out that official intervention in land tenure should be institutionalised in a manner that fully understands and takes advantage of the relationships between land tenure and land use, as well as between land and people, which are well-balanced within the local context. This study demonstrated that communal regulation plays a key role in managing land use in informal settlements but its positive impact could be easily undermined by land regularisation. Thus, an important learning from this study is that land in informal settlements should be regularised in a way that fully understands and takes advantage of communal regulation of land use to ensure a desirable living environment.

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

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

Recent advances in structure-based enzyme engineering for functional reconstruction.

Structural information can help engineer enzymes. Usually, specific amino acids in particular regions are targeted for functional reconstruction to enhance the catalytic performance, including activity, stereoselectivity, and thermostability. Appropriate selection of target sites is the key to structure-based design, which requires elucidationof the structure-function relationships. Here, we summarize the mutations of residues in different specific regions, including active center, access tunnels, and flexible loops, on fine-tuning the catalytic performance of enzymes, and discuss the effects of altering the local structural environment on the functions. In addition, we keep up with the recent progress of structure-based approaches for enzyme engineering, aiming to provide some guidance on how to take advantage of the structural information.

The HIV-1 Capsid-Targeted Inhibitor GSK878 Alters Selection of Target Sites for HIV DNA Integration.

Decades of effort have yielded highly effective antiviral agents to treat HIV, but viral strains have evolved resistance to each inhibitor type, focusing attention on the importance of developing new inhibitor classes. A particularly promising new target is the HIV capsid, the function of which can be disrupted by highly potent inhibitors that persist long term in treated subjects. Studies with such inhibitors have contributed to an evolving picture of the role of capsid itself-the inhibitors, like certain capsid protein (CA) amino acid substitutions, can disrupt intracellular trafficking to alter the selection of target sites for HIV DNA integration in cellular chromosomes. In this study, we compare effects on HIV integration targeting for two potent inhibitors-a new molecule targeting CA, GSK878, and the previously studied lenacapavir (LEN, formerly known as GS-6207). We find that both inhibitors reduce integration in active transcription units and near epigenetic marks associated with active transcription. A careful study of integration near repeated sequences indicated frequencies were also altered for integration within multiple repeat classes. One notable finding was increased integration in centromeric satellite repeats in the presence of LEN and GSK878, which is of interest because proviruses integrated in centromeric repeats have been associated with transcriptional repression, inducibility, and latency. These data add to the picture that CA protein remains associated with preintegration complexes through the point in infection during which target sites for integration are selected, and specify new aspects of the consequences of disrupting this mechanism.

Modularity and diversity of target selectors in Tn7 transposons

To spread, transposons must integrate into target sites without disruption of essential genes while avoiding host defense systems. Tn7-like transposons employ multiple mechanisms for target-site selection, including protein-guided targeting and, in CRISPR-associated transposons (CASTs), RNA-guided targeting. Combining phylogenomic and structural analyses, we conducted a broad survey of target selectors, revealing diverse mechanisms used by Tn7 to recognize target sites, including previously uncharacterized target-selector proteins found in newly discovered transposable elements (TEs). We experimentally characterized a CAST I-D system and a Tn6022-like transposon that uses TnsF, which contains an inactivated tyrosine recombinase domain, to target the comM gene. Additionally, we identified a non-Tn7 transposon, Tsy, encoding a homolog of TnsF with an active tyrosine recombinase domain, whichwe show also inserts into comM. Our findings show that Tn7 transposons employ modular architecture and co-opt target selectors from various sources to optimize target selection and drive transposon spread.

Mitral annular electrogram morphology - a balanced signal does not identify the anatomical annulus

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): British Heart Foundation Background Electrogram morphology is used to identify the atrio-ventricular annuli during catheter ablation procedures with a "balanced" signal considered to represent the annuli. However, anatomical studies highlight a significantly greater mass of ventricular myocardium compared to atrial myocardium at the valve plane suggesting the correct electrical signature of the anatomical annulus may not be a balanced signal. Purpose To identify the relationship between atrial and ventricular electrogram components at the anatomically defined mitral valve annulus. Methods Mapping of the mitral annulus was performed in patients undergoing ablation for paroxysmal atrial fibrillation. Following femoral venous access and trans-septal puncture, left atrial anatomy was defined using rotational angiography performed using a 100ml contrast injection 4 seconds prior to X-ray acquisition, during rapid pacing and apnoea while covering 200 degrees at 40 degrees/s. A SmartTouch SF ablation catheter (Biosense Webster) was advanced into the left atrium and used to record intracardiac electrograms at four positions around the mitral annulus and at five predetermined levels ranging from ventricular to atrial (Figure, panel A). Data analysis was performed using OpenEP (https://openep.io). For each electrogram, the distance to the mitral valve plane and the amplitudes of atrial and ventricular electrogram components were calculated (Figure, panel B and C). Mean electrogram component amplitudes were calculated including all points recorded within ±2.5mm of the mitral valve plane. For each patient, a linear regression model was fitted and used to identify the atrial/ventricular ratio at the anatomically defined mitral valve annulus (Figure, panel D). Results The protocol was completed in 9 patients, including analysis of 240 atrial or ventricular electrogram components. Within ±2.5mm of the anatomically defined mitral valve annulus, the mean recorded amplitudes of atrial and ventricular electrogram components was 0.15±0.17mV and 1.35±1.27mV, respectively (A/V ratio 1:9). Using the linear regression model, the ventricular electrogram component was calculated to be 16.5±11.6 times greater in amplitude than the atrial electrogram component at the mitral valve plane (A/V ratio 1:16). The use of a balanced signal to identify the mitral valve annulus resulted in a positional error of -7.9±9.5mm (i.e. identifying a level which is approximately ~8mm too "atrial" compared to the anatomically defined annulus). Conclusion The conventional use of a "balanced" electrogram signal does not identify the anatomical mitral valve annulus. An atrial/ventricular electrogram ratio between 1:9 and 1:16 best approximates the mitral valve annular plane determined by left atrial rotational angiography. These findings have relevance in target site selection for an accessory pathway and the ventricular extent of linear ablation at the mitral isthmus.

PO-04-095 ACCURATE EAM-BASED TARGET DELINEATION IN STEREOTACTIC ARRHYTHMIA RADIOABLATION OF VT

Stereotactic Arrhythmia Radiotherapy (STAR) aided by precise image-guided delivery and respiratory motion management has been used successfully for experimental treatment of refractory VT. However, accurate target selection remains challenging. Current methods often rely on manual delineation and/or visual estimation of predefined cardiac segments. Develop a systematic process for registering VT targets from electroanatomic maps (EAM) to reference imaging coordinates of the planning CT (pCT) used for radiation treatment. The goal is to minimize potential variations in target site selection as well as treated volumes. In patients on antiarrhythmic treatment and undergoing a 2nd unsuccessful VT ablation, EA maps of the LV (and LA/RV when possible) and more focal maps of VT target sites were acquired (CARTO®3, Biosense-Webster, CA, USA) and exported. In-house software was used to register these to a planning image set and convert them to DICOM structure files (Fig 1). Dice similarity coefficients were calculated between ventricular mesh data and corresponding reference contours. Initial targets (GTV) were expanded to include internal target volumes (ITV) and planning target volumes (PTV) to account for respiratory and cardiac motion and residual uncertainties, with minor manual adjustments guided by consensus among electrophysiologists, radiation oncologists and medical physicists. STAR was delivered as a single fraction of 25 Gy using volumetric-modulated arc therapy or dynamic conformal arc therapy depending on the target shape. 7 patients with refractory VT were treated by defining targets based on registering individualized EAM maps on the planning images. Dice similarity indices between transferred reference map and reference contours were 0.83 ± 0.04 and 0.72 ± 0.04 for LV and LA/RV, respectively (table 1). The median clinical target was 22cc, with PTVs ranging from 62.4 to 199.6 cc. All patients demonstrated a significant reduction in VT events by 6 weeks post STAR. We have developed an effective methodology for direct registration of VT targets selected for STAR, based on direct anatomic registration of individualized EA maps acquired during ablation procedures. This process offers a way to efficiently delineate variable target boundaries in a semi-automated way, which is advantageous when unusual contours make standard segmentation inaccurate and visual estimation difficult. However, rigorous comparison with other approaches remains to be done.Tabled 1Patient-specific metrics1234567Dice, LV0.8680.8450.7910.8060.7210.8720.794Dice, LA/RV0.7520.6670.734N/A0.382N/A0.338GTV (cc)22.313.526.520.759.863.927.2ITV (cc)45.529.142.230.959.8127.829.6PTV (cc)98.662.488.275.5166.8199.672.8 Open table in a new tab

Current Bioinformatics Tools to Optimize CRISPR/Cas9 Experiments to Reduce Off-Target Effects.

The CRISPR-Cas system has evolved into a cutting-edge technology that has transformed the field of biological sciences through precise genetic manipulation. CRISPR/Cas9 nuclease is evolving into a revolutionizing method to edit any gene of any species with desirable outcomes. The swift advancement of CRISPR-Cas technology is reflected in an ever-expanding ecosystem of bioinformatics tools designed to make CRISPR/Cas9 experiments easier. To assist researchers with efficient guide RNA designs with fewer off-target effects, nuclease target site selection, and experimental validation, bioinformaticians have built and developed a comprehensive set of tools. In this article, we will review the various computational tools available for the assessment of off-target effects, as well as the quantification of nuclease activity and specificity, including web-based search tools and experimental methods, and we will describe how these tools can be optimized for gene knock-out (KO) and gene knock-in (KI) for model organisms. We also discuss future directions in precision genome editing and its applications, as well as challenges in target selection, particularly in predicting off-target effects.

Dual specificity and target gene selection by the MADS-domain protein FRUITFULL.

How transcription factors attain their target gene specificity and how this specificity may be modulated, acquiring different regulatory functions through the development of plant tissues, is an open question. Here we characterized different regulatory roles of the MADS-domain transcription factor FRUITFULL (FUL) in flower development and mechanisms modulating its activity. We found that the dual role of FUL in regulating floral transition and pistil development is associated with its different in vivo patterns of DNA binding in both tissues. Characterization of FUL protein complexes by liquid chromatography-tandem mass spectrometry and SELEX-seq experiments shows that aspects of tissue-specific target site selection can be predicted by tissue-specific variation in the composition of FUL protein complexes with different DNA binding specificities, without considering the chromatin status of the target region. This suggests a role for dynamic changes in FUL TF complex composition in reshaping the regulatory functions of FUL during flower development.