Individual Target Sites Research Articles

One-step generation of tumor models by base editor multiplexing in adult stem cell-derived organoids

Optimization of CRISPR/Cas9-mediated genome engineering has resulted in base editors that hold promise for mutation repair and disease modeling. Here, we demonstrate the application of base editors for the generation of complex tumor models in human ASC-derived organoids. First we show efficacy of cytosine and adenine base editors in modeling CTNNB1 hot-spot mutations in hepatocyte organoids. Next, we use C > T base editors to insert nonsense mutations in PTEN in endometrial organoids and demonstrate tumorigenicity even in the heterozygous state. Moreover, drug sensitivity assays on organoids harboring either PTEN or PTEN and PIK3CA mutations reveal the mechanism underlying the initial stages of endometrial tumorigenesis. To further increase the scope of base editing we combine SpCas9 and SaCas9 for simultaneous C > T and A > G editing at individual target sites. Finally, we show that base editor multiplexing allow modeling of colorectal tumorigenesis in a single step by simultaneously transfecting sgRNAs targeting five cancer genes.

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

BackgroundIn plant genome editing, RNA-guided nucleases such as Cas9 from Streptococcus pyogenes (SpCas9) predominantly induce small insertions or deletions at target sites. This can be used for inactivation of protein-coding genes by frame shift mutations. However, in some cases, it may be advantageous to delete larger chromosomal segments. This is achieved by simultaneously inducing double strand breaks upstream and downstream of the segment to be deleted. Experimental approaches for the deletion of larger chromosomal segments have not been systematically evaluated.ResultsWe designed three pairs of guide RNAs for deletion of a ~ 2.2 kb chromosomal segment containing the Arabidopsis WRKY30 locus. We tested how the combination of guide RNA pairs and co-expression of the exonuclease TREX2 affect the frequency of wrky30 deletions in editing experiments. Our data demonstrate that compared to one pair of guide RNAs, two pairs increase the frequency of chromosomal deletions. The exonuclease TREX2 enhanced mutation frequency at individual target sites and shifted the mutation profile towards larger deletions. However, TREX2 did not elevate the frequency of chromosomal segment deletions.ConclusionsMultiplex editing with at least two pairs of guide RNAs (four guide RNAs in total) elevates the frequency of chromosomal segment deletions at least at the AtWRKY30 locus, and thus simplifies the selection of corresponding mutants. Co-expression of the TREX2 exonuclease can be used as a general strategy to increase editing efficiency in Arabidopsis without obvious negative effects.

A simple, rapid, and efficient method for generating multigene-knockout culture cells by the CRISPR/Cas9 system.

We evaluated the efficacy of simultaneous multiple-gene knockout in human culture cells. By simple co-transfection of HeLa cells with a mixture of pX330-based targeting plasmids together with a puromycin resistance plasmid, followed by transient selection of puromycin-resistant cells, Cas9/single-guide RNA (sgRNA)-transduced polyclonal cell populations were selected and grown. Western blot analyses revealed co-transfection of up to seven targeting plasmids for p38α, p38β, JNK1, JNK2, Mnk1, ERK1, and mLST8 genes, drastically reduced protein expression of these genes in the polyclonal population. Analyses of a randomly isolated group of 25 clones revealed knockout efficiencies for the seven targeted genes ranging between 68% and 100%, and in six clones (24%), all targeted genes were disrupted. Deep sequencing analyses of the individual target sites revealed that, in most cases, Cas9/sgRNA-induced nonhomologous end joining resulted in deletion or insertion of only a few base pairs at the break points. These results demonstrate that simple co-transfection-based simultaneous targeting offers an easy, rapid, and efficient method to generate multiplex gene-knockout cell lines.

Transferability of multivariate extreme value models for safety assessment by applying artificial intelligence-based video analytics

Traffic conflict techniques represent the state-of-the-art for road safety assessments. However, the lack of research on transferability of conflict-based crash risk models, which refers to applying the developed crash risk estimation models to a set of external sites, can reduce their appeal for large-scale traffic safety evaluations. Therefore, this study investigates the transferability of multivariate peak-over threshold models for estimating crash frequency-by-severity. In particular, the study proposes two transferability approaches: (i) an uncalibrated approach involving a direct application of the uncalibrated base model to the target sites and (ii) a threshold calibration approach involving calibration of conflict thresholds of the conflict indicators. In the latter approach, the conflict thresholds of the Modified Time-To-Collision (MTTC) and Delta-V indicators were calibrated using local data from the target sites. Finally, the two transferability approaches were compared with a complete re-estimation approach where all the model parameters were estimated using local data. All three approaches were tested for a target set of signalized intersections in Southeast Queensland, Australia. Traffic movements at the target intersections were observed using video cameras for two days (12 h each day). The road user trajectories and rear-end conflicts were extracted using an automated artificial intelligence-based algorithm utilizing state-of-the-art Computer Vision methods. The base models developed in an earlier study were then transferred to the target sites using the two transferability approaches and the local data from the target sites. Results show that the threshold calibration approach provides the most accurate and precise predictions of crash frequency-by-severity for target sites. Thus, for peak-over threshold models, the threshold parameter is the most important, and its calibration improves the performance of the base models. The complete re-estimation of models for individual target sites yields inferior fits and less precise crash estimates than the two transferability approaches since they utilize fewer traffic conflict extremes in their development than the larger dataset utilized in base model development. Therefore, the study results can significantly advance the applicability of traffic conflict models for crash risk estimation at transport facilities.

Atlas-informed computational processing pipeline for individual targeting of brain areas for therapeutic navigated transcranial magnetic stimulation

ObjectiveNavigated transcranial magnetic stimulation (nTMS) is targeted at different cortical sites for diagnostic, therapeutic, and neuroscientific purposes. Correct identification of the cortical target areas is important for achieving desired effects, but it is challenging when no direct responses arise upon target area stimulation. We aimed at utilizing atlas-based marking of cortical areas for nTMS targeting to present a convenient, rater-independent method for overlaying the individual target sites with brain anatomy. MethodsWe developed a pipeline, which fits a brain atlas to the individual brain and enables visualization of the target areas during the nTMS session. We applied the pipeline to our previous nTMS data, focusing on depression and schizophrenia patients. Furthermore, we included examples of Tourette syndrome and tinnitus therapies, as well as neurosurgical and motor mappings. ResultsIn depression and schizophrenia patients, the visually selected dorsolateral prefrontal cortex (DLPFC) targets were close to the border between atlas areas A9/46 and A8. In the other areas, the atlas-based areas were in agreement with the treatment targets. ConclusionsThe atlas-based target areas agreed well with the cortical targets selected by experts during the treatments. SignificanceOverlaying atlas information over the navigation view is a convenient and useful add-on for improving nTMS targeting.

CReVIS-Seq: A highly accurate and multiplexable method for genome-wide mapping of lentiviral integration sites

Lentiviruses have been widely used as a means of transferring exogenous DNAs into human cells to treat various genetic diseases. Lentiviral vectors are fundamentally integrated into the host genome, but their integration sites are generally unpredictable, which may increase the uncertainty for their use in therapeutics. To determine the viral integration sites in the host genome, several PCR-based methods have been developed. However, the sensitivities of the PCR-based methods are highly dependent on the primer sequences, and optimized primer design is required for individual target sites. In order to address this issue, we developed an alternative method for genome-wide mapping of viral insertion sites, named CReVIS-seq (CRISPR-enhanced Viral Integration Site Sequencing). The method is based on the sequential steps: fragmentation of genomic DNAs, in vitro circularization, cleavage of target sequence in a CRISPR guide RNA-specific manner, high-throughput sequencing of the linearized DNA fragments in an unbiased manner, and identification of viral insertion sites via sequence analysis. By design, CReVIS-seq is not affected by biases that could be introduced during the target enrichment step via PCR amplification using site specific primers. Furthermore, we found that multiplexed CReVIS-seq, using collections of different single-guide RNAs (sgRNAs), enables simultaneous identification of multiple target sites and structural variations (i.e., circularized viral genome), in both single cell clones and heterogeneous cell populations.

Programmed Nanococktail Based on pH-Responsive Function Switch for Self-Synergistic Tumor-Targeting Therapy.

Tumor-targeting combination chemotherapy is an important way to improve the therapeutic index and reduce the side effects as compared to traditional cancer treatments. However, one of the major challenges in surface functionalization of nanoparticle (NP) is accomplishing multiple purposes through one single ligand. Upon such consideration, methotrexate (MTX), an anticancer drug with a targeting moiety inspired by the similar structure of folate, could be used to covalently link with lipid-polymer conjugate (DSPE-PEG) via a pH-sensitive dynamic covalent imine (CH═N) bond to synthesize the acid-induced function "targeting-anticancer" switching DSPE-PEG-CH═N-MTX. We hypothesize that using this kind of MTX prodrug to functionalize NP's surface would be conductive to combine the early phase active targeting function and the late-phase anticancer function in one nanosystem. Herein, a nanococktail is programmed for codelivery of epirubicin (EPI) and MTX by co-self-assembly of acid-dissociated EPI-phospholipid (PC) complex and acid-cleavable DSPE-PEG-CH═N-MTX conjugate. The obtained nanococktail (MTX-PEG-EPI-PC NPs) could not only actively target folate receptors-overexpressing tumor cells but also respond to acidic endo/lysosomes for triggering the on-demand release of pharmaceutically active EPI/MTX. The intracellular drug distribution also demonstrated that the system could codeliver two drugs to individual target sites of action, inducing the significant synergistic anticancer efficiency based on different anticancer mechanisms. More importantly, the in vivo tumor accumulation and anticancer efficacy of MTX-PEG-EPI-PC NPs (via cleavable imine bond) were significantly enhanced as compared to the individual free drug, both free drugs, PEG-EPI-PC NPs, and MTX-PEG-EPI-PC NPs (via the uncleavable amide bond). This self-synergistic tumor-targeting therapy might represent a promising strategy for cancer treatment.

Tapping natural reservoirs of homing endonucleases for targeted gene modification

Homing endonucleases mobilize their own genes by generating double-strand breaks at individual target sites within potential host DNA. Because of their high specificity, these proteins are used for "genome editing" in higher eukaryotes. However, alteration of homing endonuclease specificity is quite challenging. Here we describe the identification and phylogenetic analysis of over 200 naturally occurring LAGLIDADG homing endonucleases (LHEs). Biochemical and structural characterization of endonucleases from one clade within the phylogenetic tree demonstrates strong conservation of protein structure contrasted against highly diverged DNA target sites and indicates that a significant fraction of these proteins are sufficiently stable and active to serve as engineering scaffolds. This information was exploited to create a targeting enzyme to disrupt the endogenous monoamine oxidase B gene in human cells. The ubiquitous presence and diversity of LHEs described in this study may facilitate the creation of many tailored nucleases for genome editing.

A two-step site and mRNA-level model for predicting microRNA targets

BackgroundDespite experiments showing that the number of microRNA (miRNA) target sites is critical for miRNA targeting, most existing methods focus on identifying individual miRNA target sites and do not model contributions of multiple target sites to miRNA regulation. To address this possible fault, we developed a miRNA target prediction model that recognizes the individual characteristics of functional binding sites and the global characteristics of miRNA-targeted mRNAs.ResultsBenchmark experiments showed that this two-step model generally had a higher overall performance than other established miRNA target prediction algorithms and that the model was especially suited to identify true miRNA targets among genes that all contain conserved target sites.ConclusionsThis improved performance could partly be explained by the model not relying on conservation when predicting targets. The critical factors for the model's performance, however, were mRNA-level features that characterized the number and strength of individual target sites within the mRNA. The model is available for online predictions or as pre-computed predictions on the human genome http://tare.medisin.ntnu.no/mirna_target.

Detection and Localization of Specific Sequences on Single Microfluidically Trapped DNA Molecules using PNA Probes

We propose a single molecule fluorescence-based approach to rapidly locate specific sequences on DNA. Using the roughly 50,000 base pair lambda-DNA as a model molecule, we demonstrate that patterns of targeted sequences can be detected using peptide nucleic acid (PNA)-based probes. These bisPNAs, modified with biotin and Tamra on opposing ends, bind to target sequences on double-stranded lambda-DNA. While PNA probes were chosen for their specificity and versatility, they are prone to bind to non-target sites that differ from the target site by one terminal base pair. PNA binding to these single-end mismatch (SEMM) sites can be minimized by a moderate amount of additional heating following the binding reaction and this step must be optimized to achieve the requisite specificity.Here we demonstrate the single-molecule analysis of the binding of two PNAs, with three and two target sites on lambda-DNA, respectively. Neutravidin-coated 40 nm fluorescent polymer spheres are attached to the DNA-bound biotinylated PNAs and the DNA is fluorescently stained. The locations of the bound beads along single DNA molecules are determined by stretching the DNA on slides and by trapping and stretching single DNA molecules in a microfluidic cross-slot, utilizing a stagnation-point extensional flow. In comparing to previously published results, we find that the end modifications have substantial effects on binding conditions. Furthermore, the effects of additional heating on individual target sites and mismatched sites were quantified.

IS 1630 of Mycoplasma fermentans , a Novel IS 30 -Type Insertion Element That Targets and Duplicates Inverted Repeats of Variable Length and Sequence during Insertion

A new insertion sequence (IS) of Mycoplasma fermentans is described. This element, designated IS1630, is 1,377 bp long and has 27-bp inverted repeats at the termini. A single open reading frame (ORF), predicted to encode a basic protein of either 366 or 387 amino acids (depending on the start codon utilized), occupies most of this compact element. The predicted translation product of this ORF has homology to transposases of the IS30 family of IS elements and is most closely related (27% identical amino acid residues) to the product of the prototype of the group, IS30. Multiple copies of IS1630 are present in the genomes of at least two M. fermentans strains. Characterization and comparison of nine copies of the element revealed that IS1630 exhibits unusual target site specificity and, upon insertion, duplicates target sequences in a manner unlike that of any other IS element. IS1630 was shown to have the striking ability to target and duplicate inverted repeats of variable length and sequence during transposition. IS30-type elements typically generate 2- or 3-bp target site duplications, whereas those created by IS1630 vary between 19 and 26 bp. With the exception of two recently reported IS4-type elements which have the ability to generate variable large duplications (B. B. Plikaytis, J. T. Crawford, and T. M. Shinnick, J. Bacteriol. 180:1037-1043, 1998; E. M. Vilei, J. Nicolet, and J. Frey, J. Bacteriol. 181:1319-1323, 1999), such large direct repeats had not been observed for other IS elements. Interestingly, the IS1630-generated duplications are all symmetrical inverted repeat sequences that are apparently derived from rho-independent transcription terminators of neighboring genes. Although the consensus target site for IS30 is almost palindromic, individual target sites possess considerably less inverted symmetry. In contrast, IS1630 appears to exhibit an increased stringency for inverted repeat recognition, since the majority of target sites had no mismatches in the inverted repeat sequences. In the course of this study, an additional copy of the previously identified insertion sequence ISMi1 was cloned. Analysis of the sequence of this element revealed that the transposase encoded by this element is more than 200 amino acid residues longer and is more closely related to the products of other IS3 family members than had previously been recognized. A potential site for programmed translational frameshifting in ISMi1 was also identified.

Electron microscopic studies of sequence‐specific recognition of duplex DNA by different ligands

The following ligands were used to study sequence specific recognition of duplex DNA by electron microscopic techniques: methyltransferases BspR1 and EcoR124 (recognition sequences GGCC and GAAN7RTCG, respectively), a biotinylated deoxyoligonucleotide 5'-CTCTCTCTCTCTCT-3' capable of forming triplex DNA, and PNA oligomer H-T10-LysNH2. For each ligand the best conditions for electron microscopic (EM) detection of stable specific complex formation were determined. It was demonstrated that EM allowed us to determine the position of the individual target site with an error of 15-20 bp, the relative affinities for individual target sites and kinetic parameters of the binding. These results open new possibilities for EM investigations of sequence-specific interactions with a wide range of other ligands of a similar nature. They also imply that a wide range of different sequences can be unambiguously and precisely mapped by EM and greatly extend the scope of EM applications for physical mapping of genomic DNA.

The constitution of a progesterone response element.

Progesterone receptors (PRs) recognize and bind to DNA in a sequence-specific manner. To define the sequence-specific determinants of PR binding to DNA, we employed a detailed series of target elements and analyzed both PR binding in vitro and PR-mediated activation of a reporter gene in vivo. These elements represent point substitution or insertion mutants of a progesterone response element derived from the mouse mammary tumor virus (MMTV). Substitution of a G for the first T in the 15-base pair (bp) MMTV progesterone response element core sequence, GTTACAAACTGTTCT, increases PR-DNA binding in vitro. All other tested mutations within this sequence either had no effect or decreased PR binding. From these analyses, we infer an optimal PR recognition sequence, -7RGNACANRNTGTNCY+7, composed of hexameric half-sites separated by precisely 3 bp and exhibiting dyad symmetry. Limited substitution of a suboptimal base for an optimal base, as in the wild type MMTV element, can be tolerated, but further suboptimal substitutions significantly decrease binding by PR. The identity of 1 bp within the hexameric half-site, position +/- 5, is unconstrained. Transition mutations, but not transversions, are permitted at position +/- 7. Here the hydrogen bond acceptor of the N-7 position of the purine ring may be involved in receptor recognition, since this feature is shared by the permitted substitutions. Insertion of a single base pair between the half-sites abolishes detectable binding, suggesting that the spatial orientation of the DNA binding domains of the monomeric receptor subunits are fixed by dimerization of the receptor. This pattern of sequence-specific recognition parallels that previously inferred for the glucocorticoid receptor, indicating that the two receptors may be unable to distinguish individual target sites. Each of the mutant response elements was also assessed for its ability to confer progestin responsiveness to a truncated MMTV promoter when introduced into a PR-containing cell line. Elements exhibiting strong receptor binding in vitro were fully inducible, whereas poorly inducible or uninducible elements displayed little or no recognition by receptor in vitro. However, some elements, though poorly bound in vitro, still activated transcription in vivo in response to hormone. In certain cases activation was as good as that seen with the wild type element. Further examination of in vitro receptor binding by this class of mutant elements using higher levels of baculovirus-expressed receptor revealed that many were indeed recognized by receptor, albeit with a lower affinity than the wild type sequence.(ABSTRACT TRUNCATED AT 400 WORDS)