Off-target Activity Research Articles

Third generation quinoline-3-carboxamide transcriptional disrupter of HDAC4, HIF-1α, and MEF-2 signaling for metastatic castration-resistant prostate cancer.

The quinoline-3-carboxamide, Tasquinimod (TasQ), is orally active as a maintenance therapy with an on-target mechanism-of-action via allosteric binding to HDAC4. This prevents formation of the HDAC4/NCoR1/HDAC3 complex, disrupting HIF-1α transcriptional activation and repressing MEF-2 target genes needed for adaptive survival signaling in the compromised tumor micro environment. In phase 3 clinical testing against metastatic castration-resistant prostate cancer(mCRPC), TasQ (1 mg/day) increased time-to-progression, but not overall survival. TasQ analogs were chemically synthesized and tested for activity compared to the parental compound. These included HDAC4 enzymatic assays, qRT-PCR and western blot analyses of gene and protein expression following treatment, in vitro and in vivo efficacy against multiple prostate cancer models including PDXs, pharmacokinetic analyses,AHR binding and agonist assays, SPR analyses of binding to HDAC4 and NCoR1, RNAseq analysis of in vivo tumors, 3D endothelial sprouting assays, and a targeted kinase screen. Genetic knockout or knockdown controls were used when appropriate. Here, we document that, on this regimen (1 mg/day), TasQ blood levels are 10-fold lower than the optimal concentration (≥2 μM) needed for anticancer activity, suggesting higher daily doses are needed. Unfortunately, we also demonstrate that TasQ is an arylhydrocarbon receptor (AHR) agonist, which binds with an EC50 of 1 μM to produce unwanted off-target side effects. Therefore, we screened a library of TasQ analogsto maximize on-target versus off-target activity. Using this approach, we identified ESATA-20, which has ~10-fold lower AHR agonism and 5-fold greater potency against prostate cancer patient-derived xenografts. This increased therapeuticindex nominates ESATA-20 as a lead candidate forclinical development as an orally active third generation quinoline-3-carboxamide analog thatretains its on-target ability to disrupt HDAC4/HIF-1α/MEF-2-dependent adaptive survival signaling in the compromisedtumor microenvironment found in mCRPC.

Off-target pharmacological activity at various kinases: Potential functional and pathological side effects

In drug discovery, during the lead optimization and candidate characterization stages, novel small molecules are frequently evaluated in a battery of in vitro pharmacology assays to identify potential unintended, off-target interactions with various receptors, transporters, ion channels, and enzymes, including kinases. Furthermore, these screening panels may also provide utility at later stages of development to provide a mechanistic understanding of unexpected safety findings. Here, we present a compendium of the most likely functional and pathological outcomes associated with interaction(s) to a panel of 95 kinases based on an extensive curation of the scientific literature. This panel of kinases was designed by AbbVie based on safety-related data extracted from the literature, as well as from over 20 years of institutional knowledge generated from discovery efforts. For each kinase, the scientific literature was reviewed using online databases and the most often reported functional and pathological effects were summarized. This work should serve as a practical guide for small molecule drug discovery scientists and clinical investigators to predict and/or interpret adverse effects related to pharmacological interactions with these kinases.

A preclinical secondary pharmacology resource illuminates target-adverse drug reaction associations of marketed drugs

In vitro secondary pharmacology assays are an important tool for predicting clinical adverse drug reactions (ADRs) of investigational drugs. We created the Secondary Pharmacology Database (SPD) by testing 1958 drugs using 200 assays to validate target-ADR associations. Compared to public and subscription resources, 95% of all and 36% of active (AC50 < 1 µM) results are unique to SPD, with bias towards higher activity in public resources. Annotating drugs with free maximal plasma concentrations, we find 684 physiologically relevant unpublished off-target activities. Furthermore, 64% of putative ADRs linked to target activity in key literature reviews are not statistically significant in SPD. Systematic analysis of all target-ADR pairs identifies several putative associations supported by publications. Finally, candidate mechanisms for known ADRs are proposed based on SPD off-target activities. Here we present a freely-available resource for benchmarking ADR predictions, explaining phenotypic activity and investigating clinical properties of marketed drugs.

Deep Characterization and Comparison of Different Retrovirus-like Particles Preloaded with CRISPR/Cas9 RNPs.

The CRISPR/Cas system has a broad range of possible medical applications, but its clinical translation has been hampered, particularly by the lack of safe and efficient vector systems mediating the short-term expression of its components. Recently, different virus-like particles (VLPs) have been introduced as promising vectors for the delivery of CRISPR/Cas genome editing components. Here, we characterized and directly compared three different types of retrovirus-based (R) VLPs, two derived from the γ-retrovirus murine leukemia virus (gRVLPs and "enhanced" egRVLPs) and one from the lentivirus human immunodeficiency virus, HIV (LVLPs). First, we unified and optimized the production of the different RVLPs. To ensure maximal comparability of the produced RVLPs, we adapted several assays, including nanoparticle tracking analysis (NTA), multi-parametric imaging flow cytometry (IFC), and Cas9-ELISA, to analyze their morphology, surface composition, size, and concentration. Next, we comparatively tested the three RVLPs targeting different genes in 293T model cells. Using identical gRNAs, we found egRVLPs to mediate the most efficient editing. Functional analyses indicated better cargo (i.e., Cas9) transfer and/or release as the underlying reason for their superior performance. Finally, we compared on- and off-target activities of the three RVLPs in human-induced pluripotent stem cells (hiPSC) exploiting the clinically relevant C-C motif chemokine receptor 5 (CCR5) as the target. Again, egRVLPs facilitated the highest, almost 100% knockout rates, importantly with minimal off-target activity. In conclusion, in direct comparison, egRVLPs were the most efficient RVLPs. Moreover, we established methods for in-depth characterization of VLPs, facilitating their validation and thus more predictable and safe application.

Increased prime edit rates in KCNQ2 and SCN1A via single nicking all-in-one plasmids

BackgroundPrime editing (PE) is the most recent gene editing technology able to introduce targeted alterations to the genome, including single base pair changes, small insertions, and deletions. Several improvements to the PE machinery have been made in the past few years, and these have been tested in a range of model systems including immortalized cell lines, stem cells, and animal models. While double nicking RNA (dncRNA) PE systems PE3 and PE5 currently show the highest editing rates, they come with reduced accuracy as undesired indels or SNVs arise at edited loci. Here, we aimed to improve single ncRNA (sncRNA) systems PE2 and PE4max by generating novel all-in-one (pAIO) plasmids driven by an EF-1α promoter, which is especially suitable for human-induced pluripotent stem cell (hiPSC) models.ResultspAIO-EF1α-PE2 and pAIO-EF1α-PE4max were used to edit the voltage gated potassium channel gene KCNQ2 and voltage gated sodium channel gene SCN1A. Two clinically relevant mutations were corrected using pAIO-EF1α-PE2 including the homozygous truncating SCN1A R612* variant in HEK293T cells and the heterozygous gain-of-function KCNQ2 R201C variant in patient-derived hiPSC. We show that sncRNA PE yielded detectable editing rates in hiPSC ranging between 6.4% and 9.8%, which was further increased to 41% after a GFP-based fluorescence-activated cell sorting (FACS) cell sorting step. Furthermore, we show that selecting the high GFP expressing population improved editing efficiencies up to 3.2-fold compared to the low GFP expressing population, demonstrating that not only delivery but also the number of copies of the PE enzyme and/or pegRNA per cell are important for efficient editing. Edit rates were not improved when an additional silent protospacer-adjacent motif (PAM)-removing alteration was introduced in hiPSC at the target locus. Finally, there were no genome-wide off-target effects using pAIO-EF1α-PE2 and no off-target editing activity near the edit locus highlighting the accuracy of snc prime editors.ConclusionTaken together, our study shows an improved efficacy of EF-1α driven sncRNA pAIO-PE plasmids in hiPSC reaching high editing rates, especially after FACS sorting. Optimizing these sncRNA PE systems is of high value when considering future therapeutic in vivo use, where accuracy will be extremely important.

Fibrinogen Cleavage by Lectin Pathway Proteases: Roles and Implications

The lectin pathway proteases (MASPs) are diverse in regard to their substrate selectivity and also have substrates outside the complement pathway. These proteases have been reported to cleave proteins of the coagulation cascade. MASP-1 has thrombin-like activity and therefore cleaves fibrinogen, fibrin and prothrombin. MASP-2 also participates in coagulation by cleaving prothrombin. Thus, these proteases are key players in the process of thrombosis and thrombolysis, therefore have implications in thrombotic vascular diseases. The cleaved fragments of fibrinogen and fibrin, consequently produced during coagulation and fibrinolysis, are important in the repair process and hence may have involvement in inflammatory and fibro-proliferative diseases. Also, altered levels of MBL and its associated serine proteases reported to be involved in different diseases such as cardiovascular diseases, diabetes, cancer, sepsis, stroke, COVID-19 etc. may have a role in thrombosis and thrombolysis. Upregulated lectin pathway-associated proteins predispose towards autoimmune disease susceptibility and may also exhibit off-target activities resulting in clot formation. Thus, the lectin pathway and its associated proteases are of great importance in normal circumstances where it plays a role in the maintenance of hemostasis and homeostasis, playing a preventive role in infections. Modulation in the level of MASPs proteases could be developed as a promising approach to treat a variety of infections and diseases.

Discovery of MK-8768, a Potent and Selective mGluR2 Negative Allosteric Modulator.

Glutamate plays a key role in cognition and mood, and it has been shown that inhibiting ionotropic glutamate receptors disrupts cognition, while enhancing ionotropic receptor activity is pro-cognitive. One approach to elevating glutamatergic tone has been to antagonize presynaptic metabotropic glutamate receptor 2 (mGluR2). A desire for selectivity over the largely homologous mGluR3 motivated a strategy to achieve selectivity through the identification of mGluR2 negative allosteric modulators (NAMs). Extensive screening and optimization efforts led to the identification of a novel series of 4-arylquinoline-2-carboxamides. This series was optimized for mGluR2 NAM potency, clean off-target activity, and desirable physical properties, which resulted in the identification of improved C4 and C7 substituents. The initial lead compound from this series was Ames-positive in a single strain with metabolic activation, indicating that a reactive metabolite was likely responsible for the genetic toxicity. Metabolic profiling and Ames assessment across multiple analogs identified key structure-activity relationships associated with Ames positivity. Further optimization led to the Ames-negative mGluR2 negative allosteric modulator MK-8768.

Prediction of on-target and off-target activity of CRISPR-Cas13d guide RNAs using deep learning.

Transcriptome engineering applications in living cells with RNA-targeting CRISPR effectors depend on accurate prediction of on-target activity and off-target avoidance. Here we design and test ~200,000 RfxCas13d guide RNAs targeting essential genes in human cells with systematically designed mismatches and insertions and deletions (indels). We find that mismatches and indels have a position- and context-dependent impact on Cas13d activity, and mismatches that result in G-U wobble pairings are better tolerated than other single-base mismatches. Using this large-scale dataset, we train a convolutional neural network that we term targeted inhibition of gene expression via gRNA design (TIGER) to predict efficacy from guide sequence and context. TIGER outperforms the existing models at predicting on-target and off-target activity on our dataset and published datasets. We show that TIGER scoring combined with specific mismatches yields the first general framework to modulate transcript expression, enabling the use of RNA-targeting CRISPRs to precisely control gene dosage.

Eyes on Topical Ocular Disposition: The Considered Design of a Lead Janus Kinase (JAK) Inhibitor That Utilizes a Unique Azetidin-3-Amino Bridging Scaffold to Attenuate Off-Target Kinase Activity, While Driving Potency and Aqueous Solubility.

An unmet medical need remains for patients suffering from dry eye disease (DED). A fast-acting, better-tolerated noncorticosteroid anti-inflammatory eye drop could improve patient outcomes and quality of life. Herein, we describe a small-molecule drug discovery effort to identify novel, potent, and water-soluble JAK inhibitors as immunomodulating agents for topical ocular disposition. A focused library of known 3-(4-(2-(arylamino)pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitriles was evaluated as a molecular starting point. Structure-activity relationships (SARs) revealed a ligand-efficient (LE) JAK inhibitor series, amenable to aqueous solubility. Subsequent in vitro analysis indicated the potential for off-target toxicity. A KINOMEscan selectivity profile of 5 substantiated the likelihood of widespread series affinity across the human kinome. An sp2-to-sp3 drug design strategy was undertaken to attenuate off-target kinase activity while driving JAK-STAT potency and aqueous solubility. Tactics to reduce aromatic character, increase fraction sp3 (Fsp3), and bolster molecular complexity led to the azetidin-3-amino bridging scaffold in 31.

Anti-infectives Developed as Racemic Drugs in the 21st Century: Norm or Exception?

This viewpoint outlines the case for developing new chemical entities (NCEs) as racemates in infectious diseases and where both enantiomers and racemate retain similar on- and off-target activities as well as similar PK profiles. There are not major regulatory impediments for the development of a racemic drug, and minimizing the manufacturing costs becomes a particularly important objective when bringing an anti-infective therapeutic to the marketplace in the endemic settings of infectious diseases.

Whole genome analysis for 163 gRNAs in Cas9-edited mice reveals minimal off-target activity

Genome editing with CRISPR-associated (Cas) proteins holds exceptional promise for “correcting” variants causing genetic disease. To realize this promise, off-target genomic changes cannot occur during the editing process. Here, we use whole genome sequencing to compare the genomes of 50 Cas9-edited founder mice to 28 untreated control mice to assess the occurrence of S. pyogenes Cas9-induced off-target mutagenesis. Computational analysis of whole-genome sequencing data detects 26 unique sequence variants at 23 predicted off-target sites for 18/163 guides used. While computationally detected variants are identified in 30% (15/50) of Cas9 gene-edited founder animals, only 38% (10/26) of the variants in 8/15 founders validate by Sanger sequencing. In vitro assays for Cas9 off-target activity identify only two unpredicted off-target sites present in genome sequencing data. In total, only 4.9% (8/163) of guides tested have detectable off-target activity, a rate of 0.2 Cas9 off-target mutations per founder analyzed. In comparison, we observe ~1,100 unique variants in each mouse regardless of genome exposure to Cas9 indicating off-target variants comprise a small fraction of genetic heterogeneity in Cas9-edited mice. These findings will inform future design and use of Cas9-edited animal models as well as provide context for evaluating off-target potential in genetically diverse patient populations.

Navitas/Optimus: A Novel Computational Tool for enhanced CRISPR/Cas Genome Editing

Clinical trials have begun using CRISPR/Cas genome editing to treat certain diseases, making safety a top priority. The CRISPR/Cas system is an efficient and versatile approach to gene editing, using a single guide RNA (sgRNA) to induce precise DNA modifications. However, screening sgRNAs for efficacy and specificity is time-consuming and resource-intensive. To streamline this process, we present Navitas/Optimus, a novel computational tool that predicts the optimal sgRNA for a given target sequence. Navitas features a unique approach of not differentiating between on- and off-target activity, and employs a combination of Machine Learning models, including XGBoost, Linear Regression, Random Forest, and Feedforward Neural Network (FNN), with FNN performing the best overall. The results of Navitas, which utilized FNN, are further optimized by data augmentation. Optimus, an Evolutionary Algorithm (EA), optimizes the prediction of the optimal sgRNA given a target DNA sequence using Navitas as its fitness function. Navitas/Optimus has the potential to contribute to the field of genome editing by offering a novel way of predicting the optimal sgRNA for a given target sequence.

The controversial abscopal effect: A single-center experience about the combination of stereotactic radiotherapy with pembrolizumab in patients with metastatic dMMR/MSI-H colorectal cancer.

2608 Background: Colorectal cancer (CRC) patients with mismatch repair system deficiency (dMMR/MSI-H) have shown impressive responses to immune checkpoint inhibitors in any lines of treatment. However, almost 30% of these patients are primary refractory and another 30% develop secondary resistance. Thus, many treatment combinations are being pursued to overcome resistance. One of the proposed strategies is the addition of radiotherapy to immunotherapy based on the potential abscopal effect and its consequent on-target and off-target activity. Methods: Between November 2019 and April 2021, we conducted a prospective interventional single-institution study to assess the feasibility, disease control and safety of the combination of pembrolizumab with stereotactic ablative radiotherapy (SABR) in a cohort of 14 consecutive dMMR CRC patients. SABR was administered in combination within the second or third cycle of Pembrolizumab 200 mg infused every 21 days. SABR was delivered on one or more targetable metastases with the ablative dose of 15 Gy in the first fraction followed by a second fraction of 5 Gy. Results: Fourteen patients were enrolled and 11 received radiotherapy in combination with Pembrolizumab as per protocol. Three patients who started Pembrolizumab never received radiotherapy due to deterioration of their general conditions, leading to death within two months. Two out of these three patients were BRAFV600E mutated. Five patients (35.7%) received Pembrolizumab as first line, 6 (43%) as second line and 3 (21%) as third or later lines. Six out of 14 patients (42.9%) harboured BRAFV600E mutation, 2 out of 14 (14.3%) RAS mutation and the others were RAS/BRAF wild type. Objective response was observed in 4 out of 14 patients (29%) and disease control was achieved in 7/14 patients (50%). Among these, six patients (42%) achieved a disease control lasting more than 15 months. This regimen caused substantial toxicity, mainly linked to SABR. Any grade TRAEs occurred in 50% of patients, with one patient with G1 event, three patients with G2 events, and two patients with G3 events (pneumonia, bowel perforation); no treatment-related deaths were observed. Conclusions: These data do not show hint of added efficacy of Pembrolizumab by SART in unresectable dMMR/MSI-H mCRC.

A phase 1/2a, multicenter, open-label study of CNA3103 (LGR5-targeted, autologous CAR-T cells) in patients with metastatic colorectal cancer (mCRC).

TPS3632 Background: Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a cancer stem cell (CSC) marker that is abundant in solid tumors, highly expressed in colorectal cancer and involved in tumor initiation, growth, and metastasis. Its expression is linked to poor survival and poor patient response to therapy in mCRC, and presents an attractive opportunity for selectively targeting a highly specific, functional CSC receptor in this disease. CNA3103 is a first-in-class cell product consisting of autologous T cells transduced to express a CAR to target LGR5. It contains the antigen-biding domain of BNC101, a humanized monoclonal LGR5 antibody previously tested in Phase 1 in mCRC subjects and demonstrated to be safe and well tolerated with no off-target binding activity. Methods: This is a first in human, multicenter, open label, Phase 1/2a dose escalation and expansion study to determine the safety of and overall best response to CNA3103 in subjects with mCRC. Up to 44 subjects (24 in Phase 1 and 20 in Phase 2a) will be enrolled. The trial follows a BOIN design with a minimum of 3 consecutively enrolled subjects per cohort, and a 28-day DLT evaluation period. Four cell dose levels (5 × 107, 1.5 × 108, 4.5 × 108, 1.5 × 109) of CNA3103 will be evaluated. A Safety Monitoring Committee (SMC) will oversee the conduct of the study. The pharmacokinetic (PK) profile of CNA3103 in blood will be assessed. Exploratory objectives include evaluation of LGR5+ cells and CNA3103 in tumor biopsies, immune cellular profile, cytokine levels and circulating tumor DNA in blood, and immunogenicity of CNA3103. Main inclusion criteria: mCRC subjects positive for LGR5 expression and failing prior treatment with 5-FU, oxaliplatin and irinotecan-based regimens for metastatic disease; &gt;1 radiologically measurable lesion per RECISTv1.1; &gt;1 biopsiable lesion at baseline; ECOG 0 or 1; serum albumin &gt;3 g/dL; and normal organ and marrow function. Main exclusion criteria: BRAF-mutated disease, clinical ascites/ pleural effusions; active autoimmune/ connective tissue disease; history of inflammatory bowel disease or active peptic ulcer disease. The trial is sponsored by Carina Biotech. Phase 1 enrolment is limited to Australian sites. Phase 2a enrolment will take place in Australia and the US. For information, contact Lina Jablonskis lina@carinabiotech.com or +61 437 891 563.

Preliminary data from the phase I study of TCMCB07, a study to assess the safety, tolerability and pharmacokinetics of the melanocortin antagonist TCMCB07 in healthy subjects.

e15195 Background: Cachexia is a complex wasting syndrome characterized by anorexia, an involuntary loss of adipose tissue and lean body mass, and a paradoxical increase in energy catabolism. TCMCB07 is an optimized peptide antagonist of the melanocortin type 3 and 4 receptors for the treatment of cachexia. Several studies have been completed on TCMCB07 including: primary efficacy studies in rodent models of cachexia, studies to assess off-target activities, PK/ADME studies, and a 28-day GLP tox program in rats and dogs. Pre-clinical animal trials show significant results in ameliorating cancer and chronic kidney disease-associated cachexia. Based on these data, a phase 1 dose escalation study was conducted in healthy volunteers. Methods: The Phase 1 study of TCMCB07 (NCT05529849) is a single center, double-blind, randomized, sequential assessment of 5 single ascending dose cohorts (SAD) and 3 multiple ascending dose cohorts of TCMCB07. Subjects received subcutaneous administration of TCMCB07 or placebo on each of 5 consecutive days. Primary endpoints were safety and tolerability. The secondary endpoint was characterization of pharmacokinetics. Results: 40 subjects completed the SAD and 36 subjects completed the MAD portions of the trial. In a preliminary analysis of the results, there were no subjects with abnormal vital signs, no subjects with abnormal electrocardiograms, and no drug-related serious adverse events. The most common study-related adverse event was injection site reaction, graded 1 mild, or 2 moderate out of 5 (NCI CTCAE). Analysis of pharmacokinetics yielded Cmax and AUC higher than what was expected based on rat and dog studies. This resulted in lowering the cohort 3 dose so that the CMax would not exceed the cohort 5 SAD CMax. A weight difference in MAD cohort 2 and 3 between placebo and control was observed with the change in weight from end-of-study to baseline of 1.07 kg (p = 0.0195). In the same groups, on a VAS hunger scale, subjects taking TCMCB07 indicated 15% greater ease in eating meals compared to controls. This measure reached significance at one time point (p = 0.0497) but not at another (p = 0.0892). Conclusions: CB07 was well tolerated by normal healthy volunteers. Though the Phase 1 study was not designed to study efficacy, a modest difference was observed between placebo and active for body weight change and hunger. Results warrant further examination in a randomized Phase 2 study in cancer patients experiencing cachexia. Clinical trial information: NCT05529849 .

AAV-vectored base editor trans-splicing delivers dystrophin repair

AAV-vectored base editor trans-splicing delivers dystrophin repair

Design and Optimization of Novel Benzimidazole- and Imidazo[4,5-b]pyridine-Based ATM Kinase Inhibitors with Subnanomolar Activities.

The ATM kinase is a promising target in cancer treatment as an important regulator of the cellular response to DNA double-strand breaks. In this work, we present a new class of specific benzimidazole-based ATM inhibitors with picomolar potency against the isolated enzyme and favorable selectivity within relative PIKK and PI3K kinases. We could identify two promising inhibitor subgroups with significantly different physicochemical properties, which we developed simultaneously. These efforts lead to numerous highly active inhibitors with picomolar enzymatic activities. Furthermore, initial low cellular activities on A549 cells could be increased significantly in numerous examples resulting in cellular IC50 values in the subnanomolar range. Further characterization of the highly potent inhibitors 90 und 93 revealed promising pharmacokinetic properties and strong activities in organoids in combination with etoposide. Additionally, 93 showed no off-target activities within a kinome-representative mini kinase panel, with favorable selectivities within the PIKK- and PI3K-families.

N-sulfonyl peptide-hybrids as a new class of dengue virus protease inhibitors.

Dengue virus (DENV) from the Flaviviridae family causes an epidemic disease that seriously threatens human life. The viral serine protease NS2B-NS3 is a promising target for drug development against DENV and other flaviviruses. We here report the design, synthesis, and in-vitro characterization of potent peptidic inhibitors of DENV protease with a sulfonyl moiety as N-terminal cap, thereby creating sulfonamide-peptide hybrids. The in-vitro target affinities of some synthesized compounds were in the nanomolar range, with the most promising derivative reaching a Ki value of 78nM against DENV-2 protease. The synthesized compounds did not have relevant off-target activity nor cytotoxicity. The metabolic stability of compounds against rat liver microsomes and pancreatic enzymes was remarkable. In general, the integration of sulfonamide moieties at the N-terminus of peptidic inhibitors proved to be a promising and attractive strategy for further drug development against DENV infections.

CasKAS: direct profiling of genome-wide dCas9 and Cas9 specificity using ssDNA mapping

Detecting and mitigating off-target activity is critical to the practical application of CRISPR-mediated genome and epigenome editing. While numerous methods have been developed to map Cas9 binding specificity genome-wide, they are generally time-consuming and/or expensive, and not applicable to catalytically dead CRISPR enzymes. We have developed CasKAS, a rapid, inexpensive, and facile assay for identifying off-target CRISPR enzyme binding and cleavage by chemically mapping the unwound single-stranded DNA structures formed upon binding of a sgRNA-loaded Cas9 protein. We demonstrate this method in both in vitro and in vivo contexts.

Transformer-based anti-noise models for CRISPR-Cas9 off-target activities prediction.

The off-target effect occurring in the CRISPR-Cas9 system has been a challenging problem for the practical application of this gene editing technology. In recent years, various prediction models have been proposed to predict potential off-target activities. However, most of the existing prediction methods do not fully exploit guide RNA (gRNA) and DNA sequence pair information effectively. In addition, available prediction methods usually ignore the noise effect in original off-target datasets. To address these issues, we design a novel coding scheme, which considers the key features of mismatch type, mismatch location and the gRNA-DNA sequence pair information. Furthermore, a transformer-based anti-noise model called CrisprDNT is developed to solve the noise problem that exists in the off-target data. Experimental results of eight existing datasets demonstrate that the method with the inclusion of the anti-noise loss functions is superior to available state-of-the-art prediction methods. CrisprDNT is available at https://github.com/gzrgzx/CrisprDNT.