Zero-shot prediction of mutation effects with multimodal deep representation learning guides protein engineering.

Mutations in amino acid sequences can provoke changes in protein function. Accurate and unsupervised prediction of mutation effects is critical in biotechnology and biomedicine, but remains a fundamental challenge. To resolve this challenge, here we present Protein Mutational Effect Predictor (ProMEP), a general and multiple sequence alignment-free method that enables zero-shot prediction of mutation effects. A multimodal deep representation learning model embedded in ProMEP was developed to comprehensively learn both sequence and structure contexts from ~160 million proteins. ProMEP achieves state-of-the-art performance in mutational effect prediction and accomplishes a tremendous improvement in speed, enabling efficient and intelligent protein engineering. Specifically, ProMEP accurately forecasts mutational consequences on the gene-editing enzymes TnpB and TadA, and successfully guides the development of high-performance gene-editing tools with their engineered variants. The gene-editing efficiency of a 5-site mutant of TnpB reaches up to 74.04% (vs 24.66% for the wild type); and the base editing tool developed on the basis of a TadA 15-site mutant (in addition to the A106V/D108N double mutation that renders deoxyadenosine deaminase activity to TadA) exhibits an A-to-G conversion frequency of up to 77.27% (vs 69.80% for ABE8e, a previous TadA-based adenine base editor) with significantly reduced bystander and off-target effects compared to ABE8e. ProMEP not only showcases superior performance in predicting mutational effects on proteins but also demonstrates a great capability to guide protein engineering. Therefore, ProMEP enables efficient exploration of the gigantic protein space and facilitates practical design of proteins, thereby advancing studies in biomedicine and synthetic biology.

nCas9 Engineering for Improved Target Interaction Presents an Effective Strategy to Enhance Base Editing.

Base editors (BEs) are a recent generation of genome editing tools that couple a cytidine or adenosine deaminase activity to a catalytically impaired Cas9 moiety (nCas9) to enable specific base conversions at the targeted genomic loci. Given their strong application potential, BEs are under active developments toward greater levels of efficiency and safety. Here, a previously overlooked nCas9-centric strategy is explored for enhancement of BE. Based on a cytosine BE (CBE), 20 point mutations associated with nCas9-target interaction are tested. Subsequently, from the initial positive X-to-arginine hits, combinatorial modifications are applied to establish further enhanced CBE variants (1.1-1.3). Parallel nCas9 modifications in other versions of CBEs including A3A-Y130F-BE4max, YEE-BE4max, CGBE, and split-AncBE4max, as well as in the context of two adenine BEs (ABE), likewise enhance their respective activities. The same strategy also substantially improves the efficiencies of high-fidelity nCas9/BEs. Further evidence confirms that the stabilization of nCas9-substrate interactions underlies the enhanced BE activities. In support of their translational potential, the engineered CBE and ABE variants respectively enable 82% and 25% higher rates of editing than the controls in primary human T-cells. This study thus demonstrates a highly adaptable strategy for enhancing BE, and for optimizing other forms of Cas9-derived tools.

CoHIT: a one-pot ultrasensitive ERA-CRISPR system for detecting multiple same-site indels.

Genetic testing is crucial for precision cancer medicine. However, detecting multiple same-site insertions or deletions (indels) is challenging. Here, we introduce CoHIT (Cas12a-based One-for-all High-speed Isothermal Test), a one-pot CRISPR-based assay for indel detection. Leveraging an engineered AsCas12a protein variant with high mismatch tolerance and broad PAM scope, CoHIT can use a single crRNA to detect multiple NPM1 gene c.863_864 4-bp insertions in acute myeloid leukemia (AML). After optimizing multiple parameters, CoHIT achieves a detection limit of 0.01% and rapid results within 30 minutes, without wild-type cross-reactivity. It successfully identifies NPM1 mutations in 30 out of 108 AML patients and demonstrates potential in monitoring minimal residual disease (MRD) through continuous sample analysis from three patients. The CoHIT method is also competent for detecting indels of KIT, BRAF, and EGFR genes. Integration with lateral flow test strips and microfluidic chips highlights CoHIT's adaptability and multiplexing capability, promising significant advancements in clinical cancer diagnostics.

Optimizing multicopy chromosomal integration for stable high-performing strains.

The copy number of genes in chromosomes can be modified by chromosomal integration to construct efficient microbial cell factories but the resulting genetic systems are prone to failure or instability from triggering homologous recombination in repetitive DNA sequences. Finding the optimal copy number of each gene in a pathway is also time and labor intensive. To overcome these challenges, we applied a multiple nonrepetitive coding sequence calculator that generates sets of coding DNA sequence (CDS) variants. A machine learning method was developed to calculate the optimal copy number combination of genes in a pathway. We obtained an engineered Yarrowia lipolytica strain for eicosapentaenoic acid biosynthesis in 6 months, producing the highest titer of 27.5 g l-1 in a 50-liter bioreactor. Moreover, the lycopene production in Escherichia coli was also greatly improved. Importantly, all engineered strains of Y. lipolytica, E. coli and Saccharomyces cerevisiae constructed with nonrepetitive CDSs maintained genetic stability.

Point-of-care testing of methamphetamine and cocaine utilizing wearable sensors.

The imperative for the point-of-care testing of methamphetamine and cocaine in drug abuse prevention necessitates innovative solutions. To address this need, we have introduced a multi-channel wearable sensor harnessing CRISPR/Cas12a system. A CRISPR/Cas12a based system, integrated with aptamers specific to methamphetamine and cocaine, has been engineered. These aptamers function as signal-mediated intermediaries, converting methamphetamine and cocaine into nucleic acid signals, subsequently generating single-stranded DNA to activate the Cas12 protein. Additionally, we have integrated a microfluidic system and magnetic separation technology into the CRISPR system, enabling rapid and precise detection of cocaine and methamphetamine. The proposed sensing platform demonstrated exceptional sensitivity, achieving a detection limit as low as 0.1 ng/mL. This sensor is expected to be used for on-site drug detection in the future.

Restoring T and B cell generation in X-linked severe combined immunodeficiency mice through hematopoietic stem cells adenine base editing.

Base editing of hematopoietic stem/progenitor cells (HSPCs) is an attractive strategy for treating immunohematologic diseases. However, the feasibility of using adenine-base-edited HSPCs for treating X-linked severe combined immunodeficiency (SCID-X1), the influence of dose-response relationships on immune cell generation, and the potential risks have not been demonstrated in vivo. Here, a humanized SCID-X1 mouse model was established, and 86.67% ± 2.52% (n = 3) of mouse hematopoietic stem cell (HSC) pathogenic mutations were corrected, with no single-guide-RNA (sgRNA)-dependent off-target effects detected. Analysis of peripheral blood over 16 weeks post-transplantation in mice with different immunodeficiency backgrounds revealed efficient immune cell generation following transplantation of different amounts of modified HSCs. Therefore, a large-scale infusion of gene-corrected HSCs within a safe range can achieve rapid, stable, and durable immune cell regeneration. Tissue-section staining further demonstrated the restoration of immune organ tissue structures, with no tumor formation in multiple organs. Collectively, these data suggest that base-edited HSCs are a potential therapeutic approach for SCID-X1 and that a threshold infusion dose of gene-corrected cells is required for immune cell regeneration. This study lays a theoretical foundation for the clinical application of base-edited HSCs in treating SCID-X1.

Biodegradable bio-film based on Cordyceps militaris and metal-organic frameworks for fruit preservation.

In this study, Cordyceps militaris matrix was employed for the first time to fabricate a biodegradable food packaging. Carmine and Ag@CuBTC were introduced to cross-link with mycelium and were uniformly dispersed within the matrix to enhance the water resistance, antimicrobial, and antioxidant properties of the bio-films. The bio-film displayed high biodegradability, with nearly 100 % degradation achieved after three weeks. The bio-film exhibited exceptional resistance to oxidation (49.30 % DPPH and 93.94 % ABTS•+), as well as effective inhibitory capabilities against E. coli and S. aureus, respectively. The composite film maintained a high CO2/O2 selective permeability, which was advantageous for mitigating fruit metabolism and extending shelf life. Simultaneously, food preservation experiments confirmed that these bio-films can decelerate the spoilage of fruits and effectively prolong the shelf-life of food. The experimental findings indicated that the prepared Bio-R-Ag@Cu film held promise as an environmentally friendly biodegradable material for food packaging.

BacPE: a versatile prime-editing platform in bacteria by inhibiting DNA exonucleases.

Prime editing allows precise installation of any single base substitution and small insertions and deletions without requiring homologous recombination or double-strand DNA breaks in eukaryotic cells. However, the applications in bacteria are hindered and the underlying mechanisms that impede efficient prime editing remain enigmatic. Here, we report the determination of vital cellular factors that affect prime editing in bacteria. Genetic screening of 129 Escherichia coli transposon mutants identified sbcB, a 3'→5' DNA exonuclease, as a key genetic determinant in impeding prime editing in E. coli, combinational deletions of which with two additional 3'→5' DNA exonucleases, xseA and exoX, drastically enhanced the prime editing efficiency by up to 100-fold. Efficient prime editing in wild-type E. coli can be achieved by simultaneously inhibiting the DNA exonucleases via CRISPRi. Our results pave the way for versatile applications of prime editing for bacterial genome engineering.

Integrated multi-omics profiling to dissect the spatiotemporal evolution of metastatic hepatocellular carcinoma.

Comprehensive molecular analyses of metastatic hepatocellular carcinoma (HCC) are lacking. Here, we generate multi-omic profiling of 257 primary and 176 metastatic regions from 182 HCC patients. Primary tumors rich in hypoxia signatures facilitated polyclonal dissemination. Genomic divergence between primary and metastatic HCC is high, and early dissemination is prevalent. The remarkable neoantigen intratumor heterogeneity observed in metastases is associated with decreased T cell reactivity, resulting from disruptions to neoantigen presentation. We identify somatic copy number alterations as highly selected events driving metastasis. Subclones without Wnt mutations show a stronger selective advantage for metastasis than those with Wnt mutations and are characterized by a microenvironment rich in activated fibroblasts favoring a pro-metastatic phenotype. Finally, metastases without Wnt mutations exhibit higher enrichment of immunosuppressive B cells that mediate terminal exhaustion of CD8+ T cells via HLA-E:CD94-NKG2A checkpoint axis. Collectively, our results provide a multi-dimensional dissection of the complex evolutionary process of metastasis.

MscI restriction enzyme cooperating recombinase-aided isothermal amplification for the ultrasensitive and rapid detection of low-abundance EGFR mutations on microfluidic chip.

The detection of low-abundance mutation genes of the epidermal growth factor receptor (EGFR) exon 21 (EGFR L858R) plays a crucial role in the diagnosis of non-small cell lung cancer (NSCLC), as it enables early cancer detection and facilitates the development of treatment strategies. A detection platform was developed by combining the MscI restriction enzyme with the recombinase-aided isothermal amplification (RAA) technique (MRE-RAA). During the RAA process, "TGG^CCA" site of the wild-type genes was cleaved by the MscI restriction enzyme, while only the low-abundance mutation genes underwent amplification. Notably, when the RAA product was combined with CRISPR-Cas system, the sensitivity of detecting the EGFR L858R mutation increased by up to 1000-fold for addition of the MscI restriction enzyme. This achievement marked the first instance of attaining an analytical sensitivity of 0.001%. Furthermore, a disk-shaped microfluidic chip was developed to automate pretreatment while concurrently analyzing four blood samples. The microfluidic features of the chip include DNA extraction, MRE-RAA, and CRISPR-based detection. The fluorescence signal is employed for detection in the microfluidic chip, which is visible to the naked eye upon exposure to blue light irradiation. Furthermore, this platform has the capability to facilitate early diagnosis for various types of cancer by enabling high-sensitivity detection of low-abundance mutation genes.

ProRefiner: an entropy-based refining strategy for inverse protein folding with global graph attention.

Inverse Protein Folding (IPF) is an important task of protein design, which aims to design sequences compatible with a given backbone structure. Despite the prosperous development of algorithms for this task, existing methods tend to rely on noisy predicted residues located in the local neighborhood when generating sequences. To address this limitation, we propose an entropy-based residue selection method to remove noise in the input residue context. Additionally, we introduce ProRefiner, a memory-efficient global graph attention model to fully utilize the denoised context. Our proposed method achieves state-of-the-art performance on multiple sequence design benchmarks in different design settings. Furthermore, we demonstrate the applicability of ProRefiner in redesigning Transposon-associated transposase B, where six out of the 20 variants we propose exhibit improved gene editing activity.

CRISPR/Cas9-mediated knockout of intracellular molecule SHP-1 enhances tumor-killing ability of CD133-targeted CAR T cells in vitro.

CAR T cell therapy has been successfully used in the treatment of hematological malignancies, and the strategy that deletion of inhibitory receptor on the CAR T cell surface, such as PD-1, greatly enhance the antitumor effects. Here, we describe a one-step electroporation for the co-transfection of Cas9:sgRNA and CAR plasmids on primary T cells to demonstrate the effect of SHP-1 deletion in CAR T cells. By using PiggyBac Transposase system, we can achieve more than 90% of T cells express CAR gene and nearly 60% SHP-1 knockout efficiency in T cells. We show that knockout of SHP-1 in CD133 CAR T cells resulted in significantly improve the cytolysis effect on CD133 positive glioma cell lines. We further demonstrate that the enhanced antitumor efficacy of SHP-1 deletion is due to the increased release of TNF-α, IL-2 and IFN-γ in vitro. Finally, we evaluated the biosafety of Cas9 genome editing and did not find any insertions of Cas9 and obvious editing in off-target sites in CAR T cells. These data provide an approach for achieving both intracellular inhibitory molecule, SHP-1 deletion and CD133 CAR gene over-expression in human T cells. And SHP-1 could be a new potential target for adoptive CAR T cells immunotherapy.

De Novo Design and Synthesis of Polypeptide Immunomodulators for Resetting Macrophage Polarization.

Modulating the extracellular matrix microenvironment is critical for achieving the desired macrophage phenotype in immune investigations or tumor therapy. Combining de novo protein design and biosynthesis techniques, herein, we designed a biomimetic polypeptide self-assembled nano-immunomodulator to trigger the activation of a specific macrophage phenotype. It was intended to be made up of (​GGS​GGP​GGG​PAS​AAA​NSA​SRA​TSN​SP)n, the RGD motif from collagen, and the IKVAV motif from laminin. The combination of these domains allows the biomimetic polypeptide to assemble into extracellular matrix-like nanofibrils, creating an extracellular matrix-like milieu for macrophages. Furthermore, changing the concentration further provides a facile route to fine-tune macrophage polarization, which enhances antitumor immune responses by precisely resetting tumor-associated macrophage immune responses into an M1-like phenotype, which is generally considered to be tumor-killing macrophages, primarily antitumor, and immune-promoting. Unlike metal or synthetic polymer-based nanoparticles, this polypeptide-based nanomaterial exhibits excellent biocompatibility, high efficacy, and precise tunability in immunomodulatory effectiveness. These encouraging findings motivate us to continue our research into cancer immunotherapy applications in the future.

A massively parallel approach for assessing CRISPR off-targets in vitro.

In a recent issue of Med, Tian et al.1 present AID-seq, an approach that enables massively parallel identification of off-targets for different CRISPR nucleases in vitro. By using a pooled strategy to simultaneously identify the on-/off-targets of multiple gRNAs, the authors could screen the most efficient and safe gRNA candidates.

Suppression of cytokine release syndrome during CAR-T-cell therapy via a subcutaneously injected interleukin-6-adsorbing hydrogel.

The infusion of chimaeric antigen receptor (CAR) T cells can trigger the release of life-threatening supraphysiological levels of pro-inflammatory cytokines. However, uncertainty regarding the timing and severity of such cytokine release syndrome (CRS) demands careful monitoring of the conditions required for the administration of neutralizing antibodies. Here we show that a temperature-sensitive hydrogel conjugated with antibodies for the pro-inflammatory cytokine interleukin-6 (IL-6) and subcutaneously injected before the infusion of CAR-T cells substantially reduces the levels of IL-6 during CRS while maintaining the therapy's antitumour efficacy. In immunodeficient mice and in mice with transplanted human haematopoietic stem cells, the subcutaneous IL-6-adsorbing hydrogel largely suppressed CAR-T-cell-induced CRS, substantially improving the animals' survival and alleviating their levels of fever, hypotension and weight loss relative to the administration of free IL-6 antibodies. The implanted hydrogel, which can be easily removed with a syringe following a cooling-induced gel-sol transition, may allow for a shift in the management of CRS, from monitoring to prevention.

A Cas12a-based fluorescent microfluidic system for rapid on-site human papillomavirus diagnostics.

Persistent infection with human papillomavirus (HPV) is the leading cause of cervical cancer, and early diagnosis is crucial for clinical management. However, the easy and rapid on-site diagnostic for HPV genotyping remains challenging. Here, we develop a Cas12a-based fluorescent microfluidic detection system for diagnosing six HPV subtypes (HPV6, HPV11, HPV16, HPV18, HPV31, and HPV33). A panel of crRNAs and recombinase polymerase amplification (RPA) primers targeting the HPV L1 gene was screened for sensitive and specific detection. Furthermore, a one-pot RPA reaction was developed to amplify the six HPV subtypes without cross-reactivity. For on-site detection, we integrated the RPA-Cas12a detection into a microfluidic device, enabling the detection of processed clinical samples within 35 minutes. The assay was validated using 112 clinical swab samples and obtained consistent results with the qPCR assay, with a concordance rate of 99.1%. Overall, our diagnostic method offers a rapid, sensitive, and easy-to-use on-site assay for detecting HPV genotypes and holds promise for improving cervical cancer screening and prevention. KEY POINTS: • The Cas12a-based fluorescent microfluidic detection system for the diagnosis of six HPV subtypes. • A one-pot RPA reaction for amplifying the six HPV subtypes without cross-reactivity. • The RPA-Cas12a-microfluidic system provides results within 35 minutes for on-site detection.

Deletion of ARGLU1 causes global defects in alternative splicing in vivo and mouse cortical malformations primarily via apoptosis.

Haploinsufficient mutation in arginine and glutamine-rich protein 1 (Arglu1), a newly identified pre-mRNA splicing regulator, may be linked to neural developmental disorders associated with mental retardation and epilepsy in human patients, but the underlying causes remain elusive. Here we show that ablation of Arglu1 promotes radial glial cell (RG) detachment from the ventricular zone (VZ), leading to ectopic localized RGs in the mouse embryonic cortex. Although they remain proliferative, ectopic progenitors, as well as progenitors in the VZ, exhibit prolonged mitosis, p53 upregulation and cell apoptosis, leading to reduced neuron production, neuronal loss and microcephaly. RNA seq analysis reveals widespread changes in alternative splicing in the mutant mouse embryonic cortex, preferentially affecting genes involved in neuronal functions. Mdm2 and Mdm4 are found to be alternatively spliced at the exon 3 and exon 5 respectively, leading to absence of the p53-binding domain and nonsense-mediated mRNA decay (NMD) and thus relieve inhibition of p53. Removal of p53 largely rescues the microcephaly caused by deletion of Arglu1. Our findings provide mechanistic insights into cortical malformations of human patients with Arglu1 haploinsufficient mutation.

Rapid and Sensitive Diagnosis of Leber Hereditary Optic Neuropathy Variants Using CRISPR/Cas12a Detection.

Leber hereditary optic neuropathy (LHON) is the most common maternally inherited mitochondrial disease, with >90% of cases harboring one of three point variants (m.3460G>A, m.11778G>A, and m.14484T>C). Rapid and sensitive diagnosis of LHON variants is urgently needed for early diagnosis and timely treatment after onset, which is currently limited. Herein, we adapted the Cas12a-based DNA detection platform for LHON mitochondrial variant diagnosis. Single-strand guide CRISPR RNAs and enzymatic recombinase amplification primers were first screened, the CRISPR/Cas12a system was then optimized with restriction enzymes, and finally compared with Sanger sequencing and next-generation sequencing (NGS) in multicenter clinical samples. This approach can be completed within 30 minutes using only one drop of blood and could reach a sensitivity of 1% of heteroplasmy. Among the 182 multicenter clinical samples, the CRISPR/Cas12a detection system showed high consistency with Sanger sequencing and NGS in both specificity and sensitivity. Notably, a sample harboring a de novo 3.78% m.11778G>A variant detected by NGS, but not by Sanger sequencing, was successfully confirmed using the CRISPR/Cas12a assay, which proved the effectiveness of our method. Overall, our CRISPR/Cas12a detection system provides an alternative for rapid, convenient, and sensitive detection of LHON variants, exhibiting great potential for clinical practice.

Age-related decline in hippocampal tyrosine phosphatase PTPRO is a mechanistic factor in chemotherapy-related cognitive impairment.

Chemotherapy-related cognitive impairment (CRCI) or "chemo brain" is a devastating neurotoxic sequela of cancer-related treatments, especially for the elderly individuals. Here we show that PTPRO, a tyrosine phosphatase, is highly enriched in the hippocampus, and its level is tightly associated with neurocognitive function but declined significantly during aging. To understand the protective role of PTPRO in CRCI, a mouse model was generated by treating Ptpro-/- female mice with doxorubicin (DOX) because Ptpro-/- female mice are more vulnerable to DOX, showing cognitive impairments and neurodegeneration. By analyzing PTPRO substrates that are neurocognition-associated tyrosine kinases, we found that SRC and EPHA4 are highly phosphorylated/activated in the hippocampi of Ptpro-/- female mice, with increased sensitivity to DOX-induced CRCI. On the other hand, restoration of PTPRO in the hippocampal CA3 region significantly ameliorate CRCI in Ptpro-/- female mice. In addition, we found that the plant alkaloid berberine (BBR) is capable of ameliorating CRCI in aged female mice by upregulating hippocampal PTPRO. Mechanistically, BBR upregulates PTPRO by downregulating miR-25-3p, which directly targeted PTPRO. These findings collectively demonstrate the protective role of hippocampal PTPRO against CRCI.