@article{GenomePAM2025,
  title = {GenomePAM directs PAM characterization and engineering of CRISPR-Cas nucleases using mammalian genome repeats},
  author = {Yu, M and Ai, L and Wang, B and et al.},
  journal = {Nature Biomedical Engineering (IF 27.7)},
  volume = {},
  number = {},
  pages = {},
  year = {2025},
  doi = {10.1038/s41551-025-01464-y},
  file = {s41551-025-01464-y.pdf},
  excerpt = {This paper is about the CRISPR protospacer adjacent motif (PAM) identification using the native repeat sequences of human genome, enabling the discovery of novel Cas nucleases and profiling the activities and fidelities in mammalian cells in parallel.}
}

Characterizing the protospacer adjacent motif (PAM) requirements of different Cas enzymes is a bottleneck in the discovery of Cas proteins and their engineered variants in mammalian cell contexts. Here, to overcome this challenge and to enable more scalable characterization of PAM preferences, we develop a method named GenomePAM that allows for direct PAM characterization in mammalian cells. GenomePAM leverages genomic repetitive sequences as target sites and does not require protein purification or synthetic oligos. GenomePAM uses a 20-nt protospacer that occurs  16,942 times in every human diploid cell and is flanked by nearly random sequences. We demonstrate that GenomePAM can accurately characterize the PAM requirement of type II and type V nucleases, including the minimal PAM requirement of the near-PAMless SpRY and extended PAM for CjCas9. Beyond PAM characterization, GenomePAM allows for simultaneous comparison of activities and fidelities among different Cas nucleases on thousands of match and mismatch sites across the genome using a single gRNA and provides insight into the genome-wide chromatin accessibility profiles in different cell types.

@article{ECM2025,
  title = {Extracellular matrix scaffold crosslinked with vancomycin for multifunctional antibacterial bone infection therapy},
  author = {Fang, B and Qiu, P and Xia, C and et al.},
  journal = {Biomaterials (IF 15.3) [*co-corresponding]},
  volume = {},
  number = {},
  pages = {},
  year = {2021},
  doi = {10.1016/j.biomaterials.2020.120603},
  file = {},
  excerpt = {This paper is about the multi-roles and mechanisms of vancomycin-corsslinked extracellular matrix in both anti-infection and osteogenesis}
}

The treatment of acute and chronic bone infections remains a major clinical challenge. The various factors released by the bacteria, acidic environment, and bacterial colonies in the bone grooves and implanted synthetic materials collectively promote the formation of biofilms. Dormant bacteria and biofilms cause infections that are difficult to cure and that can develop chronically. Therefore, a new antibacterial material was synthesized in the present study for multifunctional bone infection therapy and consists of specific demineralized extracellular cancellous bone (SDECM) crosslinked with vancomycin (Van) by means of electrostatic interactions and chemical bonds. It was verified in vitro that the new material (Van-SDECM) not only has pH-sensitive release and biofilm inhibition properties, but also maintains sustained bactericidal ability accompanied by the degradation of the scaffold, which does not affect its favorable osteogenic performance. The infectious bone defect in vivo model further confirms the comprehensive anti-infective and osteogenic ability of the Van-SDECM. Further, these favorable properties are due to the pH-sensitive sustained release sterilization and scaffold contact antibacterial properties, accompanied by osteoclast activity inhibition, osteogenesis promotion and immunoregulation effects. This study provides a new drug-scaffold composite preparation method based on a native-derived extracellular matrix scaffold.

@article{mtDNA2021,
  title = {CRISPR/Cas9-mediated mutagenesis at microhomologous regions of human mitochondrial genome},
  author = {Wang, B and Lv, X and Wang, Y and et al.},
  journal = {Science China-Life Sciences (IF 10.3)},
  volume = {},
  number = {},
  pages = {},
  year = {2021},
  doi = {10.1007/s11427-020-1819-8},
  file = {},
  excerpt = {This paper is about the microhomology-mediated end joining of human mtDNA triggered by SaCas9, which indicates the de novo mutagenesis across the hotspots of mtDNA.}
}

Genetic manipulation of mitochondrial DNA (mtDNA) could be harnessed for deciphering the gene function of mitochondria; it also acts as a promising approach for the therapeutic correction of pathogenic mutation in mtDNA. However, there is still a lack of direct evidence showing the edited mutagenesis within human mtDNA by clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9). Here, using engineered CRISPR/Cas9, we observed numerous insertion/deletion (InDel) events at several mtDNA microhomologous regions, which were triggered specifically by double-strand break (DSB) lesions within mtDNA. InDel mutagenesis was significantly improved by sgRNA multiplexing and a DSB repair inhibitor, iniparib, demonstrating the evidence of rewiring DSB repair status to manipulate mtDNA using CRISPR/Cas9. These findings would provide novel insights into mtDNA mutagenesis and mitochondrial gene therapy for diseases involving pathogenic mtDNA.

@article{efSaCas9_2020,
  title = {High-fidelity SaCas9 identified by directional screening in human cells},
  author = {Xie, H and Ge, X and Yang, F and et al.},
  journal = {PLoS Biology (IF 9.5)},
  volume = {},
  number = {},
  pages = {},
  year = {2020},
  doi = {10.1371/journal.pbio.3000747},
  file = {},
  excerpt = {This work is about screening and identification of a high-fidelity SaCas9 (efSaCas9) for safer human genome editing.}
}

CRISPR-Staphylococcus aureus Cas9 (CRISPR-SaCas9) has been harnessed as an effective in vivo genome-editing tool to manipulate genomes. However, off-target effects remain a major bottleneck that precludes safe and reliable applications in genome editing. Here, we characterize the off-target effects of wild-type (WT) SaCas9 at single-nucleotide (single-nt) resolution and describe a directional screening system to identify novel SaCas9 variants with desired properties in human cells. Using this system, we identified enhanced-fidelity SaCas9 (efSaCas9) (variant Mut268 harboring the single mutation of N260D), which could effectively distinguish and reject single base-pair mismatches. We demonstrate dramatically reduced off-target effects (approximately 2- to 93-fold improvements) of Mut268 compared to WT using targeted deep-sequencing analyses. To understand the structural origin of the fidelity enhancement, we find that N260, located in the REC3 domain, orchestrates an extensive network of contacts between REC3 and the guide RNA-DNA heteroduplex. efSaCas9 can be broadly used in genome-editing applications that require high fidelity. Furthermore, this study provides a general strategy to rapidly evolve other desired CRISPR-Cas9 traits besides enhanced fidelity, to expand the utility of the CRISPR toolkit.

@inproceedings{example,
  author = {Wang, B and Cao, Y and Jiang, Z and et al.},
  journal = {American Society of Gene & Cell Therapy (Oral presentation)},
  year = {2025},
  title = {Safe and Efficient Editing of Novel CRISPR-Cas Ribonucleoprotein Complexes Delivered by Engineered Protein Delivery Vehicle},
  file = {},
  fileurl = {https://annualmeeting.asgct.org/program/print-program},
  excerpt = {This work is about in-house novel Cas nuclease ribonucleoproteins functioning in vivo with an engineered protein delivery vehicle.}
}

Clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR-Cas) system holds great promise to revolutionize genome editing and therapy in basic and translational settings. Various CRISPR-Cas systems have been explored and engineered, such as CRISPR-Cas9 and CRISPR-Cas12a that belong to Class 2 of CRISPR-Cas system. However, therapeutic applications of these tools are still limited due to specificity of protospacer adjacent motif (PAM) and potential off-target effects.

To address these limitations, we mine new Cas nucleases from nature and use protein delivery vehicles (PDVs). With this new platform, we identified and characterized dozens of novel type II Cas nucleases with diverse PAM recognitions and protein sizes. We then packaged one high-fidelity Cas ribonucleoprotein (RNP) complex in a protein delivery vehicle (PDV). The PDV-delivered Cas RNP showed generally higher editing potency than plasmid expression and lipofectamine transfection at ten endogenous genetic loci, and with lower off-target editing at tested sites that were identified by GUIDE-seq. In vivo experiments in mice demonstrated that the Cas RNP-PDV achieved an editing efficiency of up to 69%, reaching the theoretical limit of about 70% of hepatocytes in liver tissue. The transient nature of RNP delivered by PDV avoided prolonged Cas9 exposure, reducing the risk of off-target editing.

This platform demonstrates a significant advancement in in vivo genome editing, offering a safer and more efficient alternative to some non-RNP-based delivery strategies. Furthermore, our modular combination allows adaptation to diverse Cas nucleases encapsulated by PDV, broadening applications in cell and genome editing therapies.

@inproceedings{Cas9_catalogue,
  author = {Wang, B and Cao, Y and Yu, M and et al.},
  journal = {American Society of Gene & Cell Therapy (Poster)},
  year = {2024},
  title = {A Catalogue of Novel Type II CRISPR Nucleases with Diverse PAM Preferences},
  file = {},
  fileurl = {https://annualmeeting.asgct.org/program/print-program},
  excerpt = {This work is about the identification and characterization of novel type II Cas nucleases with diverse PAM restrictions (e.g. C-riched, A-riched, ACT, etc.) and protein sizes (1000aa - 1600aa).}
}

Clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR-Cas) system holds great promise to revolutionize genome editing and therapy in basic and translational settings. Various CRISPR-Cas systems have been explored and engineered, such as CRISPR-Cas9 and CRISPR-Cas12a that belong to the Class II of CRISPR-Cas system. However, therapeutics applications of these tools can be limited due to the restriction of the protospacer adjacent motif (PAM)-related targetability and potential off-target effects.

To address these challenges, we established and streamlined an efficient, fast, high-throughput platform for screening natural novel CRISPR-Cas nucleases. With this new platform, we identified and characterized dozens of novel type II Cas nucleases with diverse PAM restrictions and protein sizes. We selected three representatives with A-rich, C-rich, and AC PAM recognitions for detailed optimization and engineering. The A-rich PAM nuclease (GEBxII108) showed generally comparable editing potency compared to the well-documented Streptococcus pyogenes (SpCas9) in human cells, and with higher fidelities at tested sites than SpCas9 as evaluated by GUIDE-seq. We optimized single guide RNA (sgRNA), which remarkably enhanced the potency in primary cell using RNA format, enabling fully functioning. For the C-rich PAM nuclease (GEBxII920), we mutated key amino acids at the endonuclease domain to be used as a nickase. The results showed that it was functional when fused with an adenine base editor, with an expanded base editing window (A4 to A16), which might be useful for base editing at the cis-regulatory region (e.g. promoter) or splicing site. There are numerous scenarios where disease-causing mutations are not in an ideal distance to NGG - the PAM sequence of the most widely used SpCas9, making it necessary to use nucleases with alternative PAM recognitions. We identified a nuclease (GEBxII901) that recognizes a 5’-NRHACT-3’ PAM, where the ‘ACT’ bases are highly restrictive. We utilized this restriction property for an allele-specific targeting application. The result showed a significant discriminative power on editing mutation-containing allele than the WT allele by around 90 folds. A base editor based on this GEBxII901-engineered nickase showed higher potencies compared to SpCas9-base editor at tested target loci.

Based on our novel Class II Cas nuclease mining platform, we have identified a collection of type II Cas nucleases with diverse PAM restrictions that could be useful alternatives to SpCas9 in cell and genome editing therapy development.

@inproceedings{Baecoded_LNP,
  author = {Xia, Q and Wang, B and Zhou, Z and et al.},
  journal = {American Society of Gene & Cell Therapy (Poster)},
  year = {2023},
  title = {High-Throughput Screening Platform of Lipid Nanoparticles for In Vivo CRISPR/Cas Gene Editing},
  file = {},
  fileurl = {https://annualmeeting.asgct.org/program/print-program},
  excerpt = {This work is about the in vivo high-throughput screening platform using barcoded LNP harboring functional CRISPR/Cas system.}
}

Gene editing mediated by CRISPR/Cas holds great promise for gene therapy. Efficient delivery of CRISPR/Cas systems to specific tissue is one of the greatest challenges in in vivo gene editing. Lipid nanoparticle (LNP)-based mRNA delivery represents a clinically proven and effective nonviral delivery system that is gaining attractions across the fields of the nucleic acid-based therapies. Previously, thousands of novel lipid components and LNP compositions were screened either in cell culture or in vivo contexts to evaluate mRNA delivery and expression level. However, these screening strategies may not be applicable on CRISPR/Cas9 mRNA-based in vivo gene editing, which needs codelivery of two components, both Cas9 mRNA and target sgRNA, and function as ribonucleoprotein complex in a cell that typically contains only two copies of target alleles. A high-throughput LNP screening platform for in vivo delivery of functional CRISPR/Cas9 system is in great demand.

Here, we describe and validate a screening platform that employs a direct quantification of barcoded LNP harboring functional CRISPR/Cas complex, enabling high-throughput and straightforward readouts for editing efficiency of individual LNPs when delivered in a pooled format in vivo. We envision that the high-throughput platform could be broadly applicable to screen delivery to other tissues beyond liver, with non-lipid nanoparticles and beyond Cas nucleases, therefore accelerating in vivo gene editing therapeutic applications.

@inproceedings{Cas_discovery,
  author = {Wang, B and Wong, N WY and Yu, M and et al.},
  journal = {American Society of Gene & Cell Therapy (Poster)},
  year = {2023},
  title = {Development of De Novo Mining Platform for Discovery and Identification of Novel Small Class II CRISPR Nucleases with Expanded PAM Preference},
  file = {},
  fileurl = {https://annualmeeting.asgct.org/program/print-program},
  excerpt = {This work is about the novel CRISPR-Cas discovery platform based-on metagenome mining, human genome PAM assay, and structural-informed engineering.}
}

Clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR-Cas) system holds great promise to revolutionize genome editing and therapy in basic and translational settings. Various CRISPR-Cas systems have been explored and engineered, such as CRISPR-Cas9 and CRISPR-Cas12a that belong to the Class 2 of CRISPR-Cas system. However, in vivo applications of these tools remain limited in certain delivery methods due to their relatively large payload size, PAM restrictions and potential off-target effects.

Here, we mined an extensive genome-resolved metagenomics database and identified an array of novel CRISPR effectors (300 to 700 amino acids) from natural environments. We streamlined the pipeline of predicting and identifying crRNA and tracrRNA from CRISPR operon by RuvC domain-based Hidden Markov Models and small RNA-seq, respectively. We also developed a robust screening method in mammalian cells for rapid identification of the recognizable PAM of each Cas effector. Our platform uncovered dozens of novel Cas nucleases or derivations with diverse PAM preferences. We selected two representative Cas and further engineered both the protein and single guide RNA based on the modeled R-loop complex structure. We show that these novel Cas nucleases discovered by our platform are potent, specific, and have translational potentials for broad use in cell and gene therapy development.

@inproceedings{EDITED-seq,
  author = {Wang, B and Yu, M and Zheng, Z},
  journal = {Cold Spring Harbor Laboratory Conference (Poster)},
  year = {2022},
  title = {Sensitive One-step Discovery and Validation of CRISPR Off-targets Using EDITED-Seq},
  file = {},
  fileurl = {},
  excerpt = {This work established a sensitive approach for profiling the off-target effects of both in vitro and in vivo CRISPR-edited genome.}
}

Targeted genome editing such as using the CRISPR-Cas system holds great promise to treat genetic diseases. However, the potential off-target effects are important issues to address, especially when in vivo gene editing using the CRISPR-Cas9 system is being tested in clinical trials. Various methods have been developed to characterize and nominate CRISPR genome-wide offtarget effects, however, methods for simultaneously discovering new off-targets and validating known potential off-targets directly following natural gene editing events are largely lacking. Here, we introduce editing event detection by sequencing (EDITED-Seq) as a versatile approach to simultaneously detect new and validate known or predicted potential off-target sites; and is compatible with regular gene editing protocols in cells without extra cell manipulations.

@unpublished{Empty,
  author = {Empty},
  title = {},
  note = {},
  doi = {},
  file = {}
}