Chengkun Wang got his PhD from Zhejiang University in 2018.
Bachelor of Science, Shandong Teachers College (2012)
Doctor of Philosophy, Zhejiang University (2018)
Doctor of Pharmacy, Zhejiang University (2018)
Bachelor of Chem Engineering, Shandong Teachers College (2018)
Machine-learning-optimized Cas12a barcoding enables the recovery of single-cell lineages and transcriptional profiles.
The development of CRISPR-based barcoding methods creates an exciting opportunity to understand cellular phylogenies. We present a compact, tunable, high-capacity Cas12a barcoding system called dual acting inverted site array (DAISY). We combined high-throughput screening and machine learning to predict and optimize the 60-bp DAISY barcode sequences. After optimization, top-performing barcodes had ∼10-fold increased capacity relative to the best random-screened designs and performed reliably across diverse cell types. DAISY barcode arrays generated ∼12 bits of entropy and ∼66,000 unique barcodes. Thus, DAISY barcodes-at a fraction of the size of Cas9 barcodes-achieved high-capacity barcoding. We coupled DAISY barcoding with single-cell RNA-seq to recover lineages and gene expression profiles from ∼47,000 human melanoma cells. A single DAISY barcode recovered up to ∼700 lineages from one parental cell. This analysis revealed heritable single-cell gene expression and potential epigenetic modulation of memory gene transcription. Overall, Cas12a DAISY barcoding is an efficient tool for investigating cell-state dynamics.
View details for DOI 10.1016/j.molcel.2022.06.001
View details for PubMedID 35752172
dCas9-based gene editing for cleavage-free genomic knock-in of long sequences.
Nature cell biology
Gene editing is a powerful tool for genome and cell engineering. Exemplified by CRISPR-Cas, gene editing could cause DNA damage and trigger DNA repair processes that are often error-prone. Such unwanted mutations and safety concerns can be exacerbated when altering long sequences. Here we couple microbial single-strand annealing proteins (SSAPs) with catalytically inactive dCas9 for gene editing. This cleavage-free gene editor, dCas9-SSAP, promotes the knock-in of long sequences in mammalian cells. The dCas9-SSAP editor has low on-target errors and minimal off-target effects, showing higher accuracy than canonical Cas9 methods. It is effective for inserting kilobase-scale sequences, with an efficiency of up to approximately 20% and robust performance across donor designs and cell types, including human stem cells. We show that dCas9-SSAP is less sensitive to inhibition of DNA repair enzymes than Cas9 references. We further performed truncation and aptamer engineering to minimize its size to fit into a single adeno-associated-virus vector for future application. Together, this tool opens opportunities towards safer long-sequence genome engineering.
View details for DOI 10.1038/s41556-021-00836-1
View details for PubMedID 35145221
CRISPR-Cas12a System With Synergistic Phage Recombination Proteins for Multiplex Precision Editing in Human Cells.
Frontiers in cell and developmental biology
2021; 9: 719705
The development of CRISPR-based gene-editing technologies has brought an unprecedented revolution in the field of genome engineering. Cas12a, a member of the Class 2 Type V CRISPR-associated endonuclease family distinct from Cas9, has been repurposed and developed into versatile gene-editing tools with distinct PAM recognition sites and multiplexed gene targeting capability. However, with current CRISPR/Cas12a technologies, it remains a challenge to perform efficient and precise genome editing of long sequences in mammalian cells. To address this limitation, we utilized phage recombination enzymes and developed an efficient CRISPR/Cas12a tool for multiplexed precision editing in mammalian cells. Through protein engineering, we were able to recruit phage recombination proteins to Cas12a to enhance its homology-directed repair efficiencies. Our phage-recombination-assisted Cas12a system achieved up to 3-fold improvements for kilobase-scale knock-ins in human cells without compromising the specificity of the enzyme. The performance of this system compares favorably against Cas9 references, the commonly used enzyme for gene-editing tasks, with improved specificity. Additionally, we demonstrated multi-target editing with similar improved activities thanks to the RNA-processing activity of the Cas12a system. This compact, multi-target editing tool has the potential to assist in understanding multi-gene interactions. In particular, it paves the way for a gene therapy method for human diseases that complements existing tools and is suitable for polygenic disorders and diseases requiring long-sequence corrections.
View details for DOI 10.3389/fcell.2021.719705
View details for PubMedID 35774104
View details for PubMedCentralID PMC9237396
Cleavage-Free dCas9 Knock-In Gene-Editing Tool Leveraging RNA-Guided Targeting of Recombineering Proteins
CELL PRESS. 2021: 107
View details for Web of Science ID 000645188700204
Microbial single-strand annealing proteins enable CRISPR gene-editing tools with improved knock-in efficiencies and reduced off-target effects.
Nucleic acids research
Several existing technologies enable short genomic alterations including generating indels and short nucleotide variants, however,engineering more significant genomic changes is more challenging due to reduced efficiency and precision. Here, we developed RecT Editor via Designer-Cas9-Initiated Targeting (REDIT), which leverages phage single-stranded DNA-annealing proteins (SSAP) RecT for mammalian genome engineering. Relative to Cas9-mediated homology-directed repair (HDR), REDIT yielded up to a 5-fold increase of efficiency to insert kilobase-scale exogenous sequences at defined genomic regions. We validated our REDIT approach using different formats and lengths of knock-in templates. We further demonstrated that REDIT tools using Cas9 nickase have efficient gene-editing activities and reduced off-target errors, measured using a combination of targeted sequencing, genome-wide indel, and insertion mapping assays. Our experiments inhibiting repair enzyme activities suggested that REDIT has the potential to overcome limitations of endogenous DNA repair steps.Finally, our REDIT method is applicable across cell types including human stem cells,and is generalizable to different Cas9 enzymes.
View details for DOI 10.1093/nar/gkaa1264
View details for PubMedID 33619540