Doctor of Philosophy, Chinese Academy Of Sciences (2017)
Stanley Qi, Postdoctoral Faculty Sponsor
Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing.
Compact and versatile CRISPR-Cas systems will enable genome engineering applications through high-efficiency delivery in a wide variety of contexts. Here, we create an efficient miniature Cas system (CasMINI) engineered from the type V-F Cas12f (Cas14) system by guide RNA and protein engineering, which is less than half the size of currently used CRISPR systems (Cas9 or Cas12a). We demonstrate that CasMINI can drive high levels of gene activation (up to thousands-fold increases), while the natural Cas12f system fails to function in mammalian cells. We show that the CasMINI system has comparable activities to Cas12a for gene activation, is highly specific, and allows robust base editing and gene editing. We expect that CasMINI can be broadly useful for cell engineering and gene therapy applications ex vivo and in vivo.
View details for DOI 10.1016/j.molcel.2021.08.008
View details for PubMedID 34480847
Anti-CRISPR-mediated control of gene editing and synthetic circuits in eukaryotic cells.
2019; 10 (1): 194
Repurposed CRISPR-Cas molecules provide a useful tool set for broad applications of genomic editing and regulation of gene expression in prokaryotes and eukaryotes. Recent discovery of phage-derived proteins, anti-CRISPRs, which serve to abrogate natural CRISPR anti-phage activity, potentially expands the ability to build synthetic CRISPR-mediated circuits. Here, we characterize a panel of anti-CRISPR molecules for expanded applications to counteract CRISPR-mediated gene activation and repression of reporter and endogenous genes in various cell types. We demonstrate that cells pre-engineered with anti-CRISPR molecules become resistant to gene editing, thus providing a means to generate "write-protected" cells that prevent future gene editing. We further show that anti-CRISPRs can be used to control CRISPR-based gene regulation circuits, including implementation of a pulse generator circuit in mammalian cells. Our work suggests that anti-CRISPR proteins should serve as widely applicable tools for synthetic systems regulating the behavior of eukaryotic cells.
View details for PubMedID 30643127
- A CRISPR-dCas Toolbox for Genetic Engineering and Synthetic Biology JOURNAL OF MOLECULAR BIOLOGY 2019; 431 (1): 34–47
CRISPR-Mediated Programmable 3D Genome Positioning and Nuclear Organization.
Programmable control of spatial genome organization is a powerful approach for studying how nuclear structure affects gene regulation and cellular function. Here, we develop a versatile CRISPR-genome organization (CRISPR-GO) system that can efficiently control the spatial positioning of genomic loci relative to specific nuclear compartments, including the nuclear periphery, Cajal bodies, and promyelocytic leukemia (PML) bodies. CRISPR-GO is chemically inducible and reversible, enabling interrogation of real-time dynamics of chromatin interactions with nuclear compartments in living cells. Inducible repositioning of genomic loci to the nuclear periphery allows for dissection of mitosis-dependent and -independent relocalization events and also for interrogation of the relationship between gene position and gene expression. CRISPR-GO mediates rapid de novo formation of Cajal bodies at desired chromatin loci and causes significant repression of endogenous gene expression over long distances (30-600 kb). The CRISPR-GO system offers a programmable platform to investigate large-scale spatial genome organization and function.
View details for PubMedID 30318144
- Streptomyces virginiae PPDC Is a New Type of Phenylpyruvate Decarboxylase Composed of Two Subunits ACS Chemical Biology 2017; 12 (8): 2008-2014
- CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum Nature Communications 2017; 8 (15179)
- Mutagenesis of Key Residues in the Binding Center of l-Aspartate-β-Semialdehyde Dehydrogenase from Escherichia coli Enhances Utilization of the Cofactor NAD(H) Chembiochem 2016; 17 (1): 56-64
Phytosulfokine Is Involved in Positive Regulation of Lotus japonicus Nodulation
MOLECULAR PLANT-MICROBE INTERACTIONS
2015; 28 (8): 847-855
Phytosulfokine (PSK) is a tyrosine-sulfated peptide that is widely distributed in plants, participating in cell proliferation, differentiation, and innate immunity. The potential role of PSK in nodulation in legumes has not been reported. In this work, five PSK precursor genes were identified in Lotus japonicas, designated as LjPSK1 to LjPSK5. Three of them (LjPSK1, LjPSK4, and LjPSK5) were found to be expressed in nitrogen-fixing root nodules. LjPSK1 and LjPSK4 were not induced at the early stage of nodulation. Interestingly, while the expression of LjPSK4 was also found in spontaneous nodules without rhizobial colonization, LjPSK1 was not induced in these pseudo nodules. Promoter-β-glucuronidase analysis revealed that LjPSK1 was highly expressed in enlarged symbiotic cells of nodules. Exogenous addition of 1 1M synthetic PSK peptide resulted in increased nodule numbers per plant. Consistently, the number of mature nodules but not the events of rhizobial infection and nodule initiation was increased by overexpressing LjPSK1 in transgenic hairy roots, in which the expression of jasmonate-responsive genes was found to be repressed. These results suggest that PSK is a new peptide signal that regulates nodulation in legumes, probably through cross-talking with other phytohormones.
View details for DOI 10.1094/MPMI-02-15-0032-R
View details for Web of Science ID 000359434700001
View details for PubMedID 25775272
- Involvement of ROP6 and clathrin in nodulation factor signaling Plant Signaling & Behavior 2015; 10 (7)