A research individual with more than ten years of extensive research experience in molecular biology techniques ranging from DNA/RNA manipulations, recombinant protein expression, purification, and biochemical characterization of DNA-cutting enzymes, needed for genetic engineering. I am a postdoctoral research fellow in the Department of Genetics, Stanford University. I work with Dr. Mike Snyder. My research involves multiplexed-molecular imaging for the precancerous and cancerous colon polyps (Familial adenomatous polyposis) using a cutting-edge technology, Codex. My previous work involved novel gene therapy approaches to cure muscle disorders, particularly Limb-girdle muscular dystrophy 2A using mouse models, and gene repair studies using modified CRISPR system through plasmid/protein-oligo based transfections, electroporation in human cells, microinjection in frog embryos and analyzing editing efficiencies using flow cytometry. I have a multi-disciplinary background, therefore I have a solid understanding and working knowledge in a broader domain within the biological sciences, be it from animal behavior, ecology, gene regulation, genetic diseases to understanding and designing “molecular switches” within DNA-cutting proteins, such as meganucleases, and CRISPR/Cas9 for genome engineering. I have strong communication skills, presented my research in various conferences and published heavily in the field of DNA-cutting enzymes and their use as genome editing tools. I have a proven ability to manage challenging research objectives, collaborated with other research teams, and delivered results effectively. I always welcome new ideas and interact with people to learn any new skills and experiences. I have also supervised several undergraduate project students, summer students and junior graduate students. My mentorship to the lab colleagues have been very productive. While I am not working, I enjoy photography.
Honors & Awards
International Graduate Student Scholarship (IGSS), CAD 8000, University of Manitoba (2012 - 2013)
Faculty of Science Graduate Scholarship (FSGS), CAD 10000 / year, University of Manitoba (2011 - 2013)
International Graduate Student Entrance Scholarship (IGSES), CAD 8000, University of Manitoba (2009 -2010)
Master of Science, University of Manitoba (2011)
Master of Science, University Of Calcutta (2006)
Bachelor of Science, University Of Calcutta (2004)
Doctor of Philosophy, University of Manitoba (2016)
Michael Snyder, Postdoctoral Faculty Sponsor
The Human Tumor Atlas Network: Charting Tumor Transitions across Space and Time at Single-Cell Resolution.
2020; 181 (2): 236–49
Crucial transitions in cancer-including tumor initiation, local expansion, metastasis, and therapeutic resistance-involve complex interactions between cells within the dynamic tumor ecosystem. Transformative single-cell genomics technologies and spatial multiplex in situ methods now provide an opportunity to interrogate this complexity at unprecedented resolution. The Human Tumor Atlas Network (HTAN), part of the National Cancer Institute (NCI) Cancer Moonshot Initiative, will establish a clinical, experimental, computational, and organizational framework to generate informative and accessible three-dimensional atlases of cancer transitions for a diverse set of tumor types. This effort complements both ongoing efforts to map healthy organs and previous large-scale cancer genomics approaches focused on bulk sequencing at a single point in time. Generating single-cell, multiparametric, longitudinal atlases and integrating them with clinical outcomes should help identify novel predictive biomarkers and features as well as therapeutically relevant cell types, cell states, and cellular interactions across transitions. The resulting tumor atlases should have a profound impact on our understanding of cancer biology and have the potential to improve cancer detection, prevention, and therapeutic discovery for better precision-medicine treatments of cancer patients and those at risk for cancer.
View details for DOI 10.1016/j.cell.2020.03.053
View details for PubMedID 32302568
Nucleofection of phiC31 Integrase Protein Mediates Sequence-Specific Genomic Integration in Human Cells.
Journal of molecular biology
The phage-derived phiC31 integrase is a useful tool for mediating sequence-specific genomic integration in mammalian cells, recombining donor plasmids bearing the attB recognition site with introduced genomic attP sites or endogeneous pseudo-attP sites having partial identity to attP. In most prior studies, phiC31 integrase has been introduced as plasmid DNA or mRNA. The current report examines whether phiC31 integrase functions efficiently in mammalian cells when co-nucleofected as a purified protein, along with attB-containing donor plasmids or PCR fragments. We describe preparation of phiC31 integrase protein and evidence that it can mediate genomic integration in human 293 cells, including PCR evidence for integration at an endogenous pseudo-attP site. This work demonstrates for the first time the ability of 605- and 613-amino acid versions of phiC31 integrase protein to mediate efficient, site-specific integration into the genome of human cells when co-nucleofected with full-sized attB-containing donor plasmids or linear 2.5-kb PCR fragments. This protein-mediated approach may be especially useful for integration of exogenous sequences into valuable therapeutic target cells, such as hematopoietic stem cells or T cells, that are sensitive to introduced DNA.
View details for DOI 10.1016/j.jmb.2020.04.019
View details for PubMedID 32339531
- Plasmid-Mediated Gene Therapy in Mouse Models of Limb Girdle Muscular Dystrophy MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT 2019; 15: 294–304
Production of Endoglucanase and Xylanase Using Food Waste by Solid-State Fermentation
Waste and Biomass Valorization
View details for DOI 10.1007/s12649-017-0192-7
Three new active members of the I-OnuI family of homing endonucleases.
Canadian journal of microbiology
In vitro characterization of 3 LAGLIDADG-type homing endonucleases (HEs) (I-CcaI, I-CcaII, and I-AstI) that belong to the I-OnuI family showed that they are functional HEs that cleave their respective cognate target sites. These endonucleases are encoded within group ID introns and appear to be orthologues that have inserted into 3 different mitochondrial genes: rns, rnl, and cox3. The endonuclease activity of I-CcaI was tested using various substrates, and its minimum DNA recognition sequence was estimated to be 26 nt. This set of HEs may provide some insight into how these types of mobile elements can migrate into new locations. This study provides additional endonucleases that can be added to the catalog of currently available HEs that may have various biotechnology applications.
View details for DOI 10.1139/cjm-2017-0067
View details for PubMedID 28414922
The intron landscape of the mtDNA cytb gene among the Ascomycota: introns and intron-encoded open reading frames.
Mitochondrial DNA. Part A, DNA mapping, sequencing, and analysis
Fungal mitochondrial genes are frequently noted for the presence of introns. These introns are self-splicing and can be assigned to either group I or II introns and they can encode open reading frames (ORFs). This study examines the introns present within the cytochrome b (cytb) gene of ascomycetes fungi. Cytochrome b gene sequences were sampled from GenBank and supplemented with our own data for species of Leptographium and Ophiostoma. Group I introns were encountered most frequently, many encoding either LAGLIDADG or GIY-YIG homing endonucleases (HEs). Numerous examples of different intron/ORF arrangements were observed including nested ORFs, multiple ORFs within a single intron and intron ORFs at various stages of erosion due to the accumulation of mutations. In addition, we noted one example of a nested intron and one complex group II intron that could potentially allow for alternative splicing. Documenting the distribution of introns within the same gene across a range of species allows for a better understanding of the evolution of introns and intronic ORFs. Intron landscapes also are a resource that can help in annotating genes and in bioprospecting for potentially active HEs, which are rare-cutting DNA endonucleases with applications in biotechnology.
View details for DOI 10.1080/24701394.2017.1404042
View details for PubMedID 29157056
Applications of Alternative Nucleases in the Age of CRISPR/Cas9.
International journal of molecular sciences
2017; 18 (12)
Breakthroughs in the development of programmable site-specific nucleases, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases (MNs), and most recently, the clustered regularly interspaced short palindromic repeats (CRISPR) associated proteins (including Cas9) have greatly enabled and accelerated genome editing. By targeting double-strand breaks to user-defined locations, the rates of DNA repair events are greatly enhanced relative to un-catalyzed events at the same sites. However, the underlying biology of each genome-editing nuclease influences the targeting potential, the spectrum of off-target cleavages, the ease-of-use, and the types of recombination events at targeted double-strand breaks. No single genome-editing nuclease is optimized for all possible applications. Here, we focus on the diversity of nuclease domains available for genome editing, highlighting biochemical properties and the potential applications that are best suited to each domain.
View details for DOI 10.3390/ijms18122565
View details for PubMedID 29186020
View details for PubMedCentralID PMC5751168
Programmable Genome Editing Tools and their Regulation for Efficient Genome Engineering.
Computational and structural biotechnology journal
2017; 15: 146-160
Targeted genome editing has become a powerful genetic tool for studying gene function or for modifying genomes by correcting defective genes or introducing genes. A variety of reagents have been developed in recent years that can generate targeted double-stranded DNA cuts which can be repaired by the error-prone, non-homologous end joining repair system or via the homologous recombination-based double-strand break repair pathway provided a suitable template is available. These genome editing reagents require components for recognizing a specific DNA target site and for DNA-cleavage that generates the double-stranded break. In order to reduce potential toxic effects of genome editing reagents, it might be desirable to control the in vitro or in vivo activity of these reagents by incorporating regulatory switches that can reduce off-target activities and/or allow for these reagents to be turned on or off. This review will outline the various genome editing tools that are currently available and describe the strategies that have so far been employed for regulating these editing reagents. In addition, this review will examine potential regulatory switches/strategies that can be employed in the future in order to provide temporal control for these reagents.
View details for DOI 10.1016/j.csbj.2016.12.006
View details for PubMedID 28179977
View details for PubMedCentralID PMC5279741
Insertion of Group II Intron-Based Ribozyme Switches into Homing Endonuclease Genes.
Methods in molecular biology (Clifton, N.J.)
2017; 1498: 135-152
Fungal mitochondrial genomes act as "reservoirs" for homing endonucleases. These enzymes with their DNA site-specific cleavage activities are attractive tools for genome editing, targeted mutagenesis and gene therapy applications. Herein, we present strategies where homing endonuclease open reading frames (HEases ORFs) are interrupted with group II intron sequences. The ultimate goal is to achieve in vivo expression of HEases that can be regulated by manipulating the splicing efficiency of the HEase ORF-embedded group II introns. That addition of exogenous magnesium chloride (MgCl2) appears to stimulate splicing of nonnative group II introns in Escherichia coli and the addition of cobalt chloride (CoCl2) to the growth medium antagonizes the expression of HEase activity (i.e., splicing). Group II introns are potentially autocatalytic self-splicing elements and thus can be used as molecular switches that allow for temporal regulated HEase expression. This should be useful in precision genome engineering, mutagenesis, and minimizing off-target activities.
View details for PubMedID 27709573
Using Group II Introns for Attenuating the In Vitro and In Vivo Expression of a Homing Endonuclease
2016; 11 (2)
In Chaetomium thermophilum (DSM 1495) within the mitochondrial DNA (mtDNA) small ribosomal subunit (rns) gene a group IIA1 intron interrupts an open reading frame (ORF) encoded within a group I intron (mS1247). This arrangement offers the opportunity to examine if the nested group II intron could be utilized as a regulatory element for the expression of the homing endonuclease (HEase). Constructs were generated where the codon-optimized ORF was interrupted with either the native group IIA1 intron or a group IIB type intron. This study showed that the expression of the HEase (in vivo) in Escherichia coli can be regulated by manipulating the splicing efficiency of the HEase ORF-embedded group II introns. Exogenous magnesium chloride (MgCl2) stimulated the expression of a functional HEase but the addition of cobalt chloride (CoCl2) to growth media antagonized the expression of HEase activity. Ultimately the ability to attenuate HEase activity might be useful in precision genome engineering, minimizing off target activities, or where pathways have to be altered during a specific growth phase.
View details for DOI 10.1371/journal.pone.0150097
View details for Web of Science ID 000371164700067
View details for PubMedID 26909494
View details for PubMedCentralID PMC4801052
I-OmiI and I-OmiII: Two intron-encoded homing endonucleases within the Ophiostoma minus rns gene
2014; 118 (8): 721-731
The mitochondrial small subunit ribosomal RNA (rns) gene of the ascomycetous fungus Ophiostoma minus [strain WIN(M)371] was found to contain a group IC2 and a group IIB1 intron at positions mS569 and mS952 respectively. Both introns have open reading frames (ORFs) embedded that encode double motif LAGLIDADG homing endonucleases (I-OmiI and I-OmiII respectively). Codon-optimized versions of I-OmiI and I-OmiII were synthesized for overexpression in Escherichia coli. The in vitro characterization of I-OmiII showed that it is a functional homing endonuclease that cleaves the rns target site two nucleotides upstream (sense strand) of the intron insertion site generating 4 nucleotide 3' overhangs. The endonuclease activity of I-OmiII was tested using linear and circular substrates and cleavage activity was evaluated at various temperatures. The I-OmiI protein was expressed in E. coli, but purification was difficult, thus the endonuclease activity of this protein was tested via in vivo assays. Overall this study showed that there are many native forms of functional homing endonucleases yet to be discovered among fungal mtDNA genomes.
View details for DOI 10.1016/j.funbio.2014.05.002
View details for Web of Science ID 000341349200007
View details for PubMedID 25110134
A homing endonuclease with a switch: Characterization of a twintron encoded homing endonuclease
FUNGAL GENETICS AND BIOLOGY
2014; 65: 57-68
The small ribosomal subunit gene residing in the mitochondrial DNA of the thermophilic fungus Chaetomium thermophilum var. thermophilum La Touche DSM 1495 is interrupted by a twintron at position mS1247. The mS1247 twintron represents the first mixed twintron found in fungal mtDNA, composed of an external group I intron encoding a LAGLIDADG open reading frame that is interrupted by an internal group II intron. Splicing of the internal group II intron reconstitutes the open reading frame and thus facilitates the expression of the encoded homing endonuclease. The cleavage assays suggest that the twintron encodes an active homing endonuclease that could potentially mobilize the twintron to rns genes that have not yet been invaded by this mobile composite element.
View details for DOI 10.1016/j.fgb.2014.01.004
View details for Web of Science ID 000333499100006
View details for PubMedID 24508098
PCR-based bioprospecting for homing endonucleases in fungal mitochondrial rRNA genes.
Methods in molecular biology (Clifton, N.J.)
2014; 1123: 37-53
Fungal mitochondrial genomes act as "reservoirs" for homing endonucleases. These enzymes with their DNA site-specific cleavage activities are attractive tools for genome editing and gene therapy applications. Bioprospecting and characterization of naturally occurring homing endonucleases offers an alternative to synthesizing artificial endonucleases. Here, we describe methods for PCR-based screening of fungal mitochondrial rRNA genes for homing endonuclease encoding sequences, and we also provide protocols for the purification and biochemical characterization of putative native homing endonucleases.
View details for DOI 10.1007/978-1-62703-968-0_3
View details for PubMedID 24510258