Publications by authors named "Snigdha Poddar"

4 Publications

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Efficient isolation of protoplasts from rice calli with pause points and its application in transient gene expression and genome editing assays.

Plant Methods 2020 Nov 12;16(1):151. Epub 2020 Nov 12.

Innovative Genomics Institute, University of California, Berkeley, CA, 94720, USA.

Background: An efficient in vivo transient transfection system using protoplasts is an important tool to study gene expression, metabolic pathways, and multiple mutagenesis parameters in plants. Although rice protoplasts can be isolated from germinated seedlings or cell suspension culture, preparation of those donor tissues can be inefficient, time-consuming, and laborious. Additionally, the lengthy process of protoplast isolation and transfection needs to be completed in a single day.

Results: Here we report a protocol for the isolation of protoplasts directly from rice calli, without using seedlings or suspension culture. The method is developed to employ discretionary pause points during protoplast isolation and before transfection. Protoplasts maintained within a sucrose cushion partway through isolation, for completion on a subsequent day, per the first pause point, are referred to as S protoplasts. Fully isolated protoplasts maintained in MMG solution for transfection on a subsequent day, per the second pause point, are referred to as M protoplasts. Both S and M protoplasts, 1 day after initiation of protoplast isolation, had minimal loss of viability and transfection efficiency compared to protoplasts 0 days after isolation. S protoplast viability decreases at a lower rate over time than that of M protoplasts and can be used with added flexibility for transient transfection assays and time-course experiments. The protoplasts produced by this method are competent for transfection of both plasmids and ribonucleoproteins (RNPs). Cas9 RNPs were used to demonstrate the utility of these protoplasts to assay genome editing in vivo.

Conclusion: The current study describes a highly effective and accessible method to isolate protoplasts from callus tissue induced from rice seeds. This method utilizes donor materials that are resource-efficient and easy to propagate, permits convenience via pause points, and allows for flexible transfection days after protoplast isolation. It provides an advantageous and useful platform for a variety of in vivo transient transfection studies in rice.
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http://dx.doi.org/10.1186/s13007-020-00692-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7663885PMC
November 2020

Engineering Kluyveromyces marxianus as a Robust Synthetic Biology Platform Host.

mBio 2018 09 25;9(5). Epub 2018 Sep 25.

Department of Molecular and Cell Biology, University of California, Berkeley, California, USA

Throughout history, the yeast has played a central role in human society due to its use in food production and more recently as a major industrial and model microorganism, because of the many genetic and genomic tools available to probe its biology. However, has proven difficult to engineer to expand the carbon sources it can utilize, the products it can make, and the harsh conditions it can tolerate in industrial applications. Other yeasts that could solve many of these problems remain difficult to manipulate genetically. Here, we engineered the thermotolerant yeast to create a new synthetic biology platform. Using CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats with Cas9)-mediated genome editing, we show that wild isolates of can be made heterothallic for sexual crossing. By breeding two of these mating-type engineered strains, we combined three complex traits-thermotolerance, lipid production, and facile transformation with exogenous DNA-into a single host. The ability to cross strains with relative ease, together with CRISPR-Cas9 genome editing, should enable engineering of isolates with promising lipid production at temperatures far exceeding those of other fungi under development for industrial applications. These results establish as a synthetic biology platform comparable to , with naturally more robust traits that hold potential for the industrial production of renewable chemicals. The yeast grows at high temperatures and on a wide range of carbon sources, making it a promising host for industrial biotechnology to produce renewable chemicals from plant biomass feedstocks. However, major genetic engineering limitations have kept this yeast from replacing the commonly used yeast in industrial applications. Here, we describe genetic tools for genome editing and breeding strains, which we use to create a new thermotolerant strain with promising fatty acid production. These results open the door to using as a versatile synthetic biology platform organism for industrial applications.
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http://dx.doi.org/10.1128/mBio.01410-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6156195PMC
September 2018

CRISPR-Cas9 Genome Engineering in Saccharomyces cerevisiae Cells.

Cold Spring Harb Protoc 2016 06 1;2016(6). Epub 2016 Jun 1.

Energy Biosciences Institute, University of California, Berkeley, California 94720; Department of Molecular and Cell Biology, University of California, Berkeley, California 94720; Department of Chemistry, University of California, Berkeley, California 94720.

This protocol describes a method for CRISPR-Cas9-mediated genome editing that results in scarless and marker-free integrations of DNA into Saccharomyces cerevisiae genomes. DNA integration results from cotransforming (1) a single plasmid (pCAS) that coexpresses the Cas9 endonuclease and a uniquely engineered single guide RNA (sgRNA) expression cassette and (2) a linear DNA molecule that is used to repair the chromosomal DNA damage by homology-directed repair. For target specificity, the pCAS plasmid requires only a single cloning modification: replacing the 20-bp guide RNA sequence within the sgRNA cassette. This CRISPR-Cas9 protocol includes methods for (1) cloning the unique target sequence into pCAS, (2) assembly of the double-stranded DNA repair oligonucleotides, and (3) cotransformation of pCAS and linear repair DNA into yeast cells. The protocol is technically facile and requires no special equipment. It can be used in any S. cerevisiae strain, including industrial polyploid isolates. Therefore, this CRISPR-Cas9-based DNA integration protocol is achievable by virtually any yeast genetics and molecular biology laboratory.
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http://dx.doi.org/10.1101/pdb.prot086827DOI Listing
June 2016

Selection of chromosomal DNA libraries using a multiplex CRISPR system.

Elife 2014 Aug 19;3. Epub 2014 Aug 19.

Energy Biosciences Institute, University of California, Berkeley, Berkeley, United States.

The directed evolution of biomolecules to improve or change their activity is central to many engineering and synthetic biology efforts. However, selecting improved variants from gene libraries in living cells requires plasmid expression systems that suffer from variable copy number effects, or the use of complex marker-dependent chromosomal integration strategies. We developed quantitative gene assembly and DNA library insertion into the Saccharomyces cerevisiae genome by optimizing an efficient single-step and marker-free genome editing system using CRISPR-Cas9. With this Multiplex CRISPR (CRISPRm) system, we selected an improved cellobiose utilization pathway in diploid yeast in a single round of mutagenesis and selection, which increased cellobiose fermentation rates by over 10-fold. Mutations recovered in the best cellodextrin transporters reveal synergy between substrate binding and transporter dynamics, and demonstrate the power of CRISPRm to accelerate selection experiments and discoveries of the molecular determinants that enhance biomolecule function.
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http://dx.doi.org/10.7554/eLife.03703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4161972PMC
August 2014