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1. A Course-Based Undergraduate Research Experience for High-Throughput Reverse Genetic Studies in Arabidopsis Thaliana with CRISPR-Cas9

   https://doi.org/10.20944/preprints202008.0619.v1  

   Mills, Alison ; Jagannatha, Venkateswari ; Cortez, Alejandro ; Guzman, Michael ; Burnette III, James M ; Collin, Matthew ; Lopez-Lopez, Berenise ; Wessler, Susan R ; Van Norman, Jaimie M ; Nelson, David C ; et al ( August 2020 , Preprints)

   Gene editing tools such as CRISPR-Cas9 have created unprecedented opportunities for genetic studies in plants and animals. We designed a course-based undergraduate research experience (CURE) to train introductory biology students in the concepts and implementation of gene editing technology as well as develop their soft skills in data management and scientific communication. We present two versions of the course that can be implemented with twice- weekly meetings over a five-week period. In the remote-learning version, students perform homology searches, design guide RNAs and primers, and learn the principles of molecular cloning. This version is appropriate when access to laboratory equipment or in-person instruction is limited, such as closures that have occurred in response to the Covid-19 pandemic. In the in-person version, students design guide RNAs, clone CRISPR-Cas9 constructs, and perform genetic transformation of the model plant Arabidopsis thaliana. The highly parallel nature of the CURE makes it possible to target dozens to hundreds of genes, depending on the number of course sections available. Applying this approach in a sensitized mutant background enables focused reverse genetic screens for genetic suppressors or enhancers. The course can be readily adapted to other organisms or projects that employ gene editing.
2. Efficient CRISPR/Cas9 gene editing in a tilapia cell line model using endogenous promoters

   Hamar, JC* ; Kültz, D ( January 2021 , SICB Virtual Annual Meeting 2021)

   Cell cultures are effective supplemental models to study specific biochemical pathways used for environmental adaption in animals. They enable isolation from system influence and facilitate control the extracellular environment. For work focusing on fish species many representative cell lines now exist, including a tilapia brain cell line (OmB) developed in our lab. CRISPR/Cas9 gene editing is an additional tool aiding these studies by allowing manipulation of specific genetic loci and evaluating their causal relationship between phenotypes of interest. However, established CRISPR/Cas9 gene targeting tools and methods often have not functioned as efficiently in fish cells as seen in other animal cell models such as mammalian cell lines, consistent with our initial attempts to apply CRISPR/Cas9 in OmB cells that failed to indicate genomic alteration at the targeted sites. Poor expression of heterologous promoters in OmB cells was hypothesized to be a primary cause for this occurrence so we constructed a custom plasmid vector based system utilizing tilapia endogenous promoters (EF1 alpha to express Cas9 and a U6 to express gRNAs). This system demonstrated substantial editing of most target sites attempted with mutational efficiency as high 80%. This work specifically highlighted the importance of phylogenetic proximity in selection of a polymerasemore »III promoter for gRNA expression as commonly used interspecies U6 promoters (human and zebrafish) yielded no detectable gene editing when applied in this system with a common gRNA target sequence. These new tools will allow generation of knockout cell lines for gene targeting studies in tilapia and other phylogenetically close fish species.« less
3. Structural and functional insights into the bona fide catalytic state of Streptococcus pyogenes Cas9 HNH nuclease domain

   https://doi.org/10.7554/eLife.46500  

   Zuo, Zhicheng ; Zolekar, Ashwini ; Babu, Kesavan ; Lin, Victor JT ; Hayatshahi, Hamed S ; Rajan, Rakhi ; Wang, Yu-Chieh ; Liu, Jin ( July 2019 , eLife)

   The DNA inside human cells provides instructions for all of the processes that happen inside the body. Errors in the DNA may lead to cancer, sickle cell disease, cystic fibrosis, Huntington’s disease, or other genetic disorders. Medical researchers are exploring whether it is possible to replace or repair the faulty DNA (an approach known as gene therapy) to reduce the symptoms, or even cure individuals, of these conditions. Over the last ten years, a new technology known as CRISPR-Cas9 gene editing has proved to be a reliable and efficient way to make small and precise changes to DNA in living cells. First, an enzyme called Cas9 searches for a segment of target DNA segment that matches a template molecule the enzyme carries. Cas9 then cuts the target DNA, which is repaired to match a new customized DNA sequence: this changes the genetic information of the cell. The Cas9 protein is made of a succession of building blocks called amino acids that create long chains which then fold to form the final three-dimensional shape of the enzyme. A region of Cas9 known as the HNH domain is responsible for cutting the target DNA. However, it remains unclear exactly which amino acidsmore »within this domain work together to sever the DNA. Here, Zuo et al. combined computational and experimental approaches to reveal the three-dimensional structure of the Cas9 enzyme when the HNH domain is poised to cut the target DNA. The findings were used to generate a computational model of Cas9 and this model predicted that the HNH domain relies on a group of three amino acids known collectively as D839-H840-N863 to cleave DNA strands. This knowledge is useful to understand exactly how Cas9 modifies genetic information. Ultimately, this may help to improve CRISPR-Cas9 technology so it could be safely used in geneediting therapies.« less
4. An efficient vector-based CRISPR/Cas9 system in an Oreochromis mossambicus cell line using endogenous promoters

   https://doi.org/10.1038/s41598-021-87068-3  

   Hamar, Jens ; Kültz, Dietmar ( April 2021 , Scientific Reports)

   CRISPR/Cas9 gene editing is effective in manipulating genetic loci in mammalian cell cultures and whole fish but efficient platforms applicable to fish cell lines are currently limited. Our initial attempts to employ this technology in fish cell lines using heterologous promoters or a ribonucleoprotein approach failed to indicate genomic alteration at targeted sites in a tilapia brain cell line (OmB). For potential use in a DNA vector approach, endogenous tilapia beta Actin (OmBAct), EF1 alpha (OmEF1a), and U6 (TU6) promoters were isolated. The strongest candidate promoter determined by EGFP reporter assay, OmEF1a, was used to drive constitutive Cas9 expression in a modified OmB cell line (Cas9-OmB1). Cas9-OmB1 cell transfection with vectors expressing gRNAs driven by the TU6 promoter achieved mutational efficiencies as high as 81% following hygromycin selection. Mutations were not detected using human and zebrafish U6 promoters demonstrating the phylogenetic proximity of U6 promoters as critical when used for gRNA expression. Sequence alteration to TU6 improved mutation rate and cloning efficiency. In conclusion, we report new tools for ectopic expression and a highly efficient, economical system for manipulation of genomic loci and evaluation of their causal relationship with adaptive cellular phenotypes by CRISPR/Cas9 gene editing in fish cells.
5. A CRISPRi-dCas9 System for Archaea and Its Use To Examine Gene Function during Nitrogen Fixation by Methanosarcina acetivorans

   https://doi.org/10.1128/AEM.01402-20  

   Dhamad, Ahmed E. ; Lessner, Daniel J. ( October 2020 , Applied and Environmental Microbiology)

   Atomi, Haruyuki (Ed.)

   ABSTRACT CRISPR-based systems are emerging as the premier method to manipulate many cellular processes. In this study, a simple and efficient CRISPR interference (CRISPRi) system for targeted gene repression in archaea was developed. The Methanosarcina acetivorans CRISPR-Cas9 system was repurposed by replacing Cas9 with the catalytically dead Cas9 (dCas9) to generate a CRISPRi-dCas9 system for targeted gene repression. To test the utility of the system, genes involved in nitrogen (N 2 ) fixation were targeted for dCas9-mediated repression. First, the nif operon ( nifHI 1 I 2 DKEN ) that encodes molybdenum nitrogenase was targeted by separate guide RNAs (gRNAs), one targeting the promoter and the other targeting nifD . Remarkably, growth of M. acetivorans with N 2 was abolished by dCas9-mediated repression of the nif operon with each gRNA. The abundance of nif transcripts was >90% reduced in both strains expressing the gRNAs, and NifD was not detected in cell lysate. Next, we targeted NifB, which is required for nitrogenase cofactor biogenesis. Expression of a gRNA targeting the coding sequence of NifB decreased nifB transcript abundance >85% and impaired but did not abolish growth of M. acetivorans with N 2 . Finally, to ascertain the ability to study genemore »regulation using CRISPRi-dCas9, nrpR1 , encoding a subunit of the repressor of the nif operon, was targeted. The nrpR1 repression strain grew normally with N 2 but had increased nif operon transcript abundance, consistent with NrpR1 acting as a repressor. These results highlight the utility of the system, whereby a single gRNA when expressed with dCas9 can block transcription of targeted genes and operons in M. acetivorans . IMPORTANCE Genetic tools are needed to understand and manipulate the biology of archaea, which serve critical roles in the biosphere. Methanogenic archaea (methanogens) are essential for the biological production of methane, an intermediate in the global carbon cycle, an important greenhouse gas, and a biofuel. The CRISPRi-dCas9 system in the model methanogen Methanosarcina acetivorans is, to our knowledge, the first Cas9-based CRISPR interference system in archaea. Results demonstrate that the system is remarkably efficient in targeted gene repression and provide new insight into nitrogen fixation by methanogens, the only archaea with nitrogenase. Overall, the CRISPRi-dCas9 system provides a simple, yet powerful, genetic tool to control the expression of target genes and operons in methanogens.« less