Plant Science & Landscape Architecture

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Author: search.filters.author.Sretenovic, Simon

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    Expanding the targeting scope of FokI-dCas nuclease systems with SpRY and Mb2Cas12a
    (Wiley, 2022-04-04) Cheng, Yanhao; Sretenovic, Simon; Zhang, Yingxiao; Pan, Changtian; Huang, Ji; Qi, Yiping
    CRISPR-Cas9 and Cas12a are widely used sequence-specific nucleases (SSNs) for genome editing. The nuclease domains of Cas proteins can induce DNA double strand breaks upon RNA guided DNA targeting. Zinc finger nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs) have been popular SSNs prior to CRISPR. Both ZFNs and TALENs are based on reconstitution of two monomers with each consisting of a DNA binding domain and a FokI nuclease domain. Inspired by the configuration of ZFNs and TALENs, dimeric FokI-dCas9 systems were previously demonstrated in human cells. Such configuration, based on a pair of guide RNAs (gRNAs), offers great improvement on targeting specificity. To expand the targeting scope of dimeric FokI-dCas systems, the PAM (protospacer adjacent motif)-less SpRY Cas9 variant and the PAM-relaxed Mb2Cas12a system were explored. Rice cells showed that FokI-dSpRY had more robust editing efficiency than a paired SpRY nickase system. Furthermore, a dimeric FokI-dMb2Cas12a system was developed that displayed comparable editing activity to Mb2Cas12a nuclease in rice cells. Finally, a single-chain FokI-FokI-dMb2Cas12a system was developed that cuts DNA outside its targeting sequence, which could be useful for many versatile applications. Together, this work greatly expanded the FokI based CRISPR-Cas systems for genome editing.
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    DEVELOPING AND IMPROVING CRISPR-BASED GENOME EDITING TECHNOLOGIES IN PLANTS
    (2022) Sretenovic, Simon; Qi, Yiping; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Earlier genome editing technologies were developed based on programmable nucleases including zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN), both requiring tedious protein engineering. By contrast, clustered regularly interspaced short palindromic repeats (CRISPR) systems, such as CRISPR-Cas9, has revolutionized the genome editing field in the past decade due to ease of guiding Cas9 endonuclease to the target site by programmable guide RNAs. However, not every target site can be edited due to Cas9 endonucleases’ recognition of so-called protospacer adjacent motif (PAM) when binding to the target site. For example, the PAM for the widely used SpCas9 is NGG (N=A, C, T or G). This drastically limits targeting scope in the genomes. Thus, researchers have developed engineered Cas9 variants recognizing more relaxed PAMs and tested them in mammalian cell lines. Repair of Cas9 endonuclease-induced double strand breaks through non-homologous end joining (NHEJ) DNA repair pathway typically generates unspecified insertions and deletions, which is a missed opportunity for introducing precise edits. To confer precise genome editing, CRISPR-Cas9 derived base editors and prime editors have been developed. In this work, expanding the plant genome editing scope with engineered Cas9 variants, improving precise cytosine and adenine base editing in plants as well as demonstrating prime editing in plant cells were pursued. The technologies were tested in the model crop, rice, in transiently transformed protoplasts and stably transformed T0 lines. Findings suggest that engineered Cas9 variants can drastically expand the targeting scope for targeted mutagenesis and base editing in plants. Additionally, newer genome editing technologies such as base editors and prime editors can be applied in plants to achieve precise genome editing with varying efficiencies. These validated and useful CRISPR-Cas9 genome editing toolkits have been deposited to the public vector depository, Addgene. Adoption of these genome editing technologies by plant scientists and breeders will enable basic research discoveries and accelerate breeding of next generation crops, ensuring global food security amidst climate change and increasing global population.
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    Application of CRISPR-Cas12a temperature sensitivity for improved genome editing in rice, maize, and Arabidopsis
    (Springer Nature, 2019-01-31) Malzahn, Aimee A.; Tang, Xu; Lee, Keunsub; Ren, Qiurong; Sretenovic, Simon; Zhang, Yingxiao; Chen, Hongqiao; Kang, Minjeong; Bao, Yu; Zheng, Xuelian; Deng, Kejun; Zhang, Tao; Salcedo, Valeria; Wang, Kan; Zhang, Yong; Qi, Yiping
    CRISPR-Cas12a (formerly Cpf1) is an RNA-guided endonuclease with distinct features that have expanded genome editing capabilities. Cas12a-mediated genome editing is temperature sensitive in plants, but a lack of a comprehensive understanding on Cas12a temperature sensitivity in plant cells has hampered effective application of Cas12a nucleases in plant genome editing. We compared AsCas12a, FnCas12a, and LbCas12a for their editing efficiencies and non-homologous end joining (NHEJ) repair profiles at four different temperatures in rice. We found that AsCas12a is more sensitive to temperature and that it requires a temperature of over 28 °C for high activity. Each Cas12a nuclease exhibited distinct indel mutation profiles which were not affected by temperatures. For the first time, we successfully applied AsCas12a for generating rice mutants with high frequencies up to 93% among T0 lines. We next pursued editing in the dicot model plant Arabidopsis, for which Cas12a-based genome editing has not been previously demonstrated. While LbCas12a barely showed any editing activity at 22 °C, its editing activity was rescued by growing the transgenic plants at 29 °C. With an early high-temperature treatment regime, we successfully achieved germline editing at the two target genes, GL2 and TT4, in Arabidopsis transgenic lines. We then used high-temperature treatment to improve Cas12a-mediated genome editing in maize. By growing LbCas12a T0 maize lines at 28 °C, we obtained Cas12a-edited mutants at frequencies up to 100% in the T1 generation. Finally, we demonstrated DNA binding of Cas12a was not abolished at lower temperatures by using a dCas12a-SRDX-based transcriptional repression system in Arabidopsis. Our study demonstrates the use of high-temperature regimes to achieve high editing efficiencies with Cas12a systems in rice, Arabidopsis, and maize and sheds light on the mechanism of temperature sensitivity for Cas12a in plants.