2014-06-05 Direct. Nat. Kieser, T., Bibb, M. J., Buttner, M. J., Chater, K. F., and Hopwood, D. A. (d) Indels and insertions observed by clonal sequence analysis of PCR amplicons after CRISPR-mediated recombination of the TCR α and β locus. Thus, increasing numbers of genetic engineering tools have been developed to explore these products (Luo et al., 2015b, 2016; Zhao et al., 2019). Engineered biosynthesis of natural products in heterologous hosts. Using multiplexed gRNAs can target multiple locations in the genome to modify, whether it be editing CRISPRi, CRISPRa, … Biotechnol. Additionally, the CRISPR-FnCas12a3 system employing the engineered FnCas12a mutant EP16, which recognizes a broad spectrum of PAM sequences, was used to precisely perform site mutations and insertions. uuid:413d1142-1dd2-11b2-0a00-ac0927edca00 Epub 2017 May 12. Impact Factor 3.644 | CiteScore 3.2More on impact ›, Development and Application of Novel Genome Engineering Tools in Microbial Biotechnology Biotechnol. 2017 Mar 7;8(10):17002-17011. doi: 10.18632/oncotarget.15218. To develop efficient and versatile genome editing tools in multiple Streptomyces strains, two FnCas12a systems were constructed: one based on the pWHU2653 system (Zeng et al., 2015) (CRISPR-FnCas12a1) and the other based on the pCRISPomyces-2 system (Cobb et al., 2015) (CRISPR-FnCas12a2). The experiments were performed in triplicate. Chen Z, Zhu S, Kindig K, Wang S, Chou SW, Davis RW, Dercoli MR, Weaver H, Stepanyan R, McDermott BM. doi: 10.1002/cbic.201700516, Will, W. R., and Frost, L. S. (2006). (2018). Together, our results provide a framework for applying CRISPR-Cas9 to R. toruloides that will facilitate rapid and high-throughput genome engineering in this industrially relevant organism. Highly efficient editing of the actinorhodin polyketide chain length factor gene in Streptomyces coelicolor M145 using CRISPR/Cas9-CodA(sm) combined system. For single cuts, one to three spacers were selected for each cluster. © 2019 Elsevier Ltd. All rights reserved. 2019 Nov 16;7(11):569. doi: 10.3390/microorganisms7110569. doi: 10.1016/j.molp.2018.03.008, Zhu, F., Qin, C., Tao, L., Liu, X., Shi, Z., Ma, X., et al. Curr Res Transl Med. R2YE medium supplemented with 20 μg/mL apramycin and 40 μg/mL nalidixic acid was used to screen potentially edited Streptomyces. ACS Synth. Received: 07 January 2020; Accepted: 09 June 2020;Published: 30 June 2020. Next, two 1- or 2-kb homologous arms corresponding to the upstream and downstream regions of the target genes or gene clusters were amplified from purified genomic DNA and fused into the XbaI site of the desired plasmid by one-step assembly. Red arrow indicates putative cleavage site. (C) The 1,726-bp PCR products amplified from the mutants were digested into 1,113-bp and 613-bp fragments by BglII, while the WT amplicons were not digested. The CRISPR-FnCas12a2 system was used to delete large fragments ranging from 21.4 to 128 kb. The editor and reviewers' affiliations are the latest provided on their Loop research profiles and may not reflect their situation at the time of review. Proc. false Other FnCas12a1 plasmids were also transformed into S. griseus and generated 95 ± 39 (pYL-rpsLp(XC)-FnCas12a1), 62 ± 4 (pYL-kasOp∗-FnCas12a1), and 143 ± 23 (pYL-ermEp∗-FnCas12a1) exconjugants, respectively (Figure 2C).  |  Figure 3. The CRISPR-FnCas12a1 system was constructed based on pWHU2653 (Zeng et al., 2015). The supernatant was then mixed with an equal volume of ethyl acetate to extract the metabolites. Front Plant Sci. ©2016 American Association for Cancer Research. All the colonies that showed double bands of WT and mutant products were calculated as wild type, and they were not included in the edited colonies. Ren J, Zhang X, Liu X, Fang C, Jiang S, June CH, Zhao Y. Oncotarget. A Multiplexed CRISPR/Cas9 Editing System Based on the Endogenous tRNA Processing. Front Immunol. false eCollection 2020. www.pnas.org The kasOp∗ promoter (Wang et al., 2013), rpsLp(XC) promoter (Shao et al., 2013), ermEp∗ promoter (Luo et al., 2015a), and Potr∗ system (Wang et al., 2016) were amplified by PCR from template plasmids. With multiple gRNAs, specific gene expression or methylation status can be efficiently controlled by dCas9 fused to activators, repressors, methyltransferase, demethylase, or other functional domains. <> Hu B, Zou Y, Zhang L, Tang J, Niedermann G, Firat E, Huang X, Zhu X. Hum Gene Ther.