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Optimizing a CRISPR-Cpf1-Based Genome Engineering System For Zhang et al. Microb Cell Fact (2019) 18:60 https://doi.org/10.1186/s12934-019-1109-x Microbial Cell Factories RESEARCH Open Access Optimizing a CRISPR-Cpf1-based genome engineering system for Corynebacterium glutamicum Jiao Zhang1, Fayu Yang1, Yunpeng Yang1,2, Yu Jiang3 and Yi‑Xin Huo1,2* Abstract Background: Corynebacterium glutamicum is an important industrial strain for the production of a diverse range of chemicals. Cpf1 nucleases are highly specifc and programmable, with efciencies comparable to those of Cas9. Although the Francisella novicida (Fn) CRISPR‑Cpf1 system has been adapted for genome editing in C. glutamicum, the editing efciency is currently less than 15%, due to false positives caused by the poor targeting efciency of the crRNA. Results: To address this limitation, a screening strategy was developed in this study to systematically evaluate crRNA targeting efciency in C. glutamicum. We quantitatively examined various parameters of the C. glutamicum CRISPR‑ Cpf1 system, including the protospacer adjacent motif (PAM) sequence, the length of the spacer sequence, and the type of repair template. We found that the most efcient C. glutamicum crRNA contained a 5′‑NYTV‑3′ PAM and a 21 bp spacer sequence. Moreover, we observed that linear DNA could be used to repair double strand breaks. Conclusions: Here, we identifed optimized PAM‑related parameters for the CRISPR‑Cpf1 system in C. glutamicum. Our study sheds light on the function of the FnCpf1 endonuclease and Cpf1‑based genome editing. This optimized system, with higher editing efciency, could be used to increase the production of bulk chemicals, such as isobu‑ tyrate, in C. glutamicum. Keywords: Corynebacterium glutamicum, CRISPR‑Cpf1, PAM, crRNA, Linear template, Isobutyrate Background with a false-positive rate of 20–40% [8–11]. CRISPR- Corynebacterium glutamicum is a facultatively anaerobic Cpf1 has attracted much attention in the feld of genome Gram-positive asporogenic bacterium with short rod-like editing because it is easy to manipulate and has a low cells that is widely distributed in soil. Generally recog- of-target rate. However, gene deletion or insertion in C. nized as safe, C. glutamicum is non-pathogenic and does glutamicum using CRISPR-Cpf1 reaches editing efcien- not produce endotoxins [1, 2]. It has been used in fer- cies of only 5–15% [12]. Hence, it is of great importance mentation for over 50 years. Under anaerobic conditions, to improve the editing efciency of CRISPR-Cpf1 in C. C. glutamicum can efciently convert glucose to various glutamicum. amino acids [3, 4], organic acids [5], higher alcohols [6], Double-strand breaks (DSBs) introduced by the and polymers [7]. Traditional genetic engineering strate- CRISPR-Cpf1 system are lethal to most bacteria, includ- gies in C. glutamicum rely on a SacB-based suicide vec- ing C. glutamicum [13], which lacks a non-homologous tor [8]. Tis process is time-consuming and inefcient, end joining mechanism. Terefore, DSBs serve as a selec- tion pressure to enrich mutations repaired using the arti- *Correspondence: [email protected] fcial donor DNA templates in this organism [14–18]. 1 Key Laboratory of Molecular Medicine and Biotherapy, School of Life Since CRISPR-Cpf1-induced DSBs are lethal in C. glu- Sciences, Beijing Institute of Technology, No. 5 South Zhongguancun Street, Beijing 100081, China tamicum, the targeting efciencies of crRNA are refected Full list of author information is available at the end of the article by survival rates. crRNAs with low targeting efciencies © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhang et al. Microb Cell Fact (2019) 18:60 Page 2 of 13 cause a high rate of false positives during genome edit- crRNA or the plasmid, pYJ1_SH_∆gene, expressing the ing, which result in the survival of many wild-type cells crRNA and donor DNA template in competent C. glu- within the population [17, 19–21]. tamicum cells. Unless repaired by HR in the presence of FnCpf1 requires a single crRNA and a thymine-rich donor DNA templates, cells that took up a plasmid with PAM. crRNA contains a 19 bp direct repeat sequence functional crRNA were killed by Cpf1-mediated diges- and a 23–25 bp spacer sequence [22–25]. PAM is a tion. Eight colonies from a culture plate were picked and 2–6 bp protospacer-adjacent DNA sequence (Fig. 1a), incubated in BHIS culture medium overnight and their which is recognized by Cpf1 and plays an important role genomic DNA was extracted individually. Gene knockout in both Cpf1 binding and cleavage [26]. In the presence was verifed by PCR, using primers that bind upstream of of a PAM, the Cpf1-crRNA complex can bind the tar- the 5′ homologous arm and downstream of the 3′ homol- get DNA and induce DSBs. Te cleavage site of Cpf1 is ogous arm. Mutants with the designed gene knockout afected by the length of the spacer sequence. When the were incubated at 37 °C in BHIS medium without antibi- spacer length is greater than or equal to 20 bp, Cpf1 tends otics for plasmid curing and were then streaked on plates to cut the 18th nucleotide of the non-complementary without antibiotics and grown at 30 °C (Fig. 1b). After strand and the 23rd nucleotide of the target DNA com- confrming the loss of the plasmid, colonies were picked plementary strand. When the spacer length is less than to prepare electrocompetent cells for the next round of 20 bp [27], Cpf1 tends to cut the 14th nucleotide of the editing. non-complementary strand. Te cohesive end introduced To investigate the editing efciencies of our system for by Cpf1 facilitates homologous recombination (HR) [28]. gene deletion in C. glutamicum, the upp gene was chosen Taken together, the PAM and the length of the spacer as the target gene, since its deletion leads to a 5-fuoro- sequence are key determinants of the efciency of Cpf1- uracil (5-FU)-resistant phenotype, which is convenient dependent genome editing. for screening [10]. We designed a crRNA targeting the Editing efciency is also infuenced by the type of upp gene and generated the plasmid, pYJ1_S_∆upp. Te repair template. In C. glutamicum, the plasmid-borne repair template was inserted into pYJ1_S_∆upp to create dsDNA template can be utilized to repair the DSBs via pYJ1_SH_∆upp. After engineering and plating, all 5-FU- HR [29]. Although linear DNA templates could be used resistant colonies on the plate were assumed to be upp to repair DSBs in the genomes of C. elegans [30], mouse, knockouts. Eight colonies were confrmed to have the and human cells [31] with high efciency, the repair of upp gene deleted by both PCR screening and sequencing, DSBs in C. glutamicum via linear DNA has, to the best of as shown in Fig. 1c, d. Te efciency of upp deletion was our knowledge, not been reported. 100%. To further evaluate the efciency of the system, we Here, we describe a CRISPR-Cpf1 based method for constructed pYJ1_SH_∆crtYe/f to delete the crtYe/f gene, iterative genome editing and metabolic engineering of since its deletion leads to a color change from yellow to C. glutamicum. We established a lethal reporter system red [32–34]. When the pYJ1_SH_∆crtYe/f plasmid was to verify the targeting efciency of the crRNA and con- introduced into competent C. glutamicum cells, approx- structed a series of crRNA plasmids to characterize their imately 17% of single colonies were correctly edited, cleavage activities in C. glutamicum. We optimized this according to their color. system with respect to PAM sequence, the length of the spacer sequence, and the type of repair templates. Finally, the central metabolic pathways of C. glutamicum were Generation of a lethal reporter system and optimization modifed by our genome editing method to improve of PAM sequence for the Cpf1‑mediated genome editing isobutyrate production. Te fndings of this study will system facilitate engineering of metabolic networks for the syn- Two genes (upp and crtYe/f) were selected to verify the thesis of value-added products using C. glutamicum. optimal PAM sequence of CRISPR-Cpf1, using a lethal reporter system. We systematically profled the target- Results ing efciencies of crRNAs by co-expressing crRNA Establishing a CRISPR‑Cpf1‑mediated genome editing with Cpf1. Diferent crRNAs were inserted into the system in C. glutamicum pYJ1 plasmid to construct a series of pYJ1_S_∆upp or Our CRISPR-Cpf1 system contained three elements: a pYJ1_S_∆crtYe/f plasmids, which targeted diferent PAM constitutive Cpf1 expression cassette, a crRNA expres- sequences. Te plasmid, pYJ1, without crRNA inser- sion cassette and a donor DNA template for repairing the tion, was used as the control. After transformation, the DSBs introduced by the Cpf1-crRNA complex (Fig. 1a). survival rate of cells in the presence of a specifc crRNA As shown in Fig. 1b, each cycle of editing started with was calculated using the formula shown in the “Methods” the transformation plasmid, pYJ1_S_∆gene, expressing section (Fig. 2a). Te efciency of crRNA targeting of Zhang et al. Microb Cell Fact (2019) 18:60 Page 3 of 13 Fig. 1 Schematic overview of the CRISPR–Cpf1‑based system for iterative genome editing in C.
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