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Fig. 2 | Microbial Cell Factories

Fig. 2

From: Optimizing a CRISPR-Cpf1-based genome engineering system for Corynebacterium glutamicum

Fig. 2

PAM sequence optimization for FnCpf1-mediated gene editing in C. glutamicum. a Illustration of the lethal reporter system for measuring Cpf1-mediated DNA cleavage efficiency in C. glutamicum. A series of crRNAs containing different PAM sequences are designed and inserted into the pYJ1 vector. Then, the C. glutamicum cells containing one of the pYJ1 derivatives are spread on plates in the presence or absence of antibiotics. The targeting efficiency of the crRNA can be calculated based on the number of colonies. b, c The effects of the last PAM sequence nucleotide (N) of 5′-XXTN-3′ on the efficiency of editing the upp (b) and crtYe/f (c) genes. All columns show survival rates (%), which are correlated with the targeting efficiency of the corresponding crRNA. d, e The effect of the second PAM sequence nucleotide (N) of 5′-XNTA-3′ on the efficiency of editing the upp (d) and crtYe/f (e) genes. X represents an unchanged nucleotide in the same set and N represents a changeable nucleotide. f, g The effect of the first PAM sequence nucleotide of 5′-NTTA-3′ on the efficiency of editing the upp (f) and crtYe/f (g) genes. Spacer and PAM sequences are shown in black and red, respectively. The gray column represents the control, the N in the purple column represents C, the N in the green column represents G, the N in the orange column represents A, and the N in the pink column represents T. The spacer sequences are also shown in Additional file 1: Table S2. Error bars; n = 3; data are analyzed using a paired-sample t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

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