Increasing on-target cleavage efficiency for CRISPR/Cas9-induced large fragment deletion in Myxococcus xanthus

Table 1 Information of the 5′-N20-NGG-3′ spacer candidates for the deletion of the biosynthetic gene clusters for myxovirescin and myxalamids

Name^a	20 nt spacer sequence^b	Hairpin	GC (%)	ΔG^c
11-1	TGCAAATCGGAGTAGAAGCG CGG	N	50	−39.6
11-2	GCCGACGCCGCCCTTGATGG TGG	N	75	−49.0
11-3	GCGTGAGGAGGAAGCGGCGG TGG	N	75	−47.3
11-4	TCCTGTCCGAACCGCCCGCC CGG	N	75	−48.4
17-4	GACGAGGCACCTAGAGACAC TGG	N	60	−35.8
17-5	CCACTCATCCAGGACGCTAC TGG	N	60	−37.8
17-6	CGCGAGGCTGCGGCTGCCAC TGG	Y	80	−49.3
17-7	GACGCCCCATCCCCAACCAC TGG	N	70	−44.7
17-8	CGAGAGGTCGCTCTCCAAAC TGG	Y	60	−39.3
17-9	GGCGATGATGAGGAACCAAC TGG	N	55	−39.2
17-10	CCTGCTCCTGCGCCTTCCAC TGG	N	70	−44.0
Degenerate^d	NVH-N2-BNH-N2-H-N5-HHAC TGG

^aThe locations are referred to Fig. 4 for 11-1 to 11-4 and Fig. 5 for 17-4 to 17-10
^bDisfavored nucleotides are shown in italics
^cΔG indicates free energy (kcals/mol). The calculation of free energy and prediction of hairpin in 20 nt spacer were performed by primer premier 5.00
^dH: A/T/C, V: G/A/C, B: G/T/C, N: any nucleotide; the C-terminus TGG or CGG is the PAM motif

ISSN: 1475-2859