Result Tracking|sgRNA Online

Job information description: Example data, referenced id: 6ffc5bee490e0e2dee3353a1665a7552

Status: Complete

Submitted by:

Yongbing Zhao (yongbing.zhao@nih.gov)

Date Submitted:

2018-03-19 20:32:00

Screening Parameters:

PAM type: xCas9 3.7 (PAM: NG, GAA, GAT)

Target genome: Mouse (mm10)

sgRNA Size: 20bp

Sequence number: 3

sequence #1
sequence #2
sequence #3

Table 1: Guide sgRNAs for the sequence [example1]

Guide #	sgRNA Sequence(+PAM)	GC content	Strand	Position on sequence	Off-target Number	Off-target Number at TSS\|Exon\|Intergenic	Top3 Off-target Sites Scores	sgRNA Score (Based on Off-target)	PAM Score	Cas9 Loading Score	Forward oligo primer	Reverse oligo primer
#1	GGGCAATGCTGCGTCGTCGTAGT	65%	+	95	66	3\|4\|59	0.51 \| 0.47 \| 0.45	95	★★☆☆	34.5	caccGGGCAATGCTGCGTCGTCGT	aaacACGACGACGCAGCATTGCCC
#2	CCAAAAAACTACGACGACGCAGC	50%	-	-4	55	3\|3\|49	0.64 \| 0.64 \| 0.44	94	★★☆☆	49	caccgCCAAAAAACTACGACGACGC	aaacGCGTCGTCGTAGTTTTTTGGc
#3	GCTGCGTCGTCGTAGTTTTTTGG	50%	+	102	49	2\|2\|45	0.74 \| 0.72 \| 0.49	92	★★☆☆	13.5	caccGCTGCGTCGTCGTAGTTTTT	aaacAAAAACTACGACGACGCAGC
#4	AGGGGGCAATGCTGCGTCGTCGT	65%	+	92	128	6\|7\|115	0.91 \| 0.71 \| 0.51	90	★★★★	34.5	caccgAGGGGGCAATGCTGCGTCGT	aaacACGACGCAGCATTGCCCCCTc
#5	AAACTACGACGACGCAGCATTGC	50%	-	-9	68	2\|10\|56	0.9 \| 0.7 \| 0.67	89	★★☆☆	45	caccgAAACTACGACGACGCAGCAT	aaacATGCTGCGTCGTCGTAGTTTc
#6	CCCCCAAAAAACTACGACGACGC	50%	-	-1	204	3\|6\|195	1.38 \| 1.22 \| 0.81	89	★★☆☆	57	caccgCCCCCAAAAAACTACGACGA	aaacTCGTCGTAGTTTTTTGGGGGc
#7	AGTCCAGCACTCGCTCGCGCCGG	70%	-	-82	100	5\|25\|70	1.57 \| 0.84 \| 0.66	87	★★★☆	54	caccgAGTCCAGCACTCGCTCGCGC	aaacGCGCGAGCGAGTGCTGGACTc
#8	GTCCCTGCAGCTCCGACGAGCGA	70%	-	-104	212	16\|20\|176	3.26 \| 0.84 \| 0.64	84	★★★★	55	caccGTCCCTGCAGCTCCGACGAG	aaacCTCGTCGGAGCTGCAGGGAC
#9	AACAGTCCAGCACTCGCTCGCGC	60%	-	-79	229	2\|19\|208	0.86 \| 0.82 \| 0.71	84	★★☆☆	43	caccgAACAGTCCAGCACTCGCTCG	aaacCGAGCGAGTGCTGGACTGTTc
#10	GACCGGCGCGAGCGAGTGCTGGA	75%	+	22	69	8\|30\|31	1.68 \| 1.29 \| 1.29	83	★☆☆☆	26.5	caccGACCGGCGCGAGCGAGTGCT	aaacAGCACTCGCTCGCGCCGGTC
#11	GTCCAGCACTCGCTCGCGCCGGT	75%	-	-83	107	10\|31\|66	2.6 \| 2.54 \| 0.82	83	★★★☆	42	caccGTCCAGCACTCGCTCGCGCC	aaacGGCGCGAGCGAGTGCTGGAC
#12	CGGTCCCTGCAGCTCCGACGAGC	75%	-	-102	128	6\|32\|90	1.34 \| 1.27 \| 1.11	82	★★☆☆	52	caccgCGGTCCCTGCAGCTCCGACG	aaacCGTCGGAGCTGCAGGGACCGc
#13	GCGTCGTCGTAGTTTTTTGGGGG	50%	+	105	59	3\|2\|54	1.38 \| 1.38 \| 1.34	81	★★★☆	41.5	caccGCGTCGTCGTAGTTTTTTGG	aaacCCAAAAAACTACGACGACGC
#14	GGACCGGCGCGAGCGAGTGCTGG	80%	+	21	130	16\|57\|57	1.46 \| 1.25 \| 0.91	80	★★☆☆	46	caccGGACCGGCGCGAGCGAGTGC	aaacGCACTCGCTCGCGCCGGTCC
#15	TCGCTCGCGCCGGTCCCTGCAGC	80%	-	-92	117	19\|32\|66	1.63 \| 1.59 \| 1.31	80	★★☆☆	42	caccgTCGCTCGCGCCGGTCCCTGC	aaacGCAGGGACCGGCGCGAGCGAc
#16	TGCGTCGTCGTAGTTTTTTGGGG	45%	+	104	84	3\|3\|78	1.46 \| 1.29 \| 0.91	78	★★★☆	28.5	caccgTGCGTCGTCGTAGTTTTTTG	aaacCAAAAAACTACGACGACGCAc
#17	CTGCGTCGTCGTAGTTTTTTGGG	45%	+	103	69	2\|2\|65	1.56 \| 1.35 \| 1.31	78	★★★☆	13.5	caccgCTGCGTCGTCGTAGTTTTTT	aaacAAAAAACTACGACGACGCAGc
#18	CTAAGGGGGCAATGCTGCGTCGT	60%	+	89	143	3\|11\|129	2.17 \| 1.19 \| 1.04	77	★★★★	47	caccgCTAAGGGGGCAATGCTGCGT	aaacACGCAGCATTGCCCCCTTAGc
#19	AGCCACATGGGTCCCCTCGGAGC	70%	-	-48	252	8\|24\|220	1.36 \| 0.94 \| 0.93	76	★★☆☆	49.5	caccgAGCCACATGGGTCCCCTCGG	aaacCCGAGGGGACCCATGTGGCTc
#20	GGCGCGAGCGAGTGCTGGACTGT	75%	+	26	129	15\|25\|89	2.33 \| 1.36 \| 1.27	75	★★★☆	51	caccGGCGCGAGCGAGTGCTGGAC	aaacGTCCAGCACTCGCTCGCGCC
#21	CGACGCAGCATTGCCCCCTTAGC	65%	-	-18	93	2\|10\|81	2.42 \| 1.38 \| 1.31	75	★★☆☆	30	caccgCGACGCAGCATTGCCCCCTT	aaacAAGGGGGCAATGCTGCGTCGc
#22	TGCAGGGACCGGCGCGAGCGAGT	80%	+	16	161	27\|38\|96	2.46 \| 1.5 \| 1.25	72	★★☆☆	49	caccgTGCAGGGACCGGCGCGAGCG	aaacCGCTCGCGCCGGTCCCTGCAc
#23	GTCGCTCGTCGGAGCTGCAGGGA	70%	+	1	95	12\|15\|68	1.68 \| 1.44 \| 1.31	72	★☆☆☆	60	caccGTCGCTCGTCGGAGCTGCAG	aaacCTGCAGCTCCGACGAGCGAC
#24	GAGCTGCAGGGACCGGCGCGAGC	80%	+	12	306	36\|82\|188	1.69 \| 1.43 \| 1.35	71	★★☆☆	50	caccGAGCTGCAGGGACCGGCGCG	aaacCGCGCCGGTCCCTGCAGCTC
#25	GCTGCAGGGACCGGCGCGAGCGA	80%	+	14	250	27\|58\|165	2.33 \| 2.33 \| 1.65	69	★★★★	55	caccGCTGCAGGGACCGGCGCGAG	aaacCTCGCGCCGGTCCCTGCAGC
#26	CGAGCGAGTGCTGGACTGTTTGT	60%	+	30	183	5\|6\|172	2.39 \| 1.31 \| 1.29	68	★★★☆	20.5	caccgCGAGCGAGTGCTGGACTGTT	aaacAACAGTCCAGCACTCGCTCGc
#27	CAAACAGTCCAGCACTCGCTCGC	55%	-	-77	372	8\|22\|342	3.86 \| 1.39 \| 1.31	66	★★☆☆	35	caccgCAAACAGTCCAGCACTCGCT	aaacAGCGAGTGCTGGACTGTTTGc
#28	GGCTCCGAGGGGACCCATGTGGC	70%	+	57	251	13\|25\|213	1.67 \| 1.67 \| 1.07	66	★★★☆	34	caccGGCTCCGAGGGGACCCATGT	aaacACATGGGTCCCCTCGGAGCC
#29	CGGAGCTGCAGGGACCGGCGCGA	80%	+	10	324	31\|92\|201	5.54 \| 2.36 \| 1.72	64	★★★★	54	caccgCGGAGCTGCAGGGACCGGCG	aaacCGCCGGTCCCTGCAGCTCCGc
#30	CAGGGACCGGCGCGAGCGAGTGC	80%	+	18	398	35\|40\|323	1.41 \| 1.09 \| 0.87	62	★★☆☆	55	caccgCAGGGACCGGCGCGAGCGAG	aaacCTCGCTCGCGCCGGTCCCTGc
#31	CTGTTTGTGCAGGGCTCCGAGGG	60%	+	45	476	10\|29\|437	1.34 \| 1.07 \| 1.04	62	★★★☆	58	caccgCTGTTTGTGCAGGGCTCCGA	aaacTCGGAGCCCTGCACAAACAGc
#32	ACTGTTTGTGCAGGGCTCCGAGG	60%	+	44	416	12\|29\|375	4.56 \| 2.52 \| 1.42	56	★★☆☆	42.5	caccgACTGTTTGTGCAGGGCTCCG	aaacCGGAGCCCTGCACAAACAGTc
#33	AGCGAGTGCTGGACTGTTTGTGC	55%	+	32	333	13\|13\|307	2.39 \| 1.63 \| 1.48	56	★★☆☆	28.5	caccgAGCGAGTGCTGGACTGTTTG	aaacCAAACAGTCCAGCACTCGCTc
#34	GTCGGAGCTGCAGGGACCGGCGC	75%	+	8	409	22\|81\|306	4.75 \| 2.37 \| 2.37	55	★★☆☆	58	caccGTCGGAGCTGCAGGGACCGG	aaacCCGGTCCCTGCAGCTCCGAC
#35	GGGCTCCGAGGGGACCCATGTGG	75%	+	56	348	21\|35\|292	2.27 \| 1.73 \| 1.51	55	★★☆☆	42	caccGGGCTCCGAGGGGACCCATG	aaacCATGGGTCCCCTCGGAGCCC
#36	GTTTGTGCAGGGCTCCGAGGGGA	65%	+	47	476	17\|47\|412	6.38 \| 3.29 \| 2.5	55	★☆☆☆	59	caccGTTTGTGCAGGGCTCCGAGG	aaacCCTCGGAGCCCTGCACAAAC
#37	TGTTTGTGCAGGGCTCCGAGGGG	60%	+	46	534	12\|37\|485	4.21 \| 2.52 \| 2.36	54	★★★☆	55	caccgTGTTTGTGCAGGGCTCCGAG	aaacCTCGGAGCCCTGCACAAACAc
#38	CGCGCCGGTCCCTGCAGCTCCGA	80%	-	-97	233	28\|69\|136	5.45 \| 1.67 \| 1.67	53	★★★★	41	caccgCGCGCCGGTCCCTGCAGCTC	aaacGAGCTGCAGGGACCGGCGCGc
#39	TCGTCGGAGCTGCAGGGACCGGC	70%	+	6	229	7\|34\|188	2.6 \| 2.42 \| 2.28	53	★★★☆	44	caccgTCGTCGGAGCTGCAGGGACC	aaacGGTCCCTGCAGCTCCGACGAc
#40	CACTCGCTCGCGCCGGTCCCTGC	80%	-	-89	296	21\|43\|232	1.39 \| 1.27 \| 1.09	52	★★☆☆	34.5	caccgCACTCGCTCGCGCCGGTCCC	aaacGGGACCGGCGCGAGCGAGTGc
#41	GCCGGTCCCTGCAGCTCCGACGA	75%	-	-100	362	15\|46\|301	6.38 \| 2.39 \| 2.39	52	★★★★	58	caccGCCGGTCCCTGCAGCTCCGA	aaacTCGGAGCTGCAGGGACCGGC
#42	TGAGCCACATGGGTCCCCTCGGA	65%	-	-46	492	20\|38\|434	5.83 \| 4.56 \| 4.32	50	★☆☆☆	37	caccgTGAGCCACATGGGTCCCCTC	aaacGAGGGGACCCATGTGGCTCAc
#43	CGAGGGGACCCATGTGGCTCAGG	70%	+	62	323	15\|23\|285	1.63 \| 1.54 \| 1.48	50	★★☆☆	41	caccgCGAGGGGACCCATGTGGCTC	aaacGAGCCACATGGGTCCCCTCGc
#44	CCTCGGAGCCCTGCACAAACAGT	65%	-	-62	397	8\|38\|351	6.38 \| 5.89 \| 2.36	49	★★☆☆	44	caccgCCTCGGAGCCCTGCACAAAC	aaacGTTTGTGCAGGGCTCCGAGGc
#45	CTCGTCGGAGCTGCAGGGACCGG	70%	+	5	385	10\|35\|340	2.28 \| 1.67 \| 1.62	47	★★★☆	51	caccgCTCGTCGGAGCTGCAGGGAC	aaacGTCCCTGCAGCTCCGACGAGc
#46	CTGAGCCACATGGGTCCCCTCGG	65%	-	-45	626	25\|49\|552	1.65 \| 1.56 \| 1.44	47	★★★☆	36	caccgCTGAGCCACATGGGTCCCCT	aaacAGGGGACCCATGTGGCTCAGc
#47	GAGCCCTGCACAAACAGTCCAGC	60%	-	-67	560	10\|26\|524	2.51 \| 2.46 \| 2.28	46	★★☆☆	39	caccGAGCCCTGCACAAACAGTCC	aaacGGACTGTTTGTGCAGGGCTC
#48	TGCACAAACAGTCCAGCACTCGC	50%	-	-73	544	18\|21\|505	1.52 \| 1.52 \| 1.52	46	★★☆☆	33	caccgTGCACAAACAGTCCAGCACT	aaacAGTGCTGGACTGTTTGTGCAc
#49	CATGGGTCCCCTCGGAGCCCTGC	75%	-	-53	437	21\|46\|370	38.7 \| 4.33 \| 3.29	46	★★☆☆	47	caccgCATGGGTCCCCTCGGAGCCC	aaacGGGCTCCGAGGGGACCCATGc
#50	ATGTGGCTCAGGGTGGCTAAGGG	55%	+	73	712	18\|26\|668	3.91 \| 2.39 \| 2.27	44	★★★☆	48	caccgATGTGGCTCAGGGTGGCTAA	aaacTTAGCCACCCTGAGCCACATc
#51	CATTGCCCCCTTAGCCACCCTGA	65%	-	-26	503	8\|20\|475	17.2 \| 3 \| 2.36	43	★★★☆	42	caccgCATTGCCCCCTTAGCCACCC	aaacGGGTGGCTAAGGGGGCAATGc
#52	GGTGGCTAAGGGGGCAATGCTGC	65%	+	84	848	14\|31\|803	6.38 \| 1.52 \| 1.49	43	★★☆☆	41	caccGGTGGCTAAGGGGGCAATGC	aaacGCATTGCCCCCTTAGCCACC
#53	GAGGGGACCCATGTGGCTCAGGG	65%	+	63	563	14\|45\|504	7.08 \| 5.72 \| 3.26	39	★★★☆	43	caccGAGGGGACCCATGTGGCTCA	aaacTGAGCCACATGGGTCCCCTC
#54	GTGGCTCAGGGTGGCTAAGGGGG	65%	+	75	729	23\|59\|647	5.83 \| 5.38 \| 5.14	39	★★★☆	54	caccGTGGCTCAGGGTGGCTAAGG	aaacCCTTAGCCACCCTGAGCCAC
#55	TGGCTCAGGGTGGCTAAGGGGGC	65%	+	76	791	28\|49\|714	14.9 \| 3.86 \| 3.26	39	★★★☆	56	caccgTGGCTCAGGGTGGCTAAGGG	aaacCCCTTAGCCACCCTGAGCCAc
#56	TTGCCCCCTTAGCCACCCTGAGC	65%	-	-28	539	9\|38\|492	6.01 \| 3.89 \| 3.59	38	★★☆☆	45	caccgTTGCCCCCTTAGCCACCCTG	aaacCAGGGTGGCTAAGGGGGCAAc
#57	GAGTGCTGGACTGTTTGTGCAGG	55%	+	35	705	16\|25\|664	3.07 \| 2.28 \| 1.56	37	★★☆☆	46	caccGAGTGCTGGACTGTTTGTGC	aaacGCACAAACAGTCCAGCACTC
#58	TGTGGCTCAGGGTGGCTAAGGGG	60%	+	74	942	28\|43\|871	6.43 \| 6.24 \| 5.21	36	★★★☆	44.5	caccgTGTGGCTCAGGGTGGCTAAG	aaacCTTAGCCACCCTGAGCCACAc
#59	CAGGGCTCCGAGGGGACCCATGT	75%	+	54	611	22\|79\|510	6.94 \| 4.25 \| 3.86	36	★★★☆	51	caccgCAGGGCTCCGAGGGGACCCA	aaacTGGGTCCCCTCGGAGCCCTGc
#60	GGACTGTTTGTGCAGGGCTCCGA	60%	+	42	660	11\|28\|621	6.69 \| 4.27 \| 4.03	35	★★★★	41	caccGGACTGTTTGTGCAGGGCTC	aaacGAGCCCTGCACAAACAGTCC
#61	AGTGCTGGACTGTTTGTGCAGGG	50%	+	36	689	23\|34\|632	60.5 \| 5.21 \| 1.72	33	★★★☆	41.5	caccgAGTGCTGGACTGTTTGTGCA	aaacTGCACAAACAGTCCAGCACTc
#62	TGGCTAAGGGGGCAATGCTGCGT	60%	+	86	1647	8\|37\|1602	2.34 \| 2.34 \| 2.03	30	★★★★	49	caccgTGGCTAAGGGGGCAATGCTG	aaacCAGCATTGCCCCCTTAGCCAc
#63	CATGTGGCTCAGGGTGGCTAAGG	60%	+	72	1608	47\|73\|1488	3.07 \| 2.52 \| 2.36	30	★★☆☆	49	caccgCATGTGGCTCAGGGTGGCTA	aaacTAGCCACCCTGAGCCACATGc
#64	GTGCTGGACTGTTTGTGCAGGGC	55%	+	37	1367	20\|20\|1327	4.46 \| 2.44 \| 1.71	30	★★★☆	60	caccGTGCTGGACTGTTTGTGCAG	aaacCTGCACAAACAGTCCAGCAC
#65	CTTAGCCACCCTGAGCCACATGG	60%	-	-35	934	28\|49\|857	4.12 \| 2.66 \| 2.52	26	★★☆☆	48	caccgCTTAGCCACCCTGAGCCACA	aaacTGTGGCTCAGGGTGGCTAAGc
#66	CAGGGTGGCTAAGGGGGCAATGC	65%	+	81	1150	25\|45\|1080	100 \| 5.89 \| 4.32	25	★★☆☆	60	caccgCAGGGTGGCTAAGGGGGCAA	aaacTTGCCCCCTTAGCCACCCTGc
#67	AGGGGACCCATGTGGCTCAGGGT	65%	+	64	784	25\|39\|720	68.3 \| 7.41 \| 7.41	24	★★★☆	47.5	caccgAGGGGACCCATGTGGCTCAG	aaacCTGAGCCACATGGGTCCCCTc
#68	GGGACCCATGTGGCTCAGGGTGG	70%	+	66	1121	38\|47\|1036	100 \| 6.38 \| 3.7	22	★★☆☆	56	caccGGGACCCATGTGGCTCAGGG	aaacCCCTGAGCCACATGGGTCCC
#69	TTAGCCACCCTGAGCCACATGGG	55%	-	-36	1016	37\|25\|954	2.6 \| 2.6 \| 2.6	22	★★★☆	40	caccgTTAGCCACCCTGAGCCACAT	aaacATGTGGCTCAGGGTGGCTAAc
#70	GGACCCATGTGGCTCAGGGTGGC	65%	+	67	1095	29\|32\|1034	100 \| 100 \| 6.01	16	★★★☆	46	caccGGACCCATGTGGCTCAGGGT	aaacACCCTGAGCCACATGGGTCC
#71	TAGCCACCCTGAGCCACATGGGT	60%	-	-37	1735	55\|28\|1652	5.72 \| 4.79 \| 4.58	14	★★★☆	42	caccgTAGCCACCCTGAGCCACATG	aaacCATGTGGCTCAGGGTGGCTAc

Table 2: Guide sgRNAs for the sequence [example2]

Guide #	sgRNA Sequence(+PAM)	GC content	Strand	Position on sequence	Off-target Number	Off-target Number at TSS\|Exon\|Intergenic	Top3 Off-target Sites Scores	sgRNA Score (Based on Off-target)	PAM Score	Cas9 Loading Score	Forward oligo primer	Reverse oligo primer
#1	CCGGCCGCCACTCAAGTGGGGAA	75%	+	9	17	0\|3\|14	0.35 \| 0.35 \| 0.24	97	★★☆☆	48.5	caccgCCGGCCGCCACTCAAGTGGG	aaacCCCACTTGAGTGGCGGCCGGc
#2	AGTGGGGAAGCTTCCGCGACTGC	65%	+	23	142	6\|16\|120	1.07 \| 0.71 \| 0.56	89	★★☆☆	47	caccgAGTGGGGAAGCTTCCGCGAC	aaacGTCGCGGAAGCTTCCCCACTc
#3	GGCCGCTGAGCAGTCGCGGAAGC	75%	-	-2	156	15\|33\|108	0.82 \| 0.55 \| 0.54	87	★★☆☆	57	caccGGCCGCTGAGCAGTCGCGGA	aaacTCCGCGACTGCTCAGCGGCC
#4	CACTTGAGTGGCGGCCGGCCCGC	75%	-	-30	124	14\|18\|92	3.59 \| 1.06 \| 0.91	82	★★☆☆	46	caccgCACTTGAGTGGCGGCCGGCC	aaacGGCCGGCCGCCACTCAAGTGc
#5	GGCGGGCCGGCCGCCACTCAAGT	85%	+	3	104	11\|37\|56	1.63 \| 1.33 \| 1.29	81	★★☆☆	43	caccGGCGGGCCGGCCGCCACTCA	aaacTGAGTGGCGGCCGGCCCGCC
#6	CGGGCCGGCCGCCACTCAAGTGG	80%	+	5	83	13\|22\|48	1.46 \| 1.29 \| 1.03	80	★★☆☆	53	caccgCGGGCCGGCCGCCACTCAAG	aaacCTTGAGTGGCGGCCGGCCCGc
#7	GCCGGCCGCCACTCAAGTGGGGA	75%	+	8	97	3\|13\|81	0.98 \| 0.96 \| 0.94	80	★☆☆☆	54	caccGCCGGCCGCCACTCAAGTGG	aaacCCACTTGAGTGGCGGCCGGC
#8	GTCGCGGAAGCTTCCCCACTTGA	65%	-	-14	91	6\|9\|76	1.51 \| 1.38 \| 1.31	79	★★★☆	33	caccGTCGCGGAAGCTTCCCCACT	aaacAGTGGGGAAGCTTCCGCGAC
#9	GGCCGGCCGCCACTCAAGTGGGG	75%	+	7	84	6\|12\|66	2.46 \| 2.34 \| 1.62	77	★★★☆	43	caccGGCCGGCCGCCACTCAAGTG	aaacCACTTGAGTGGCGGCCGGCC
#10	TTCCCCACTTGAGTGGCGGCCGG	65%	-	-25	220	8\|13\|199	2.33 \| 0.99 \| 0.9	75	★★★☆	49	caccgTTCCCCACTTGAGTGGCGGC	aaacGCCGCCACTCAAGTGGGGAAc
#11	TCCCCACTTGAGTGGCGGCCGGC	70%	-	-26	190	6\|19\|165	2.52 \| 1.31 \| 1.29	74	★★★☆	46	caccgTCCCCACTTGAGTGGCGGCC	aaacGGCCGCCACTCAAGTGGGGAc
#12	GGAAGCTTCCGCGACTGCTCAGC	65%	+	28	204	9\|27\|168	1.27 \| 1.22 \| 1.06	71	★★☆☆	41	caccGGAAGCTTCCGCGACTGCTC	aaacGAGCAGTCGCGGAAGCTTCC
#13	CGCGGAAGCTTCCCCACTTGAGT	65%	-	-16	135	7\|10\|118	1.71 \| 1.63 \| 1.38	67	★★☆☆	37	caccgCGCGGAAGCTTCCCCACTTG	aaacCAAGTGGGGAAGCTTCCGCGc
#14	CGGAAGCTTCCCCACTTGAGTGG	60%	-	-18	220	7\|8\|205	1.52 \| 1.29 \| 1.11	66	★★☆☆	46.5	caccgCGGAAGCTTCCCCACTTGAG	aaacCTCAAGTGGGGAAGCTTCCGc
#15	CTCAAGTGGGGAAGCTTCCGCGA	60%	+	19	295	18\|16\|261	1.11 \| 1.1 \| 0.85	66	★★★★	42.5	caccgCTCAAGTGGGGAAGCTTCCG	aaacCGGAAGCTTCCCCACTTGAGc
#16	GGGCCGGCCGCCACTCAAGTGGG	75%	+	6	155	4\|19\|132	1.56 \| 1.41 \| 1.29	65	★★★☆	39	caccGGGCCGGCCGCCACTCAAGT	aaacACTTGAGTGGCGGCCGGCCC
#17	GGCCGCCACTCAAGTGGGGAAGC	70%	+	11	209	9\|16\|184	1.73 \| 1.63 \| 1.46	63	★★☆☆	61	caccGGCCGCCACTCAAGTGGGGA	aaacTCCCCACTTGAGTGGCGGCC
#18	GGAAGCTTCCCCACTTGAGTGGC	55%	-	-19	320	11\|15\|294	1.5 \| 1.38 \| 1.32	60	★★★☆	44	caccGGAAGCTTCCCCACTTGAGT	aaacACTCAAGTGGGGAAGCTTCC
#19	AAGCTTCCCCACTTGAGTGGCGG	55%	-	-21	369	14\|25\|330	4.6 \| 1.63 \| 1.54	55	★★★☆	54	caccgAAGCTTCCCCACTTGAGTGG	aaacCCACTCAAGTGGGGAAGCTTc
#20	AAGCTTCCGCGACTGCTCAGCGG	60%	+	30	239	11\|24\|204	3.9 \| 2.47 \| 1.56	55	★★★☆	47.5	caccgAAGCTTCCGCGACTGCTCAG	aaacCTGAGCAGTCGCGGAAGCTTc
#21	AGCTTCCGCGACTGCTCAGCGGC	65%	+	31	280	12\|27\|241	6.16 \| 4.6 \| 3.67	49	★★★☆	47	caccgAGCTTCCGCGACTGCTCAGC	aaacGCTGAGCAGTCGCGGAAGCTc
#22	AGCTTCCCCACTTGAGTGGCGGC	60%	-	-22	459	18\|27\|414	2.52 \| 1.78 \| 1.73	47	★★★☆	40.5	caccgAGCTTCCCCACTTGAGTGGC	aaacGCCACTCAAGTGGGGAAGCTc
#23	CACTCAAGTGGGGAAGCTTCCGC	55%	+	17	562	9\|20\|533	3.56 \| 2.39 \| 2.34	41	★★☆☆	29	caccgCACTCAAGTGGGGAAGCTTC	aaacGAAGCTTCCCCACTTGAGTGc

Table 3: Guide sgRNAs for the sequence [example3]

Guide #	sgRNA Sequence(+PAM)	GC content	Strand	Position on sequence	Off-target Number	Off-target Number at TSS\|Exon\|Intergenic	Top3 Off-target Sites Scores	sgRNA Score (Based on Off-target)	PAM Score	Cas9 Loading Score	Forward oligo primer	Reverse oligo primer
#1	CCGACGCTGTTCATGGCGGTGAT	65%	-	-16	15	0\|2\|13	1.48 \| 0.76 \| 0.32	96	★★☆☆	42	caccgCCGACGCTGTTCATGGCGGT	aaacACCGCCATGAACAGCGTCGGc
#2	GCAGCTGCCATCACCGCCATGAA	65%	+	7	66	0\|9\|57	1.42 \| 0.84 \| 0.8	89	★★☆☆	41	caccGCAGCTGCCATCACCGCCAT	aaacATGGCGGTGATGGCAGCTGC
#3	ACCGCCATGAACAGCGTCGGGGA	65%	+	19	90	1\|18\|71	1.11 \| 0.81 \| 0.74	88	★☆☆☆	49.5	caccgACCGCCATGAACAGCGTCGG	aaacCCGACGCTGTTCATGGCGGTc
#4	CACCGCCATGAACAGCGTCGGGG	65%	+	18	109	2\|29\|78	0.53 \| 0.53 \| 0.51	88	★★★☆	47	caccgCACCGCCATGAACAGCGTCG	aaacCGACGCTGTTCATGGCGGTGc
#5	CGCCATGAACAGCGTCGGGGAGG	70%	+	21	102	3\|31\|68	0.91 \| 0.73 \| 0.56	87	★★☆☆	61	caccgCGCCATGAACAGCGTCGGGG	aaacCCCCGACGCTGTTCATGGCGc
#6	AGTGCAAGCCTCCCCGACGCTGT	70%	-	-3	129	5\|20\|104	3.59 \| 0.71 \| 0.43	86	★★★☆	49	caccgAGTGCAAGCCTCCCCGACGC	aaacGCGTCGGGGAGGCTTGCACTc
#7	CCCGACGCTGTTCATGGCGGTGA	70%	-	-15	99	2\|21\|76	1.71 \| 1.48 \| 1.46	84	★★★☆	62	caccgCCCGACGCTGTTCATGGCGG	aaacCCGCCATGAACAGCGTCGGGc
#8	ATCACCGCCATGAACAGCGTCGG	55%	+	16	185	6\|12\|167	0.92 \| 0.81 \| 0.54	80	★★★☆	47	caccgATCACCGCCATGAACAGCGT	aaacACGCTGTTCATGGCGGTGATc
#9	GCCATGAACAGCGTCGGGGAGGC	65%	+	22	260	10\|38\|212	0.86 \| 0.83 \| 0.82	76	★★★☆	61	caccGCCATGAACAGCGTCGGGGA	aaacTCCCCGACGCTGTTCATGGC
#10	TCACCGCCATGAACAGCGTCGGG	60%	+	17	201	2\|26\|173	1.27 \| 1.02 \| 0.89	75	★★★☆	36	caccgTCACCGCCATGAACAGCGTC	aaacGACGCTGTTCATGGCGGTGAc
#11	AGCCTCCCCGACGCTGTTCATGG	65%	-	-9	176	5\|32\|139	2.11 \| 1.31 \| 0.85	72	★★☆☆	34.5	caccgAGCCTCCCCGACGCTGTTCA	aaacTGAACAGCGTCGGGGAGGCTc
#12	GCCTCCCCGACGCTGTTCATGGC	65%	-	-10	160	3\|18\|139	4.6 \| 1.46 \| 1.09	71	★★★☆	32.5	caccGCCTCCCCGACGCTGTTCAT	aaacATGAACAGCGTCGGGGAGGC
#13	AGCGTCGGGGAGGCTTGCACTGA	70%	+	31	174	6\|27\|141	1.43 \| 0.91 \| 0.9	71	★★★☆	52	caccgAGCGTCGGGGAGGCTTGCAC	aaacGTGCAAGCCTCCCCGACGCTc
#14	TCCCCGACGCTGTTCATGGCGGT	65%	-	-13	211	6\|28\|177	1.56 \| 1.38 \| 1.35	67	★★★☆	40.5	caccgTCCCCGACGCTGTTCATGGC	aaacGCCATGAACAGCGTCGGGGAc
#15	ACGCTGTTCATGGCGGTGATGGC	55%	-	-19	192	5\|19\|168	4.04 \| 2.37 \| 2.37	67	★★★☆	31.5	caccgACGCTGTTCATGGCGGTGAT	aaacATCACCGCCATGAACAGCGTc
#16	CTCCCCGACGCTGTTCATGGCGG	65%	-	-12	206	6\|30\|170	2.17 \| 1.42 \| 0.92	66	★★★☆	49	caccgCTCCCCGACGCTGTTCATGG	aaacCCATGAACAGCGTCGGGGAGc
#17	TGAACAGCGTCGGGGAGGCTTGC	65%	+	26	209	6\|19\|184	1.42 \| 1.32 \| 1.31	62	★★☆☆	39	caccgTGAACAGCGTCGGGGAGGCT	aaacAGCCTCCCCGACGCTGTTCAc
#18	CTGCCATCACCGCCATGAACAGC	60%	+	11	392	6\|29\|357	2.66 \| 1.42 \| 1.4	58	★★☆☆	49	caccgCTGCCATCACCGCCATGAAC	aaacGTTCATGGCGGTGATGGCAGc
#19	CGCAGCTGCCATCACCGCCATGA	70%	+	6	425	19\|84\|322	1.5 \| 1.38 \| 1.29	52	★★★☆	55	caccgCGCAGCTGCCATCACCGCCA	aaacTGGCGGTGATGGCAGCTGCGc
#20	GCCATCACCGCCATGAACAGCGT	60%	+	13	383	8\|39\|336	5.89 \| 1.56 \| 1.53	51	★★★★	55	caccGCCATCACCGCCATGAACAG	aaacCTGTTCATGGCGGTGATGGC
#21	GACGCTGTTCATGGCGGTGATGG	60%	-	-18	280	4\|42\|234	6.01 \| 4.04 \| 2.42	50	★★☆☆	54	caccGACGCTGTTCATGGCGGTGA	aaacTCACCGCCATGAACAGCGTC
#22	CTGTTCATGGCGGTGATGGCAGC	60%	-	-22	483	10\|45\|428	6.38 \| 1.56 \| 1.56	49	★★☆☆	40.5	caccgCTGTTCATGGCGGTGATGGC	aaacGCCATCACCGCCATGAACAGc
#23	TTCATGGCGGTGATGGCAGCTGC	60%	-	-25	501	11\|53\|437	4.21 \| 3.17 \| 2.56	39	★★☆☆	47	caccgTTCATGGCGGTGATGGCAGC	aaacGCTGCCATCACCGCCATGAAc
#24	GTCACCGCAGCTGCCATCACCGC	65%	+	1	556	12\|81\|463	100 \| 4.32 \| 1.69	29	★★☆☆	39.5	caccGTCACCGCAGCTGCCATCAC	aaacGTGATGGCAGCTGCGGTGAC
#25	CATGGCGGTGATGGCAGCTGCGG	65%	-	-27	762	14\|99\|649	100 \| 4.04 \| 3.41	27	★★★☆	49	caccgCATGGCGGTGATGGCAGCTG	aaacCAGCTGCCATCACCGCCATGc
#26	ATGGCGGTGATGGCAGCTGCGGT	65%	-	-28	854	19\|126\|709	3.71 \| 2.52 \| 2.52	26	★★★☆	42	caccgATGGCGGTGATGGCAGCTGC	aaacGCAGCTGCCATCACCGCCATc
#27	GGCGGTGATGGCAGCTGCGGTGA	75%	-	-30	1229	118\|388\|723	100 \| 6.24 \| 6.24	24	★★★☆	62	caccGGCGGTGATGGCAGCTGCGG	aaacCCGCAGCTGCCATCACCGCC

sequence #1
sequence #2
sequence #3

Sequence: example1(127 bp)

GTCGCTCGTCGGAGCTGCAGGGACCGGCGCGAGCGAGTGCTGGACTGTTTGTGCAGGGCTCCGAGGGGACCCATGTGGCTCAGGGTGGCTAAGGGGGCAATGCTGCGTCGTCGTAGTTTTTTGGGGG

Potential guide sgRNA number: 71

These two kinds of sgRNA will be removed automatically: 1) GC content >80% or <20%; 2) >=50% nucleotides are As, Gs, Gs or Ts.

#	Score	Sequence
#1	95	GGGCAATGCTGCGTCGTCGTAGT
#2	94	CCAAAAAACTACGACGACGCAGC
#3	92	GCTGCGTCGTCGTAGTTTTTTGG
#4	90	AGGGGGCAATGCTGCGTCGTCGT
#5	89	AAACTACGACGACGCAGCATTGC
#6	89	CCCCCAAAAAACTACGACGACGC
#7	87	AGTCCAGCACTCGCTCGCGCCGG
#8	84	GTCCCTGCAGCTCCGACGAGCGA
#9	84	AACAGTCCAGCACTCGCTCGCGC
#10	83	GACCGGCGCGAGCGAGTGCTGGA
#11	83	GTCCAGCACTCGCTCGCGCCGGT
#12	82	CGGTCCCTGCAGCTCCGACGAGC
#13	81	GCGTCGTCGTAGTTTTTTGGGGG
#14	80	GGACCGGCGCGAGCGAGTGCTGG
#15	80	TCGCTCGCGCCGGTCCCTGCAGC
#16	78	TGCGTCGTCGTAGTTTTTTGGGG
#17	78	CTGCGTCGTCGTAGTTTTTTGGG
#18	77	CTAAGGGGGCAATGCTGCGTCGT
#19	76	AGCCACATGGGTCCCCTCGGAGC
#20	75	GGCGCGAGCGAGTGCTGGACTGT
#21	75	CGACGCAGCATTGCCCCCTTAGC
#22	72	TGCAGGGACCGGCGCGAGCGAGT
#23	72	GTCGCTCGTCGGAGCTGCAGGGA
#24	71	GAGCTGCAGGGACCGGCGCGAGC
#25	69	GCTGCAGGGACCGGCGCGAGCGA
#26	68	CGAGCGAGTGCTGGACTGTTTGT
#27	66	CAAACAGTCCAGCACTCGCTCGC
#28	66	GGCTCCGAGGGGACCCATGTGGC
#29	64	CGGAGCTGCAGGGACCGGCGCGA
#30	62	CAGGGACCGGCGCGAGCGAGTGC
#31	62	CTGTTTGTGCAGGGCTCCGAGGG
#32	56	ACTGTTTGTGCAGGGCTCCGAGG
#33	56	AGCGAGTGCTGGACTGTTTGTGC
#34	55	GTCGGAGCTGCAGGGACCGGCGC
#35	55	GGGCTCCGAGGGGACCCATGTGG
#36	55	GTTTGTGCAGGGCTCCGAGGGGA
#37	54	TGTTTGTGCAGGGCTCCGAGGGG
#38	53	CGCGCCGGTCCCTGCAGCTCCGA
#39	53	TCGTCGGAGCTGCAGGGACCGGC
#40	52	CACTCGCTCGCGCCGGTCCCTGC
#41	52	GCCGGTCCCTGCAGCTCCGACGA
#42	50	TGAGCCACATGGGTCCCCTCGGA
#43	50	CGAGGGGACCCATGTGGCTCAGG
#44	49	CCTCGGAGCCCTGCACAAACAGT
#45	47	CTCGTCGGAGCTGCAGGGACCGG
#46	47	CTGAGCCACATGGGTCCCCTCGG
#47	46	GAGCCCTGCACAAACAGTCCAGC
#48	46	TGCACAAACAGTCCAGCACTCGC
#49	46	CATGGGTCCCCTCGGAGCCCTGC
#50	44	ATGTGGCTCAGGGTGGCTAAGGG
#51	43	CATTGCCCCCTTAGCCACCCTGA
#52	43	GGTGGCTAAGGGGGCAATGCTGC
#53	39	GAGGGGACCCATGTGGCTCAGGG
#54	39	GTGGCTCAGGGTGGCTAAGGGGG
#55	39	TGGCTCAGGGTGGCTAAGGGGGC
#56	38	TTGCCCCCTTAGCCACCCTGAGC
#57	37	GAGTGCTGGACTGTTTGTGCAGG
#58	36	TGTGGCTCAGGGTGGCTAAGGGG
#59	36	CAGGGCTCCGAGGGGACCCATGT
#60	35	GGACTGTTTGTGCAGGGCTCCGA
#61	33	AGTGCTGGACTGTTTGTGCAGGG
#62	30	TGGCTAAGGGGGCAATGCTGCGT
#63	30	CATGTGGCTCAGGGTGGCTAAGG
#64	30	GTGCTGGACTGTTTGTGCAGGGC
#65	26	CTTAGCCACCCTGAGCCACATGG
#66	25	CAGGGTGGCTAAGGGGGCAATGC
#67	24	AGGGGACCCATGTGGCTCAGGGT
#68	22	GGGACCCATGTGGCTCAGGGTGG
#69	22	TTAGCCACCCTGAGCCACATGGG
#70	16	GGACCCATGTGGCTCAGGGTGGC
#71	14	TAGCCACCCTGAGCCACATGGGT

Legend

High quality

Low quality

Guide sgRNA # 1

sgRNA score: 95

GC content: 65%

Strand: Forward

Potential off-target sites: 66 (Mismatch site number <=4)

Oligos to order: FWD: caccGGGCAATGCTGCGTCGTCGT REV: aaacACGACGACGCAGCATTGCCC

Top 20 off-target sites were shown here.

#	Sequence	Score	Mismatch	Genomic locus	Genomic annotation
#1	GGGCggcGCcGCGTCGTCGT CGT	0.51	4MMs [5:6:7:10]	chr18\|38418891\|+	Ndfip1\|TSS upstream +2kb,Ndfip1\|exon
#2	GGGacATGtTtCGTCGTCGT CGT	0.47	4MMs [4:5:9:11]	chr8\|119874183\|-
#3	aGGCAAaGCaGgGTCGTCGT GGT	0.45	4MMs [1:7:10:12]	chr17\|86347110\|-
#4	caGCAcTGCTGCcTCGTCGT TGA	0.35	4MMs [1:2:6:13]	chr17\|50086767\|-
#5	GaaCAATGCTGaGTCGaCGT GGC	0.29	4MMs [2:3:12:17]	chr7\|55988953\|+
#6	GGcCAAgGaTGCcTCGTCGT AGA	0.23	4MMs [3:7:9:13]	chr13\|40800542\|+
#7	aGGaAAgGCTGCGTCGTgGT GGT	0.21	4MMs [1:4:7:18]	chr13\|107793431\|-
#8	GGcCAcTGaTGCGTCGTCtT CGA	0.18	4MMs [3:6:9:19]	chr10\|93162133\|-	Mir1931\|TSS upstream +2kb
#9	GGtCAATGgaGCGTCGTgGT GGA	0.17	4MMs [3:9:10:18]	chr3\|122440975\|+
#10	GGatAATGCTGgGTCGTgGT AGG	0.15	4MMs [3:4:12:18]	chr3\|86889856\|-
#11	GGaCAATGgTGgGTCGTCcT GGG	0.15	4MMs [3:9:12:19]	chr3\|10357387\|-
#12	GGGCgAcGgTGCGTCGTaGT CGG	0.12	4MMs [5:7:9:18]	chr1\|171065434\|-	Mir6546\|TSS upstream +2kb
#13	GGGCgAcGgTGCGTCGTaGT CGG	0.12	4MMs [5:7:9:18]	chr1\|171080683\|-
#14	GGGCgAcGgTGCGTCGTaGT CGG	0.12	4MMs [5:7:9:18]	chr1\|171073069\|-
#15	GGGacATGCTGCGTCagCGT GGG	0.1	4MMs [4:5:16:17]	chr17\|29389361\|+
#16	GGGCAgaGCTGgGTCGTCcT AGC	0.09	4MMs [6:7:12:19]	chr11\|107881324\|-
#17	GGGCAAgGgcGCGTCcTCGT GGG	0.09	4MMs [7:9:10:16]	chr17\|34197773\|+	Psmb8\|TSS upstream +2kb
#18	aGGCAATGCTtgGTCtTCGT TGC	0.07	4MMs [1:11:12:16]	chr7\|52027194\|-
#19	GGGCtcTGCTGCGTCtTCGa TGG	0.06	4MMs [5:6:16:20]	chr12\|32849634\|-
#20	GGGCAATcCTcCGTaGTCaT AGA	0.06	4MMs [8:11:15:19]	chrY\|9658244\|-

Sequence: example2(55 bp)

GAGGCGGGCCGGCCGCCACTCAAGTGGGGAAGCTTCCGCGACTGCTCAGCGGCCG

Potential guide sgRNA number: 23

These two kinds of sgRNA will be removed automatically: 1) GC content >80% or <20%; 2) >=50% nucleotides are As, Gs, Gs or Ts.

#	Score	Sequence
#1	97	CCGGCCGCCACTCAAGTGGGGAA
#2	89	AGTGGGGAAGCTTCCGCGACTGC
#3	87	GGCCGCTGAGCAGTCGCGGAAGC
#4	82	CACTTGAGTGGCGGCCGGCCCGC
#5	81	GGCGGGCCGGCCGCCACTCAAGT
#6	80	CGGGCCGGCCGCCACTCAAGTGG
#7	80	GCCGGCCGCCACTCAAGTGGGGA
#8	79	GTCGCGGAAGCTTCCCCACTTGA
#9	77	GGCCGGCCGCCACTCAAGTGGGG
#10	75	TTCCCCACTTGAGTGGCGGCCGG
#11	74	TCCCCACTTGAGTGGCGGCCGGC
#12	71	GGAAGCTTCCGCGACTGCTCAGC
#13	67	CGCGGAAGCTTCCCCACTTGAGT
#14	66	CGGAAGCTTCCCCACTTGAGTGG
#15	66	CTCAAGTGGGGAAGCTTCCGCGA
#16	65	GGGCCGGCCGCCACTCAAGTGGG
#17	63	GGCCGCCACTCAAGTGGGGAAGC
#18	60	GGAAGCTTCCCCACTTGAGTGGC
#19	55	AAGCTTCCCCACTTGAGTGGCGG
#20	55	AAGCTTCCGCGACTGCTCAGCGG
#21	49	AGCTTCCGCGACTGCTCAGCGGC
#22	47	AGCTTCCCCACTTGAGTGGCGGC
#23	41	CACTCAAGTGGGGAAGCTTCCGC

Legend

High quality

Low quality

Guide sgRNA # 1

sgRNA score: 97

GC content: 75%

Strand: Forward

Potential off-target sites: 17 (Mismatch site number <=4)

Oligos to order: FWD: caccgCCGGCCGCCACTCAAGTGGG REV: aaacCCCACTTGAGTGGCGGCCGGc

Top 20 off-target sites were shown here.

#	Sequence	Score	Mismatch	Genomic locus	Genomic annotation
#1	CtGGtCtCCACTCAAGTGtG GAA	0.35	4MMs [2:5:7:19]	chr11\|55236692\|-
#2	CCcGCCGCCgggCAAGTGGG GAA	0.35	4MMs [3:10:11:12]	chr4\|21679605\|-	Prdm13\|exon
#3	CaGcCtGCCACTCAtGTGGG GAA	0.24	4MMs [2:4:6:15]	chr5\|144889654\|+
#4	CtGGCCtCCAgTCAAGcGGG GAT	0.23	4MMs [2:7:11:17]	chr12\|110352230\|-
#5	CCtGCtGCCcCTCAtGTGGG GAA	0.22	4MMs [3:6:10:15]	chr19\|5422722\|+	4930481A15Rik\|exon
#6	CCGcCCcCCAtTCAAGaGGG GAA	0.22	4MMs [4:7:11:17]	chr11\|94698538\|+
#7	CCGGCCtgCACaCAAGaGGG GAT	0.18	4MMs [7:8:12:17]	chr5\|18799114\|+
#8	CtGGCCaCCAgTCAtGTGGG GAA	0.15	4MMs [2:7:11:15]	chr17\|52744784\|-
#9	CtGGCCaCCACTCAtGTGGt GAA	0.12	4MMs [2:7:15:20]	chr8\|88583467\|-
#10	CaGGCCcCCACTCAAGgtGG GAA	0.08	4MMs [2:7:17:18]	chr10\|58231365\|-	Dux\|exon
#11	CaGGCCcCCACTCAAGgtGG GAA	0.08	4MMs [2:7:17:18]	chr10\|58228571\|-
#12	CaGGCCcCCACTCAAGgtGG GAA	0.08	4MMs [2:7:17:18]	chr10\|58228022\|-
#13	CaGGCCcCCACTCAAGgtGG GAA	0.08	4MMs [2:7:17:18]	chr10\|58223686\|-
#14	CaGGCCcCCACTCAAGgtGG GAA	0.08	4MMs [2:7:17:18]	chr10\|58223137\|-
#15	CCaGaCGCCACTCAAcTGaG GAA	0.08	4MMs [3:5:16:19]	chr7\|14997154\|+
#16	CCGcCaGCCACTCAAGTaGt GAT	0.07	4MMs [4:6:18:20]	chr10\|36855326\|-
#17	CCGGCCtCCACTCAtGTctG GAT	0.01	4MMs [7:15:18:19]	chr8\|95729990\|-

Sequence: example3(53 bp)

GTCACCGCAGCTGCCATCACCGCCATGAACAGCGTCGGGGAGGCTTGCACTGA

Potential guide sgRNA number: 27

These two kinds of sgRNA will be removed automatically: 1) GC content >80% or <20%; 2) >=50% nucleotides are As, Gs, Gs or Ts.

#	Score	Sequence
#1	96	CCGACGCTGTTCATGGCGGTGAT
#2	89	GCAGCTGCCATCACCGCCATGAA
#3	88	ACCGCCATGAACAGCGTCGGGGA
#4	88	CACCGCCATGAACAGCGTCGGGG
#5	87	CGCCATGAACAGCGTCGGGGAGG
#6	86	AGTGCAAGCCTCCCCGACGCTGT
#7	84	CCCGACGCTGTTCATGGCGGTGA
#8	80	ATCACCGCCATGAACAGCGTCGG
#9	76	GCCATGAACAGCGTCGGGGAGGC
#10	75	TCACCGCCATGAACAGCGTCGGG
#11	72	AGCCTCCCCGACGCTGTTCATGG
#12	71	GCCTCCCCGACGCTGTTCATGGC
#13	71	AGCGTCGGGGAGGCTTGCACTGA
#14	67	TCCCCGACGCTGTTCATGGCGGT
#15	67	ACGCTGTTCATGGCGGTGATGGC
#16	66	CTCCCCGACGCTGTTCATGGCGG
#17	62	TGAACAGCGTCGGGGAGGCTTGC
#18	58	CTGCCATCACCGCCATGAACAGC
#19	52	CGCAGCTGCCATCACCGCCATGA
#20	51	GCCATCACCGCCATGAACAGCGT
#21	50	GACGCTGTTCATGGCGGTGATGG
#22	49	CTGTTCATGGCGGTGATGGCAGC
#23	39	TTCATGGCGGTGATGGCAGCTGC
#24	29	GTCACCGCAGCTGCCATCACCGC
#25	27	CATGGCGGTGATGGCAGCTGCGG
#26	26	ATGGCGGTGATGGCAGCTGCGGT
#27	24	GGCGGTGATGGCAGCTGCGGTGA

Legend

High quality

Low quality

Guide sgRNA # 1

sgRNA score: 96

GC content: 65%

Strand: Reverse

Potential off-target sites: 15 (Mismatch site number <=4)

Oligos to order: FWD: caccgCCGACGCTGTTCATGGCGGT REV: aaacACCGCCATGAACAGCGTCGGc

Top 20 off-target sites were shown here.

#	Sequence	Score	Mismatch	Genomic locus	Genomic annotation
#1	CtGACaCTGaTCATGGCGGT GAT	1.48	3MMs [2:6:10]	chr9\|120895933\|-
#2	CCaAaGCTagTCATGGCGGT GAA	0.76	4MMs [3:5:9:10]	chr11\|96856457\|+
#3	CCtACcCTGaTCATGGaGGT GAA	0.32	4MMs [3:6:10:17]	chr5\|73688707\|+
#4	CCcAtGCTGTgCATGGtGGT GAT	0.32	4MMs [3:5:11:17]	chr7\|64895781\|-
#5	CCGAaGCTGgTCATGGaGcT GAT	0.17	4MMs [5:10:17:19]	chr11\|61678556\|-	Slc5a10\|exon
#6	CaGACaCTGTTgATGGCGaT GAT	0.15	4MMs [2:6:12:19]	chr8\|23985881\|-
#7	CCtcCaCTGTTCATGtCGGT GAT	0.15	4MMs [3:4:6:16]	chr15\|4753125\|+
#8	CCGACctTcTTCATGGtGGT GAA	0.14	4MMs [6:7:9:17]	chr7\|70491153\|-
#9	CtGAaGCTGTTCcTGGCaGT GAT	0.12	4MMs [2:5:13:18]	chr8\|46920746\|-
#10	CCcAgGCTGTTCATGGgtGT GAA	0.11	4MMs [3:5:17:18]	chr17\|14287838\|+
#11	CaGAaGCTGTTCAaGGtGGT GAA	0.09	4MMs [2:5:14:17]	chr9\|83679650\|+
#12	CaGACGCTGTaaAaGGCGGT GAT	0.06	4MMs [2:11:12:14]	chr4\|148327336\|-
#13	gCGAtGCTGTTCAcGGCaGT GAT	0.04	4MMs [1:5:14:18]	chr7\|134723194\|+
#14	CCaACtCTGTTCAaGGCaGT GAT	0.02	4MMs [3:6:14:18]	chr3\|96931594\|-
#15	CCGACGCTGTcCAgGGtGcT GAA	0.01	4MMs [11:14:17:19]	chr17\|72603314\|-	Alk\|exon

Here is the zip file for all results.

#	Sequence	Score	Mismatch	Genomic locus	Genomic annotation
#1	CaAAcAAACgACGACGACGa TGA	0.64	4MMs [2:5:10:20]	chr3\|96292542\|+
#2	CaAAcAAACaACGACGACGa CGA	0.64	4MMs [2:5:10:20]	chr1\|126263535\|-
#3	gCAAcAAAtTAtGACGACGC TGC	0.44	4MMs [1:5:9:12]	chr4\|85513331\|-
#4	CaAAgAAAaTAaGACGACGC TGT	0.43	4MMs [2:5:9:12]	chr11\|95629627\|-
#5	CaAAAAgACgACGACGACGa CGA	0.43	4MMs [2:7:10:20]	chr15\|83617998\|+
#6	CCAcAAcACgAgGACGACGC GGC	0.43	4MMs [4:7:10:12]	chr9\|122886971\|-	Zkscan7\|TSS upstream +2kb
#7	CCAgAAcACcACGACGACGa CGA	0.42	4MMs [4:7:10:20]	chr3\|100568615\|+
#8	CaAAAtAACgACGACGACGa CGA	0.38	4MMs [2:6:10:20]	chr8\|96286064\|-
#9	CaAAAcAACgACGACGACGa CGA	0.38	4MMs [2:6:10:20]	chr12\|11006049\|+
#10	CaAAAAAACacCGAgGACGC TGG	0.2	4MMs [2:10:11:15]	chr2\|69732227\|-	Ppig\|exon
#11	CaAAAAAACaACcACGACtC AGG	0.18	4MMs [2:10:13:19]	chr4\|72351118\|-
#12	CaAtAAAACTACGAtGACcC AGG	0.13	4MMs [2:4:15:19]	chr9\|8155154\|-
#13	CCAAAAAtCcAaGACGAgGC AGA	0.12	4MMs [8:10:12:18]	chr12\|90548173\|+
#14	CCAAAgAAgTAtGACGACGa CGA	0.11	4MMs [6:9:12:20]	chr1\|44059786\|-
#15	CCAAAcAACaACGACGACaa TGA	0.11	4MMs [6:10:19:20]	chr19\|33800943\|+
#16	CCAAAAAtaTACtACGACtC AGG	0.11	4MMs [8:9:13:19]	chr9\|104333642\|+
#17	CCAAcAAACaACGACaACGa CGA	0.1	4MMs [5:10:16:20]	chr2\|128137732\|-
#18	gCAAAAAAgTAtGACGAaGC CGA	0.09	4MMs [1:9:12:18]	chr19\|34574087\|+	Ifit2\|exon
#19	CtgAAAAACTACGAaGAtGC AGA	0.08	4MMs [2:3:15:18]	chr9\|108393242\|+	1700102P08Rik\|exon
#20	CCAgAAAACcACGAgGAaGC AGA	0.07	4MMs [4:10:15:18]	chr16\|11388202\|-

#	Sequence	Score	Mismatch	Genomic locus	Genomic annotation
#1	tCTGCGTtGgtGTAGTTTTT TGT	0.74	4MMs [1:8:10:11]	chr1\|62315726\|+
#2	aCTGtGTtGTCtTAGTTTTT GGA	0.72	4MMs [1:5:8:12]	chr9\|113336860\|-
#3	tCTGaGTCcTgGTAGTTTTT AGT	0.49	4MMs [1:5:9:11]	chr5\|37651314\|-
#4	ctTGCGTCcTCGTAGTTTTc AGA	0.43	4MMs [1:2:9:20]	chr7\|125914609\|+
#5	GCTtCtTaGTCcTAGTTTTT TGC	0.41	4MMs [4:6:8:12]	chr3\|105824597\|+
#6	GCgGCGTtGTttTAGTTTTT TGA	0.38	4MMs [3:8:11:12]	chr8\|114472022\|-
#7	GCTGCtTtGaCcTAGTTTTT AGT	0.37	4MMs [6:8:10:12]	chrX\|73415786\|-
#8	GCTGCtTgGaCtTAGTTTTT TGT	0.37	4MMs [6:8:10:12]	chr3\|83948320\|+
#9	aCTGCGTgGTCtTAGTTTTg GGC	0.34	4MMs [1:8:12:20]	chr14\|20378101\|-
#10	GCTGgGTaGTaGTAGTgTTT GGG	0.32	4MMs [5:8:11:17]	chr1\|80301009\|+
#11	GtTGtGTCGTgGaAGTTTTT TGT	0.31	4MMs [2:5:11:13]	chr14\|76852954\|+
#12	GCTtCGTgGTCaTAGTgTTT TGT	0.28	4MMs [4:8:12:17]	chr9\|47377696\|+
#13	GCTGaGaCGTgGTAGTTTTg TGT	0.25	4MMs [5:7:11:20]	chr3\|121996581\|+
#14	GCTaCtTCtTCGTAGTTTTc TGA	0.24	4MMs [4:6:9:20]	chr13\|81703507\|+
#15	GCTGCGTaGgCtgAGTTTTT GGC	0.23	4MMs [8:10:12:13]	chr7\|141222983\|-	Mir210\|TSS upstream +2kb
#16	GaTGCGTaGTgGTAcTTTTT AGA	0.22	4MMs [2:8:11:15]	chrY\|2457114\|+
#17	GCTGaGTCtTgGTAGTTTTa GGT	0.22	4MMs [5:9:11:20]	chr14\|9568660\|+
#18	GCTGCcTtGTacTAGTTTTT AGA	0.22	4MMs [6:8:11:12]	chr16\|34439927\|-
#19	GgTGCGTCtTgGTAGTcTTT GGC	0.2	4MMs [2:9:11:17]	chr18\|15126684\|-
#20	GCTGtGTaGTCaTAtTTTTT TGC	0.19	4MMs [5:8:12:15]	chr3\|85714357\|-

#	Sequence	Score	Mismatch	Genomic locus	Genomic annotation
#1	AGGGaGCAAgGCTGCGTCcT TGT	0.91	3MMs [5:10:19]	chr4\|107590116\|+
#2	gGctGGCAATGtTGCGTCGT GGC	0.71	4MMs [1:3:4:12]	chr13\|107887608\|-
#3	AaGtGGCcATGCTGCGTCtT AGG	0.51	4MMs [2:4:8:19]	chr17\|34095552\|-	BC051537\|TSS upstream +2kb
#4	AGGcGGCtcTaCTGCGTCGT TGC	0.47	4MMs [4:8:9:11]	chr8\|24993149\|-
#5	AGGGGGaAAgGCTGgGTCGT GGT	0.45	3MMs [7:10:15]	chr5\|113740025\|-	Ficd\|exon
#6	tGGtGGCcATGCTGgGTCGT AGA	0.41	4MMs [1:4:8:15]	chr15\|87991611\|-
#7	AGGtGGCtcTGCaGCGTCGT CGG	0.33	4MMs [4:8:9:13]	chr10\|70194591\|-
#8	AGaGtGCAAgGCTGCGTgGT GGA	0.28	4MMs [3:5:10:18]	chr2\|168649330\|+
#9	AGGatGCAAgGCTGCGTgGT AGC	0.28	4MMs [4:5:10:18]	chr15\|95332291\|-
#10	AGGGGGCAgTGtgGCGTCGT GGG	0.28	3MMs [9:12:13]	chr17\|46680398\|-	Mea1\|TSS upstream +2kb
#11	AGGGaGCcAcGCTGCGTgGT AGA	0.27	4MMs [5:8:10:18]	chr3\|37569744\|-
#12	AGGcGGaAAcGCTGaGTCGT TGG	0.24	4MMs [4:7:10:15]	chr13\|51651715\|+	Secisbp2\|TSS upstream +2kb,Secisbp2\|exon
#13	tGGGGGaAATtCTGCGaCGT TGT	0.23	4MMs [1:7:11:17]	chr8\|17369623\|-
#14	AGGGGtagATGCTGCGTCcT CGT	0.19	4MMs [6:7:8:19]	chr16\|57462082\|-
#15	AGtGGGgAATaCTGCGTCaT AGA	0.18	4MMs [3:7:11:19]	chr1\|169419737\|-
#16	tGGGGatAATGCTGgGTCGT GGT	0.17	4MMs [1:6:7:15]	chr3\|86889859\|-
#17	tGGGGGCcATGCTGtGcCGT GGG	0.16	4MMs [1:8:15:17]	chr11\|50653721\|+
#18	tGGGGGCAgTGCTGCGcCGc CGC	0.16	4MMs [1:9:17:20]	chr7\|120835863\|+
#19	AaGGacCAATGCTGCcTCGT CGT	0.16	4MMs [2:5:6:16]	chr5\|77746596\|-
#20	AGaGGtCAATGgaGCGTCGT GGT	0.16	4MMs [3:6:12:13]	chr3\|122440972\|+

#	Sequence	Score	Mismatch	Genomic locus	Genomic annotation
#1	AAtaTAaaACGACGCAGCAT GGG	0.9	4MMs [3:4:7:8]	chr14\|100149979\|+	Klf12\|TSS upstream +2kb
#2	AAcCcACGAaaACGCAGCAT CGA	0.7	4MMs [3:5:10:11]	chr14\|59907659\|+
#3	AAAtcACcACGACGCAGCAc AGT	0.67	4MMs [4:5:8:20]	chr3\|103717617\|+
#4	AcACTACaAaGgCGCAGCAT TGG	0.65	4MMs [2:8:10:12]	chr4\|117653126\|-
#5	tAACcACGAgGAgGCAGCAT AGA	0.53	4MMs [1:5:10:13]	chr13\|51423831\|-
#6	AAAggACGAaGAgGCAGCAT TGT	0.5	4MMs [4:5:10:13]	chr6\|72748876\|+
#7	AAAgTACcAaGAtGCAGCAT GGA	0.5	4MMs [4:8:10:13]	chr16\|74548095\|+
#8	gAACcAtGACGgCGCAGCAT TGA	0.49	4MMs [1:5:7:12]	chr18\|68765793\|+
#9	AAAtTAaGAgGAgGCAGCAT AGG	0.34	4MMs [4:7:10:13]	chr12\|80846769\|+
#10	AAACTAaaAaGAtGCAGCAT GGT	0.33	4MMs [7:8:10:13]	chr6\|21327904\|-
#11	AAACTcCtAgGAaGCAGCAT AGA	0.29	4MMs [6:8:10:13]	chr2\|53141242\|+
#12	cAACTACaACGAgGCAGCAa AGA	0.27	4MMs [1:8:13:20]	chr17\|80715256\|+
#13	AgACgACGACGAaGCAGCAa TGG	0.26	4MMs [2:5:13:20]	chr1\|86333361\|+
#14	AAACTACaAatAgGCAGCAT GGA	0.26	4MMs [8:10:11:13]	chr2\|141382055\|+
#15	AtACTtaGACGAaGCAGCAT AGA	0.23	4MMs [2:6:7:13]	chr6\|94604448\|-	Slc25a26\|exon
#16	AAACaACGgCatCGCAGCAT TGG	0.23	4MMs [5:9:11:12]	chr11\|67055738\|+	Sco1\|exon
#17	AAACTAgcACGtCGCAGCAg AGG	0.21	4MMs [7:8:12:20]	chr8\|47612948\|+
#18	AAAtTACGAaGAaGCAcCAT GGA	0.2	4MMs [4:10:13:17]	chr16\|27469888\|+
#19	AAACTggGAgGAaGCAGCAT GGG	0.2	4MMs [6:7:10:13]	chr8\|71930633\|-
#20	AAAgTACaAaGACaCAGCAT GGG	0.19	4MMs [4:8:10:14]	chr18\|88579727\|+

#	Sequence	Score	Mismatch	Genomic locus	Genomic annotation
#1	CCCCCAgAAAACTAtGACGA CGG	1.38	2MMs [7:15]	chr8\|58911669\|+	BC030500\|TSS upstream +2kb,Galntl6\|TSS upstream +2kb
#2	CCaCCAAAAcAtTACGACGA AGT	1.22	3MMs [3:10:12]	chr3\|139117226\|-
#3	CaCCCAAtAAACTAgGACGA TGA	0.81	3MMs [2:8:15]	chr4\|94371165\|+
#4	ttCgCAAAAAACTACGACcA TGA	0.52	4MMs [1:2:4:19]	chr4\|105707379\|-
#5	aCCtCAAgAAACTAaGACGA GGT	0.41	4MMs [1:4:8:15]	chr7\|113625190\|-
#6	CaCatAAAAAACTAtGACGA TGT	0.4	4MMs [2:4:5:15]	chr8\|17720088\|+
#7	CaCaCAcAAAACaACGACGA TGA	0.39	4MMs [2:4:7:13]	chr18\|5237344\|-
#8	CCCaCAcAAcACaACGACGA CGA	0.34	4MMs [4:7:10:13]	chr16\|40967878\|-
#9	CttCCAgAAAACTAgGACGA GGG	0.27	4MMs [2:3:7:15]	chr4\|68002596\|+
#10	CtaCCAAAAtACTACcACGA GGA	0.24	4MMs [2:3:10:16]	chr14\|87958292\|-
#11	CtCtCAAAAAACTAaGACGg GGA	0.18	4MMs [2:4:15:20]	chr19\|33172480\|-
#12	CaCCCAAAAcACaACGACtA AGA	0.18	4MMs [2:10:13:19]	chr15\|90758207\|-
#13	CtCCtAAAAAtCTACGAgGA TGA	0.17	4MMs [2:5:11:18]	chr15\|28560655\|-
#14	CtCCCAgAcAACTAgGACGA GGG	0.17	4MMs [2:7:9:15]	chr7\|57182268\|+
#15	CtCCCAgAcAACTAaGACGA AGG	0.17	4MMs [2:7:9:15]	chr3\|65879847\|+
#16	aCaCCAAAAAACgAtGACGA AGG	0.15	4MMs [1:3:13:15]	chr13\|34152720\|+
#17	aCCCtAAAAAcCTACcACGA GGG	0.15	4MMs [1:5:11:16]	chr16\|96448826\|-
#18	CtCCCtAAAAAaTACGACtA AGG	0.15	4MMs [2:6:12:19]	chr7\|101752334\|+
#19	CCCCCAAAcAACTAaGACcA TGA	0.14	3MMs [9:15:19]	chr18\|69328237\|+
#20	CCtCaAAAAAACTAtGACaA AGC	0.13	4MMs [3:5:15:19]	chr6\|64484310\|-

#	Sequence	Score	Mismatch	Genomic locus	Genomic annotation
#1	CAAtCAGTtCAGCACTCGCT AGC	3.86	2MMs [4:9]	chr1\|176153564\|-
#2	CAAAaAGTtgAGCACTCGCT GGA	1.39	3MMs [5:9:10]	chr10\|12632521\|-
#3	ggAAtAGTCtAGCACTCGCT GGA	1.31	4MMs [1:2:5:10]	chr6\|145860988\|-	Bhlhe41\|exon
#4	CAAACAGgtgAGCACTCGCT AGA	1.3	3MMs [8:9:10]	chr9\|66855859\|+
#5	CAgACAGcCCAGCACTgGCT GGC	1.19	3MMs [3:8:17]	chr5\|129521641\|+
#6	CctcCAGTCCAcCACTCGCT GGT	0.7	4MMs [2:3:4:12]	chr4\|56661027\|-
#7	aAAAaAGTCCAGCACTCtCT TGG	0.67	3MMs [1:5:18]	chr16\|34602651\|-
#8	CcAtCAGTCCAGCACTCtCT TGG	0.65	3MMs [2:4:18]	chr14\|9361256\|+
#9	CAtcCAGTCtAcCACTCGCT GGT	0.63	4MMs [3:4:10:12]	chr5\|65601953\|-
#10	gcAACAGgCCAGCACTgGCT GGA	0.62	4MMs [1:2:8:17]	chr4\|43341981\|+
#11	gAAtCAGgCCAGCACTgGCT TGT	0.62	4MMs [1:4:8:17]	chr12\|103209944\|+
#12	aAAAgAGaCCAGCACTgGCT TGG	0.62	4MMs [1:5:8:17]	chr19\|25700716\|-
#13	CAcAgAGTCCAGCACTCtCT AGA	0.62	3MMs [3:5:18]	chr4\|142707594\|+
#14	aAtAaAGTCCAGCACTtGCT GGT	0.61	4MMs [1:3:5:17]	chr11\|89411992\|+	Ankfn1\|exon
#15	tAAAaAGTCaAGCACTaGCT TGC	0.57	4MMs [1:5:10:17]	chr11\|52210464\|-
#16	CAtACAGgCaAGCACTCGtT CGT	0.46	4MMs [3:8:10:19]	chr7\|80110044\|+
#17	CAgcCAtTCCAGCACTCGCa GGA	0.46	4MMs [3:4:7:20]	chr1\|171276795\|-	B4galt3\|exon
#18	CAgACAGaCaAGCACTCGgT GGT	0.46	4MMs [3:8:10:19]	chr3\|15978323\|+
#19	CAAcCtGaCCtGCACTCGCT TGG	0.46	4MMs [4:6:8:11]	chr10\|117461255\|-
#20	gtAACtGTCCAcCACTCGCT CGT	0.44	4MMs [1:2:6:12]	chr16\|62803903\|+	Arl13b\|exon