CRISPR 编辑靶点设计

输入基因 ID、CDS、cDNA 或 DNA 序列，快速筛选 SpCas9（NGG PAM）候选靶点并按公开规则排序。页面只提供靶点与评分，不生成载体相关引物，也不将评分视作实验编辑效率。

真实数据示例

TkA01G000070 · TK1151 · CDS 1170 bp

直接运行示例下载示例FASTA

示例序列取自本站TK1151参考CDS，可查看基因卡片及参考来源。

查看算法、评分及证据边界

单次最多 250,000 bp；在完整输入上扫描两条链，超限会明确提示，不会删除歧义碱基或静默截断。

设计与评分规则

工具在正、负链扫描 20 nt guide 紧邻的 SpCas9 NGG PAM；切割位点按 SpCas9 规则标为 PAM 上游 3 bp。每条候选的总分限制在 0–100，仅用于本工具内的优先排序。

组成	规则与分值
基础与 GC 平衡	基础分 38；加 max(0, 24 − 1.15 × \|GC% − 50\|)，因此 GC 接近 50% 时最高加 24 分。
PAM / 5′ 偏好	NGG PAM 加 10；guide 首位为 G 加 6；在输入序列较靠前的位置加 0–18 分，用于优先靠近基因或 CDS/cDNA 5′端的候选。
序列惩罚	guide 含 TTTT 减 12；任一单碱基连续 5 个及以上减 8。最后将结果截断为 0–100。

评分是可复现的序列优先级规则，不是体内编辑效率或脱靶风险预测。后续载体、启动子、递送方式和靶外验证应由具体实验体系单独确定。

输入解析

记录	参考	来源	长度
`TkA01G000070`	TK1151	CDS	1170 bp

候选靶点

候选靶点: 80PAM: NGG脱靶：未进行全基因组搜索

位置为输入序列上1起始、闭区间，包含20 nt guide及3 nt PAM；切点表示输入上链相邻碱基之间的位置，不直接当作染色体坐标。CDS/cDNA跨外显子连接处的候选需回到基因组核对。

#	靶点	位置 / 切割位点	Guide (20 nt)	PAM	GC%	序列排序分
1	`TkA01G000070`	- 58-80 / cut 63	`GTGACTCCAACTAATGTGCT`	`TGG`	45.0	87.7
2	`TkA01G000070`	+ 125-147 / cut 141	`GATTTGTGGTGAGATCAGAG`	`AGG`	45.0	84.8
3	`TkA01G000070`	+ 129-151 / cut 145	`TGTGGTGAGATCAGAGAGGA`	`TGG`	50.0	84.4
4	`TkA01G000070`	+ 30-52 / cut 46	`GTGCATAAAAGGTAATGGAG`	`GGG`	40.0	83.2
5	`TkA01G000070`	+ 52-74 / cut 68	`GTAGTTCCAAGCACATTAGT`	`TGG`	40.0	82.3
6	`TkA01G000070`	+ 83-105 / cut 99	`GCATATACCCTCTTCTATAC`	`AGG`	40.0	80.9
7	`TkA01G000070`	+ 89-111 / cut 105	`ACCCTCTTCTATACAGGGAA`	`AGG`	45.0	80.4
8	`TkA01G000070`	+ 102-124 / cut 118	`CAGGGAAAGGTTAAGTGAAG`	`GGG`	45.0	79.8
9	`TkA01G000070`	+ 103-125 / cut 119	`AGGGAAAGGTTAAGTGAAGG`	`GGG`	45.0	79.8
10	`TkA01G000070`	+ 140-162 / cut 156	`CAGAGAGGATGGAAAGCAAA`	`AGG`	45.0	78.1
11	`TkA01G000070`	+ 763-785 / cut 779	`GCCGCATTTGTGGTGTATGT`	`GGG`	50.0	78.0
12	`TkA01G000070`	+ 846-868 / cut 862	`GACCAGTCAACATGACCTCT`	`CGG`	50.0	78.0
13	`TkA01G000070`	+ 111-133 / cut 127	`GTTAAGTGAAGGGGGATTTG`	`TGG`	45.0	77.4
14	`TkA01G000070`	+ 177-199 / cut 193	`CTACAGACGTACAACCATTG`	`TGG`	45.0	76.5
15	`TkA01G000070`	+ 181-203 / cut 197	`AGACGTACAACCATTGTGGA`	`AGG`	45.0	76.3
16	`TkA01G000070`	- 90-112 / cut 95	`ACCTTTCCCTGTATAGAAGA`	`GGG`	40.0	74.6
17	`TkA01G000070`	- 91-113 / cut 96	`AACCTTTCCCTGTATAGAAG`	`AGG`	40.0	74.5
18	`TkA01G000070`	+ 100-122 / cut 116	`TACAGGGAAAGGTTAAGTGA`	`AGG`	40.0	74.1
19	`TkA01G000070`	+ 101-123 / cut 117	`ACAGGGAAAGGTTAAGTGAA`	`GGG`	40.0	74.1
20	`TkA01G000070`	+ 537-559 / cut 553	`GAAACAGCAATCAGAGTTGG`	`TGG`	45.0	72.2
21	`TkA01G000070`	+ 781-803 / cut 797	`GTGGGTGAGGTGCATAAATT`	`TGG`	45.0	72.2
22	`TkA01G000070`	- 848-870 / cut 853	`GTCCGAGAGGTCATGTTGAC`	`TGG`	55.0	72.2
23	`TkA01G000070`	- 881-903 / cut 886	`GGATGGTGAAAAGAAGCTGA`	`TGG`	45.0	72.2
24	`TkA01G000070`	- 16-38 / cut 21	`TTTATGCACTTAAGGGCTAA`	`TGG`	35.0	72.1
25	`TkA01G000070`	+ 487-509 / cut 503	`CAGACCAATTCCCAACAGCA`	`AGG`	50.0	72.0
26	`TkA01G000070`	- 491-513 / cut 496	`CAAGCCTTGCTGTTGGGAAT`	`TGG`	50.0	72.0
27	`TkA01G000070`	+ 540-562 / cut 556	`ACAGCAATCAGAGTTGGTGG`	`AGG`	50.0	72.0
28	`TkA01G000070`	+ 541-563 / cut 557	`CAGCAATCAGAGTTGGTGGA`	`GGG`	50.0	72.0
29	`TkA01G000070`	+ 762-784 / cut 778	`TGCCGCATTTGTGGTGTATG`	`TGG`	50.0	72.0
30	`TkA01G000070`	+ 1082-1104 / cut 1098	`CAGCTTCTTCTGCTGATCGA`	`TGG`	50.0	72.0
31	`TkA01G000070`	+ 19-41 / cut 35	`TTAGCCCTTAAGTGCATAAA`	`AGG`	35.0	72.0
32	`TkA01G000070`	+ 28-50 / cut 44	`AAGTGCATAAAAGGTAATGG`	`AGG`	35.0	71.6
33	`TkA01G000070`	+ 29-51 / cut 45	`AGTGCATAAAAGGTAATGGA`	`GGG`	35.0	71.5
34	`TkA01G000070`	- 314-336 / cut 319	`GAGTTGTTCTGAAGTCATCT`	`CGG`	40.0	70.7
35	`TkA01G000070`	+ 84-106 / cut 100	`CATATACCCTCTTCTATACA`	`GGG`	35.0	69.1
36	`TkA01G000070`	+ 245-267 / cut 261	`TTGATGATGAAGAAGGTGAG`	`AGG`	40.0	67.8
37	`TkA01G000070`	+ 254-276 / cut 270	`AAGAAGGTGAGAGGATTTCA`	`CGG`	40.0	67.4
38	`TkA01G000070`	+ 562-584 / cut 578	`GGTGAAGCATACAATGTATC`	`TGG`	40.0	66.5
39	`TkA01G000070`	- 654-676 / cut 659	`GAGTTGAAGATGGAGGTAAT`	`GGG`	40.0	66.5
40	`TkA01G000070`	+ 451-473 / cut 467	`CTTATGAGAGTCAGAGTCGT`	`TGG`	45.0	66.2
41	`TkA01G000070`	- 528-550 / cut 533	`CTGATTGCTGTTTCTCGGTT`	`GGG`	45.0	66.2
42	`TkA01G000070`	- 529-551 / cut 534	`TCTGATTGCTGTTTCTCGGT`	`TGG`	45.0	66.2
43	`TkA01G000070`	+ 534-556 / cut 550	`CGAGAAACAGCAATCAGAGT`	`TGG`	45.0	66.2
44	`TkA01G000070`	- 746-768 / cut 751	`TGCGGCAACATCAAATTCAC`	`TGG`	45.0	66.2
45	`TkA01G000070`	- 764-786 / cut 769	`ACCCACATACACCACAAATG`	`CGG`	45.0	66.2
46	`TkA01G000070`	+ 768-790 / cut 784	`ATTTGTGGTGTATGTGGGTG`	`AGG`	45.0	66.2
47	`TkA01G000070`	+ 1083-1105 / cut 1099	`AGCTTCTTCTGCTGATCGAT`	`GGG`	45.0	66.2
48	`TkA01G000070`	+ 25-47 / cut 41	`CTTAAGTGCATAAAAGGTAA`	`TGG`	30.0	65.9
49	`TkA01G000070`	+ 291-313 / cut 307	`AGCTGAACCGGAAGTTATTA`	`TGG`	40.0	65.8
50	`TkA01G000070`	- 298-320 / cut 303	`ATCTCGGCCATAATAACTTC`	`CGG`	40.0	65.4
51	`TkA01G000070`	- 191-213 / cut 196	`AGACATAATTCCTTCCACAA`	`TGG`	35.0	64.4
52	`TkA01G000070`	+ 403-425 / cut 419	`AATAAGGCGTTAGATGAAGC`	`TGG`	40.0	60.8
53	`TkA01G000070`	+ 987-1009 / cut 1003	`GGACCATTTGAATCGTATAT`	`GGG`	35.0	60.8
54	`TkA01G000070`	- 497-519 / cut 502	`AGTTATCAAGCCTTGCTGTT`	`GGG`	40.0	60.5
55	`TkA01G000070`	- 498-520 / cut 503	`TAGTTATCAAGCCTTGCTGT`	`TGG`	40.0	60.5
56	`TkA01G000070`	+ 503-525 / cut 519	`AGCAAGGCTTGATAACTATC`	`TGG`	40.0	60.5
57	`TkA01G000070`	- 533-555 / cut 538	`CAACTCTGATTGCTGTTTCT`	`CGG`	40.0	60.5
58	`TkA01G000070`	- 655-677 / cut 660	`TGAGTTGAAGATGGAGGTAA`	`TGG`	40.0	60.5
59	`TkA01G000070`	- 661-683 / cut 666	`AAGTGTTGAGTTGAAGATGG`	`AGG`	40.0	60.5
60	`TkA01G000070`	+ 753-775 / cut 769	`ATTTGATGTTGCCGCATTTG`	`TGG`	40.0	60.5
61	`TkA01G000070`	- 861-883 / cut 866	`TGGCAAGCAGTGAGTCCGAG`	`AGG`	60.0	60.5
62	`TkA01G000070`	- 898-920 / cut 903	`ATGGAATCACAGTCAATGGA`	`TGG`	40.0	60.5
63	`TkA01G000070`	- 902-924 / cut 907	`TACGATGGAATCACAGTCAA`	`TGG`	40.0	60.5
64	`TkA01G000070`	+ 937-959 / cut 953	`AATCTTGTAGGATCAACGGT`	`TGG`	40.0	60.5
65	`TkA01G000070`	+ 938-960 / cut 954	`ATCTTGTAGGATCAACGGTT`	`GGG`	40.0	60.5
66	`TkA01G000070`	+ 990-1012 / cut 1006	`CCATTTGAATCGTATATGGG`	`TGG`	40.0	60.5
67	`TkA01G000070`	- 990-1012 / cut 995	`CCACCCATATACGATTCAAA`	`TGG`	40.0	60.5
68	`TkA01G000070`	+ 279-301 / cut 295	`ACTAGAAAAAGCAGCTGAAC`	`CGG`	40.0	58.3
69	`TkA01G000070`	+ 205-227 / cut 221	`ATTATGTCTGAGTTTCAAAG`	`AGG`	30.0	58.0
70	`TkA01G000070`	+ 823-845 / cut 839	`GTTTTTGTGGCAGATGGGAG`	`TGG`	50.0	58.0
71	`TkA01G000070`	+ 206-228 / cut 222	`TTATGTCTGAGTTTCAAAGA`	`GGG`	30.0	58.0
72	`TkA01G000070`	+ 238-260 / cut 254	`ATCGAAATTGATGATGAAGA`	`AGG`	30.0	56.6
73	`TkA01G000070`	- 592-614 / cut 597	`GTAGATGAATCTGAGTTAAT`	`TGG`	30.0	55.0
74	`TkA01G000070`	+ 413-435 / cut 429	`TAGATGAAGCTGGATTAAGT`	`AGG`	35.0	54.8
75	`TkA01G000070`	+ 613-635 / cut 629	`ACTATTTATTTGCGTGCAAG`	`AGG`	35.0	54.8
76	`TkA01G000070`	+ 629-651 / cut 645	`CAAGAGGATCTTCAAACAAA`	`TGG`	35.0	54.8
77	`TkA01G000070`	- 664-686 / cut 669	`AGAAAGTGTTGAGTTGAAGA`	`TGG`	35.0	54.8
78	`TkA01G000070`	- 739-761 / cut 744	`ACATCAAATTCACTGGATAC`	`GGG`	35.0	54.8
79	`TkA01G000070`	- 917-939 / cut 922	`ATTGTAGTTGATTGGTACGA`	`TGG`	35.0	54.8
80	`TkA01G000070`	+ 925-947 / cut 941	`CCAATCAACTACAATCTTGT`	`AGG`	35.0	54.8