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	GENOMIC DNA	1858 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	88.7
2	`TkA01G000070`	+ 197-219 / cut 213	`GATTTGTGGTGAGATCAGAG`	`AGG`	45.0	84.8
3	`TkA01G000070`	+ 201-223 / cut 217	`TGTGGTGAGATCAGAGAGGA`	`TGG`	50.0	84.5
4	`TkA01G000070`	+ 30-52 / cut 46	`GTGCATAAAAGGTAATGGAG`	`GGG`	40.0	83.7
5	`TkA01G000070`	+ 52-74 / cut 68	`GTAGTTCCAAGCACATTAGT`	`TGG`	40.0	83.1
6	`TkA01G000070`	+ 83-105 / cut 99	`GCATATACCCTCTTCTATAC`	`AGG`	40.0	82.2
7	`TkA01G000070`	+ 89-111 / cut 105	`ACCCTCTTCTATACAGGGAA`	`AGG`	45.0	81.8
8	`TkA01G000070`	+ 212-234 / cut 228	`CAGAGAGGATGGAAAGCAAA`	`AGG`	45.0	78.4
9	`TkA01G000070`	+ 754-776 / cut 770	`GTAGCAATCAGAGTTGGTGG`	`AGG`	50.0	78.0
10	`TkA01G000070`	+ 1115-1137 / cut 1131	`GCCGCATTTGTGGTGTATGT`	`GGG`	50.0	78.0
11	`TkA01G000070`	+ 1198-1220 / cut 1214	`GACCAGTCAACATGACCTCT`	`CGG`	50.0	78.0
12	`TkA01G000070`	+ 249-271 / cut 265	`CTACAGACGTACAACCATTG`	`TGG`	45.0	77.4
13	`TkA01G000070`	+ 253-275 / cut 269	`AGACGTACAACCATTGTGGA`	`AGG`	45.0	77.3
14	`TkA01G000070`	+ 183-205 / cut 199	`GTTAAGTGAAGGGGGATTTG`	`TGG`	45.0	77.2
15	`TkA01G000070`	- 90-112 / cut 95	`ACCTTTCCCTGTATAGAAGA`	`GGG`	40.0	76.0
16	`TkA01G000070`	- 91-113 / cut 96	`TACCTTTCCCTGTATAGAAG`	`AGG`	40.0	76.0
17	`TkA01G000070`	+ 174-196 / cut 190	`GACATTTAGGTTAAGTGAAG`	`GGG`	35.0	74.0
18	`TkA01G000070`	- 386-408 / cut 391	`GAGTTGTTCTGAAGTCATCT`	`CGG`	40.0	73.8
19	`TkA01G000070`	+ 172-194 / cut 188	`CCGACATTTAGGTTAAGTGA`	`AGG`	40.0	73.8
20	`TkA01G000070`	+ 629-651 / cut 645	`CAGACCAATTCCCAACAGCA`	`AGG`	50.0	72.6
21	`TkA01G000070`	- 633-655 / cut 638	`CAAGCCTTGCTGTTGGGAAT`	`TGG`	50.0	72.5
22	`TkA01G000070`	- 16-38 / cut 21	`TTTATGCACTTAAGGGCTAA`	`TGG`	35.0	72.3
23	`TkA01G000070`	+ 19-41 / cut 35	`TTAGCCCTTAAGTGCATAAA`	`AGG`	35.0	72.3
24	`TkA01G000070`	+ 1133-1155 / cut 1149	`GTGGGTGAGGTGCATAAATT`	`TGG`	45.0	72.2
25	`TkA01G000070`	- 1200-1222 / cut 1205	`GTCCGAGAGGTCATGTTGAC`	`TGG`	55.0	72.2
26	`TkA01G000070`	- 1233-1255 / cut 1238	`GGATGGTGAAAAGAAGCTGA`	`TGG`	45.0	72.2
27	`TkA01G000070`	+ 28-50 / cut 44	`AAGTGCATAAAAGGTAATGG`	`AGG`	35.0	72.0
28	`TkA01G000070`	+ 1114-1136 / cut 1130	`TGCCGCATTTGTGGTGTATG`	`TGG`	50.0	72.0
29	`TkA01G000070`	+ 1519-1541 / cut 1535	`CAGCTTCTTCTGCTGATCGA`	`TGG`	50.0	72.0
30	`TkA01G000070`	+ 29-51 / cut 45	`AGTGCATAAAAGGTAATGGA`	`GGG`	35.0	72.0
31	`TkA01G000070`	+ 84-106 / cut 100	`CATATACCCTCTTCTATACA`	`GGG`	35.0	70.5
32	`TkA01G000070`	+ 317-339 / cut 333	`TTGATGATGAAGAAGGTGAG`	`AGG`	40.0	69.8
33	`TkA01G000070`	+ 326-348 / cut 342	`AAGAAGGTGAGAGGATTTCA`	`CGG`	40.0	69.5
34	`TkA01G000070`	+ 363-385 / cut 379	`AGCTGAACCGGAAGTTATTA`	`TGG`	40.0	68.5
35	`TkA01G000070`	- 370-392 / cut 375	`ATCTCGGCCATAATAACTTC`	`CGG`	40.0	68.3
36	`TkA01G000070`	- 172-194 / cut 177	`CCTTCACTTAACCTAAATGT`	`CGG`	35.0	68.0
37	`TkA01G000070`	+ 173-195 / cut 189	`CGACATTTAGGTTAAGTGAA`	`GGG`	35.0	68.0
38	`TkA01G000070`	+ 175-197 / cut 191	`ACATTTAGGTTAAGTGAAGG`	`GGG`	35.0	67.9
39	`TkA01G000070`	+ 593-615 / cut 609	`CTTATGAGAGTCAGAGTCGT`	`TGG`	45.0	67.9
40	`TkA01G000070`	+ 776-798 / cut 792	`GGTGAAGCATACAATGTATC`	`TGG`	40.0	66.5
41	`TkA01G000070`	- 868-890 / cut 873	`GAGTTGAAGATGGAGGTAAT`	`GGG`	40.0	66.5
42	`TkA01G000070`	+ 1623-1645 / cut 1639	`GCAGATGATTGAGAGACTAA`	`AGG`	40.0	66.5
43	`TkA01G000070`	+ 25-47 / cut 41	`CTTAAGTGCATAAAAGGTAA`	`TGG`	30.0	66.3
44	`TkA01G000070`	+ 664-686 / cut 680	`CTGGAACCCAACCGAGAAAC`	`AGG`	55.0	66.2
45	`TkA01G000070`	+ 751-773 / cut 767	`CATGTAGCAATCAGAGTTGG`	`TGG`	45.0	66.2
46	`TkA01G000070`	+ 755-777 / cut 771	`TAGCAATCAGAGTTGGTGGA`	`GGG`	45.0	66.2
47	`TkA01G000070`	+ 1018-1040 / cut 1034	`TGGGAGAAGTTCCCGTATCC`	`AGG`	55.0	66.2
48	`TkA01G000070`	- 1116-1138 / cut 1121	`ACCCACATACACCACAAATG`	`CGG`	45.0	66.2
49	`TkA01G000070`	+ 1120-1142 / cut 1136	`ATTTGTGGTGTATGTGGGTG`	`AGG`	45.0	66.2
50	`TkA01G000070`	+ 1520-1542 / cut 1536	`AGCTTCTTCTGCTGATCGAT`	`GGG`	45.0	66.2
51	`TkA01G000070`	+ 1538-1560 / cut 1554	`ATGGGCTAAAGCCTCTACTA`	`TGG`	45.0	66.2
52	`TkA01G000070`	- 263-285 / cut 268	`AGACATAATTCCTTCCACAA`	`TGG`	35.0	65.5
53	`TkA01G000070`	+ 545-567 / cut 561	`AATAAGGCGTTAGATGAAGC`	`TGG`	40.0	63.4
54	`TkA01G000070`	- 639-661 / cut 644	`AGTTATCAAGCCTTGCTGTT`	`GGG`	40.0	60.8
55	`TkA01G000070`	- 640-662 / cut 645	`TAGTTATCAAGCCTTGCTGT`	`TGG`	40.0	60.8
56	`TkA01G000070`	+ 351-373 / cut 367	`ACTAGAAAAAGCAGCTGAAC`	`CGG`	40.0	60.8
57	`TkA01G000070`	+ 1424-1446 / cut 1440	`GGACCATTTGAATCGTATAT`	`GGG`	35.0	60.8
58	`TkA01G000070`	+ 645-667 / cut 661	`AGCAAGGCTTGATAACTATC`	`TGG`	40.0	60.7
59	`TkA01G000070`	- 869-891 / cut 874	`TGAGTTGAAGATGGAGGTAA`	`TGG`	40.0	60.5
60	`TkA01G000070`	- 875-897 / cut 880	`AAGTGTTGAGTTGAAGATGG`	`AGG`	40.0	60.5
61	`TkA01G000070`	+ 1105-1127 / cut 1121	`ATTTGATGTTGCCGCATTTG`	`TGG`	40.0	60.5
62	`TkA01G000070`	- 1213-1235 / cut 1218	`TGGCAAGCAGTGAGTCCGAG`	`AGG`	60.0	60.5
63	`TkA01G000070`	- 1250-1272 / cut 1255	`ATGGAATCACAGTCAATGGA`	`TGG`	40.0	60.5
64	`TkA01G000070`	- 1254-1276 / cut 1259	`TACGATGGAATCACAGTCAA`	`TGG`	40.0	60.5
65	`TkA01G000070`	+ 1427-1449 / cut 1443	`CCATTTGAATCGTATATGGG`	`TGG`	40.0	60.5
66	`TkA01G000070`	- 1427-1449 / cut 1432	`CCACCCATATACGATTCAAA`	`TGG`	40.0	60.5
67	`TkA01G000070`	+ 1658-1680 / cut 1674	`CTATGGTTGCTAGATAGGTA`	`TGG`	40.0	60.5
68	`TkA01G000070`	- 454-476 / cut 459	`GAAGGGCATTTACGTCTTTT`	`TGG`	40.0	60.0
69	`TkA01G000070`	+ 277-299 / cut 293	`ATTATGTCTGAGTTTCAAAG`	`AGG`	30.0	59.4
70	`TkA01G000070`	+ 278-300 / cut 294	`TTATGTCTGAGTTTCAAAGA`	`GGG`	30.0	59.3
71	`TkA01G000070`	+ 310-332 / cut 326	`ATCGAAATTGATGATGAAGA`	`AGG`	30.0	58.4
72	`TkA01G000070`	+ 1175-1197 / cut 1191	`GTTTTTGTGGCAGATGGGAG`	`TGG`	50.0	58.0
73	`TkA01G000070`	+ 555-577 / cut 571	`TAGATGAAGCTGGATTAAGT`	`AGG`	35.0	57.4
74	`TkA01G000070`	+ 420-442 / cut 436	`TTCTTATCAATCAAAACTCG`	`AGG`	30.0	55.4
75	`TkA01G000070`	- 806-828 / cut 811	`GTAGATGAATCTGAGTTAAT`	`TGG`	30.0	55.0
76	`TkA01G000070`	+ 1812-1834 / cut 1828	`GCTTTAACTTCTGAATAGTT`	`TGG`	30.0	55.0
77	`TkA01G000070`	+ 748-770 / cut 764	`TTTCATGTAGCAATCAGAGT`	`TGG`	35.0	54.8
78	`TkA01G000070`	+ 827-849 / cut 843	`ACTATTTATTTGCGTGCAAG`	`AGG`	35.0	54.8
79	`TkA01G000070`	+ 843-865 / cut 859	`CAAGAGGATCTTCAAACAAA`	`TGG`	35.0	54.8
80	`TkA01G000070`	- 878-900 / cut 883	`AGAAAGTGTTGAGTTGAAGA`	`TGG`	35.0	54.8