Development of a graphical user interface for sgRNAcas9 and its application.
The CRISPR/Cas9 genome editing technique is a powerful tool for researchers. However, off-target effects of the Cas9 nuclease activity is a recurrent concern of the CRISPR system. Thus, designing sgRNA (single guide RNA) with minimal off-target effects is very important. sgRNAcas9 is a software package, which can be used to design sgRNA and to evaluate potential off-target cleavage sites. In this study, a graphical user interface for sgRNAcas9 was developed using the Java programming language. In addition, off-target effect for sgRNAs was evaluated according to mismatched number and "seed sequence" specification. Moreover, sgRNAcas9 software was used to design 34 124 sgRNAs, which can target 4691 microRNA (miRNA) precursors from human, mouse, rat, pig, and chicken. In particular, the off-target effect of a sgRNA targeting to human miR-206 precursor was analyzed, and the on/off-target activity of this sgRNA was validated by T7E1 assay in vitro. Taken together, these data showed that the interface can simplify the usage of the sgRNAcas9 program, which can be used to design sgRNAs for the majority of miRNA precursors. We also found that the GC% of those sgRNAs ranged from 40% to 60%. In summary, the sgRNAcas9 software can be easily used to design sgRNA with minimal off-target effects for any species. The software can be downloaded from BiooTools website (http://www.biootools.com/).
Key Lab of Agricultural Animal Genetics, Breeding, and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan 430070, China.
Chang-zhi
CZ
Zhao
Department of Obstetrics and Gynecology, No.161 Hospital of PLA, Wuhan 430010, China.
Yi
Y
Zhang
Key Lab of Agricultural Animal Genetics, Breeding, and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan 430070, China.
Guang-lei
GL
Li
Department of Animal Physiology and Biochemistry, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China.
Ji-liang
JL
Chen
Key Lab of Agricultural Animal Genetics, Breeding, and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan 430070, China.
Jing-Jin
JJ
Li
Key Lab of Agricultural Animal Genetics, Breeding, and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan 430070, China.
Rui-min
RM
Ren
Key Lab of Agricultural Animal Genetics, Breeding, and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan 430070, China.
Pan
P
Ni
Key Lab of Agricultural Animal Genetics, Breeding, and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan 430070, China.
Shu-hong
SH
Zhao
1. Key Lab of Agricultural Animal Genetics, Breeding, and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan 430070, China; 2. Department of Animal Physiology and Biochemistry, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China;
Sheng-song
SS
Xie
D000818:Animals; D064113:CRISPR-Cas Systems; D002645:Chickens; D019295:Computational Biology / Q000379:methods; D004720:Endonucleases / Q000378:metabolism*; D006801:Humans; D020407:Internet; D051379:Mice; D035683:MicroRNAs / Q000235:genetics; D011381:Programming Languages; D012322:RNA Precursors / Q000235:genetics; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000378:metabolism*; D051381:Rats; D015203:Reproducibility of Results; D012984:Software; D013552:Swine; D014584:User-Computer Interface
D035683:MicroRNAs; D012322:RNA Precursors; D000094704:RNA, Guide, CRISPR-Cas Systems; D004720:Endonucleases