Development and application of dual-fluorescence reporter systems for measuring specific nuclease activity based on SSA repair mechanism.
Reporter vector system has become an important method for measuring activity of specific nucleases because of its fast construction, simple modification, easy operation, economic effectiveness as well as its role in enriching positive cells with genomic modification through mediating screen of specific nucleases positive cells. After introducing double strand breaks (DSBs), a reporter system based on non-homologous end joining (NHEJ)-mediated repair can only repair maximally two thirds of reporter genes after optimization, while single strand annealing (SSA)-mediated repair can repair all reporter genes theoretically which has higher sensitivity and facilitates the detection of specific nuclease with low activity and provides an effective way to detect specific nuclease activity in genome modification studies. In this study, we designed and constructed three sets of dual-fluorescence reporter systems based on SSA repair mechanism and applied the mRFP-eGFP system in measuring the effective activity of three pairs of ZFNs, which was 8.9%, 9.3% and 5.0%, respectively. Our study provides an effective way to detect the activity of nucleases.
College of Animal Science & Technology, Northwest A&F University, Yangling 712100, China.
Fu-rong
FR
Han
1. College of Animal Science & Technology, Northwest A&F University, Yangling 712100, China; 2. School of Biological Science and Engineering, Shaanxi University of technology, Hanzhong 723000, China;
Ling
L
Wang
College of Animal Science & Technology, Northwest A&F University, Yangling 712100, China.
Kun
K
Xu
College of Animal Science & Technology, Northwest A&F University, Yangling 712100, China.
Zhi-ying
ZY
Zhang
College of Animal Science & Technology, Northwest A&F University, Yangling 712100, China.
Xin
X
Wang
D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000235:genetics; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods; D005434:Flow Cytometry / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D015203:Reproducibility of Results; D016335:Zinc Fingers / Q000235:genetics; D000097573:Red Fluorescent Protein
D004277:DNA, Single-Stranded; D004720:Endonucleases; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein