PMID- 28416245
OWN - NLM
STAT- MEDLINE
DCOM- 20171225
LR  - 20220408
IS  - 1673-8527 (Print)
IS  - 1673-8527 (Linking)
VI  - 44
IP  - 4
DP  - 2017 Apr 20
TI  - A simple and efficient method for CRISPR/Cas9-induced mutant screening.
PG  - 207-213
LID - S1673-8527(17)30057-7 [pii]
LID - 10.1016/j.jgg.2017.03.005 [doi]
AB  - The clustered regularly interspaced short palindromic repeats 
      (CRISPR)/CRISPR-associated protein 9 (Cas9) system provides a technological 
      breakthrough in mutant generation. Several methods such as the polymerase chain 
      reaction (PCR)/restriction enzyme (RE) assay, T7 endonuclease I (T7EI) assay, 
      Surveyor nuclease assay, PAGE-based genotyping assay, and high-resolution melting 
      (HRM) analysis-based assay have been developed for screening CRISPR/Cas9-induced 
      mutants. However, these methods are time- and labour-intensive and may also be 
      sequence-limited or require very expensive equipment. Here, we described a 
      cost-effective and sensitive screening technique based on conventional PCR, 
      annealing at critical temperature PCR (ACT-PCR), for identifying mutants. ACT-PCR 
      requires only a single PCR step followed by agarose gel electrophoresis. We 
      demonstrated that ACT-PCR accurately distinguished CRISPR/Cas9-induced mutants 
      from wild type in both rice and zebrafish. Moreover, the method can be adapted 
      for accurately determining mutation frequency in cultured cells. The simplicity 
      of ACT-PCR makes it particularly suitable for rapid, large-scale screening of 
      CRISPR/Cas9-induced mutants in both plants and animals.
CI  - Copyright (c) 2017 Institute of Genetics and Developmental Biology, Chinese Academy 
      of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights 
      reserved.
FAU - Hua, Yufeng
AU  - Hua Y
AD  - State Key Laboratory of Rice Biology, China National Rice Research Institute, 
      Chinese Academy of Agricultural Sciences, Hangzhou 310006, China.
FAU - Wang, Chun
AU  - Wang C
AD  - State Key Laboratory of Rice Biology, China National Rice Research Institute, 
      Chinese Academy of Agricultural Sciences, Hangzhou 310006, China.
FAU - Huang, Jian
AU  - Huang J
AD  - School of Biology & Basic Medical Sciences, Soochow University, Suzhou 215123, 
      China.
FAU - Wang, Kejian
AU  - Wang K
AD  - State Key Laboratory of Rice Biology, China National Rice Research Institute, 
      Chinese Academy of Agricultural Sciences, Hangzhou 310006, China. Electronic 
      address: wangkejian@caas.cn.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20170404
PL  - China
TA  - J Genet Genomics
JT  - Journal of genetics and genomics = Yi chuan xue bao
JID - 101304616
SB  - IM
MH  - Animals
MH  - CRISPR-Cas Systems/*genetics
MH  - DNA Mutational Analysis/*methods
MH  - Oryza/genetics
MH  - Polymerase Chain Reaction
MH  - Zebrafish/genetics
OTO - NOTNLM
OT  - ACT-PCR
OT  - CRISPR/Cas9
OT  - Genome editing
OT  - Mutant screening
EDAT- 2017/04/19 06:00
MHDA- 2017/12/26 06:00
CRDT- 2017/04/19 06:00
PHST- 2017/01/09 00:00 [received]
PHST- 2017/02/17 00:00 [revised]
PHST- 2017/03/07 00:00 [accepted]
PHST- 2017/04/19 06:00 [pubmed]
PHST- 2017/12/26 06:00 [medline]
PHST- 2017/04/19 06:00 [entrez]
AID - S1673-8527(17)30057-7 [pii]
AID - 10.1016/j.jgg.2017.03.005 [doi]
PST - ppublish
SO  - J Genet Genomics. 2017 Apr 20;44(4):207-213. doi: 10.1016/j.jgg.2017.03.005. Epub 
      2017 Apr 4.