High-throughput genotyping of CRISPR/Cas9-mediated mutants using fluorescent PCR-capillary gel electrophoresis.
Recent advances in the engineering of sequence-specific synthetic nucleases provide enormous opportunities for genetic manipulation of gene expression in order to study their cellular function in vivo. However, current genotyping methods to detect these programmable nuclease-induced insertion/deletion (indel) mutations in targeted human cells are not compatible for high-throughput screening of knockout clones due to inherent limitations and high cost. Here, we describe an efficient method of genotyping clonal CRISPR/Cas9-mediated mutants in a high-throughput manner involving the use of a direct lysis buffer to extract crude genomic DNA straight from cells in culture, and fluorescent PCR coupled with capillary gel electrophoresis. This technique also allows for genotyping of multiplexed gene targeting in a single clone. Overall, this time- and cost-saving technique is able to circumvent the limitations of current genotyping methods and support high-throughput screening of nuclease-induced mutants.
Cancer and Stem Cell Biology Program, Duke-NUS Graduate Medical School, Singapore 169857.
Muhammad Khairul
MK
Ramlee
Cancer and Stem Cell Biology Program, Duke-NUS Graduate Medical School, Singapore 169857.
Tingdong
T
Yan
Cancer and Stem Cell Biology Program, Duke-NUS Graduate Medical School, Singapore 169857.
Alice M S
AM
Cheung
Department of Haematology, Singapore General Hospital, Singapore 169608.
Charles T H
CT
Chuah
Cancer and Stem Cell Biology Program, Duke-NUS Graduate Medical School, Singapore 169857.|Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597.
Shang
S
Li
D064113:CRISPR-Cas Systems; D002460:Cell Line; D002999:Clone Cells; D019075:Electrophoresis, Capillary / Q000379:methods*; D018390:Gene Targeting; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D054643:INDEL Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*