High-throughput genotyping of CRISPR/Cas9-mediated mutants using fluorescent PCR-capillary gel electrophoresis

Muhammad Khairul Ramlee,Tingdong Yan,Alice M S Cheung,Shang Li,Charles T H Chuah

doi:10.1038/srep15587

Abstract

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.

Highlights

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
An integral function of these programmable nucleases is the targeted knockout of specific genes via the introduction of insertion/deletion mutations in the coding region of these genes. These potentially frameshift-causing mutations are mediated by the error-prone non-homologous end joining (NHEJ) repair induced by double strand breaks (DSBs) by the nucleases
There are currently several techniques available for detecting engineered nuclease-mediated mutations in cells: SURVEYOR or T7E1 assay which detects mismatches in double-stranded DNA6,7; DNA melting analysis which differentiates fragments based on their melting curve[8,9]; restriction fragment length polymorphism (RFLP) assay which reports the disappearance of restriction sequences at nuclease target sites[10]; and Sanger and deep sequencing

Summary

Introduction

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. 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 allows for genotyping of multiplexed gene targeting in a single clone. Using fluorescent PCR coupled with capillary gel electrophoresis, we are able to effectively and efficiently screen for CRISPR/ Cas9-mediated mutant clones harbouring indels at three targeted genes (ATRX, TP53 and MIR615) in a high-throughput fashion This technique supports multiplexed targeting of genes which is useful in synergistic and redundancy studies. The method we describe here is not merely an alternative to the existing genotyping techniques available but a superior one on the basis that it allows for high-throughput screening of mutants, which renders it more time- and cost-effective

Methods

Results

Conclusion