A qPCR method for genome editing efficiency determination and single-cell clone screening in human cells

Bo Li,Qilai Huang,Lele Yang,Naixia Ren,Junhao Liu

doi:10.1038/s41598-019-55463-6

Bo Li, Qilai Huang + Show 3 more

Open Access

https://doi.org/10.1038/s41598-019-55463-6

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Abstract

CRISPR/Cas9 technology has been widely used for targeted genome modification both in vivo and in vitro. However, an effective method for evaluating genome editing efficiency and screening single-cell clones for desired modification is still lacking. Here, we developed this real time PCR method based on the sensitivity of Taq DNA polymerase to nucleotide mismatch at primer 3′ end during initiating DNA replication. Applications to CRISPR gRNAs targeting EMX1, DYRK1A and HOXB13 genes in Lenti-X 293 T cells exhibited comprehensive advantages. Just in one-round qPCR analysis using genomic DNA from cells underwent CRISPR/Cas9 or BE4 treatments, the genome editing efficiency could be determined accurately and quickly, for indel, HDR as well as base editing. When applied to single-cell clone screening, the genotype of each cell colony could also be determined accurately. This method defined a rigorous and practical way in quantify genome editing events.

Highlights

CRISPR/Cas[9] technology has been widely used for targeted genome modification both in vivo and in vitro
Many other alternatives have been developed with improvement in certain aspects, including qEva-CRISPR21, engineered nuclease-induced translocations(ENIT)[22], Cas[9] nuclease based restriction fragment length polymorphism (RFLP) analysis[23], Indel Detection by Amplicon Analysis (IDAA)[24] and the gene-editing frequency digital PCR (GEF-dPCR)[25]
Applications in Lenti-X 293 T cells on 9 single guide RNA (sgRNA) targets indicate that this technique could determine the genome editing efficiency accurately in all cases of genome editing including non-homologous end joining (NHEJ) induced indels, homology-directed repair (HDR) and base editing

Summary

Introduction

CRISPR/Cas[9] technology has been widely used for targeted genome modification both in vivo and in vitro. An effective method for evaluating genome editing efficiency and screening single-cell clones for desired modification is still lacking We developed this real time PCR method based on the sensitivity of Taq DNA polymerase to nucleotide mismatch at primer 3′ end during initiating DNA replication. This multistep method can provide detailed information of each mutation event induced by nuclease, but is quite time-consuming, costly and laborious[10] To overcome these disadvantages, computational algorism was introduced to realize editing efficiency quantification based on direct Sanger sequencing of amplicon mixture of target DNA region. Applications in Lenti-X 293 T cells on 9 sgRNA targets indicate that this technique could determine the genome editing efficiency accurately in all cases of genome editing including NHEJ induced indels, HDR and base editing This method exhibited great power in single-cell clone genotyping by its ability in telling exactly how many alleles were modified. This technique described here provides the most robust strategy by far that can be used in genome editing efficiency quantification and single-cell clone genotyping in a high throughput way

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Results

Conclusion