Abstract
The T7 endonuclease 1 (T7E1) mismatch detection assay is a widely used method for evaluating the activity of site-specific nucleases, such as the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system. To determine the accuracy and sensitivity of this assay, we compared the editing estimates derived by the T7E1 assay with that of targeted next-generation sequencing (NGS) in pools of edited mammalian cells. Here, we report that estimates of nuclease activity determined by T7E1 most often do not accurately reflect the activity observed in edited cells. Editing efficiencies of CRISPR-Cas9 complexes with similar activity by T7E1 can prove dramatically different by NGS. Additionally, we compared editing efficiencies predicted by the Tracking of Indels by Decomposition (TIDE) assay and the Indel Detection by Amplicon Analysis (IDAA) assay to that observed by targeted NGS for both cellular pools and single-cell derived clones. We show that targeted NGS, TIDE, and IDAA assays predict similar editing efficiencies for pools of cells but that TIDE and IDAA can miscall alleles in edited clones.
Highlights
Advances in genome engineering have ushered in exciting new opportunities for scientists to understand and enhance biology
We explored the accuracy of four assays (T7E1, Tracking of Indels by Decomposition (TIDE), Indel Detection by Amplicon Analysis (IDAA) and next-generation sequencing (NGS)) frequently used to determine the level of activity for a given single guide RNA (sgRNA) when complexed with Cas[9]
The presence of inaccurate frequencies and unexpected indels can confound clone verification and suggest contamination with other clonal populations arising from the same edited pool. Both TIDE and IDAA analysis did correctly predict that all 8 edited clones were knockout clones, neither assay specifies the sequence identity of called indels and, does not eliminate the need for laborious TA cloning and Sanger sequencing procedures to obtain the accurate genotype of a given clone. These results suggest that the nonhomologous end-joining (NHEJ) frequency estimated by the T7 endonuclease 1 (T7E1) mismatch detection assay is often inconsistent with the actual frequency of editing in pools of cells
Summary
Advances in genome engineering have ushered in exciting new opportunities for scientists to understand and enhance biology. The popularity of the CRISPR-Cas[9] platform can be attributed to its conceptually straightforward design: a single cloning step generates a single guide RNA (sgRNA) that directs Cas9-mediated endonuclease activity to the site of interest. The only design constraint limiting the commonly used S. pyogenes Cas[9] nuclease is the requirement of a 5′-NGG-3′ protospacer adjacent motif (PAM) on the target template immediately following the sgRNA target sequence. This prerequisite can be made more flexible by using Cas[9] orthologs with different PAM sequence specificities[2,3]. Target H1a H2 H3 H4 H5 H6 H7 H8 H9 M1b M2 M3 M4 M5 M6 M7 M8 M9 M10
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