Unexpectedly High Levels of Inverted Re-Insertions Using Paired sgRNAs for Genomic Deletions.

Joseph Blayney,Matteo Morotti,Philip Hublitz,Julie Xiao,Marta Jagielowicz,Mira Kreuzer,Katharina Reglinski,Evangeline Foster

doi:10.3390/mps3030053

Abstract

Use of dual sgRNAs is a common CRISPR/Cas9-based strategy for the creation of genetic deletions. The ease of screening combined with a rather high rate of success makes this approach a reliable genome engineering procedure. Recently, a number of studies using CRISPR/Cas9 have revealed unwanted large-scale rearrangements, duplications, inversions or larger-than-expected deletions. Strict quality control measures are required to validate the model system, and this crucially depends on knowing which potential experimental outcomes to expect. Using the dual sgRNA deletion approach, our team discovered high levels of excision, inversion and re-insertion at the site of targeting. We detected those at a variety of genomic loci and in several immortalized cell lines, demonstrating that inverted re-insertions are a common by-product with an overall frequency between 3% and 20%. Our findings imply an inherent danger in the misinterpretation of screening data when using only a single PCR screening. While amplification of the region of interest might classify clones as wild type (WT) based on amplicon size, secondary analyses can discover heterozygous (HET) clones among presumptive WTs, and events deemed as HET clones could potentially be full KO. As such, screening for inverted re-insertions helps in decreasing the number of clones required to obtain a full KO. With this technical note, we want to raise awareness of this phenomenon and suggest implementing a standard secondary PCR while screening for deletions.

Highlights

Since the discovery of the CRISPR/Cas9 genome engineering technology platform [1], the mostSince the discovery of thehasCRISPR/Cas9 genome engineering technology platform [1],The thecellular most commonly used application been generation of microdeletions using a single sgRNA.commonly used application has been generation of microdeletions using a singleThe cellular non‐homologous end‐joining (NHEJ) pathway repairs the double‐strand breaks, and this can lead to non-homologous (NHEJ)pathway repairs theFunctional double-strand breaks, can lead generation of anend-joining out‐of‐frame gene knockout (KO) [2].outcomes ofand thisthis process needtoto generation of an out-of-frame gene knockoutFunctional of this process need to be be properly screened for
Using a dual sgRNA-based strategy has the potential to double the number of potential OFF-target modifications; using highly selected sgRNAs, we have successfully generated many model systems and have been unable to detect significant numbers of aberrantly generated models. This can potentially be attributed to the fact that we generally apply careful initial PCR screening procedures that always take into account potential larger-than-expected deletions [18]
Investigating this matter by molecular cloning and Sanger sequencing, we found that a substantial number of clonal cell lines displayed inverted re-insertion of the excised fragment (Table 1)

Summary

Introduction

Since the discovery of the CRISPR/Cas genome engineering technology platform [1], the mostSince the discovery of thehasCRISPR/Cas genome engineering technology platform [1],The thecellular most commonly used application been generation of microdeletions using a single sgRNA.commonly used application has been generation of microdeletions using a singleThe cellular non‐homologous end‐joining (NHEJ) pathway repairs the double‐strand breaks, and this can lead to non-homologous (NHEJ)pathway repairs theFunctional double-strand breaks, can lead generation of anend-joining out‐of‐frame gene knockout (KO) [2].outcomes ofand thisthis process needtoto generation of an out-of-frame gene knockoutFunctional of this process need to be be properly screened for. Since the discovery of the CRISPR/Cas genome engineering technology platform [1], the most. CRISPR/Cas genome engineering technology platform [1],The thecellular most commonly used application been generation of microdeletions using a single sgRNA. Commonly used application has been generation of microdeletions using a single. The cellular non‐homologous end‐joining (NHEJ) pathway repairs the double‐strand breaks, and this can lead to non-homologous (NHEJ). Pathway repairs theFunctional double-strand breaks, can lead generation of anend-joining out‐of‐frame gene knockout (KO) [2]. Outcomes ofand thisthis process needtoto generation of an out-of-frame gene knockout. Functional of this process need to be be properly screened for. DNA motifs, outcomes such as transcription factor‐binding properly. Disruption of functional motifs, or such as transcription factor-binding sites sites or screened splicing for.

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Conclusion