LATE–a novel sensitive cell-based assay for the study of CRISPR/Cas9-related long-term adverse treatment effects

Dawid Głów,Simon Meyer,Irene García Roldán,Lara Marie Akingunsade,Kristoffer Riecken,Boris Fehse

doi:10.1016/j.omtm.2021.07.004

Abstract

Since the introduction of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), genome editing has been broadly applied in basic research and applied biotechnology, whereas translation into clinical testing has raised safety concerns. Indeed, although frequencies and locations of off-target events have been widely addressed, little is known about their potential biological consequences in large-scale long-term settings. We have developed a long-term adverse treatment effect (LATE) in vitro assay that addresses potential toxicity of designer nucleases by assessing cell transformation events. In small-scale proof-of-principle experiments we reproducibly detected low-frequency (<0.5%) growth-promoting events in primary human newborn foreskin fibroblasts (NUFF cells) resulting from off-target cleavage in the TP53 gene. Importantly, the LATE assay detected not only off-target effects in TP53 not predicted by popular online tools but also growth-promoting mutations in other tumor suppressor genes, such as p21 and PLZF. It convincingly verified strongly reduced off-target activities of high fidelity compared with first-generation Cas9. Finally, the LATE assay was readily adapted to other cell types, namely clinically relevant human mesenchymal stromal cells (hMSCs) and retinal pigmented epithelial (RPE-1) cells. In conclusion, the LATE assay allows assessment of physiological adverse effects of the CRISPR/Cas system and might therefore be useful for preclinical safety studies.

Highlights

In 2011, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) designer nucleases were first described for use in other than the original bacteria.[1]
Deploying a worst-case scenario this assay aims at assessing the risk of growth-promoting off-target events based on the use of permanently genetically marked cells as targets of CRISPR/Cas[9] delivery and sensitive readout methods including flow cytometry (FC) and digital PCR. In this proof-of-concept study we demonstrate the potential of the long-term adverse treatment effect (LATE) assay to identify low-frequency growth-promoting off-target events and to compare off-target toxicity of different Cas[9] proteins
LATE assay principle: design and proof of concept It was previously shown that TP53 knockout in primary human newborn foreskin fibroblasts (NUFFs) leads to relative growth advantage resulting in positive selection and eventual outgrowth of affected clones.[30]

Summary

Introduction

In 2011, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) designer nucleases were first described for use in other than the original bacteria.[1]. The latest remarkable innovations were the introduction of base editors that allow direct correction of single-point mutations without the need of DNA strand breaks[6,7] and the invention of prime editing, a “search-and-replace” genome editing technology that facilitates a large variety of targeted genomic changes independent of cellular repair mechanisms. Prime editing was shown to mediate targeted insertions, deletions, all 12 possible base-to-base conversions, and combinations thereof in human cells without requiring doublestrand breaks (DSBs) or donor DNA templates.[8] Altogether, genome editing and CRISPR/Cas[9] have become a broadly used tool in many biotech fields and in biomedicine.[9]

Methods

Results

Conclusion