GERG Study 2019: Reproducibility of indel formation rates by comparing guideRNA format and delivery method

Abstract

Recent advances in genome engineering are allowing scientists to better understand biology by precisely deleting, editing, or tagging genomic DNA. The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas) system was first used to edit mammalian cells in 2013 and has grown in popularity ever since. Multiple guideRNA and Cas9 reagent formats can be used for editing cells. In this study, we compared three popular methods: 1. a plasmid expressing both the guideRNA and Cas9, 2. Cas9 protein combined with a synthetic single guideRNA, and 3. Cas9 combined with a 2-part guideRNA. In addition, the CRISPR/Cas system can be delivered to cells via lipofection or nucleofection transfection methods. This study aims to compare the efficiency of gene editing outcomes at 3 different genomic targets, 3 unique guideRNA reagent formats, and 2 delivery method across multiple labs. For the 2018 GERG study, the group performed a pilot study and found that the results varied considerably across the 4 sites. Three possible sources of the variation are: 1. researchers had different levels of experience with the different methods 2. the provided protocols (from the companies) were challenging to understand, and 3. each researcher only performed one replicate. In 2019, we wrote a standard protocol and repeated the experiments multiple times to more accurately evaluate the reproducibility of these methods. Determining which CRISPR reagent format is the most reproducible and has the highest gene editing outcomes will be beneficial for core facilities or research labs getting started with genome editing.