Barcoded Landing Pads at Safe Harbor Sites for Future Custom Engineering

This study aims to develop robust pipelines for creating standardized cell lines for research, inserting landing pads at safe harbor sites for future genome editing, utilizing established pipelines in multiple labs for increased efficiency.

Background:   

Targeted knockins are an often-requested gene modification in cell lines and mice. In order to test various gene and protein functions, researchers add epitope tags, gene fusions, and transgenes to defined sites in the genome. New-age genome editing tools such as CRISPR facilitate knockins by generating a blunt-ended DNA double strand break (DSB) at a specific site in the genome. To repair the DSB, cells will utilize one of two main repair pathways; the first, non-homologous end joining (NHEJ), is fast but error prone. The second, homology-directed repair (HDR), uses a DNA template with homology to each side of the DSB to repair the break. Researchers can manipulate HDR to incorporate a sequence of interest into the cut chromosome by supplying the cell with an engineered donor DNA molecule with homologous sequences to the cut site on each side of the desired insert.

Some companies such as Applied Stem Cell have combined CRISPR/Cas9 genome editing with site-specific recombinases such as PhiC31 to streamline large insertions into known safe harbor sites with high efficiency. 

Targeted knockin of large cargos remain a critical need for GERG users, while simultaneously reflecting a challenging, inefficient prospect for GERG research groups. This difficulty can lead to increased costs shouldered by users or research groups and frustrated relationships. Our proposed study aims to fix this problem in two ways: (1) generation of shareable resources to enable facile large-cargo KIs into safe-harbor sites and (2) evaluate ideal “safe-harbor sites” across multiple cell lines. We will accomplish our first aim by generating cell lines carrying recombinase sites across X safe harbor loci and across Y cell lines. These cell lines can be distributed and be used to readily incorporate synthetic constructs into safe-harbor sites using more efficient and reliable recombase-mediated modality rather than homology-directed repair. The bank of cell lines will be generated in a multitude of orthogonal cell lines selected to meet the needs of a maximum number of users. Our second aim will be accomplished by incorporating reporter constructs into the landing pads made in phase I, and evaluating the resultant expression levels in consistency, epigenetic silencing, etc.

Study Plan:   

For the study, each participating lab will insert an identical landing pad into 5 separate safe harbor sites in the cell line most highly requested by their users, to immediately generate usable resources to serve our clients.

CRISPR materials will be designed at St. Jude Children’s Research Hospital. The designs will consist of a sgRNA to  a safe harbor site, many of which are published, and ssODN donors for the selected landing pad. A short random oligonucleotide sequence will be included in the ssODN donor for future identification of the generated cell lines.

Safe harbor sites included in this study are H11, AAVS1, Rogi1, and CLYBL. 

In the following year, we will use these landing pads to compare the stability of gene expression at each site using a reporter gene driven by common promoters of different strengths.

CCoRRe: Lab reproducibility survey not described as a group activity – please add this label/link to their page, following the information on the AACR Technical Reviewer Initiative….(I don’t know how to add a “box” similar to the others…..)

2024 Rigor, Reproducibility, and Transparency Survey

Dear ABRF community,

Genome Editing Research Group (GERG) is pleased to invite interested researchers to participate in its research study.

The GERG study: Generating cell lines containing specific point mutations using CRISPR-Cas9 is a frequent request for genome engineering core facilities. In this study, we set out to compare the efficiency of point mutation generation using CRISPR with Cas9, Cas12a/Cpf1, and prime editing. This will be a multi-center study to evaluate the application of these newer tools for routine use at core facility laboratories to develop genome-edited cell lines.

Current status of the project: The experimental plan is completely designed, and the necessary reagents are procured. The experiments are being initiated at 5 independent laboratories.

What are we seeking from you? We hope to conduct this study at a greater number of laboratories, preferably up to 10. A larger number of participants and experiments will help evaluate the systems more robustly.

Participation details: We will provide the details of the cell culture experiments involving transfection, including all the necessary reagents and protocols. As a participant laboratory, you will do the cell culture experiments, collect samples, and ship them to a central laboratory for analysis. The central laboratory will perform the downstream experiments (such as NGS and other methods), and the data will be analyzed.

Benefits of participating in the study: We intend to publish the results, preferably in a peer-reviewed open-access journal. Researcher/s from the participating laboratories will have a chance to be included as authors in the article.

To get started or if you need more information: Contact abrf@abrf.org

We look forward to your participation!

Best Regards,

GERG

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.

Results were presented in a poster at ABRF 2020:  GERG Study 2019-2020: Reproducibility of indel formation rates by comparing guideRNA format and delivery method

In 2018, GERG initiated a study to evaluate the reproducibility of indel formation rates by comparing guideRNA format and cell delivery methods across multiple labs.  Various configurations of guideRNA and Cas9 components can be used for editing cells.  A few options include: a plasmid expressing both the guideRNA and Cas9, Cas9 protein combined with a synthetic single guideRNA, and Cas9 combined with a synthetic 2-part guideRNA.  In addition, delivering these components to cells can be done using lipofection or nucleofection transfection methods.  In the GERG 2017 survey (CRISPR/Cas9 Methods:  Preferences from the Field), plasmid format and lipofection delivery were favored among cell culture users.  Meanwhile, RNP format for the guideRNA and Cas9 is gaining in popularity in combination with nucleofection delivery.  This study aims to evaluate cutting efficiency at 3 different guideRNA targets based on the guideRNA format and delivery method across multiple labs.  Determining which method or format is the most reproducible will be beneficial.  Core facilities or research labs getting started with genome editing could use these results as a benchmark for optimizing their own protocols.

Results were presented in a presentation at ABRF 2019:  Reproducibility of indel formation rates by comparing guideRNA format and delivery method

Results were also presented in a poster at ABRF 2019:  Reproducibility of indel formation rates by comparing guideRNA format and delivery method

The Genome Editing Research Group surveyed users of the CRISPR/Cas9 technology to help establish an understanding of preferred methods being used. As new core facilities are being formed to support the CRISPR/Cas9 technology, or existing cores have adapted their services to fit the technology into their workflows, considering what other cores are using is important. Questions regarding preferred guideRNA design tools, format of reagents, mutation analysis methods, and other relevant topics were included.

The collected data can be viewed here: https://www.surveymonkey.com/results/SM-ZBM5L6768/

Results were summarized in a poster and presented at ABRF 2018: CRISPR/Cas9 Methods: Preferences from the Field
gerg_survey_poster_2018_final.pdf
abrf_gergstudyslides_2019.pdf
abrf_gerg_poster2019_final.pdf
abrf_gerg_poster2020.pdf