nf-core/crisprseq: a versatile pipeline for comprehensive analysis of CRISPR gene editing and screening assays

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nf-core/crisprseq: a versatile pipeline for comprehensive analysis of CRISPR gene editing and screening assays

Abstract

In recent years, CRISPR technology has become widely applied in scientific research, being simpler, cheaper, and more precise than previous gene-editing techniques. This editing technology can be used for various applications, such as gene knockout, gene knock-in, CRISPR activation, CRISPR interference, CRISPR screens, base editing, and prime editing. The share of pipelines to analyze the variety of CRISPR editing methods is low, and until now, none of them caters to both gene editing and CRISPR-based functional genomics. Here, we introduce nf-core/crisprseq, a Nextflow DSL two pipeline for the assessment of CRISPR gene editing and screening assays. The workflow is written in a modularized fashion to allow the easy incorporation of new steps. nf-core/crisprseq is the first generic pipeline enabling the analysis of the broad spectrum of CRISPR designs. We show the performance and usability of the software using publicly available datasets.

Introduction

In recent years, CRISPR technology has become widely applied in scientific research, being simpler, cheaper, and more precise than previous gene-editing techniques. CRISPR-Cas technology enables precise DNA editing and can be applied to a wide range of research areas, including drug discovery, molecular biology, functional genomics, disease modeling, agriculture, and gene therapies. Its utility extends to understanding gene function, identifying therapeutic targets, and advancing precision medicine.

This editing technology can be used for various applications, such as gene knockout, gene knock-in, CRISPR activation

(CRISPRi), CRISPR screens, base editing, and prime editing.

To manipulate a single gene for a knockout, a Cas nine enzyme is guided to the specific gene sequence by a complementary single guide RNA. Cas nine then cuts the DNA at the target site, leading to gene depletion. This approach is widely employed in functional genomics, drug discovery, and disease modeling.

In CRISPR knock-ins, double-strand breaks are repaired by homology-directed repair to insert new DNA or entire genes. This technique is used in biotechnology, recombinant protein production, cell line viability, and disease modeling.

CRISPR activation and CRISPR interference enable gene expression regulation for diverse research applications, such as developmental biology and drug resistance screening.

More recent techniques include base editing and prime editing, which enable precise single nucleotide substitutions and small insertions and deletions for the latter.

Such techniques can be applied to a single gene of interest or used in CRISPR screens. Screening experiments consist of a library of single guide RNAs designed to target a wide range of genes simultaneously. Through Next-Generation Sequencing, the effects of gene depletion or activation on cellular phenotypes can be assessed in a comprehensive manner. This technique facilitates large-scale studies, unraveling genotype-phenotype relationships for drug discovery, as it allows to determine gene essentiality. A gene is considered essential in a cell line if its loss of function leads to the individual's compromised viability or significant reduction in overall fitness. On a population scale, essential genes are identified by observing intolerance to loss-of-function variants.

Analyzing these various experiments involving CRISPR editing requires a collection of tools. Many alternatives for every step of the analysis have been presented in recent years. This complexity challenges the analysis, interpreting the results and extracting meaningful insights from the data. Most importantly, it makes tool benchmarking difficult and, thus, no community agreement for the choice of tools has been established.

Many tools have been developed for the assessment of genome editing. For the analysis of targeted gene editing, these include ampliCan, Cas-analyzer, CRISPR- Analytics, CRISPResso, CRISPResso two, CRISPR- GA, CRISPRnano, CRISPRpic, cris.py, and CrispRVariants. From these, only CRISPR-Analytics and CRISPResso two are able to analyze all kinds of CRISPR editions, knockout, knock-in, base editing, and prime editing, and only CRISPR-Analytics is a pipeline built using a workflow manager.

For the analysis of screening experiments, many tools have been developed, such as BAGEL two, CRISPRcleanR, MAGeCK, Chronos, and DrugZ, which are often then used in custom in-house scripts, sequentially. Custom in-house scripts, being not open source, limit ease of deployment, optimal use of computational resources, and cluster scalability. However, workflow management systems, such as Nextflow or Snakemake, allow scalable, reproducible, and portable workflows, and simplify application. To this date, only one workflow, MAGeCK-VISPR, exists that makes use of a workflow management system, i.e. Snakemake, for CRISPR-Cas nine screening analysis. Comprehensive analysis workflows are essential to ensuring the reliability and interpretability of research findings, and should achieve several critical objectives to ensure robust and reliable outcomes in scientific investigations. Currently, no analysis workflow allows analysis of both screening and targeted gene editing experiments. The number of CRISPR-mediated research datasets deposited in repositories, such as the European Nucleotide Archive or Gene Expression Omnibus, and the BioGRID ORCs, is continuously increasing.

Here, we introduce nf-core/crisprseq, a Nextflow DSL two pipeline for the assessment of CRISPR gene editing and screening assays. DSL two allows data analysis pipelines to be scaled and modularized. nf-core/crisprseq is the first generic pipeline enabling the analysis of the broad spectrum of CRISPR designs, from targeted gene edits to large-scale functional screens, allowing the characterization of knockouts, knock-ins, base, and prime editing, and gene modulation experiments. It is the first workflow available that is containerized, improving portability, reproducibility, and easy scalability, thanks to being coded in Nextflow. The workflow is part of the nf-core collection of community-curated, best-practice pipelines. In addition to its broad applicability, we show additional performance in the analysis by contributing new state-of-the-art software and easier usability.

Materials and methods Implementation

Applicability to the analysis of publicly available datasets

Results accuracy benchmarking

Computing performance benchmarking

Results

Pipeline output and quality control

Benchmarking

Screening

Discussion

Supplementary data

Funding

Overview

The nf-core/crisprseq pipeline is introduced as a robust tool for comprehensive analysis of CRISPR technologies, catering to both targeted gene editing and screening experiments. The modular design enhances its adaptability for current and future analysis needs in genomics.

Key Points

1nf-core/crisprseq allows for scalable and reproducible analysis of CRISPR editing techniques
2The pipeline caters to both targeted gene editing and functional genomic screens
3It includes extensive documentation and community support for users
4Performance benchmarking shows competitive analysis capabilities compared to existing tools
5The modular design enables easy integration of new analytical steps.

Details

Authors: Júlia Mir-Pedrol, Laurence Kuhlburger, Marta Sanvicente-García, Metin Yazar, Colm J Ryan, Sabrina Krakau, Gisela Gabernet, Marc Guell, Matteo Bonfanti, nf-core community, Sven Nahnsen
Category: Biology and Natural Sciences

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