Jaraba-Wallace, J. | bioRxiv Science

bioRxiv2022

CRISPR-Analytics (CRISPR-A): a platform for precise analytics and simulations for gene editing

Gene editing characterization with currently available tools does not always give precise relative proportions among the different types of gene edits present in an edited bulk of cells. We have developed CRISPR-Analytics, CRISPR-A, which is a comprehensive and versatile genome editing web application tool and a nextflow pipeline to give support to gene editing experimental design and analysis. CRISPR-A provides a robust gene editing analysis pipeline composed of data analysis tools and simulation. It achieves higher accuracy than current tools and expands the functionality. The analysis includes mock-based noise correction, spike-in calibrated amplification bias reduction, and advanced interactive graphics. This expanded robustness makes this tool ideal for analyzing highly sensitive cases such as clinical samples or experiments with low editing efficiencies. It also provides an assessment of experimental design through the simulation of gene editing results. Therefore, CRISPR-A is ideal to support multiple kinds of experiments such as double-stranded DNA break-based engineering, base editing (BE), primer editing (PE), and homology-directed repair (HDR), without the need of specifying the used experimental approach.

bioinformatics↗

bioRxiv2022

INSERT-seq enables high resolution mapping of genomically integrated DNA using nanopore sequencing

Comprehensive characterization of genome engineering with viral vectors, transposons, CRISPR/Cas mediated DNA integration and other DNA editors remains relevant for their development and safe use in human gene therapy. Currently, described methods for measuring DNA integration in edited cells rely on short read based technologies. Due to the repetitive nature of the human genome, short read based methods can potentially overlook insertion events in repetitive regions. We modelled the impact of read length in resolving insertion sites, which suggested a significant drop in insertion site detection with shorter read length. Based on that, we developed a method that combines targeted amplification of integrated DNA, UMI-based correction of PCR bias and Oxford Nanopore long-read sequencing for robust analysis of DNA integration in a genome. This method, called INSERT-seq, is capable of detecting events occurring at a frequency of up to 0.1%. INSERT-seq presents a complete handling of all insertions independently of repeat size. The experimental pipeline improves the number mappable insertions at repetitive regions by 7.3% and repeats larger than the long read sequencing size are processed computationally to perform a peak calling in a repeat database. INSERT-seq is a simple, cheap and robust method to quantitatively characterise DNA integration in diverse ex-vivo and in-vivo samples.

genomics↗