Identification of genome edited cells using CRISPRnano

Abstract

Genome engineering-induced cleavage sites can be resolved by non-homologous end joining (NHEJ) or homology-directed repair (HDR). Identifying genetically modified clones at the target locus remains an intensive and laborious task. Different workflows and software that rely on deep sequencing data have been developed to detect and quantify targeted mutagenesis. Nevertheless, these pipelines require high-quality reads generated by Next Generation Sequencing (NGS) platforms. Here, we have developed a robust, versatile, and easy-to-use computational webserver named CRISPRnano (www.CRISPRnano.de) that enables the analysis of low-quality reads generated by affordable and portable sequencers including Oxford Nanopore Technologies (ONT) devices. CRISPRnano allows fast and accurate identification, quantification, and visualization of genetically modified cell lines, it is compatible with NGS and ONT sequencing reads, and it can be used without an internet connection.

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

“…Some tools developed for the genome editing assessment include CRISPR-GA 13 , CRISPResso 14 , CrispRVariants 15 , CasAnalyzer 16 , cris.py 17 , CRISPResso2 18 , ampliCan 19 and CRISPRpic 20 . Other tools are focused on the analysis of Oxford Nanopore Technologies (ONT) data, instead of Illumina-based NGS data 21 . Moreover, new analysis tools have recently been developed to cover specificity assessment related to off-targets and translocations 22 .…”

mtDNA analysis using Mitopore

“…ONT devices and low-depth Flongle flow cells are cost-effective and can be used without extensive training. ONT sequencing facilitates the identification of SNVs/indels 26 and of larger deletions that may not be captured by short-read sequencing due to fragmentation and assembly challenges. 27 Finally, multiplexing (e.g., of PCR amplicons) is enabled through barcoding by ligation and can be optimized to fit individual experimental needs, such as the number of necessary reads.…”

“…Long-read sequencing analysis was conducted as previously described, 26 using a MinION Mk1C sequencer and a Flongle adaptor (ONT, Oxford, UK). To prepare the sequencing library, the pooled PCR products were subjected to library preparation using the Sequencing by Ligation Kit (ONT, SQK-LSK109) in combination with the Native Barcoding kit (ONT, EXP-NBD104) according to the manufacturer’s instructions.…”

Genome Editing in Translational Medicine: An Inventory

“…Two major issues are important in this regard: (1) off-target cleavage caused by unspecific sgRNA binding [109][110][111][112][113], and (2) predominant activation of errorprone NHEJ repair following the induction of genomic DSBs [114,115]. Although there is currently no consensus about an off-target edit threshold and this is likely cell type-dependent, testing the sgRNA in patient-derived cells followed by targeted deep sequencing [116] is most likely necessary since human lives are at stake.…”