New software tool detects, evaluates, and quantifies off-target editing activity

CRISPR technology enables researchers to edit genomes by modifying DNA sequences and hence gene function. Its numerous potential uses include treating and reducing disease transmission, repairing genetic flaws, and enhancing crops.

CRISPR

CRISPR. Image Credit: vchal/Shutterstock.com

CRISPR-Cas9 technology, for example, may be programmed to produce exceptionally well-defined modifications to the intended target on a chromosome where a certain gene or functional region is situated.

One possible difficulty is that CRISPR editing might result in unexpected genetic alterations. This is referred to as off-target activity. When many sites in the genome are targeted, off-target activity might result in translocations, atypical chromosomal rearrangement, and other unexpected genomic alterations.

One of the most difficult issues in making CRISPR-Cas9 technology precise and usable in medical practice is controlling off-target editing activity.

Current measuring assays and data analysis approaches for assessing off-target activity do not offer statistical evaluation, are insufficiently sensitive in distinguishing signal from noise in studies with low editing rates and need time-consuming attempts to find translocations.

A multidisciplinary group of researchers from the Interdisciplinary Center Herzliya and Bar-Ilan University describes the invention of a novel software tool to detect, analyze, and quantify off-target editing activity, including unfavorable translocation events that might cause cancer, in the latest issue of the journal Nature Communications.

The software is based on data from a standard measuring assay, which includes Next-Generation Sequencing (NGS) and multiplexed PCR amplification.

CRISPECTOR is a tool that analyzes next-generation sequencing data from CRISPR-Cas9 studies and uses statistical modeling to estimate and quantify editing activity. CRISPECTOR precisely measures off-target activity at all interrogated loci.

It also allows for lower false-negative rates in places with low but considerable off-target activity. Importantly, one of CRISPECTOR’s new properties is its capacity to identify unfavorable translocation events that occur during an editing experiment.

In genome editing, especially for clinical applications, it is critical to identify low level off-target activity and adverse translocation events. Even a small number of cells with carcinogenic potential, when transplanted into a patient in the context of gene therapy, can have detrimental consequences in terms of cancer pathogenesis. As part of treatment protocols, it is therefore important to detect these potential events in advance.”

Dr Ayal Hendel, Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University

Dr. Hendel oversaw the research alongside Prof. Zohar Yakhini of the Arazi School of Computer Science at the Interdisciplinary Center (IDC) Herzliya.

CRISPECTOR provides an effective method to characterize and quantify potential CRISPR-induced errors, thereby significantly improving the safety of future clinical use of genome editing.”

Dr Ayal Hendel, Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University

Hendel’s team used CRISPR-Cas9 technology to modify genes in stem cells that are linked to blood and immune system illnesses.

While examining the data, they discovered flaws in the existing methods for assessing off-target activity, as well as holes that need to be filled to increase applicability. This experience led to a cooperation with Prof. Yakhini’s world-renowned computational biology and bioinformatics group.

In experiments utilizing deep sequencing techniques that have significant levels of background noise, low levels of true off-target activity can get lost under the noise. The need for a measurement approach and related data analysis that are capable of seeing beyond the noise, as well as of detecting adverse translocation events occurring in an editing experiment, is evident to genome editing scientists and practitioners.”

Zohar Yakhini, Professor, IDC Herzliya and Technion

CRISPECTOR is a tool that can sift through the background noise to identify and quantify true off-target signal. Moreover, using statistical modeling and careful analysis of the data CRISPECTOR can also identify a wider spectrum of genomic aberrations. By characterizing and quantifying potential CRISPR-induced errors our methods will support the safer clinical use of genome editing therapeutic approaches,” concluded Yakhini.

Source:
Journal reference:

Amit, I., et al. (2021) CRISPECTOR provides accurate estimation of genome editing translocation and off-target activity from comparative NGS data. Nature Communications. doi.org/10.1038/s41467-021-22417-4.

Comments

The opinions expressed here are the views of the writer and do not necessarily reflect the views and opinions of AZoLifeSciences.
You might also like... ×
Study shows how mitochondrial function impacts schizophrenia in patients with genetic disorder