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A Bioinformatics Pipeline to Accurately and Efficiently Analyze the MicroRNA Transcriptomes in Plants

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Published on: January 21, 2020

iGUIDE: an improved pipeline for analyzing CRISPR cleavage specificity.

Christopher L Nobles¹, Shantan Reddy¹, January Salas-McKee^2,3,4,5

¹Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, 3610 Hamilton Walk, Philadelphia, PA, 19104-6076, USA.

|January 19, 2019

Summary

We improved the GUIDE-seq method to iGUIDE, accurately identifying genome editing cleavage sites and deletions. This tool helps distinguish true editing events from background DNA breaks.

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Area of Science:

Molecular Biology
Genomics
Biotechnology

Background:

Programmable nucleases have advanced genome engineering.
Quantifying on- and off-target cleavage sites and deletions is challenging.

Purpose of the Study:

To improve the GUIDE-seq method for accurate cleavage site identification.
To characterize deletions associated with genome editing.
To investigate the nature of background DNA double-strand breaks.

Main Methods:

Developed iGUIDE, an enhanced GUIDE-seq method.
Filtered out mispriming events to clarify cleavage signals.
Applied iGUIDE to Cas9-guided cleavage with four guide RNAs.

Main Results:

Specified locations of Cas9-guided cleavage.
Characterized deletions at cleavage sites.
Showed background DNA double-strand breaks associate with open chromatin, gene-dense regions, and fragile sites.

Conclusions:

iGUIDE enhances the accuracy of genome editing analysis.
The method provides insights into DNA double-strand break origins.
iGUIDE is a valuable tool for genome engineering research.