CRISPR-broad: combined design of multi-targeting gRNAs and broad, multiplex target finding

Alaguraj Veluchamy*, Kaian Teles, Wolfgang Fischle*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


In CRISPR-Cas and related nuclease-mediated genome editing, target recognition is based on guide RNAs (gRNAs) that are complementary to selected DNA regions. While single site targeting is fundamental for localized genome editing, targeting to expanded and multiple chromosome elements is desirable for various biological applications such as genome mapping and epigenome editing that make use of different fusion proteins with enzymatically dead Cas9. The current gRNA design tools are not suitable for this task, as these are optimized for defining single gRNAs for unique loci. Here, we introduce CRISPR-broad, a standalone, open-source application that defines gRNAs with multiple but specific targets in large continuous or spread regions of the genome, as defined by the user. This ability to identify multi-targeting gRNAs and corresponding multiple targetable regions in genomes is based on a novel aggregate gRNA scoring derived from on-target windows and off-target sites. Applying the new tool to the genomes of two model species, C. elegans and H. sapiens, we verified its efficiency in determining multi-targeting gRNAs and ranking potential target regions optimized for broad targeting. Further, we demonstrated the general usability of CRISPR-broad by cellular mapping of a large human genome element using dCas9 fused to green fluorescent protein.

Original languageEnglish (US)
Article number19717
JournalScientific Reports
Issue number1
StatePublished - Dec 2023

Bibliographical note

Publisher Copyright:
© 2023, The Author(s).

ASJC Scopus subject areas

  • General


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