CRISPR/Cas9-Enabled Multiplex Genome Editing and Its Application

Bastian Minkenberg, Matthew Wheatley, Yinong Yang

Research output: Chapter in Book/Report/Conference proceedingChapter

64 Scopus citations


The CRISPR/Cas9 system is a prevalent and versatile genome-editing tool of choice for basic and applied biological research. An exchange of a 20-bp spacer sequence in the gRNA can easily reprogram Cas9 to target a different DNA site. By expressing or providing multiple gRNAs, the system also enables multiplex genome editing at high efficiencies. Current approaches for providing multiple gRNAs in vivo include the use of multigene cassettes to express several gRNAs, Csy4-based excision, arrays of crRNAs, ribozyme-flanked gRNAs, tRNA-dependent cleavage of gRNAs, and direct introduction of Cas9 proteins preloaded with different gRNAs. By simultaneously targeting multiple DNA sequences, multiplex genome editing can be used to knockout multiple genes or delete chromosomal fragments. Off-target risk can also be reduced by Cas9-dimers that require the simultaneous expression of two gRNAs. With multiple gRNAs, specific gene expression or methylation status can be efficiently controlled by dCas9 fused to activators, repressors, methyltransferase, demethylase, or other functional domains. As a result, multiplex genome editing is expected to accelerate functional discovery of plant genes as well as genetic improvement of agricultural crops.

Original languageEnglish (US)
Title of host publicationProgress in Molecular Biology and Translational Science
PublisherElsevier B.V.
Number of pages22
StatePublished - 2017

Publication series

NameProgress in Molecular Biology and Translational Science
ISSN (Print)1877-1173
ISSN (Electronic)1878-0814

All Science Journal Classification (ASJC) codes

  • Molecular Medicine
  • Molecular Biology


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