Using Synthetically Engineered Guide RNAs to Enhance CRISPR Genome Editing Systems in Mammalian Cells

Daniel Allen, Michael Rosenberg, Ayal Hendel

Research output: Contribution to journalReview articlepeer-review

44 Scopus citations


CRISPR-Cas9 is quickly revolutionizing the way we approach gene therapy. CRISPR-Cas9 is a complexed, two-component system using a short guide RNA (gRNA) sequence to direct the Cas9 endonuclease to the target site. Modifying the gRNA independent of the Cas9 protein confers ease and flexibility to improve the CRISPR-Cas9 system as a genome-editing tool. gRNAs have been engineered to improve the CRISPR system's overall stability, specificity, safety, and versatility. gRNAs have been modified to increase their stability to guard against nuclease degradation, thereby enhancing their efficiency. Additionally, guide specificity has been improved by limiting off-target editing. Synthetic gRNA has been shown to ameliorate inflammatory signaling caused by the CRISPR system, thereby limiting immunogenicity and toxicity in edited mammalian cells. Furthermore, through conjugation with exogenous donor DNA, engineered gRNAs have been shown to improve homology-directed repair (HDR) efficiency by ensuring donor proximity to the edited site. Lastly, synthetic gRNAs attached to fluorescent labels have been developed to enable highly specific nuclear staining and imaging, enabling mechanistic studies of chromosomal dynamics and genomic mapping. Continued work on chemical modification and optimization of synthetic gRNAs will undoubtedly lead to clinical and therapeutic benefits and, ultimately, routinely performed CRISPR-based therapies.

Original languageEnglish
Article number617910
JournalFrontiers in Genome Editing
StatePublished - 2020

Bibliographical note

Publisher Copyright:
Copyright © 2021 Allen, Rosenberg and Hendel.


  • CRISPR therapeutics
  • CRISPR-Cas9
  • chemical modifications
  • engineered nuclease
  • gRNA
  • gene therapy
  • genome editing


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