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Ribozymes pp 205-224 | Cite as

Design and Evaluation of Guide RNA Transcripts with a 3′-Terminal HDV Ribozyme to Enhance CRISPR-Based Gene Inactivation

  • Ben BerkhoutEmail author
  • Zongliang Gao
  • Elena Herrera-CarrilloEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2167)

Abstract

The recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)-Cpf1 system, now reclassified as Cas12a, is a DNA-editing platform analogous to the widely used CRISPR-Cas9 system. The Cas12a system exhibits several distinct features over the CRISPR-Cas9 system, such as increased specificity and a smaller gene size to encode the nuclease and the matching CRISPR guide RNA (crRNA), which could mitigate off-target and delivery problems, respectively, described for the Cas9 system. However, the Cas12a system exhibits reduced gene editing efficiency compared to Cas9. A closer inspection of the crRNA sequence raised some uncertainty about the actual 5′ and 3′-ends. RNA Polymerase (Pol) III promoters are generally used for the production of small RNAs with a precise 5′ terminus, but the Pol III enzyme generates small RNAs with 3’ U-tails of variable length. To optimize the CRISPR-Cas12a system, we describe the inclusion of a self-cleaving ribozyme in the vector design to facilitate accurate 3′-end processing of the crRNA transcript to produce precise molecules. This optimized design enhanced not only the gene editing efficiency, but also the activity of the catalytically inactive Cas12a-based CRISPR gene activation platform. We thus generated an improved CRISPR-Cas12a system for more efficient gene editing and gene regulation purposes.

Key words

Gene therapy CRISPR-Cas Cas12a HDV ribozyme Luciferase reporter assay FACS northern blot Surveyor nuclease assay 

Notes

Acknowledgments

手机体育投注平台This research was supported by the National Institutes of Health (NIH) under award number 1R01AI145045-01.

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© Springer Science+Business Media, LLC, part of Springer Nature 2021

Authors and Affiliations

  1. 1.Laboratory of Experimental Virology, Department of Medical Microbiology, Amsterdam UMC, Academic Medical CenterUniversity of AmsterdamAmsterdamThe Netherlands

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