@inbook{06f027cac73a4c67b1f2d8196d0d20f5,
title = "Design and evaluation of guide RNA transcripts with a 3′-terminal HDV ribozyme to enhance CRISPR-based gene inactivation",
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{\textquoteright} 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.",
keywords = "CRISPR-Cas, Cas12a, FACS, Gene therapy, HDV ribozyme, Luciferase reporter assay, Northern blot, Surveyor nuclease assay",
author = "Ben Berkhout and Zongliang Gao and Elena Herrera-Carrillo",
note = "Funding Information: This research was supported by the National Institutes of Health (NIH) under award number 1R01AI145045-01. Publisher Copyright: {\textcopyright} Springer Science+Business Media, LLC, part of Springer Nature 2021.",
year = "2021",
doi = "https://doi.org/10.1007/978-1-0716-0716-9_12",
language = "English",
volume = "2167",
series = "Methods in Molecular Biology",
publisher = "Humana Press Inc.",
pages = "205--224",
booktitle = "Methods in Molecular Biology",
}