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.
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This research was supported by the National Institutes of Health (NIH) under award number 1R01AI145045-01.
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Berkhout, B., Gao, Z., Herrera-Carrillo, E. (2021). Design and Evaluation of Guide RNA Transcripts with a 3′-Terminal HDV Ribozyme to Enhance CRISPR-Based Gene Inactivation. In: Scarborough, R.J., Gatignol, A. (eds) Ribozymes. Methods in Molecular Biology, vol 2167. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0716-9_12
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DOI: https://doi.org/10.1007/978-1-0716-0716-9_12
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