1887

Abstract

The discovery and adaptation of CRISPR/Cas systems within molecular biology has provided advances across biological research, agriculture and human health. Genomic manipulation through use of a CRISPR nuclease and programmed guide RNAs has become a common and widely accessible practice. The identification and introduction of new engineered variants and orthologues of Cas9 as well as alternative CRISPR systems such as the type V group have provided additional molecular options for editing. These include distinct PAM requirements, staggered DNA double-strand break formation, and the ability to multiplex guide RNAs from a single expression construct. Use of CRISPR/Cas has allowed for the construction and testing of a powerful genetic architecture known as a gene drive within eukaryotic model systems. Our previous work developed a drive within budding yeast using Cas9. Here, we installed the type V Cas12a (Cpf1) nuclease gene and its corresponding guide RNA to power a highly efficient artificial gene drive in diploid yeast. We examined the consequence of altering guide length or introduction of individual mutational substitutions to the crRNA sequence. Cas12a-dependent gene-drive function required a guide RNA of at least 18 bp and could not tolerate most changes within the 5′ end of the crRNA.

Keyword(s): Cas12a , CRISPR , gene drive and yeast
Funding
This study was supported by the:
  • National Institute of Food and Agriculture (Award Hatch Project 1013520)
    • Principle Award Recipient: GregoryC Finnigan
  • National Institutes of Health (Award P20 GM103418)
    • Principle Award Recipient: NotApplicable
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
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2021-12-17
2022-01-29
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