Analysis of a Cas12a-based gene-drive system in budding yeast

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 Streptococcus pyogenes Cas9. Here, we installed the type V Francisella novicida 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.

Modelling homing suppression gene drive in haplodiploid organisms

“…Though the most rapid progress has been in Anopheles mosquitoes for reduction of vector-borne disease [5][6][7] (for which modification drives have also been developed [8][9][10]), suppression gene drives could also be used to remove invasive species or agricultural pests. Thus far, gene drives have been demonstrated in a variety of organisms, including yeast [11,12], flies [13][14][15][16][17][18], mice [19] and plants [20]. Most of these have been homing type drives, where a nuclease, usually CRISPR/Cas9, cuts a wild-type chromosome at a site-directed by its guide RNA (gRNA) [21].…”

Modelling homing suppression gene drive in haplodiploid organisms

“…Though the most rapid progress has been in Anopheles mosquitoes for reduction of vector-borne disease 5–7 (for which modification drives have also been developed 8–10 ), suppression gene drives could also be used to remove invasive species or agricultural pests. Thus far, gene drives have been demonstrated in a variety of organisms, including yeast 11,12 , flies 13–1516–18 , mice 19 , and plants 20 . Most of these have been homing types drives, where a nuclease, usually CRISPR/Cas9, cuts a wild-type chromosome at a site directed by its guide RNA (gRNA) 21 .…”

Next-generation CRISPR gene-drive systems using Cas12a nuclease

“…Both gene drives, driven by validated gRNAs targeting the ebony gene for disruption 20 , displayed the desirably lower super-Mendelian rates at 18°C and increased gene-drive efficiency at 25°C and 29°C. Our results demonstrate that Cas12a-based HDR, which has been extensively used in other organisms 17,18,22 , can also be achieved in a gene drive context using Drosophila melanogaster. This work shows the functionality of a Cas12a gene-drive system triggering super-Mendelian inheritance rates in an insect germline, and the first presenting a dual gene-drive in any organism, both advances addressing concerns in Cas9-based vector control strategies.…”

“…Furthermore, all current gene-drive methods employ a Cas9 nuclease 1 , limiting the creation of novel arrangements towards next-generation gene-drive systems. Yet, other nucleases, such as Cas12a, have demonstrated efficient genome editing in a broad range of organisms [15][16][17][18] . In Drosophila melanogaster, Cas12a-based genome editing produced efficient gene disruption 19,20 ; however, the DNA homology-directed repair (HDR) triggered by Cas12a has yet not been explored in insects.…”