Next-generation CRISPR gene-drive systems using Cas12a nuclease

Abstract

One method for reducing the impact of vector-borne diseases is through the use of CRISPR-based gene drives, which manipulate insect populations due to their ability to rapidly propagate desired genetic traits into a target population. However, all current gene drives employ a Cas9 nuclease that is constitutively active, impeding our control over their propagation abilities and limiting the generation of alternative gene drive arrangements. Yet, other nucleases such as the temperature sensitive Cas12a have not been explored for gene drive designs in insects. To address this, we herein present a proof-of-concept gene-drive system driven by Cas12a that can be regulated via temperature modulation. Furthermore, we combined Cas9 and Cas12a to build double gene drives capable of simultaneously spreading two independent engineered alleles. The development of Cas12a-mediated gene drives provides an innovative option for designing next-generation vector control strategies to combat disease vectors and agricultural pests.

Un1Cas12f1 and Cas9 gene drive in HSV1: viruses that ‘infect’ viruses

“…In line with these properties of Cas12a, Víctor López Del Amo et al reported that Cas12a gene drive produced resistant alleles in 5-8% of the target population 39 .…”

“…In this proof-of-concept study, we showed HSV1 viruses could act as gene drive vectors, allowing both Cas9 and Cas12f1 gene drives to invade into viruses with WT alleles. Taking advantage of the fast replication rate of HSV1, spread of gene drive reached plateau in one week, a speed not achieved by any other organisms tested so far 13,16,17,39 . Cas12f1 gene drives effectively converted almost all viruses and rarely produced resistant variants.…”

“…In this study, we generated for the first time HSV1 viruses that carried functional Cas9 and Un1Cas12f1 gene drives. Due to the rapid replication of HSV1, the spread of gene drive proceeded at a speed not achieved in any other organisms tested thus far 12,15,16,24 . Both Cas9 and Un1Cas12f1 gene drives were highly effective and capable of converting targets even when their initial presence was low.…”

“…However, Cas12a cuts DNA within the sgRNA target sequence 25 and is very sensitive to mismatches between the sgRNA and the target DNA 26 . In line with these properties of Cas12a, Víctor López Del Amo et al reported that the Cas12a gene drive produced resistant alleles in 5-8% of the target population 24 .…”

“…Another option is to explore Cas nucleases that would mechanistically induce fewer mutations at the target site after DSB repair. Cas12a is the only other Cas nuclease implemented for gene drive thus far and has been shown to propagate efficiently in yeast and fruit flies 23 24 . However, Cas12a cuts DNA within the sgRNA target sequence 25 and is very sensitive to mismatches between the sgRNA and the target DNA 26 .…”