Reducing resistance allele formation in CRISPR gene drive

Abstract

CRISPR homing gene drives can convert heterozygous cells with one copy of the drive allele into homozygotes, thereby enabling super-Mendelian inheritance. Such a mechanism could be used, for example, to rapidly disseminate a genetic payload in a population, promising effective strategies for the control of vector-borne diseases. However, all CRISPR homing gene drives studied in insects thus far have produced significant quantities of resistance alleles that would limit their spread. In this study, we provide an experimental demonstration that multiplexing of guide RNAs can both significantly increase the drive conversion efficiency and reduce germline resistance rates of a CRISPR homing gene drive in We further show that an autosomal drive can achieve drive conversion in the male germline, with no subsequent formation of resistance alleles in embryos through paternal carryover of Cas9. Finally, we find that the promoter significantly lowers somatic Cas9 expression compared with the promoter, suggesting that provides a superior choice in drive strategies where gene disruption in somatic cells could have fitness costs. Comparison of drive parameters among the different constructs developed in this study and a previous study suggests that, while drive conversion and germline resistance rates are similar between different genomic targets, embryo resistance rates can vary significantly. Taken together, our results mark an important step toward developing effective gene drives capable of functioning in natural populations and provide several possible avenues for further control of resistance rates.

Tree‐sequence recording in SLiM opens new horizons for forward‐time simulation of whole genomes

“…, and for exploring theoretical ideas about evolution (Assaf, Petrov, & Blundell, 2015;Champer et al, 2018;Haller, Mazzucco, & Dieckmann, 2013;Mafessoni & Lachmann, 2015), among other purposes. Because of this broad utility, there is a growing desire to run simulations with increased realism in a variety of areas: longer genomic regions up to the scale of full genome sequences, large populations, selection at multiple loci with linkage effects, complex demography, ecological interactions with other organisms and the environment, explicit space with continuous landscapes, spatial variation in environmental variables, spatial interactions such as competition and mate choice between organisms, and so forth.…”

Resistance is futile: A CRISPR homing gene drive targeting a haplolethal gene

“…The starting plasmids pCFD3 23 (Addgene plasmid #49410), pCFD4 23 (Addgene plasmid # 49411), and pCFD5 24 (Addgene plasmid #73914) were kindly supplied by Simon Bullock. A starting plasmid similar to BHDcN1 was constructed in a previous study 13 (see Supplemental Information). Restriction enzymes for plasmid digestion, Q5 Hot Start DNA Polymerase for PCR, and Assembly Master Mix for Gibson assembly were acquired from New England Biolabs.…”

“…In the progeny of females with at least one drive allele, additional resistance allele formation occurs in the offspring of females carrying the drive due to maternally deposited Cas9 and gRNAs. If cleavage occurs later in development of the embryo, mosaic resistance allele formation is possible, with different cells potentially containing a mixture of wild-type, r1, and r2 alleles 12,13 .…”

“…Two tRNA sequences at the beginning and in between the gRNAs are endogenously spliced out from the pre-RNA 24 . The multiplexed gRNAs increase efficiency of the drive by providing two opportunities for cleavage, thus potentially increasing the rate of homology-directed repair and reducing the rate at which r1 alleles are formed 13 . The drive construct is flanked by two homology arms with wild-type sequences that abut the left and right gRNA target sites.…”

“…Homing gene drives based on the CRISPR/Cas9 system have shown promise in several organisms including yeast [5][6][7][8] , flies [9][10][11][12][13][14][15] , mosquitoes [16][17][18] , and mice 19 . The mechanism of such homing drives involves RNA-guided Cas9 cleavage at a target site, allowing the drive allele to be copied to the wild-type chromosome in the germline through homology-directed repair.…”