A CRISPR endonuclease gene drive reveals two distinct mechanisms of inheritance bias

RNA guided CRISPR gene drives have shown the capability of biasing transgene inheritance in multiple species. Among these, homing endonuclease drives are the most developed. In this study, we report the functioning of sds3, bgcn, and nup50 expressed Cas9 in an Aedes aegypti homing split drive system targeting the white gene. We report their inheritance biasing capability, propensity for maternal deposition, and zygotic/somatic expression. Additionally, by making use of the tight linkage of white to the sex-determining locus, we were able to elucidate mechanisms of inheritance bias. We find inheritance bias through homing in double heterozygous males, but find that a previous report of the same drive occurred through meiotic drive. We propose that other previously reported 'homing' design gene drives may in fact bias their inheritance through other mechanisms with important implications for gene drive design.

M1.MboII and M2.MboII type IIS methyltransferases: different specificities, the same target

Methylation of a base in a specific DNA sequence protects the DNA from nucleolytic cleavage by restriction enzymes recognizing the same sequence. The MboII restriction–modification (R–M) system of Moraxella bovis ATCC 10900 consists of a restriction endonuclease gene and two methyltransferase genes. The enzymes encoded by this system recognize an asymmetrical sequence 5′-GAAGA-3′/3′-CTTCT-5′. M1.MboII modifies the last adenine in the recognition sequence 5′-GAAGA-3′ to N 6-methyladenine. A second methylase, M2.MboII, was cloned and purified to electrophoretic homogeneity using a four-step chromatographic procedure. It was demonstrated that M2.MboII modifies the internal cytosine in the recognition sequence 3′-CTTCT-5′, yielding N 4-methylcytosine, and moreover is able to methylate single-stranded DNA. The protein exists in solution as a monomer of molecular mass 30 000±1000 Da under denaturing conditions. Divalent cations (Ca2+, Mg2+, Mn2+ and Zn2+) inhibit M2.MboII methylation activity. It was found that the isomethylomer M2.NcuI from Neisseria cuniculi ATCC 14688 behaves in the same manner. Functional analysis showed that the complete MboII R–M system, consisting of two methyltransferases genes and the mboIIR gene, is the most stable and the least harmful to bacterial cells.