Paper of the month October 2019

Super-Mendelian inheritance mediated by CRISPR-Cas9 in the female mouse germline
Hannah A. Grunwald^1,5, Valentino M. Gantz^1,5, Gunnar Poplawski^2,4,5, Xiang-Ru S. Xu¹, Ethan Bier^1,3 and Kimberly L. Cooper^1,3*

Mendelian inheritance is characterized by 50% chance of an offspring to inherit a certain trait in a given population. However, CRISPR-Cas9 technique changed the game on how a genetic element can be transmitted to offspring. It starts with the insertion of an active genetic element in one of the alleles (mutant chromosome) which contains a sgRNA targeting cleavage of such trait in an animal. Upon breeding with another animal carrying a Cas9 protein, a double-strand break (DSB) is generated and corrected by a homology-directed repair (HDR) mechanism, where the wild-type cleaved chromosome (receiver) will use the mutant pair (donor) as a “mold”. This way, a resulting heterozygous animal has a higher chance to be converted in a homozygous one for the specific active genetic element altered, increasing the chance of transmitting a certain trait to the offspring. This process is called super-Mendelian inheritance.

Grunwald et al have decided to introduce this mechanism as a proof-of-concept in mice, a novelty when compared to the innumerous tests ran already for other species like insects. The active genetic element named CopyCat possesses only a gRNA which will be knocked-in into the Tyrosinase gene (Tyr) at exon 4, disrupting its activity (TyrCopyCat/-). This gene disruption is responsible for the albino phenotype in mice. TyrCopyCat/- male mice were then crossed with female mice carrying Cas9 on two safe-harbour locus (Rosa26 and H11) and a hypomorphic point mutation in exon 5 of Tyr gene (Tyrch/-), resembling partial activity of this allele, which should translate to grey-coloured offspring. Since all F2 TyrCopycat/Ch mice presented a white (Rosa26) and grey-white mosaic color (H11), offspring inheritance had to be tested for CRISPR-Cas9 transmission to offspring and assess the number of non-homologous end-joining (NHEJ) and HDR events. Hence, F2 TyrCopyCat/ch male mice were bred with female CD-1 strain mice carrying a loss-of-function point mutation in Tyr exon 1 (Tyrnull/null). F3 mice geno- and phenotypes post-breeding expected would be:

• Tyrch/null –  no Cas9 (grey) – if no NHEJ or HDR
• Tyrch/null –  +Cas9 (white) – if NHEJ
• Tyrch/Copycat –  +Cas9 (white and red-fluorescent) – if HDR

All F3 mice appeared as white-colored revealing Cas9 transmission to offspring. Also, no red-fluorescence has been observed in F3 mice, revealing that CopyCat allele was not present, meaning that all recombination events were NHEJ rather than HDR.

Non-copying of CopyCat allele in F3 white mice and therefore HDR events absence has been hypothesized as being related with chromosome non-alignment for HDR and NHEJ prevalence during meiosis. Grunwald et al tested this by delaying Cas9 expression during meiosis in germline development in Tyrch/Copycat mice. By crossing of female TyrCh/Ch carrying a conditional Cas9 allele with male TyrCopyCat/- mice carrying Vasa-cre or Stra8-cre genes, F3 progeny this time, were mostly grey-colored. Cre protein is expected to accumulate before Cas9 is actually expressed upon conditional allele activation by opposition to normal Vasa and Stra8, increasing inter-homologue HDR during meiosis. To test for CRISPR-Cas9 germline transmission, F3 female were crossed with CD-1 (Tyrnull). Same F3 phenotype as previously was expected and it was observed that, this time, most of female TyrCopycat/ch presented HDR conversion, where offspring carried CopyCat allele. Moreover, a higher number of mice and HDR events in Vasa-cre, H11 compared to Vasa-cre, Rosa26 locus has been observed. No male mice experienced HDR events, therefore not carrying CopyCat allele. Furthermore, a gene conversion of 72% was achieved in one of the female progenies. This phenomenon has a very low likelihood to happen in a natural meiotic recombination event, supporting the genetic inheritance on a super-Mendelian fashion, contrary to normal chromosomal crossover.

This paper elucidates about the timing of Cas9 expression as an influence on DSB repairing mechanism choice between HDR over NHEJ in germ cells and how this could be modelled in vivo in rodent models. The unbalance of these events between male and female sexes is also another factor to take into account and although the reason is still unknown, HDR prevalence could be further studied using such systems.