Research Projects

Isogenicity in laboratory animals represents the cornerstone of reproducible biomedical research in both humans and livestock. Experiments using non-isogenic animals require larger sample sizes to achieve equivalent statistical power, ultimately increasing total research costs. This is also a violation of the 4Rs principles i.e., Reduce, Refine, Replace, and Responsibility in lab animal ethics. In mice, traditional inbred strain development with 99.8% genetic uniformity (or inbreeding coefficient (F)) requires a minimum of 20 generations of brother-sister mating, taking 3-4 years and a minimum of 320-360 individuals involved in this process, i.e., 16-18 individuals per generation. Hence, there is scope to speed up the inbreeding process in fewer generations to achieve maximum genetic uniformity. In populations with higher F, haplotypes tend to be longer due to the decreased frequency of recombination events over time. So we hypothesize that, by erasing meiotic recombination events, longer and intact haplotypes can be preserved, ultimately leading to increased F. In a simulation study conducted by us setting zero recombination rate (c), we found that ~100% F can be achieved in only 9 generations (see technical document). Two main outcomes during Meiosis-I are: a) crossover, which results in exchange of chromosome arms through recombination, resulting in smaller haplotypes, and b) non-crossover, where homologous chromosomes remain mostly unchanged except for the repaired regions. Non-crossovers are the result of a process known as synthesis-dependent strand annealing (SDSA). We propose an innovative method termed Selective Meiotic Aptamer-Regulated Targeting for Inbreeding (SMART-In) to accelerate inbreeding by biasing Holliday junction resolution toward non-crossover pathways involving SDSA, which will set recombination rate to zero. In conventional gene editing platforms that rely on nuclease activity, such as CRISPR-Cas9, modifications occur via the generation of DNA double-strand breaks and can lead to unwanted genomic alterations and genotoxicity. Alternatively, RNA aptamer-based gene editing involves single-strand breaks, offers genomic stability by minimizing chromosomal translocations, reduces off-target effects, and has negligible disruption to cellular functions. The proposed SMART-In technology has broader applications (in terms of time and resources saved): a) development of genetically uniform laboratory animals, b) producing inbred parental lines in livestock and poultry, which maximizes heterosis in progeny generation upon crossing such parental lines, c) mapping of recessive deleterious alleles, and d) creation of recombinant inbred lines for high-resolution QTL mapping. Overall, this project will reduce both the time and number of animals needed to breed inbred strains, thereby advancing the 4Rs principles of laboratory animal ethics.