Applications of CRISPR/Cas9 in Reproductive Biology Faheem Ahmed Khan, Nuruliarizki Shinta Pandupuspitasari, Huang ChunJie, Hafz Ishfaq Ahmad, Kai Wang, Muhammad Jamil Ahmad and ShuJun Zhang* Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of the Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, Te People’s Republic of China. *Correspondence: [email protected] htps://doi.org/10.21775/cimb.026.093 Abstract into genes that interact with environment to develop Genome editing is unravelling its benefts in wide a specifc phenotype. Although extensive studies areas of scientifc development and understanding. have been carried out but still the understanding Te advances of genome editing from ZFNs and of genes and its function is far from understanding TALLENs to CRISPRs defnes its wide applicabil- because genes are diferentially expressed. Te pre- ity. Reproduction is the fundamental process by sent advances in genome editing technologies takes which all organisms maintain their generations. a leap towards endogenous genome modifcation, CRISPR/Cas9, a new versatile genome editing which gives rise to several opportunities to deal tool has been recently tamed to correct several with diseases and undesired phenotypes (Khan et disease causing genetic mutations, spreading its al., 2016). arms to improve reproductive health. It not only Te continuous update to knowledge reservoir edits harmful genetic mutations but is also applied owes to molecular studies unfolding several impor- to control the spread of parasitic diseases such as tant aspects of genes and their interactions with malaria by introducing selfsh genetic elements, the environment enabling scientists to do forward propagated through generations and population via and reverse genetics model cells and organisms reproduction. Tese applications led us to review to uncover the intricate mechanisms that may be the recent developments of CRISPRs use in repro- taking place. Such studies also open avenues of its ductive biology. practical applications, especially in medicine. Te genome manipulation was frst made possible in the 1970s when E. coli was frst manipulated for thera- Introduction peutic protein production (Itakura et al., 1977). Te blueprint of life and heredity is writen in Te advent of gene silencing methods further genome. Te human body consists of trillions increased the precise understanding of gene func- of cells which are divided in autosomal cells and tion that rely on homologous template. It is very gametes (Soh et al., 2014). Te autosomes consists precise but its application on a large scale remains of two genomes while gametes comprises a single inefcient. To overcome these limitations ZFNs genome. During the process of fertilization the and TALENs are developed which are proteins with two gametes, one from each partner, unite to form DNA-binding domains (Dhanasekaran et al., 2006; a zygote (Hughes and Page, 2015). Te genome is Reyon et al., 2012) that are much more efcient built up of billions of DNA base pairs subdivided than the gene silencing technologies; however, its Curr. Issues Mol. Biol. Vol. 26 94 | Khan et al. wider application is limited by unavailability of genetic studies of fertility, development and control skilled technicians who can make a special targeting of disease-causing vectors. Tis review will focus on protein for each genomic target. applications of the most versatile CRISPR/Cas9 Clustered regularly interspaced short palindro- in reproduction that is the fundamental process of mic repeats (CRISPR/Cas) is the most recent and species survival. accurate tool to target nucleic acids of any nature to modify, edit or disrupt any gene. Te CRISPR/ Cas system can virtually be reprogrammed against Gene drives and CRISPR/Cas9 any genome of interest to achieve required goals. Te selfsh genetic elements constitute gene drive Tis special immune response is developed by systems that are biased towards their own inherit- a unique set of CRISPR array which contains a ance in super-Mendelian fashion (Noble et al., protospacer from invading nucleic acid recognized 2016; Champer et al., 2016). Tese gene drives by short protospacer adjacent motif (PAM) which are considered as means to eradicate insect-borne helps the host resistant to its own immune response diseases including malaria, zika and dengue (Ham- (Bolotin et al., 2005; Barrangou et al., 2007). Te mond et al., 2015). Naturally found gene drives simple machinery of CRISPR/Cas system makes it are in the form of transposons, commonly known a technology of choice in various biotechnological as jumping genes (Charlesworth et al., 1989) or platforms including gene therapy, genome editing those causing segregation distortion (Tao et al., and therapeutic production. Te CRISPR/Cas9 2001) and Medea elements (Chen et al., 2007). provides an efcient way to make targeted double- Te discovery of CRISPR/Cas9 genome editing stranded breaks (DSBs) in DNA that can knock out technology has made it possible to create synthetic any gene through non-homologous DNA repair gene drives that can change the dynamics of natural pathway (NHEJ) and can stimulate template-based populations (Champer et al., 2016). homologous repair through homology directed It is noteworthy that synthetic gene drives are repair (HDR) (Khan et al., 2016). Te CRISPR/ widely studied and have potential to widely eradi- Cas9 system requires only the design of the new cate the protozoal, viral and helminthic diseases sgRNA against target, as compared to ZFNs and that causes a huge human disease burden (Zama- TALENs that needs special protein for each target. nian and Andersen 2016). Synthetic gene drives to Gene drives is a technique that can enhance the control vector-borne diseases have two principal inheritance of a particular gene to increase its preva- objectives: population modifcation or population lence in the population (Windbichler et al., 2011). suppression (Burt, 2003, 2014). Te eradication In natural populations, genes sometimes gain a of the vector, for example mosquitos carrying ftness bias that does not depend on the organism. the malaria parasite, is an example of population Te genes in a sexually reproducing organisms have suppression. In contrast, if an allele important to a 50% chance of being inherited by ofspring, but disease resistance is introduced into a vector, or some genes somehow gain an evolutionary advan- if an allele is transferred to a vector that makes it tage such that they are passed on to more than unable to transfer pathogen to host, this is a form 50% of ofspring. Tis phenomenon is considered of population modifcation gene drives. Te con- as natural ‘gene drives’ that initially is found in cept of such synthetic gene drives is derived from single organism and then is slowly transferred, over natural gene drives but its progress was very slow generations, to the whole population. Artifcial until the use of the versatile CRISPR/Cas9 system gene drives can be a very useful way to control (Zamanian and Andersen, 2016). Te recent the vectors of several diseases, including malaria applications of CRISPR/Cas9 in Drosophila for (Hammond et al., 2015), dengue and zika trans- mutagenic chain reaction (Gantz and Bier, 2015) mited by mosquitoes. Te CRISPR/Cas9 system and to achieve gene drives in malarial mosquito vec- can be efciently utilized to create artifcial gene tors that converts autocatalytic heterozygous locus drives that can render mosquitoes sterile. Apart to homozygous locus rendering mosquito unable to from disease control, CRISPR/Cas9 can be used to transmit malaria by spreading antimalarial efector study several fertility-related genes in males, hence genes through mutagenic chain reaction (Gantz providing an important platform for molecular et al., 2015). Furthermore, this mutagenic chain Curr. Issues Mol. Biol. Vol. 26 Applications of CRISPR/Cas9 in Reproductive Biology | 95 reaction was utilized recently against Plasmodium the population. Ofen an endonuclease targeting falciparum vector Anopheles gambiae to spread X chromosome is designed for such drives in het- female sterility genes, hence targeting the reproduc- erogametic X/Y species. A schematic illustrating tion capability of Anopheles gambiae (Hammond et endonuclease gene drives is shown in Fig. 8.1. al., 2016). Tese studies open powerful avenues Tese approaches emphasizes the role of to control several other mosquito borne vector CRISPR/Cas9 genome editing technology beyond diseases including flarial nematodes by eradicating the study of fertility related genes and mechanistic LF genes in wild populations that can be done by pathways but provide a robust platform to spread creating population suppression drives by spread- desired gene drives efciently in natural popula- ing genes that compromise reproductive capacity tions to control various vector-borne diseases of and limit the mosquito populations transmiting human health importance. Wuchereria and Brugia nematodes, the main aetio- logical agents of LF transfer in humans (Zamanian and Andersen, 2016). Genome engineering and Te other kind of gene drive is sex-linked mei- reproductive biology otic drive that occurs when certain alleles are biased Te gene modifcation in primates remains a chal- at meiotic level and hence are transferred to gam- lenge because of their long life cycles and hence etes and ultimately to ofspring. Several such kinds make editing impractical. Te gene introduction of meiotic