Revolutionizing male fertility factor research in mice by using the genome editing tool CRISPR/Cas9

Abstract

Background: Reproductive research is quintessential in understanding not only the cause of infertility, but also for creating family planning tools. The knockout (KO) system approach is conducive to discovering genes that are essential for fertility in mice. However, in vivo research has been limited due to its high cost and length of time needed to establish KO mice.

Methods: The mechanisms behind the CRISPR/Cas9 system and its application in investigating male fertility in mice are described by using original and review articles.

Results: The CRISPR/CAS9 SYSTEM has enabled researchers to rapidly, efficiently, and inexpensively produce genetically modified mice to study male fertility. Several genes have been highlighted that were found to be indispensable for male fertility by using the CRISPR/Cas9 system, as well as more complicated gene manipulation techniques, such as point mutations, tag insertions, and double knockouts, which have become easier with this new technology.

Conclusion: In order to increase efficiency and usage, new methods of CRISPR/Cas9 integration are being developed, such as electroporation and applying the system to embryonic stem cells. The hidden mysteries of male fertility will be unraveled with the help of this new technology.

Keywords: CRISPR/Cas9; fertility; gene editing; reproduction; spermatozoa.

Figure 1

Conventional method of gene disruption by using homologous recombination in embryonic stem (ES) cells. A, Design and construction of a mutant vector transfected into ES cells; those cells carrying the mutation were screened. B, The injection of ES cells carrying the mutation into embryos. The chimeric mice are bred and the heterozygous mice are obtained through germ‐line transmission. The process takes 1‐2 years until completion. drug^r, drug resistance gene; pA, polyadenylation signal; Prom, promoter

Figure 2

Genome editing after double‐strand cleavage by using the CRISPR/Cas9 complex. A, A double‐strand cleavage event occurs at a targeted region. There are two main DNA repair pathways, non‐homologous end‐joining (NHEJ), resulting in indel mutations, and homologous recombination (HR), resulting in a knock‐in homologous to a co‐transfected template strand. B, The single‐guide RNA (sgRNA) (black) and the Cas9 endonuclease (purple) form the CRISPR/Cas9 complex. It binds to the complementary sequence on the DNA and the Cas9 endonuclease cleaves the DNA. crRNA, CRISPR RNA; tracrRNA, transactivating crRNA

Figure 3

Four methods of the CRISPR/Cas9 system to generate gene‐modified mice. A, Cytoplasmic injection method (eg RNA): co‐injection of a single‐guide RNA (sgRNA) and the Cas9 messenger (m)RNA into the cytoplasm of zygotes. B, Pronuclear injection method (eg, DNA): plasmid‐based delivery system (a plasmid expressing sgRNA and Cas9 is injected into the pronucleus of the fertilized eggs). C, Electroporation method (eg protein): a pulse of high‐voltage electricity opens the oocyte's membrane pore to incorporate the Cas9 protein–RNA complex. D, Embryonic stem (ES) cell method: a modified plasmid with a puromycin resistance cassette (Puro^r) is transfected into ES cells and drug‐selected. The mutant ES cells are injected into eight‐cell‐stage embryos or blastocysts. CBh promoter, chicken β‐actin hybrid promoter; hCas9, humanized Cas9; polyA, polyadenylation signal; T2A, Thosea asigna virus 2A peptide; U6 promoter, commonly used for driving small RNA expression

Figure 4

Complicated gene manipulation techniques. A, Point mutation: Co‐injecting a single‐stranded DNA (ssDNA) or double‐stranded DNA (dsDNA) can cause a single nucleotide base insertion, deletion, or substitution through homologous recombination (HR). B, Tag insertion: tags, such as a FLAG‐tag, can be inserted similarly to point mutations by using ssDNA or dsDNA and HR. Reporter genes, such as enhanced green fluorescent protein (EGFP), can also be knocked in. C, Double knockout: by using a combination of different sgRNAs, Cas9 can cleave multiple target genes to generate complex KO mice. D, Large deletion: the CRISPR/Cas9 system can mediate large genomic deletions by transfecting two or more sgRNAs