Gene editing of the multi-copy H2A.B gene and its importance for fertility

• Published in: Genome Biology Vol. 20; no. 1; p. 23
• Main Authors: Anuar, Nur Diana, Kurscheid, Sebastian, Field, Matt, Zhang, Lei, Rebar, Edward, Gregory, Philip, Buchou, Thierry, Bowles, Josephine, Koopman, Peter, Tremethick, David J., Soboleva, Tatiana A.
• Format: Journal Article
• Language: English
• Published: London BioMed Central 31.01.2019; Springer Nature B.V; BMC
• Subjects: Animal Genetics and Genomics; Animals; Base Sequence; Bioinformatics; Biomedical and Life Sciences; Chromatin; Chromosomal Proteins, Non-Histone - metabolism; CRISPR; Deoxyribonucleic acid; DNA; DNA sequencing; DNA-directed RNA polymerase; Evolutionary Biology; Experiments; family; Female; Fertility - genetics; Gene Editing - methods; Gene Expression; genes; Genome editing; Genomes; H2A.B; haploidy; Histone variants; histones; Histones - genetics; Human Genetics; Hypotheses; Infertility, Male - etiology; Infertility, Male - metabolism; Infertility, Male - pathology; Insights from Genome Editing; knockout mutants; Life Sciences; Male; males; mice; Mice, Knockout; Microbial Genetics and Genomics; Mutation; Parameter estimation; Plant Genetics and Genomics; Pre-mRNA splicing; Proteins; RNA; RNA polymerase; RNA polymerase II; RNA Polymerase II - metabolism; RNA processing; Spermatogenesis; spermatozoa; Spermatozoa - metabolism; Spermatozoa - pathology; Splicing; Splicing speckles; testes; Transcription; Transcription Activator-Like Effector Nucleases; Tubules; Chromatin; H2A.B; Splicing speckles; TALENs; Histone variants; Pre-mRNA splicing; Genome editing; RNA polymerase II
• ISSN: 1474-760X; 1474-7596; 1474-760X
• DOI: 10.1186/s13059-019-1633-3

Background Altering the biochemical makeup of chromatin by the incorporation of histone variants during development represents a key mechanism in regulating gene expression. The histone variant H2A.B, H2A.B.3 in mice, appeared late in evolution and is most highly expressed in the testis. In the mouse, it is encoded by three different genes. H2A.B expression is spatially and temporally regulated during spermatogenesis being most highly expressed in the haploid round spermatid stage. Active genes gain H2A.B where it directly interacts with polymerase II and RNA processing factors within splicing speckles. However, the importance of H2A.B for gene expression and fertility are unknown. Results Here, we report the first mouse knockout of this histone variant and its effects on fertility, nuclear organization, and gene expression. In view of the controversy related to the generation of off-target mutations by gene editing approaches, we test the specificity of TALENs by disrupting the H2A.B multi-copy gene family using only one pair of TALENs. We show that TALENs do display a high level of specificity since no off-target mutations are detected by bioinformatics analyses of exome sequences obtained from three consecutive generations of knockout mice and by Sanger DNA sequencing. Male H2A.B.3 knockout mice are subfertile and display an increase in the proportion of abnormal sperm and clogged seminiferous tubules. Significantly, a loss of proper RNA Pol II targeting to distinct transcription–splicing territories and changes to pre-mRNA splicing are observed. Conclusion We have produced the first H2A.B knockout mouse using the TALEN approach.