Heterozygous mutation of the splicing factor Sf3b4 affects development of the axial skeleton and forebrain in mouse.

Background: Splicing factor 3B subunit 4 (SF3B4) is a causative gene of an acrofacial dysostosis, Nager syndrome. Although in vitro analyses of SF3B4 have proposed multiple noncanonical functions unrelated to splicing, less information is available based on in vivo studies using model animals.

Results: We performed expression and functional analyses of Sf3b4 in mice.

CLICK: one-step generation of conditional knockout mice.

Background: CRISPR/Cas9 enables the targeting of genes in zygotes; however, efficient approaches to create loxP-flanked (floxed) alleles remain elusive.

Results: Here, we show that the electroporation of Cas9, two gRNAs, and long single-stranded DNA (lssDNA) into zygotes, termed CLICK (CRISPR with lssDNA inducing conditional knockout alleles), enables the quick generation of floxed alleles in mice and rats.

Conclusions: The high efficiency of CLICK provides homozygous knock-ins in oocytes carrying tissue-specific Cre, which allows the one-step generation of conditional knockouts in founder (F0) mice.

Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ.

Background: Although CRISPR/Cas enables one-step gene cassette knock-in, assembling targeting vectors containing long homology arms is a laborious process for high-throughput knock-in. We recently developed the CRISPR/Cas-based precise integration into the target chromosome (PITCh) system for a gene cassette knock-in without long homology arms mediated by microhomology-mediated end-joining.

Results: Here, we identified exonuclease 1 (Exo1) as an enhancer for PITCh in human cells.

Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice.

Although the CRISPR/Cas system has enabled one-step generation of knockout mice, low success rates of cassette knock-in limit its application range. Here we show that cloning-free, direct nuclear delivery of Cas9 protein complex with chemically synthesized dual RNAs enables highly efficient target digestion, leading to generation of knock-in mice carrying a functional cassette with up to 50% efficiency, compared with just 10% by a commonly used method consisting of Cas9 mRNA and single guide RNA. Our cloning-free CRISPR/Cas system facilitates rapid one-step generation of cassette knock-in mice, accelerating functional genomic research by providing various in vivo genetic tools.