Switchable genome editing via genetic code expansion

Suzuki T*, Asami M*, Patel SG, Luk LYP, Tsai YH, Perry ACF.

Scientific Reports volume 8, Article number: 10051 (2018)

Multiple applications of genome editing by CRISPR-Cas9 necessitate stringent regulation and Cas9 variants have accordingly been generated whose activity responds to small ligands, temperature or light. However, these approaches are often impracticable, for example in clinical therapeutic genome editing in situ or gene drives in which environmentally-compatible control is paramount. With this in mind, we have developed heritable Cas9-mediated mammalian genome editing that is acutely controlled by the cheap lysine derivative, Lys(Boc) (BOC). Genetic code expansion permitted non-physiological BOC incorporation such that Cas9 (Cas9BOC) was expressed in a full-length, active form in cultured somatic cells only after BOC exposure. Stringently BOC-dependent, heritable editing of transgenic and native genomic loci occurred when Cas9BOC was expressed at the onset of mouse embryonic development from cRNA or Cas9BOC transgenic females. The tightly controlled Cas9 editing system reported here promises to have broad applications and is a first step towards purposed, spatiotemporal gene drive regulation over large geographical ranges.

Schematic diagrams depicting natural translation and translation in the BOC system. (A) The natural incorporation of lysine (Lys, K) and (B) termination of translation at a stop codon. (C) When introduced ectopically, the orthogonal aminoacyl-tRNA synthetase, PylRS, can attach the non-physiological amino acid, BOC to its orthogonal tRNA, which decodes the stop codon, UAG, allowing BOC incorporation into a nascent polypeptide chain during translation. (D) Chemical structures of Lys and BOC.

Asymmetric Parental genome engineering by Cas9 during mouse meiotic exit

Toru Suzuki*, Maki Asami* & Anthony C.F. Perry

*co-first author

Scientific Reports 4, Article number:7621 (2014)

Mammalian genomes can be edited by injecting pronuclear embryos with Cas9 cRNA and guide RNA (gRNA) but it is unknown whether editing can also occur during the onset of embryonic development, prior to pronuclear embryogenesis. We here report Cas9-mediated editing during sperm-induced meiotic exit and the initiation of development. Injection of unfertilized, mouse metaphase II (mII) oocytes with Cas9 cRNA, gRNA and sperm enabled efficient editing of transgenic and native alleles. Pre-loading oocytes with Cas9 increased sensitivity to gRNA ~100-fold. Paternal allelic editing occurred as an early event: single embryo genome analysis revealed editing within 3 h of sperm injection, coinciding with sperm chromatin decondensation during the gamete-to-embryo transition but prior to pronucleus formation. Maternal alleles underwent editing after the first round of DNA replication, resulting in mosaicism. Asymmetric editing of maternal and paternal alleles suggests a novel strategy for discriminatory targeting of parental genomes.

Schematic depicting a model for Cas9-mediated editing following injection of mII oocytes (mII). Limitted editing of maternal alleles during the gamete-to-embryo transition is inherent to the system, whereas limitted editing of paternal alleles in zygotes is because available targets have already been removed. Pb2, second polar body; pn, pronucleus.

Il2rg gene-targeted severe combined immunodeficiency pigs

Suzuki S, Iwamoto M, Saito Y, Fuchimoto D, Sembon S, Suzuki M, Mikawa S, Hashimoto M, Aoki Y, Najima Y, Takagi S, Suzuki N, Suzuki E, Kubo M, Mimuro J, Kashiwakura Y, Madoiwa S, Sakata Y, Perry AC, Ishikawa F, Onishi A.

Cell Stem Cell 10 (6), 753-8 (2012)

Abstract

A porcine model of severe combined immunodeficiency (SCID) promises to facilitate human cancer studies, the humanization of tissue for xenotransplantation, and the evaluation of stem cells for clinical therapy, but SCID pigs have not been described. We report here the generation and preliminary evaluation of a porcine SCID model. Fibroblasts containing a targeted disruption of the X-linked interleukin-2 receptor gamma chain gene, Il2rg, were used as donors to generate cloned pigs by serial nuclear transfer. Germline transmission of the Il2rg deletion produced healthy Il2rg(+/-) females, while Il2rg(-/Y) males were athymic and exhibited markedly impaired immunoglobulin and T and NK cell production, robustly recapitulating human SCID. Following allogeneic bone marrow transplantation, donor cells stably integrated in Il2rg(-/Y) heterozygotes and reconstituted the Il2rg(-/Y) lymphoid lineage. The SCID pigs described here represent a step toward the comprehensive evaluation of preclinical cellular regenerative strategies.

The SCID pigs are the latest of several sophisticated approaches to genome manipulation Prof. Onishi is taking to harness the biology of pigs in the development of human (and perhaps pig!) clinical strategies.