Switchable genome editing via genetic code expansion

Toru Suzuki,Maki Asami,Louis Y P Luk,Anthony C F Perry,Sanjay G Patel,Yu-Hsuan Tsai

doi:10.1038/s41598-018-28178-3

Abstract

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.

Highlights

Many applications of genome editing by CRISPR-Cas[91–4] would benefit from being tightly controllable to minimize unintended, potentially harmful genome cleavage due to leaky nuclease activity
Because we were interested in modulating protein activity in a mouse model, we designed an eGFPN150B transgene to express each of the components required for ubiquitous, constitutive expression of the eGFPN150B system (PylRS, Pyl tRNA and eGFPN150B; Supplementary Figure S1A) and coinjected it with wild-type mouse B6D2F1 sperm into B6D2F1 metaphase II oocytes (Supplementary Figure S1B)
To enhance spatiotemporally regulated embryonic expression, we synthesized all RNA components of the BOC system in vitro and coinjected them into B6D2F1 metaphase II (mII) oocytes followed by exposure to different concentrations of BOC (Supplementary Figure S2A)

Summary

Introduction

Many applications of genome editing by CRISPR-Cas[91–4] would benefit from being tightly controllable to minimize unintended, potentially harmful genome cleavage due to leaky nuclease activity Such regulation would likely impact multiple contexts, including restricting genome editing to given anatomical sites in clinical somatic cell gene therapy[5,6] and gene drives[4,7]. Gene drives duplicate a segment of genomic DNA in vivo independently of selection and in principle work in any sexually reproducing species so that all offspring inherit the gene drive segment[7] Given their speed and efficiency, gene drives have the potential to accelerate the dissemination of beneficial genetics in insects, crops and animals; they might streamline the introduction of homozygous mutations to study recessive alleles, eliminate destructive invasive species and agricultural pests, or to improve livestock rapidly and cheaply (for example, to prevent disease transmission), eliminating the need for protracted breeding programs[4].

Methods

Results

Conclusion