The combination of Cas9, guide RNA and repair template DNA can induce precise gene editing and the correction of genetic diseases in adult mammals. However, clinical implementation of this technology requires safe and effective delivery of all of these components into the nuclei of the target tissue. Here, we combine lipid nanoparticle–mediated delivery of Cas9 mRNA with adeno-associated viruses encoding a sgRNA and a repair template to induce repair of a disease gene in adult animals. We applied our delivery strategy to a mouse model of human hereditary tyrosinemia and show that the treatment generated fumarylacetoacetate hydrolase (Fah)-positive hepatocytes by correcting the causative Fah-splicing mutation. Treatment rescued disease symptoms such as weight loss and liver damage. The efficiency of correction was >6% of hepatocytes after a single application, suggesting potential utility of Cas9-based therapeutic genome editing for a range of diseases.

Efficient genome editing with Cas9–sgRNA in vivo has required the use of viral delivery systems, which have limitations for clinical applications. Translational efforts to develop other RNA therapeutics have shown that judicious chemical modification of RNAs can improve therapeutic efficacy by reducing susceptibility to nuclease degradation. Guided by the structure of the Cas9–sgRNA complex, we identify regions of sgRNA that can be modified while maintaining or enhancing genome-editing activity, and we develop an optimal set of chemical modifications for in vivo applications. Using lipid nanoparticle formulations of these enhanced sgRNAs (e-sgRNA) and mRNA encoding Cas9, we show that a single intravenous injection into mice induces >80% editing of Pcsk9 in the liver. Serum Pcsk9 is reduced to undetectable levels, and cholesterol levels are significantly lowered about 35% to 40% in animals. This strategy may enable non-viral, Cas9-based genome editing in the liver in clinical settings.

CRISPR–Cas9 is a versatile RNA-guided genome editing tool. Here we demonstrate that partial replacement of RNA nucleotides with DNA nucleotides in CRISPR RNA (crRNA) enables efficient gene editing in human cells. This strategy of partial DNA replacement retains on-target activity when used with both crRNA and sgRNA, as well as with multiple guide sequences. Partial DNA replacement also works for crRNA of Cpf1, another CRISPR system. We find that partial DNA replacement in the guide sequence significantly reduces off-target genome editing through focused analysis of off-target cleavage, measurement of mismatch tolerance and genome-wide profiling of off-target sites. Using the structure of the Cas9–sgRNA complex as a guide, the majority of the 3′ end of crRNA can be replaced with DNA nucleotide, and the 5 - and 3′-DNA-replaced crRNA enables efficient genome editing. Cas9 guided by a DNA–RNA chimera may provide a generalized strategy to reduce both the cost and the off-target genome editing in human cells.

Summary CAG/CTG trinucleotide repeats are unstable, fragile sequences that strongly position nucleosomes, but little is known about chromatin modifications required to prevent genomic instability at these or other structure-forming sequences. We discovered that regulated histone H4 acetylation is required to maintain CAG repeat stability and promote gap-induced sister chromatid recombination. CAG expansions in the absence of H4 HATs NuA4 and Hat1 and HDACs Sir2, Hos2, Hst1 depended on Rad52, Rad57, and Rad5, and were therefore arising through homology-mediated post-replication repair (PRR) events. H4K12 and H4K16 acetylation were required to prevent Rad5-dependent CAG repeat expansions, and H4K16 acetylation was enriched at CAG repeats during S-phase. Genetic experiments placed the RSC chromatin remodeler in the same PRR pathway, and Rsc2 recruitment was coincident with H4K16 acetylation. Here we have utilized a repetitive DNA sequence that induces endogenous DNA damage to identify histone modifications that regulate recombination efficiency and fidelity during post-replication gap-repair.

Globally, approximately one out of three people become infected with the obligate intracellular parasite Toxoplasma. These infections are typically asymptomatic but can cause severe disease and mortality in immunocompromised individuals. Infections can also be passed on from mother to fetus during pregnancy, potentially causing miscarriage or stillbirth.