In vivo gene correction with targeted sequence substitution through microhomology-mediated end joining.

Genome editing technology using programmable nucleases has rapidly evolved in recent years. The primary mechanism to achieve precise integration of a transgene is mainly based on homology-directed repair (HDR). However, an HDR-based genome-editing approach is less efficient than non-homologous end-joining (NHEJ). Recently, a microhomology-mediated end-joining (MMEJ)-based transgene integration approach was developed, showing feasibility both in vitro and in vivo. We expanded this method to achieve targeted sequence substitution (TSS) of mutated sequences with normal sequences using double-guide RNAs (gRNAs), and a donor template flanking the microhomologies and target sequence of the gRNAs in vitro and in vivo. Our method could realize more efficient sequence substitution than the HDR-based method in vitro using a reporter cell line, and led to the survival of a hereditary tyrosinemia mouse model in vivo. The proposed MMEJ-based TSS approach could provide a novel therapeutic strategy, in addition to HDR, to achieve gene correction from a mutated sequence to a normal sequence.

DNA double-strand break-free CRISPR interference delays Huntington's disease progression in mice.

Huntington's disease (HD) is caused by a CAG repeat expansion in the huntingtin (HTT) gene. CRISPR-Cas9 nuclease causes double-strand breaks (DSBs) in the targeted DNA that induces toxicity, whereas CRISPR interference (CRISPRi) using dead Cas9 (dCas9) suppresses the target gene expression without DSBs. Delivery of dCas9-sgRNA targeting CAG repeat region does not damage the targeted DNA in HEK293T cells containing CAG repeats. When this study investigates whether CRISPRi can suppress mutant HTT (mHTT), CRISPRi results in reduced expression of mHTT with relative preservation of the wild-type HTT in human HD fibroblasts. Although both dCas9 and Cas9 treatments reduce mHTT by sgRNA targeting the CAG repeat region, CRISPRi delays behavioral deterioration and protects striatal neurons against cell death in HD mice. Collectively, CRISPRi can delay disease progression by suppressing mHtt, suggesting DNA DSB-free CRISPRi is a potential therapy for HD that can compensate for the shortcoming of CRISPR-Cas9 nuclease.

Application of prime editing to the correction of mutations and phenotypes in adult mice with liver and eye diseases.

The use of prime editing-a gene-editing technique that induces small genetic changes without the need for donor DNA and without causing double strand breaks-to correct pathogenic mutations and phenotypes needs to be tested in animal models of human genetic diseases. Here we report the use of prime editors 2 and 3, delivered by hydrodynamic injection, in mice with the genetic liver disease hereditary tyrosinemia, and of prime editor 2, delivered by an adeno-associated virus vector, in mice with the genetic eye disease Leber congenital amaurosis. For each pathogenic mutation, we identified an optimal prime-editing guide RNA by using cells transduced with lentiviral libraries of guide-RNA-encoding sequences paired with the corresponding target sequences. The prime editors precisely corrected the disease-causing mutations and led to the amelioration of the disease phenotypes in the mice, without detectable off-target edits. Prime editing should be tested further in more animal models of genetic diseases.

Sequence-specific prediction of the efficiencies of adenine and cytosine base editors.

Base editors, including adenine base editors (ABEs)1 and cytosine base editors (CBEs)2,3, are widely used to induce point mutations. However, determining whether a specific nucleotide in its genomic context can be edited requires time-consuming experiments. Furthermore, when the editable window contains multiple target nucleotides, various genotypic products can be generated. To develop computational tools to predict base-editing efficiency and outcome product frequencies, we first evaluated the efficiencies of an ABE and a CBE and the outcome product frequencies at 13,504 and 14,157 target sequences, respectively, in human cells. We found that there were only modest asymmetric correlations between the activities of the base editors and Cas9 at the same targets. Using deep-learning-based computational modeling, we built tools to predict the efficiencies and outcome frequencies of ABE- and CBE-directed editing at any target sequence, with Pearson correlations ranging from 0.50 to 0.95. These tools and results will facilitate modeling and therapeutic correction of genetic diseases by base editing.