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CRISPR Researchers Develop First In Vivo Editing System Targeting Non-Coding DNA

NEW YORK – An international team of researchers led by the Salk Institute for Biological Studies' Juan Carlos Izpisua Belmonte has developed the first in vivo gene editing system that targets non-coding DNA in multiple tissue types.

As the researchers recently reported in the Nature journal Cell Research, a big challenge in gene editing research is the creation of a universal editing tool that will work in cell types that are both in dividing and non-dividing states. Their new method — called SATI (intercellular linearized Single homology Arm donor mediated intron-Targeting Integration) — enables the targeting of a broad range of mutations and cell types by inserting a minigene at an intron of the target gene locus, using an intracellularly linearized single homology arm donor. 

The team had previously developed a method called HITI — homology-independent targeted integration — which allowed for efficient targeted knock-in in both dividing and non-dividing cells in vitro and in vivo. HITI relied on non-homologous end joining (NHEJ) rather than homology-directed repair (HDR). The researchers were able to use HITI to integrate transgene cassettes in many organs, including non-dividing tissues, such as the brain. They used this system to restore visual function in a rat model of retinitis pigmentosa by targeted insertion of a functional copy of exon 2 of the Mertk gene to correct the gene's loss-of-function.

However, they noted, HITI had some limitations. For example, it can't be used to repair genetic point and frameshift mutations because it can't remove pre-existing mutations.

In order to improve on the HITI system, the researchers developed an NHEJ- and HDR-mediated targeted gene knock-in method that requires a DNA double-strand break induction site within a single stretch of homologous sequence on the donor. This new system, SATI, allowed for DNA knock-in via single homology arm-mediated HDR or homology-independent NHEJ-based HITI. They said that this improvement enabled the targeting of a broad range of mutations and cell types.

To evaluate the effectiveness of this new approach, the researchers targeted the Tubb3 gene in non-dividing cultured mouse primary neurons using a series of donor DNAs, gRNAs, and Streptococcus pyogenes Cas9 (SpCas9). They found that the intronic gene knock-in approach worked in non-dividing neurons.

Further experiments showed that non-canonical HDR occurred in neurons when a single-homology arm donor cut at least either the donor or chromosomal target sequence. Cutting both the donor and chromosomal target significantly increased knock-in efficiency.

The team then used SATI to correct a dominant mutation in exon 11 of the LMNA gene using a mouse model of progeria. This mutation results in the production of an abnormal form of the lamin A protein progerin, whose accumulation causes pathological changes in multiple tissues.

To correct the mutation, the researchers constructed a gene editing complex that contained a SATI-mediated gene-correction donor containing one 1.9-kb homology arm sandwiched by the intron 10 gRNA target sequence.

They observed gene-corrected events, and found that both one-armed HDR (oaHDR) and HITI were evident in the gene-corrected cells, suggesting that SATI-mediated gene correction had been achieved for the dominant point mutation causing progeria. Further, the researchers noted that the oaHDR-mediated integration for the SATI donor was predominant in these cell types.

To test SATI's ability to correct a dominant mutation in vivo, the researchers then systemically delivered an adeno-associated virus (AAV) expressing Cas9 along with AAV-Progeria-SATI into neonatal Lmna G609G/G609G progeria mice. When they performed genomic PCR and Sanger sequence analyses at day 100, they found that SATI-mediated targeted gene knock-in occurred in several tissues, including the liver, heart, muscle, kidney, and aorta, though the efficiency varied.

"We estimated that the percentage of gene correction was 2.07 percent in the liver and 0.14 percent in the heart," the authors wrote. "Moreover, oaHDR events were observed in liver and heart analyses by paired-end sequencing after in vivo systemic SATI treatment. Although this number may seem low, it is important to note that the gene-corrected cells are still present in some organs even 100 days after treatment and that correction efficiency was sufficient to elicit SATI-mediated phenotypic rescue in several tissues and organs."

Progeria mice typically exhibit progressive weight loss and shortened lifespan, but these phenotypes were delayed by SATI treatment, the investigators added. The mice exhibited a slowdown of progressive weight loss and their median survival time was significantly extended by 1.45-fold. Further, three-month old progeria mice typically exhibit age-associated pathological changes in multiple organs, including skin, spleen, and kidneys, but these aging phenotypes were diminished in 17-week-old progeria mice that received the SATI treatment.

"The unique vector structure for SATI has a bipotential capacity to achieve efficient gene knock-in by choosing the predominant DSB repair machinery (i.e. non-canonical HDR mediated by single homology arm or NHEJ) in the target cell," the authors wrote. "Regarding applicability, SATI is a versatile in vivo genome-editing method that can target a broad range of mutations and cell types."

They further added that SATI could be used to advance both basic and translational neuroscience research — for example, by inserting optogenetic activators downstream of a relevant genetic locus to gain precise cell type-specific control of neuronal activity.

"SATI-mediated genome editing in the adult mouse brain and muscle in vivo brings about the possibility to generate knock-in reporters to trace cell lineages in non-dividing tissues other species," the researchers concluded. "This would be particularly useful in animal models where transgenic tools are limited (e.g., non-human primates). … Once better understood, this advanced gene-repair approach may prove instrumental in developing effective strategies for in vivo target-gene replacement of a broad range of mutation types, including dominant mutations, as well as devastating genetic multi-organ and systemic pathologies."