CRISPR/Cas9 is a robust genome engineering tool due to its high editing efficiency and simple design principle. Taking advantage of DNA repair mechanism, namely homology directed recombination (HDR) with donor templates provided, this technology has a great promise to correct genetic defects at the molecular level for therapeutic purposes.

Haemophilia B (HB) is an inherited disease caused by multiple loss-of-function mutations in the factor IX (FIX) gene. CRISPR mediated genome editing provides a potential treatment for HB, yet hardly any attempts have achieved efficacy in vivo so far. In this context, we developed a therapeutic strategy with CRISPR/SaCas9 targeted genome editing in combination with HDR events, and demonstrate efficient corrective replacement of a mutant fragment in the murine FIX (m FIX) genome in vivo .

For the study, a mouse model of HB was employed, in which the part between 1kb upstream of the transcription start site (TSS) and intron 3 of the m FIX gene was replaced by an artificial mutant fragment. Through AAV vector delivery, CRISPR/SaCas9 targeted at specific sites around the mutant fragment in the m FIX gene and generated double-strand breaks (DSB) efficiently both in vitro and in vivo . Among all CRISPR candidates, a distinctive CRISPR efficiently inducing DSB at the site within intron 4 was chosen. Subsequently a comprehensive design of targeting vectors was performed. The replacement constructs contain a common insert of m FIX exons 1 to 4 coding sequence under the control of a synthetic liver-specific regulatory element to re-gain the full-length mRNA transcription. Multiple targeting constructs shared the 3' arm adjacent to the targeting site, whilst differed in the 5' arms either right adjacent to the targeting site, or containing the 5' UTR region bypassing the mutant fragment. In presence of CRISPR/SaCas9 genome editing, both targeting vectors mediated correction of the defective m FIX gene. As a consequence, the plasma levels of FIX coagulant activity were covered to over 15-20% of normal murine plasma level, whereas the mice receiving targeting vectors harboring the 5' arm bypassing the mutant fragment achieved higher activity compared to other groups. In addition, incorporation of miRNA622, which supposed to suppress non-homologous end joining and facilitate HR-mediated DSB repair, into SaCas9-expressing vector further improved the plasma FIX activity levels in the HB mice.

In total, our results demonstrate that CRISPR/SaCas9 mediated genome targeting combining homology-directed recombination has the capability to correct genetic defects in the m FIX genome in vivo to re-gain gene expression and recover haemostasis in the mouse model of HB. It provides a cure solution in treatment of inherited diseases including haemophilia B as well as the rationale for clinic translation of the technology.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.