Generation of Mouse Model of Hemophilia A by Introducing Novel Mutations, Using CRISPR/Nickase Gene Targeting System

Document Type : Short Communication


1 Department of Animal Biotechnology, Institute Agricultural Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran

2 Department of Molecular Medicine, Institute of Medical Genetics, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran

3 Department of Pharmaceutics, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran


Developing mouse models of hemophilia A has been shown to facilitate in vivo studies to explore the probable
mechanism(s) underlying the disease and to examine the efficiency of the relevant potential therapeutics. This study
aimed to knockout (KO) the coagulation factor viii (fviii) gene in NMRI mice, using CRISPR/Cas9 (D10A/nickase) system,
to generate a mouse model of hemophilia A. Two single guide RNAs (sgRNAs), designed from two distinct regions on
NMRI mouse FVIII (mFVIII) exon 3, were designed and inserted in the pX335 vector, expressing both sgRNAs and
nickase. The recombinant construct was delivered into mouse zygotes and implanted into the pseudopregnant female
mice’s uterus. Mutant mice were identified by genotyping, genomic sequencing, and mFVIII activity assessment. Two
separate lines of hemophilia A were obtained through interbreeding the offspring of the female mice receiving potential
CRISPR-Cas9-edited zygotes. Genomic DNA analysis revealed disruptions of the mfviii gene reading frame through
a 22-bp deletion and a 23-bp insertion in two separate founder mice. The founder mice showed all the clinical signs
of hemophilia A including; excessive bleeding after injuries, and spontaneous bleeding in joints and other organs.
Coagulation test data showed that mFVIII coagulation activity was significantly diminished in the mFVIII knockout
(FVIIIKO) mice compared to normal mice. The CRISPR/nickase system was successfully applied to generate mouse
lines with the knockout fviii gene. The two novel FVIIIKO mice demonstrated all clinical symptoms of hemophilia A, which
could be successfully inherited. Therefore, both of the developed FVIIIKO mouse lines are eligible for being considered
as proper mouse models of hemophilia A for in vivo therapeutic studies.


Main Subjects

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