Polynucleotide constructs and methods of gene editing using CPF1

Schaart, J.G.; Smulders, M.J.M.


Described is a repair process for genetic material which is Complementarity Directed End Joining (CDEJ). CDEJ essentially follows the same repair mechanism as alternative Non- Homologous End Joining (aNHEJ), except the 5' extension gives complementarity. A specifically designed excision construct is provided whereby after cutting with Cpf1 there is a joining of DNA via perfect matching complementary 5'-ends. Annealing and ligation generates a precise and predictable repair. Employing the viability of precise CDEJ repair systems, there is provided a completely removable marker selection system which can be removed by stable or (ideally) transient expression of CRISPR Cpf1 targeting the distal ends of the marker sequence. Molecular analysis of the gene edited products produced using the selection system show that neither the length of crRNA, nor the nature of the target sequence affected precision or efficiency