Efficient CRISPR/HDR-mediated knock-in system and method of use

A method of genome editing to knock in a large DNA fragment into a target gene of a host in the absence of a non-homologous end joining (NHEJ) inhibitor, comprising:

• providing at least one clustered regularly interspaced short palindromic repeat (CRISPR) system comprising
• a first plasmid nucleic acid vector comprising
• a DNA encoding a single guide RNA (sgRNA) wherein the single guide RNA comprising
• a CRISPR-related (crRNA) segment complementary to a target sequence in the target gene of the host;
• and
• a trans-activating CRISPR-related sequence (tracrRNA) required for function of a CRISPR endonuclease;
• and
• a DNA encoding the CRISPR endonuclease wherein the CRISPR endonuclease is Cas9;
• a second plasmid nucleic acid vector comprising
• a donor DNA sequence comprising
• a DNA fragment of at least 4 kb in length wherein the DNA fragment replaces the target sequence in the target gene of the host or is inserted into a target site in the target gene of the host;
• and
• two homologous arms with each arm flanking opposing sides of the DNA fragment wherein each of the homologous arms is between 3.4 kb and 9 kb in size;
• inserting the at least one CRISPR system into a cell wherein the sgRNA guides the CRISPR endonuclease to a cleavage site in the target DNA sequence of the target gene of the host and the CRISPR endonuclease introduces a double stranded break in the target DNA sequence in the target gene of the host;
• and
• allowing the cell to undergo homology-directed-repair using the two homologous arms flanking the DNA fragment to knock in the DNA fragment at the cleavage site of the target DNA sequence in the target gene of the host in the absence of a non-homologous end joining (NHEJ) inhibitor.