Methods and compositions for cloning into large vectors

A method comprising:

• synthesizing an sgRNA having a crRNA sequence operatively linked to a tracRNA sequence, where the crRNA sequence is complementary to a target sequence in a substrate vector;
• incubating the sgRNA with an amount of a Cas9 endonuclease and an amount of the substrate vector to produce a linearized cleaved substrate vector having a cleavage point;
• and
• incubating an amount of linearized cleaved substrate vector with an amount of an insert polynucleotide and an amount of at least one of the following:
• a DNA ligase, a DNA exonuclease, a DNA polymerase, or a combination thereof,
• where the insert polynucleotide comprises a 5′ end sequence that is complementary with a first polynucleotide sequence in the substrate vector and a 3′ end sequence that is complementary with a second polynucleotide sequence in the substrate vector, and
• where the first polynucleotide sequence and the second polynucleotide sequence are on opposite sides of the cleavage point.