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Technology

The current invention relies on RNA-guided endonuclease such as Cpf1 to create a sticky ended DNA break at desired sites. This is performed simply by incubating Cpf1 protein, guide RNAs, and a dsDNA template together. This results in 4-5 nucleotide overhangs, which can be used to assemble and ligate DNA fragments. This reaction is efficient in ligase buffer, allowing for “one-pot” DNA assembly with ligation happening in the same reaction mixture. A variety of engineered Cpf1 proteins are available with different protospacer adjacent motif (PAM) requirements able to be targeted by the endonuclease. Since guide RNAs can be designed against almost any sequence, this invention removes many of the sequence-based constraints of current technologies.