Site specific crrna

In vitro DNA cleavage reaction mixtures consisted of 500 ng (0.014 µM) of pHSG299 (Takara Bio Company, Shiga, Japan) plasmid DNA and 10 pmol RNP in a reaction buffer (100 mM NaCl, 20 mM Tris-HCl, 10 mM MgCl₂ and 100 µg/ml BSA) at a final volume of 20 µl. RNP complexes consisted of purified AsCas12a or SpCas9 protein (Integrated DNA Technologies, Coralville, Iowa) and site-specific crRNA (Integrated DNA Technologies, Coralville, Iowa). Each reaction was incubated for 15 min at 37 °C, after which DNA was isolated from reaction mixtures and recovered using Select-a-Size DNA Clean & Concentrator (Zymo Research, Irvine, CA). Secondary in vitro HDR reactions included DNA recovered from the initial cleavage reaction, 100 pmol of single-stranded donor DNA (Integrated DNA Technologies, Coralville, Iowa) 1364-S 5′-GACCTGCAGGCATGCAAGCTTGGCACTGGCCGTCGGCGGCCGCTTTTACAACGTCGTGACTGGGAAAACC-3′ or 1364-NS 5′-GGTTTTCCCAGTCACGACGTTGTAAAAGCGGCCGCCGACGGCCAGTGCCAAGCTTGCATGCCTGCAGGTC-3′ and 175 µg of cell-free extract supplemented with 400 cohesive end units of Quick T4 Ligase (New England Biolabs, Ipswich, MA) in a reaction buffer (20 mM TRIS, 15 mM MgCl₂, 0.4 mM DTT and 1.0 mM ATP) at a final volume of 25 µl. Each reaction was then incubated for 15 min at 37 °C. Modified plasmid DNA from the final reaction mixture was then isolated and purified during spin column recovery.

In vitro HDI reactions, to generate NotI-MT plasmids, and HDC reactions, to correct the NotI-MT plasmids, both underwent cleavage reactions consisting of 500 ng (0.014 µM) of pHSG299 (Takara Bio Company, Shiga, Japan) or NotI-MT plasmid DNA and 10 pmol RNP in a reaction buffer (100 mM NaCl, 20 mM Tris–HCl, 10 mM MgCl2 and 100 µg/ml BSA) at a final volume of 20 µl. RNP complexes consisted of purified AsCas12a (AsCpf1) protein (Integrated DNA Technologies, Coralville, Iowa) and site-specific crRNA (Integrated DNA Technologies, Coralville, Iowa). Each reaction was incubated for 15 min at 37 °C, after which DNA was isolated from reaction mixtures and recovered using DNA Clean & Concentrator (Zymo Research, Irvine, CA). Secondary in vitro re-circularization reactions included DNA recovered from the initial cleavage reactions, 175 µg of cell-free extract supplemented with 400 cohesive end units of Quick T4 Ligase (New England Biolabs, Ipswich, MA), and 100 pmol of single-stranded donor DNA, integration template for HDI and correction template for HDC (Fig. 1), in a reaction buffer (20 mM TRIS, 15 mM MgCl₂, 0.4 mM DTT and 1.0 mM ATP) at a final volume of 25 µl. Each reaction was then incubated for 15 min at 37 °C. Modified plasmid DNA from the final reaction mixture was then isolated and purified during spin column recovery.