A 20 μl reaction in 1xCas12a Nuclease Reaction Buffer containing 60 ng linearized plasmid, 10 ng crRNA, and 100 ng GenCRISPR™ Cas12a (Cpf1) Nuclease for 30 mins at 37°C results in a digestion efficiency of linearized plasmid higher than 90%, as determined by agarose gel electrophoresis.
GenCRISPR™ Cas12a (Cpf1) Nuclease
The clustered regularly interspaced short palindromic repeats, known as CRISPR systems are adaptive immune mechanisms commonly present in archaea and bacteria. The CRISPR systems enable the host to specifically target and cleave foreign nucleic acids thus targeting infectiousviruses and plasmids. Recently, a type V CRISPR system has been identified in several bacteria, the Cpf1 CRISPR from Prevotella and Francisella 1. In contrast to Cas9 systems, CRISPR/Cpf1 systems are smaller in size, do not require an additional trans-activating crRNA (tracrRNA), and allow for targeting of additional genomic regions by cleaveing the target DNA proceeded by a short T-rich protospacer-adjacent motif (PAM). On the other hand, the Cas9 system requires a G-rich PAM following the target DNA. Furthermore, Cas12a/Cpf1 introduces a staggered DNA double stranded break with a 4 or 5-nt 5’ overhang. Recombinant Acidaminococcus sp. BV3L6 Cas12a (cpf1) nuclease is expressed in E. coli and purified. The nuclease contains nuclear localization sequence (NLS) at the C-terminus and 6× His-tag at the C-terminus.
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