Li Shi-Yuan, Zhao Guo-Ping, Wang Jin
Institute of Plant Physiology and Ecology, Shanghai Insititutes for Biological Sciences, Chinese Academy of Sciences.
Institute of Plant Physiology and Ecology, Shanghai Insititutes for Biological Sciences, Chinese Academy of Sciences;
J Vis Exp. 2017 Jun 15(124):55775. doi: 10.3791/55775.
CRISPR-associated protein Cpf1 cleaves double-stranded DNA under the guidance of CRISPR RNA (crRNA), generating sticky ends. Because of this characteristic, Cpf1 has been used for the establishment of a DNA assembly standard called C-Brick, which has the advantage of long recognition sites and short scars. On a standard C-Brick vector, there are four Cpf1 recognition sites - the prefix (T1 and T2 sites) and the suffix (T3 and T4 sites) - flanking biological DNA parts. The cleavage of T2 and T3 sites produces complementary sticky ends, which allow for the assembly of DNA parts with T2 and T3 sites. Meanwhile, a short "GGATCC" scar is generated between parts after assembly. As the newly formed plasmid once again contains the four Cpf1 cleavage sites, the method allows for the iterative assembly of DNA parts, which is similar to those of BioBrick and BglBrick standards. A procedure outlining the use of the C-Brick standard to assemble DNA parts is described here. The C-Brick standard can be widely used by scientists, graduate and undergraduate students, and even amateurs.
CRISPR相关蛋白Cpf1在CRISPR RNA（crRNA）的引导下切割双链DNA，产生粘性末端。由于这一特性，Cpf1已被用于建立一种名为C-Brick的DNA组装标准，该标准具有识别位点长和疤痕短的优点。在标准的C-Brick载体上，有四个Cpf1识别位点——前缀（T1和T2位点）和后缀（T3和T4位点）——位于生物DNA片段两侧。T2和T3位点的切割产生互补的粘性末端，这使得具有T2和T3位点的DNA片段能够组装在一起。同时，组装后片段之间会产生一个短的“GGATCC”疤痕。由于新形成的质粒再次包含四个Cpf1切割位点，该方法允许DNA片段进行迭代组装，这与BioBrick和BglBrick标准类似。这里描述了一个概述使用C-Brick标准组装DNA片段的程序。C-Brick标准可被科学家、研究生和本科生甚至业余爱好者广泛使用。
