The most common methods used in constructing closed for CRISPR-Cas9 depend
on traditional means such as using Gateway reaction or specific type IIS
restriction enzymes and DNA ligation, based on several steps of polymerase
chain reaction. To ease the process, scientists created a system that can be
used to introduce sgRNA expression cassettes directly into plant binary
vectors in a single step.



When using the new system, one sgRNA expression cassette(s) is generated by
an optimized multiplex PCR, in which an overlapping PCR takes place. Thus,
two sgRNA expression cassettes were amplified in just one run of PCR. At the
same time, an LR or Golden gate reaction was placed with unpurified PCR
product and matching destination vector. This leads to the construction of
expression cloned in just 36 hours. The efficiency of the system was
confirmed through an Agrobacterium-mediated genetic transformation in rice.



[peerj.com]