A method for determining the cutting efficiency of the CRISPR/Cas system in birch and poplar

Abstract

Determination of Cas9 cutting efficiency to the target sites is important for genome editing. However, this determination can only be made via an in vitro method, as the purification of Cas protein and synthesis of gRNA are necessary. Here, we developed an in vivo method, called transient CRISPR/Cas editing in plants (TCEP) to determine Cas9 cutting efficiency. The CRISPR/Cas vector for plant transformation mediated by Agrobacterium tumefaciens was constructed as normal. Using the transient transformation method we built, the Cas9 protein and gRNA were transiently expressed and formed a complex to cut its target sites, resulting in dynamic DNA breakage. The broken DNA was quantified using qPCR to measure the efficiencies of Cas9 cutting. We studied the Cas9 cutting efficiencies to different target sites in Betula platyphylla and Populus davidiana×P. bolleana plants using TCEP and an in vitro method. The results of TCEP were consistent with those of the in vitro method, suggesting that the TCEP method is reliable in determining cutting efficiency. Additionally, using the TCEP method, we showed that both heat and sonication treatment significantly improved CRISPR/Cas efficiency. Therefore, the TCEP method has broad application value and can not only be used to analyze the CRISPR/Cas efficiency but also to determine the factors involved in Cas9 cutting.