Screening experiments in E coli
For the guide RNA activity screening experiment, competent cells were
prepared as described above (Electrocompetent cell preparation and
transformation). We collected clones per sample to achieve a proper
(>60×) coverage over the design library.
Because the DSB lethality caused by Cpf1 cleavage made the direct
calculation of transformation efficiency impossible, the respective
control group without inducer was used to evaluate the transformation
efficiency for Cpf1 transformed with gene-targeting crRNA library.
Two screening experiments (SE1 and SE2) were conducted parallelly. In
each experiment, this library was delivered to the competent cells
expressing Cpf1 driven by an inducible promoter to evaluated guide RNA
activity. After the E.coli cells were cultured with/without
inducer, the remaining cells were collected as select/control. In SE1,
Cpf1 was driven by a strong promoter (tet promoter) while in SE2 by a
weak promoter. Through a synthetical consideration of two groups of
results, we could eliminate the influence of the known leakage
expression of tet promoter.