Interrogation Of Crispr Dynamics With Fluorescent Single Guide Rnas In Live Cells

Although the CRISPR-Cas9 system has been widely used for genome editing, little is known about its dynamics in live cells such as the kinetics of sgRNA:Cas9 assembly and DNA recognition. By stably expressing both fluorescent dCas9 and sgRNAs that specifically targets on chromosome 3 in live human cells we monitored the assembly of sgRNA:dCas9 complexes and tracked its dynamics of interactions with the target gene. In the absence of dCas9 expression, the sgRNA was so unstable as to not be detectable. In the presence of dCas9 the sgRNA was primarily localized in the nucleus and displayed a half-life within 15 minutes estimated by actinomycin chase experiments. These results are in agreement with sgRNA stability and sgRNA:dCas9 complex formation being the limiting step for DNA targeting. We next monitored the dynamics of on-target dCas9:sgRNA complexes by fluorescence recovery after photobleaching (FRAP), employing different lengths of sgRNA seed sequence and found that the recovery times of dCas9:sgRNA complexes on the target were about 6, 12 and 60 min. for a 7mer, 8mer and 11mer, respectively. This suggests that the complexes having shorter sgRNA seeds exchange with the target at a higher rate, implying a weaker affinity. We also compared the PAM sequences AG vs. GG, with the same length of the sgRNA seed sequence. The fluorescence recovery times were the same (u003e 60 min.), indicating similar affinities to the target and that the reported lower cleavage efficiency of AG PAM is possibly due to its inefficient targeting specificity.Our use of dCas9 in these in vivo studies has allowed us to deconvolve the overall CRISPR cycle to extract the DNA binding step and further to facilitate the optimization of Cas9-based gene editing.