Rapidly inducible Cas9 and DSB-ddPCR to probe editing kinetics

Abstract

To investigate the kinetics of Cas9-mediated double strand break generation and repair in vivo, we developed two new tools. The first, chemically inducible Cas9 (ciCas9), is a rapidly-activated, single-component Cas9 variant engineered using a novel domain replacement strategy. ciCas9 can be activated in a matter of minutes, and the level of ciCas9 specificity and activity can be tuned. The second tool, DSB-ddPCR, is a droplet digital PCR-based assay for double strand breaks. DSB-ddPCR is the first assay to demonstrate time-resolved, highly quantitative and targeted measurement of DSBs. Combining these tools facilitated an unprecedented exploration of the kinetics of Cas9-mediated DNA cleavage and repair. We find that sgRNAs targeting different sites generally produce cleavage within minutes and repair within an hour or two. However, we observe distinct kinetic profiles, even for proximal sites, suggesting that target sequence and chromatin state modulate cleavage and repair kinetics.

Suppression of unwanted CRISPR/Cas9 editing by co-administration of catalytically inactivating truncated guide RNAs

“…Other approaches for minimizing off-target editing are also imperfect, as they reduce on-target efficiency [6][7][8][9]21,22 , introduce new off-target sites 11,14,15 , limit the number of potential target sites 11,[14][15][16][17] , or demand difficult Cas9 engineering [18][19][20][21][22]46,47 . Moreover, many of these approaches are laborious to implement in experimental models where Cas9 or a variant thereof has already been stably integrated into the genome [6][7][8][9][16][17][18][19][20][21][22]46,47 . Finally, these existing methods are generally incompatible with each other, meaning they cannot be used in concert to minimize limitations and improve performance.…”

“…HEK-293T cells were treated according to previous methods 6 . Briefly, HEK-293T cells were plated in 12 well plates at 3.0 x 10 5 cells/well.…”

“…Genomic DNA isolation, sequencing, and analysis were performed as previously described 6 . Briefly, genomic DNA was isolated using the DNEasy Blood and Tissue Kit (Qiagen) according to the manufacturer's instructions except that the proteinase K digestion was conducted for 1 hr at 56°C.…”

“…This multipartite target recognition system is imperfect, and most sgRNAs direct significant cleavage and subsequent unwanted editing at off-target sites whose sequence is similar to the target site [2][3][4][5] . Numerous approaches to reduce off-target editing have been devised, yet are hampered by various limitations [6][7][8][9][10][11][12][13][14][15][16][17] . For example, SpCas9 variants with improved specificity have been engineered [18][19][20] .…”

Spatiotemporal Control of CRISPR/Cas9 Function in Cells and Zebrafish using Light-Activated Guide RNA

“…Aiming at higher genomic editing precision by limiting the window of CRISPR/Cas9 activity as well as probing of spatiotemporally controlled gene function, researchers have endeavored to broaden the CRISPR/Cas9 toolkit for conditional control of its activity. 14–16 Such efforts include small molecule-induced Cas9 protein activation 17–20 or reassembly, 21 light activation of caged Cas9, 22 reconstitution of single-chain Cas9 23 and split-Cas9, 24 as well as optically controlled recruitment of transcription factors to catalytically dead Cas9 (dCas9). 25–26 Amongst these developments, much effort was put into the regulation of the Cas9 protein to restore its function upon external stimulation.…”