Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery
- University of California, Berkeley, United States
- Howard Hughes Medical Institute, University of California, Berkeley, United States
- Lawrence Berkeley National Laboratory, United States
Figures
Figure 1 with 1 supplement
The effect of cell cycle synchronization on total editing and homology-directed repair frequencies in HEK293T cells.
(A) Experimental schematic of timed delivery of Cas9-guide RNA ribonucleoprotein (RNP) into human cells for genome editing. (B) Chemical inhibitors used to arrest cells at specific phases of cell …
Figure 1—figure supplement 1
FACS analysis reveals cell cycle blocks and the DNA content in the cells that are arrested at different phases of cell cycle.
Bivariate cell cycle, BrdU (S-phase), 7-AAD (DNA content) FACS analysis reveals cells are arrested at different phases of cell cycle. Chemical inhibitors were used to arrest cells at specific phases …
Figure 2 with 1 supplement
The enhancement of TE and HDR at the EMX1, DYRK1 and CXCR4 loci by nocodazole synchronization in HEK293T cells.
(A) The effect of nocodazole on the TE and HDR frequencies at EMX1 locus. HEK293T cells were synchronized at M phase with 200 ng/ml of nocodazole for 17 hr before nucleofection. To determine the …
Figure 2—figure supplement 1
On-target NHEJ and HDR and off-target cleavage analyses by deep sequencing.
(A) The genomic DNAs from Figure 2A,B experiments were analyzed for NHEJ and HDR frequencies, at the on-target and off-target sites, by deep sequencing. The TE frequency (indels/total reads) was …
Figure 3
Systematic investigation of DNA templates for efficient HDR at the EMX1 locus in HEK293T cells.
(A) Segment of human EMX1 exon 3 shows the 20 nt target sequence (highlighted in blue), the TGG PAM region (in red) and the Cas9 cleavage site at three bases upstream from PAM. Seven HDR templates …
Figure 4
The enhancement of TE and HDR frequencies at the EMX1 locus by cell cycle synchronization in human primary neonatal fibroblast and embryonic stem cells.
(A) Screening of cell cycle inhibitors for enhancement of TE and HDR frequencies in human primary neonatal fibroblast cells. For each inhibitor condition (color coded), two doses of Cas9 RNP, 30 and …
Additional files
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Supplementary file 1
PCR primers for target loci amplification and indexing for deep sequencing of on-target and off-target sites. Also included is deep sequencing data and analysis.
- https://doi.org/10.7554/eLife.04766.008
