Table 1. CRISPR/Cas-mediated single-gene targeting in hESC.
| Gene | CRISPR | Delivery method | Mutant alleles per clone |
|||
|---|---|---|---|---|---|---|
| 1 | 2 |
|||||
| Compound heterozygotes |
Homozygotes | Total biallelic mutant clones |
||||
| NGN3 | Cr5 | gRNA transfection | 2/48 (4.2%) | 2/48 | 10/48 | 12/48 (25%) |
| Cr6 | gRNA transfection | 1/36 (2.8%) | 1/36 | 5/36 | 6/36 (16.7%) | |
| GATA4 | Cr2 | gRNA transfection | 2/96 (2.1%) | 19/96 | 5/96 | 24/96 (25%) |
| GATA6 | Cr1 | gRNA transfection | 14/40 (35%) | 7/40 | 0/40 | 7/40 (17.5%) |
| Cr8 | gRNA transfection | 6/48 (12.5%) | 13/48 | 1/48 | 14/48 (29.2%) | |
| Cr8 | Plasmid electroporation | 2/96 (2.1%) | 0/96 | 0/96 | 0/96 (0%) | |
| Cr8 | Plasmid electroporation* | 17/288 (5.9%) | 0/288 | 0/288 | 0/288 (0%) | |
| TET1 | Cr2 | gRNA transfection | 4/48 (8.3%) | 10/48 | 3/48 | 13/48 (27.1%) |
| TET2 | Cr4 | gRNA transfection | 6/48 (12.5%) | 14/48 | 14/48 | 28/48 (58.3%) |
| TET3 | Cr4 | gRNA transfection | 4/48 (8.3%) | 24/48 | 8/48 | 32/48 (66.6%) |
In vitro transcribed gRNAs targeting NGN3, GATA4, GATA6, NGN3, TET1, TET2, or TET3 were transfected in doxycycline-induced iCas9 hESC, or electroporated as plasmids (GATA6 Cr8) in wild type HUES8 or HUES9 (*) hESCs. The number of lines containing each specific number of mutated alleles (1 or 2) is shown in relation to the total number of lines screened in each experiment.