TABLE 1:
Summary of KO cell lines established by our knock-in systems.
| Gene | gRNA | KI system | Genotyping | KO clones (mono-KI: bi-KI) | KO clones/genotyping (%) | Independent KO (mono-KI: bi-KI) | Independent KO/genotyping (%) |
|---|---|---|---|---|---|---|---|
| IFT88 | #1 | Version 1 | 15 | 6 (5:1) | 40.0 | 4 (3:1) | 26.7 |
| IFT20 | #1 | Version 1 | 6 | 1 (1:0) | 16.7 | 1 (1:0) | 16.7 |
| IFT20 | #2 | Version 1 | 6 | 3 (3:0) | 50.0 | 3 (3:0) | 50.0 |
| IFT56 | #1 | Version 1 | 17 | 5 (5:0) | 29.4 | 3 (3:0) | 17.6 |
| IFT56 | #2 | Version 1 | 14 | 3 (3:0) | 21.4 | 2 (2:0) | 14.3 |
| IFT139 | #2 | Version 1 | 9 | 8 (7:1) | 88.9 | 3 (2:1) | 33.3 |
| IFT144 | #2 | Version 1 | 4 | 3 (3:0) | 75.0 | 3 (3:0) | 75.0 |
| ARL13B | #1 | Version 1 | 7 | 5 (5:0) | 71.4 | 5 (5:0) | 71.4 |
| ARL13B | #2 | Version 1 | 6 | 4 (4:0) | 66.7 | 4 (4:0) | 66.7 |
| IFT88 | #1 | Version 2 | 9 | 5 (4:1) | 55.6 | 3 (2:1) | 33.3 |
| IFT22 | #2 | Version 2 | 12 | 10 (10:0) | 83.3 | 3 (3:0) | 25.0 |
| IFT27 | #2 | Version 2 | 4 | 2 (2:0) | 50.0 | 1 (1:0) | 25.0 |
RPE1 cells were transfected with an expression vector for Cas9 and sgRNA together with the donor vector. After drug selection, single colonies expressing nuclear tBFP were isolated. PCR and Sanger sequencing were performed on genomic DNA of the isolated clones. Genotyping column shows the number of clones checked by genomic PCR and Sanger sequencing. KO clones column shows the number of biallelic KO clones confirmed by Sanger sequencing. Numbers enclosed in parentheses indicate the numbers of monoallelic and biallelic knock-in clones. In monoallelic knock-in clones, small indels on another allele causing gene disruption were confirmed by Sanger sequencing. Independent KO column shows the number of KO clones each having different genotypes.