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. 2017 Feb 24;8(3):491–499. doi: 10.1016/j.stemcr.2017.01.021

Figure 4.

Figure 4

Simultaneous Dual Modification using CTS

(A) Dual modification was attempted on hB53 hiPS6 using components for targeting MTERF4:c.693delATA and CRYAB:c.358A>G. Following CTS, 50 clones were sequenced and, of those, 14 had knockin at CRYAB (depicted in the red circle) and 6 had knockin at MTERF4 (depicted in the blue circle). All 6 of the cells with MTERF4 knockin also had knockin at CRYAB. Given individual editing rates, the likelihood of co-occurrence assuming random distribution of events is 5%. We observed a disproportionate co-occurrence of dual modification with a FET < 0.001.

(B) Representative chromatograms of dual-targeted clones at each loci and WT sequence. Black arrows indicate variant of interest position. Silent, engineered blocking mutations that prevent re-targeting by Cas9 are indicated by gray arrows.

(C) Table including genotypes of individual clones at both (MTERF4 and CRYAB) loci. WT, unmodified; KI, knockin; Indel, insertion or deletion. See also Table S3.