The giant sarcomeric protein titin has numerous important roles in cardiomyocytes, including providing passive tension and facilitating myofibril formation. An internal promoter has recently been identified in the titin gene (TTN) that is predicted to regulate expression of a previously unstudied isoform, Cronos. To study the role of Cronos titin in human hearts, we genetically engineered human induced pluripotent stem cells (hiPSCs) using the CRISPR/Cas9 system to introduce homozygous frameshift mutations in the TTN gene. TTN was mutated upstream of the Cronos start site in Exon 2 (Ex2 KO hiPSCs) to prevent translation of full-length titin but leave the Cronos isoform intact, and downstream of the Cronos start site in Exon 326 to prevent translation of both full-length and Cronos titin (Ex326 KO hiPSCs). Ex2 KO hiPSC-derived cardiomyocytes (hiPSC-CMs) visibly contract, and immunofluorescence studies indicate the formation of short, dispersed myofibrils. These myofibrils stain positively for the C-terminal MIR and M8-M10 regions of titin but not the N-terminal Z1Z2 domain, consistent with Cronos titin being the only isoform expressed in these cells. Additionally, twitch forces of Ex2 KO hiPSC-CMs in multicellular engineered heart tissues (EHTs) were ∼10% of isogenic wildtype controls (p<0.01), and single cell forces were reduced by ∼20% compared to wildtype (p=0.03). Interestingly, Ex326 KO hiPSC-CMs do not visibly beat, form sarcomeres, or produce measurable twitches in EHTs, but they do exhibit regular calcium transients. Based on these data, we conclude that Ex2 KO hiPSC-CMs express predominantly Cronos titin, which is sufficient for sarcomere formation but not normal function. Ongoing studies will investigate the role of Cronos titin during normal human heart development and maturation. This work was supported by NIH/NIBIB T32EB1650.