The study investigates the widespread off-target transcript silencing mediated by siRNAs, which occurs due to sequence complementarity with the seed region of the siRNA. This off-target silencing is sequence-dependent and independent of the delivery method, similar to the regulation by microRNAs (miRNAs). The seed region, particularly positions 2-7, plays a crucial role in determining which transcripts are silenced. Base mismatches within the seed region can reduce the number of off-target transcripts but generate new sets of silenced transcripts complementary to the mismatched seed sequence. The study also demonstrates that off-target transcript silencing is accompanied by loss of the corresponding protein, and this silencing is dose-dependent. Additionally, shRNAs, another form of RNAi, exhibit similar off-target transcript regulation. The findings suggest that the seed region is a key determinant of siRNA and shRNA-mediated off-target transcript silencing, and that this process is a fundamental aspect of RNAi biology.The study investigates the widespread off-target transcript silencing mediated by siRNAs, which occurs due to sequence complementarity with the seed region of the siRNA. This off-target silencing is sequence-dependent and independent of the delivery method, similar to the regulation by microRNAs (miRNAs). The seed region, particularly positions 2-7, plays a crucial role in determining which transcripts are silenced. Base mismatches within the seed region can reduce the number of off-target transcripts but generate new sets of silenced transcripts complementary to the mismatched seed sequence. The study also demonstrates that off-target transcript silencing is accompanied by loss of the corresponding protein, and this silencing is dose-dependent. Additionally, shRNAs, another form of RNAi, exhibit similar off-target transcript regulation. The findings suggest that the seed region is a key determinant of siRNA and shRNA-mediated off-target transcript silencing, and that this process is a fundamental aspect of RNAi biology.