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TE buffer was used to adjust to final concentrations of 18.1 M for the sgRNA and 2.5 M for Cas9

Under physiological conditions, their generation is controlled by the antioxidant system, which consists of enzymes such as superoxide dismutase (SOD), catalase, and glutathione peroxidases (GPXs) and their antioxidant substrates (such as glutathione and -tocopherol).[] Generated in abnormally high levels, ROS have destructive effects which can affect lipids, proteins, and nucleic acids by causing fragmentation products and cross-linked metabolism products[] that can lead to lipid peroxides formation, enzymatic dysfunction, and DNA strand break products.[] These destructive effects can be removed by antioxidant balance, which acts like free radical scavengers or cellular oxidation inhibitors,[] which can be categorized in plasma and intracellular antioxidant mechanisms.[] It has also been shown that oxidative stress plays an important role in gastrointestinal diseases

Alternatively, the final chimeric nucleic acid molecule may be generated by assembling all the designated building blocks at once in one step

Still, some protocols include breaks every 1216 weeks to prevent plateauing and maintain long-term responsiveness