Split-tracrRNA as an efficient tracrRNA system with an improved potential of scalability.

Due to the relatively long sequence, tracrRNAs are chemically less synthesizable than crRNAs, leading to limited scalability of RNA guides for CRISPR-Cas9 systems. To develop shortened versions of RNA guides with improved cost-effectiveness, we have developed a split-tracrRNA system by nicking the 67-mer tracrRNA (tracrRNA(67)). Cellular gene editing assays and in vitro DNA cleavage assays revealed that the position of the nick is critical for maintaining the activity of tracrRNA(67). TracrRNA(41 + 23), produced by nicking in stem loop 2, showed gene editing efficiency and specificity comparable to those of tracrRNA(67). Removal of the loop of stem loop 2 was further possible without compromising the efficiency and specificity when the stem duplex was stabilized via a high GC content. Binding assays and single-molecule experiments suggested that efficient split-tracrRNAs could be engineered as long as their binding affinity to Cas9 and their reaction kinetics are similar to those of tracrRNA(67).