FIG 6.

Proposed mechanism of crRNA biogenesis in artificial CRISPR mutants. Arrows indicate primary processing cleavage sites. (A) In the case of unmodified repeat sequence between artificial spacers 5 and 6, primary processing of pre-crRNA results in series of single repeat-spacer units, which are further trimmed at their 3′ ends during secondary processing. (B) Any modification into repeat sequence between artificial spacers 5 and 6 abolished cleavage within modified repeat sequence. This is evident by the lack of observable single repeat-spacer units for probes complementary to those spacers. Secondary processing, which is probably independent from the primary cleavage, separates spacer 5 crRNA from the spacer 6 fragment, which is degraded during the process. As a result, functional spacer 5 crRNA molecule is generated.