Self-targeting CRISPR/Cas genome editing allows for the iterative modification of guide RNA (gRNA) sequences. This process continuously mutates a specific genetic locus, rendering the system useful as a directed evolution platform in the context of protein engineering and for phenotypic screening and selection.
Directed evolution is used in protein engineering to generate a library of functional variants from which to screen for desired traits. The process entails subjecting a gene to iterative rounds of mutagenesis and selection in order to mimic natural evolution. Approaches fordirected mutagenesis are often restricted to microorganisms (e.g., E. coli, S. cerevisiae) and are limited in terms of scope, control, and efficiency. Self-targeting CRISPR/Cas9 genome editing can be used in human cells to efficiently generate genetic diversity at a specific locus. The system can be conditionally regulated by placing the gRNA under inducible control (e.g., UV light, small molecule, etc.) or under the control of a developmentally timed promoter.
CRISPR/Cas genome editing systems are used to target and edit specific genomic sequences. They are comprised of two components: 1) a gRNA and 2) a Cas9 endonuclease. Once transcribed, the gRNA forms a complex with Cas9 and is recruited to a target DNA locus as determined by a 20 base-pair sequence in the gRNA termed the specificity determining sequence (SDS). Once bound, Cas9 induces a double-stranded break in the genomic target upstream of a protospacer adjacent motif (PAM). This PAM sequence is not present in the DNA sequence from which the gRNAs are transcribed and as such, gRNAs cannot target this sequence. By preventing self-targeting, the system ensures that the gRNA transcripts remain unaltered and thus continue to bind their target locus. However, introducing either a PAM motif into the gRNA or a PAM interacting (PI) domain into the Cas9 protein abolishes this self vs. non-self discrimination and allows the sequence from which the gRNA is transcribed to be self-targeted. In this system, the gRNA forms a complex with Cas9 and is directed to the sequence from which it was transcribed. Cas9 generates an insertion or a deletion in the DNA template, and the next round of transcription produces a gRNA with this mutation. This continues in an iterative manner, permitting continuous rounds of targeted evolution at a defined locus. The system can be further customized by using multiple gRNAs and/or by using inducible controls to generate conditional mutations.
with human cells
to conditional regulation
system; does not require constant intervention