Efficient target cleavage by Type V Cas12a effectors programmed with split CRISPR RNA.

Area of Science:

• Molecular Biology
• Biochemistry
• Gene Editing Technologies

Background:

• CRISPR RNAs (crRNAs) are essential for guiding Type V Cas12a nucleases to target DNA.
• crRNAs typically comprise a constant scaffold and a variable spacer region.

Purpose of the Study:

• To investigate the functional impact of removing the scaffold moiety from crRNAs.
• To explore the efficacy of split crRNAs (separate scaffold and spacer) in directing Cas12a activity.
• To determine if split crRNAs are compatible with different Cas12a orthologs and cellular contexts.

Main Methods:

• In vitro assays using Acidaminococcus sp. Cas12a (AsCas12a) with truncated and split crRNAs.
• Testing split crRNA activity in human cell lysates.
• Evaluating collateral ssDNA cleavage and degradation activities of AsCas12a with split crRNAs.
• Assessing the functionality of split crRNAs with other Cas12a variants (FnCas12a, LbCas12a).

Main Results:

• Removal of most of the crRNA scaffold minimally impacted in vitro DNA cleavage by AsCas12a.
• Split crRNAs, comprising separate scaffold and spacer RNAs, efficiently and specifically cleaved target DNA in vitro and in human cell lysates.
• AsCas12a programmed with split crRNAs exhibited both specific dsDNA cleavage and ssDNA cleavage/degradation (collateral activity).
• Other Cas12a variants (FnCas12a, LbCas12a) also functioned with split crRNAs, indicating a general property of V-A effectors.

Conclusions:

• The scaffold moiety of crRNAs is largely dispensable for Cas12a-mediated DNA cleavage.
• Split crRNAs are functional and enable specific DNA targeting by Cas12a nucleases.
• Split crRNAs offer a versatile platform for Cas12a applications, potentially enhancing multiplex diagnostics and enabling new mechanistic studies.