Cpf1 proteins excise CRISPR RNAs from mRNA transcripts in mammalian cells

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR-Cas systems are powerful genome-editing tools.
• Streptococcus pyogenes Cas9 (SpCas9) is widely used but can have specificity issues.
• Cpf1 offers greater specificity compared to SpCas9.

Purpose of the Study:

• To investigate the genome-editing capabilities of Cpf1 orthologs from Lachnospiraceae bacterium (Lb) and Acidaminococus sp. (As).
• To explore the potential of Cpf1 in simplifying multiplex genome modification.
• To enhance the efficiency of Cpf1-mediated genome editing.

Main Methods:

• Expression of LbCpf1 and AsCpf1 in mammalian cells.
• Analysis of RNase activity of Cpf1 orthologs.
• Demonstration of excision of multiple CRISPR RNAs (crRNAs) from a single RNA transcript.
• Assessment of Cpf1-mediated genome editing efficiency.

Main Results:

• LbCpf1 and AsCpf1 possess RNase activities.
• These Cpf1 orthologs can excise multiple crRNAs from a single RNA polymerase II-driven transcript.
• This capability simplifies the modification of multiple genomic targets simultaneously.
• The observed property enhances the efficiency of Cpf1-mediated genome editing.

Conclusions:

• Cpf1 orthologs from Lb and As exhibit unique RNase activities beneficial for genome editing.
• The ability to process multiple crRNAs from a single transcript streamlines multiplex genome engineering.
• This finding offers a more efficient approach for Cpf1-based genome editing applications.