CRISPR-based technologies for large DNA insertions

Area of Science:

• Molecular Biology
• Biotechnology
• Genetics

Background:

• CRISPR-Cas9 and Cas12 revolutionized precise DNA mutation generation.
• Current methods are limited to small-scale mutagenesis via DNA nicks and double-stranded breaks (DSBs).
• There is a growing need for advanced genome editing tools capable of large-scale DNA manipulations.

Purpose of the Study:

• To review emerging CRISPR-based technologies for large-scale DNA manipulation.
• To highlight advanced tools that expand payload options and enable DSB-free editing.
• To discuss the translational potential of these technologies in medicine and agriculture.

Main Methods:

• Exploration of CRISPR-associated transposons and site-specific recombinases for larger DNA integrations.
• Analysis of sophisticated combinations like PASTE, PASSIGE, and PrimeRoot.
• Review of DSB-free editing modalities and expanded payload capacities.

Main Results:

• Emerging effectors enable larger DNA integrations by combining Cas enzymes with other functions.
• Advanced combinations (PASTE, PASSIGE, PrimeRoot) offer expanded payload options.
• New modalities facilitate DSB-free editing, broadening genome editing applications.

Conclusions:

• Sophisticated CRISPR-based tools are advancing genome editing beyond local mutagenesis.
• These technologies hold significant translational potential for gene and cell therapies in personalized medicine.
• The development of these tools is crucial for next-generation crop breeding in sustainable agriculture.