Improved CRISPR-Cas12a-assisted one-pot DNA editing method enables seamless DNA editing

Area of Science:

• Molecular Biology
• Biotechnology
• Gene Editing

Background:

• The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system is a powerful tool for DNA manipulation.
• Current CRISPR-Cas12a systems have limitations in targeting range due to specific protospacer adjacent motif (PAM) requirements.

Purpose of the Study:

• To engineer FnCas12a variants with expanded PAM recognition for broader DNA targeting.
• To develop an efficient one-pot DNA editing method utilizing CRISPR-Cas12a for seamless DNA assembly and modification.

Main Methods:

• Engineering of FnCas12a proteins to create variants (FnCas12a-EP15 and EP16) with altered PAM specificities.
• Development of the improved CRISPR-Cas12a-assisted one-pot DNA editing (iCOCO) method, integrating transcription, digestion, and ligation.
• Testing iCOPE efficiency for DNA fragment assembly and large DNA construct modification.

Main Results:

• Two engineered FnCas12a variants significantly expanded the targeting range, recognizing PAM sequences like YN, TAC, and CAA.
• The iCOPE method demonstrated high editing efficiencies (72-100%) for DNA fragment assembly and 100% for 21 kb large DNA construct modification.
• iCOPE facilitates one-pot cloning with minimal sequence constraints, potentially replacing multiple restriction enzymes.

Conclusions:

• Engineered CRISPR-Cas12a variants offer an expanded DNA targeting scope.
• The iCOPE method provides an efficient, seamless, and versatile tool for one-pot DNA editing and cloning.