Novel and Engineered Type II CRISPR Systems from Uncultivated Microbes with Broad Genome Editing Capability

Area of Science:

• Genomics
• Molecular Biology
• Biotechnology

Background:

• Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 nucleases are vital tools in biotechnology and therapeutics.
• Current CRISPR-Cas9 systems face limitations in size, targetability, and off-target effects, hindering broader applications.

Purpose of the Study:

• To discover novel Type II CRISPR-Cas9 effectors with improved characteristics.
• To expand the targeting capabilities and reduce off-target effects of CRISPR-Cas9 systems.

Main Methods:

• Mining a large, genome-resolved metagenomics database to identify CRISPR Type II effectors.
• Developing a high-throughput pipeline for predicting tracrRNA, designing single guide RNAs, and demonstrating in vitro nuclease activity.
• Engineering PAM-interacting domains to enhance targetability.

Main Results:

• Identification of thousands of CRISPR Type II effectors, including 41 newly described subgroups with demonstrated in vitro activity.
• Discovery of nucleases with diverse protein sequences, guide RNA structures, and varied protospacer adjacent motifs (PAMs).
• Several novel nucleases exhibited high activity comparable to or exceeding SpCas9, with smaller sizes and low off-target editing in mammalian cells.

Conclusions:

• The newly discovered CRISPR nucleases offer significant advantages over existing systems, including enhanced activity, smaller size, and improved specificity.
• Engineered chimeras further expanded the targeting range of these novel nucleases.
• These findings present a diverse toolkit of CRISPR enzymes with considerable potential for translation into various applications, particularly in therapeutics.