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Expanding the Genome-Editing Toolbox with Abyssicoccus albus Cas9 Using a Unique Protospacer Adjacent Motif Sequence
Akiyoshi Nakamura1, Hiroshi Yamamoto1, Tsubasa Yano2
1Bioproduction Research Institute, The National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan.
The CRISPR Journal
|August 7, 2024
Summary
Researchers discovered Abyssicoccus albus Cas9 (AalCas9), a smaller CRISPR-Cas9 enzyme recognizing a novel 5'-NNACR-3' PAM sequence. This expands genome editing tools for plants and human cells.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- CRISPR-Cas9 genome editing relies on protospacer adjacent motif (PAM) recognition for DNA binding.
- The widely used Streptococcus pyogenes Cas9 (SpyCas9) targets a 5'-NGG-3' PAM, limiting its application in diverse genomic regions.
- Engineering SpyCas9 and exploring Cas9 orthologs are key strategies to broaden genome editing capabilities.
Purpose of the Study:
- To identify and characterize novel Cas9 orthologs with distinct PAM specificities.
- To evaluate the genome editing efficiency of a newly identified Cas9 enzyme in plant and human cells.
- To investigate the structural basis of PAM recognition and engineer Cas9 for altered sequence preferences.
Main Methods:
- Bioinformatic identification and characterization of the Abyssicoccus albus Cas9 (AalCas9) ortholog.
- Experimental validation of AalCas9's 5'-NNACR-3' PAM sequence recognition in vitro and in vivo.
- Optimization of guide RNA sequences for enhanced AalCas9-mediated genome editing.
- Structure prediction and site-directed mutagenesis to probe PAM recognition mechanisms.
Main Results:
- A smaller Cas9 ortholog, AalCas9, was identified, recognizing a unique 5'-NNACR-3' PAM sequence.
- Modified guide RNAs significantly improved AalCas9 editing efficiency in both plant and human cell lines.
- A point mutation in the putative PAM recognition site altered AalCas9's sequence preference, confirmed by structural predictions.
Conclusions:
- AalCas9 represents a novel tool for genome editing, expanding the range of targetable genomic sites.
- Optimization of guide RNA and protein engineering can enhance the utility of new Cas9 variants.
- Understanding Cas9 diversity provides valuable insights for developing next-generation genome editing technologies.
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