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Efficient cleavage resolves PAM preferences of CRISPR-Cas in human cells
Lianchao Tang1, Fayu Yang1, Xiaoxue He1
1School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, State Key Laboratory and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang 325027 China.
Cell Regeneration (London, England)
|November 12, 2019
Summary
Researchers developed PAM-DOSE to identify functional protospacer adjacent motifs (PAMs) for CRISPR-Cas genome editing in human cells. This system aids in discovering precise DNA targets for advanced gene editing applications.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- CRISPR-Cas systems are powerful tools for genome manipulation in research and medicine.
- Protospacer adjacent motifs (PAMs) are crucial for Cas protein DNA target recognition.
- Identifying functional PAMs in human cells is challenging for precise genome editing.
Purpose of the Study:
- To develop a novel system for delineating functional PAM sequences in human cells.
- To identify functional PAMs for various CRISPR-Cas enzymes, including SpCas9 and Cas12a variants.
- To investigate the influence of spacer sequences on Cas protein PAM preference.
Main Methods:
- Developed PAM-DOSE (PAM Definition by Observable Sequence Excision), a positive screen system.
- Generated PAM libraries for multiple CRISPR-Cas enzymes (SpCas9, SpCas9-NG, FnCas12a, AsCas12a, LbCas12a, MbCas12a).
- Utilized DNA sequencing, fluorescence microscopy, and flow cytometry to track and visualize cleaved fragments in human cells.
Main Results:
- Successfully identified functional PAM sequences for several CRISPR-Cas members in human cells.
- Demonstrated that the spacer sequence can influence the PAM preference of Cas proteins.
- Validated the efficacy of the PAM-DOSE system for PAM discovery.
Conclusions:
- The PAM-DOSE system enables efficient identification of functional PAMs for CRISPR-Cas in human cells.
- Understanding PAM specificity is critical for optimizing CRISPR-Cas for human genome editing.
- This method advances the application of precise gene editing technologies in human therapeutics.
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