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Can SpRY recognize any PAM in human cells?
Jinbin Ye1, Haitao Xi1, Yilu Chen1
1Reproduction Center, Department of Obstetrics and Gynecology, the Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou 325000, China.
Abstract:
The application of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) can be limited due to a lack of compatible protospacer adjacent motif (PAM) sequences in the DNA regions of interest. Recently, SpRY, a variant of Streptococcus pyogenes Cas9 (SpCas9), was reported, which nearly completely fulfils the PAM requirement. Meanwhile, PAMs for SpRY have not been well addressed. In our previous study, we developed the PAM Definition by Observable Sequence Excision (PAM-DOSE) and green fluorescent protein (GFP)-reporter systems to study PAMs in human cells. Herein, we endeavored to identify the PAMs of SpRY with these two methods. The results indicated that 5'-NRN-3', 5'-NTA-3', and 5'-NCK-3' could be considered as canonical PAMs. 5'-NCA-3' and 5'-NTK-3' may serve as non-priority PAMs. At the same time, PAM of 5'-NYC-3' is not recommended for human cells. These findings provide further insights into the application of SpRY for human genome editing.
Insights
SpRY, a CRISPR-associated protein variant, expands genome editing capabilities. This study identifies its canonical and non-canonical protospacer adjacent motif sequences for precise human cell applications.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) are powerful genome editing tools.
- The utility of CRISPR-Cas systems is often constrained by the availability of specific protospacer adjacent motif (PAM) sequences.
- SpRY, a variant of Streptococcus pyogenes Cas9 (SpCas9), shows promise for overcoming PAM limitations in genome editing.
Purpose of the Study:
- To comprehensively identify and characterize the protospacer adjacent motif (PAM) sequences recognized by the SpRY variant.
- To evaluate the efficiency and specificity of SpRY binding to various PAM sequences in human cells.
- To provide essential data for optimizing SpRY-based genome editing strategies in human therapeutics and research.
Main Methods:
- Utilized the established PAM Definition by Observable Sequence Excision (PAM-DOSE) system.
- Employed a green fluorescent protein (GFP)-reporter assay for evaluating SpRY activity.
- Tested a range of potential PAM sequences in human cell lines to determine SpRY recognition.
Main Results:
- Identified 5'-NRN-3', 5'-NTA-3', and 5'-NCK-3' as canonical PAM sequences for SpRY.
- Determined 5'-NCA-3' and 5'-NTK-3' as potential non-priority PAM sequences.
- Found that the 5'-NYC-3' PAM sequence is not recommended for SpRY in human cells.
Conclusions:
- The identified PAMs provide crucial guidance for the effective application of SpRY in human genome editing.
- This research expands the toolkit for CRISPR-Cas genome engineering by detailing SpRY's PAM compatibility.
- Findings facilitate more precise and versatile SpRY-mediated gene targeting in human cells.
