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Scalable characterization of the PAM requirements of CRISPR-Cas enzymes using HT-PAMDA.
- Source :
-
Nature protocols [Nat Protoc] 2021 Mar; Vol. 16 (3), pp. 1511-1547. Date of Electronic Publication: 2021 Feb 05. - Publication Year :
- 2021
-
Abstract
- The continued expansion of the genome-editing toolbox necessitates methods to characterize important properties of CRISPR-Cas enzymes. One such property is the requirement for Cas proteins to recognize a protospacer-adjacent motif (PAM) in DNA target sites. The high-throughput PAM determination assay (HT-PAMDA) is a method that enables scalable characterization of the PAM preferences of different Cas proteins. Here, we provide a step-by-step protocol for the method, discuss experimental design considerations, and highlight how the method can be used to profile naturally occurring CRISPR-Cas9 enzymes, engineered derivatives with improved properties, orthologs of different classes (e.g., Cas12a), and even different platforms (e.g., base editors). A distinguishing feature of HT-PAMDA is that the enzymes are expressed in a cell type or organism of interest (e.g., mammalian cells), permitting scalable characterization and comparison of hundreds of enzymes in a relevant setting. HT-PAMDA does not require specialized equipment or expertise and is cost effective for multiplexed characterization of many enzymes. The protocol enables comprehensive PAM characterization of dozens or hundreds of Cas enzymes in parallel in <2 weeks.
- Subjects :
- Animals
CRISPR-Associated Protein 9 metabolism
CRISPR-Cas Systems genetics
Clustered Regularly Interspaced Short Palindromic Repeats genetics
DNA genetics
Humans
Nucleotide Motifs genetics
RNA, Guide, CRISPR-Cas Systems genetics
Research Design
CRISPR-Cas Systems physiology
Gene Editing methods
High-Throughput Nucleotide Sequencing methods
Subjects
Details
- Language :
- English
- ISSN :
- 1750-2799
- Volume :
- 16
- Issue :
- 3
- Database :
- MEDLINE
- Journal :
- Nature protocols
- Publication Type :
- Academic Journal
- Accession number :
- 33547443
- Full Text :
- https://doi.org/10.1038/s41596-020-00465-2