Abstract
Cpf1, a CRISPR endonuclease discovered in Prevotella and Francisella 1 bacteria, offers an alternative platform for CRISPR-based genome editing beyond the commonly used CRISPR–Cas9 system originally discovered in Streptococcus pyogenes. This protocol enables the design of engineered CRISPR–Cpf1 components, both CRISPR RNAs (crRNAs) to guide the endonuclease and Cpf1 mRNAs to express the endonuclease protein, and provides experimental procedures for effective genome editing using this system. We also describe quantification of genome-editing activity and off-target effects of the engineered CRISPR–Cpf1 in human cell lines using both T7 endonuclease I (T7E1) assay and targeted deep sequencing. This protocol enables rapid construction and identification of engineered crRNAs and Cpf1 mRNAs to enhance genome-editing efficiency using the CRISPR–Cpf1 system, as well as assessment of target specificity within 2 months. This protocol may also be appropriate for fine-tuning other types of CRISPR systems.
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Acknowledgements
We acknowledge F. Zhang and his laboratory at the Broad Institute of MIT and Harvard for providing Cpf1 plasmids and technical assistance. This work was supported by the National Institutes of Health through the National Heart, Lung, and Blood Institute (NHLBI; R01HL136652), as well as by the start-up fund from the College of Pharmacy at The Ohio State University.
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B.L., C.Z., and Y.D. developed the protocol.
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Integrated supplementary information
Supplementary Figure 1 Representative examples of chemically modified nucleotides.
(a) Linkage (backbone)-modified nucleotides. (b) Ribose-modified nucleotides. (c) Nitrogenous base (cytosine, uracil, adenine, and guanine)-modified nucleotides. Natural nucleotides are shown in blue. The modified groups are shown in red.
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Supplementary Figure 1 and Supplementary Table 1. (PDF 742 kb)
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Li, B., Zeng, C. & Dong, Y. Design and assessment of engineered CRISPR–Cpf1 and its use for genome editing. Nat Protoc 13, 899–914 (2018). https://doi.org/10.1038/nprot.2018.004
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DOI: https://doi.org/10.1038/nprot.2018.004
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