Massively parallel kinetic profiling of natural and engineered CRISPR nucleases.

Autor: Jones SK Jr; Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, USA. skjonesjr@utexas.edu.; Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, USA. skjonesjr@utexas.edu.; Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX, USA. skjonesjr@utexas.edu., Hawkins JA; Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, USA. john.hawkins@embl.de.; Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, USA. john.hawkins@embl.de.; Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX, USA. john.hawkins@embl.de.; Oden Institute for Computational Engineering and Science, University of Texas at Austin, Austin, TX, USA. john.hawkins@embl.de., Johnson NV; Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, USA.; Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, USA.; Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX, USA., Jung C; Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul, Republic of Korea., Hu K; Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, USA.; Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, USA.; Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX, USA., Rybarski JR; Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, USA.; Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, USA.; Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX, USA., Chen JS; Department of Molecular and Cell Biology, Berkeley, CA, USA., Doudna JA; Department of Molecular and Cell Biology, Berkeley, CA, USA.; Department of Chemistry, University of California, Berkeley, CA, USA.; Howard Hughes Medical Institute, University of California, Berkeley, CA, USA.; Lawrence Berkeley National Laboratory, Physical Biosciences Division, Berkeley, CA, USA., Press WH; Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, USA.; Oden Institute for Computational Engineering and Science, University of Texas at Austin, Austin, TX, USA., Finkelstein IJ; Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, USA. ilya@finkelsteinlab.org.; Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, USA. ilya@finkelsteinlab.org.; Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX, USA. ilya@finkelsteinlab.org.
Jazyk: angličtina
Zdroj: Nature biotechnology [Nat Biotechnol] 2021 Jan; Vol. 39 (1), pp. 84-93. Date of Electronic Publication: 2020 Sep 07.
DOI: 10.1038/s41587-020-0646-5
Abstrakt: Engineered SpCas9s and AsCas12a cleave fewer off-target genomic sites than wild-type (wt) Cas9. However, understanding their fidelity, mechanisms and cleavage outcomes requires systematic profiling across mispaired target DNAs. Here we describe NucleaSeq-nuclease digestion and deep sequencing-a massively parallel platform that measures the cleavage kinetics and time-resolved cleavage products for over 10,000 targets containing mismatches, insertions and deletions relative to the guide RNA. Combining cleavage rates and binding specificities on the same target libraries, we benchmarked five SpCas9 variants and AsCas12a. A biophysical model built from these data sets revealed mechanistic insights into off-target cleavage. Engineered Cas9s, especially Cas9-HF1, dramatically increased cleavage specificity but not binding specificity compared to wtCas9. Surprisingly, AsCas12a cleavage specificity differed little from that of wtCas9. Initial DNA cleavage sites and end trimming varied by nuclease, guide RNA and the positions of mispaired nucleotides. More broadly, NucleaSeq enables rapid, quantitative and systematic comparisons of specificity and cleavage outcomes across engineered and natural nucleases.
Databáze: MEDLINE
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