skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Synthetic chimeric nucleases function for efficient genome editing

Abstract

CRISPR-Cas systems have revolutionized genome editing across a broad range of biotechnological endeavors. Many CRISPR-Cas nucleases have been identified and engineered for improved capabilities. Given the modular structure of such enzymes, we hypothesized that engineering chimeric sequences would generate non-natural variants that span the kinetic parameter landscape, and thus provide for the rapid selection of nucleases fit for a particular editing system. Here, we design a chimeric Cas12a-type library with approximately 560 synthetic chimeras, and select several functional variants. We demonstrate that certain nuclease domains can be recombined across distantly related nuclease templates to produce variants that function in bacteria, yeast, and human cell lines. We further characterize selected chimeric nucleases and find that they have different protospacer adjacent motif (PAM) preferences and the M44 chimera has higher specificity relative to wild-type (WT) sequences. This demonstration opens up the possibility of generating nuclease sequences with implications across biotechnology.

Authors:
 [1];  [1];  [1];  [1];  [1];  [1];  [2]; ORCiD logo [3];  [4]
  1. Univ. of Colorado, Boulder, CO (United States)
  2. Inscripta, Inc., Boulder, CO (United States)
  3. Univ. of Colorado, Boulder, CO (United States); National Renewable Energy Lab. (NREL), Golden, CO (United States)
  4. Univ. of Colorado, Boulder, CO (United States); Danish Technical Univ., Lyngby (Denmark)
Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1580024
Report Number(s):
NREL/JA-2700-75556
Journal ID: ISSN 2041-1723
Grant/Contract Number:  
AC36-08GO28308; SC0018368
Resource Type:
Accepted Manuscript
Journal Name:
Nature Communications
Additional Journal Information:
Journal Volume: 10; Journal Issue: 1; Journal ID: ISSN 2041-1723
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; CRISPR; genome editing; Cas12a; genetic techniques; high-throughput screening; synthetic biology

Citation Formats

Liu, R. M., Liang, L. L., Freed, E., Chang, H., Oh, E., Liu, Z. Y., Garst, A., Eckert, Carrie A., and Gill, R. T. Synthetic chimeric nucleases function for efficient genome editing. United States: N. p., 2019. Web. doi:10.1038/s41467-019-13500-y.
Liu, R. M., Liang, L. L., Freed, E., Chang, H., Oh, E., Liu, Z. Y., Garst, A., Eckert, Carrie A., & Gill, R. T. Synthetic chimeric nucleases function for efficient genome editing. United States. doi:10.1038/s41467-019-13500-y.
Liu, R. M., Liang, L. L., Freed, E., Chang, H., Oh, E., Liu, Z. Y., Garst, A., Eckert, Carrie A., and Gill, R. T. Wed . "Synthetic chimeric nucleases function for efficient genome editing". United States. doi:10.1038/s41467-019-13500-y. https://www.osti.gov/servlets/purl/1580024.
@article{osti_1580024,
title = {Synthetic chimeric nucleases function for efficient genome editing},
author = {Liu, R. M. and Liang, L. L. and Freed, E. and Chang, H. and Oh, E. and Liu, Z. Y. and Garst, A. and Eckert, Carrie A. and Gill, R. T.},
abstractNote = {CRISPR-Cas systems have revolutionized genome editing across a broad range of biotechnological endeavors. Many CRISPR-Cas nucleases have been identified and engineered for improved capabilities. Given the modular structure of such enzymes, we hypothesized that engineering chimeric sequences would generate non-natural variants that span the kinetic parameter landscape, and thus provide for the rapid selection of nucleases fit for a particular editing system. Here, we design a chimeric Cas12a-type library with approximately 560 synthetic chimeras, and select several functional variants. We demonstrate that certain nuclease domains can be recombined across distantly related nuclease templates to produce variants that function in bacteria, yeast, and human cell lines. We further characterize selected chimeric nucleases and find that they have different protospacer adjacent motif (PAM) preferences and the M44 chimera has higher specificity relative to wild-type (WT) sequences. This demonstration opens up the possibility of generating nuclease sequences with implications across biotechnology.},
doi = {10.1038/s41467-019-13500-y},
journal = {Nature Communications},
number = 1,
volume = 10,
place = {United States},
year = {2019},
month = {12}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Save / Share:

Works referenced in this record:

A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity
journal, June 2012


Multiplex Genome Engineering Using CRISPR/Cas Systems
journal, January 2013


RNA-Guided Human Genome Engineering via Cas9
journal, January 2013


Cas9 as a versatile tool for engineering biology
journal, September 2013

  • Mali, Prashant; Esvelt, Kevin M.; Church, George M.
  • Nature Methods, Vol. 10, Issue 10
  • DOI: 10.1038/nmeth.2649

Genome-wide mapping of mutations at single-nucleotide resolution for protein, metabolic and genome engineering
journal, December 2016

  • Garst, Andrew D.; Bassalo, Marcelo C.; Pines, Gur
  • Nature Biotechnology, Vol. 35, Issue 1, p. 48-55
  • DOI: 10.1038/nbt.3718

CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes
journal, July 2013


The new frontier of genome engineering with CRISPR-Cas9
journal, November 2014


Development and Applications of CRISPR-Cas9 for Genome Engineering
journal, June 2014


High-throughput functional genomics using CRISPR–Cas9
journal, April 2015

  • Shalem, Ophir; Sanjana, Neville E.; Zhang, Feng
  • Nature Reviews Genetics, Vol. 16, Issue 5
  • DOI: 10.1038/nrg3899

CRISPR-Cas systems for editing, regulating and targeting genomes
journal, March 2014

  • Sander, Jeffry D.; Joung, J. Keith
  • Nature Biotechnology, Vol. 32, Issue 4
  • DOI: 10.1038/nbt.2842

Diversity, classification and evolution of CRISPR-Cas systems
journal, June 2017


Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System
journal, October 2015

  • Zetsche, Bernd; Gootenberg, Jonathan S.; Abudayyeh, Omar O.
  • Cell, Vol. 163, Issue 3, p. 759-771
  • DOI: 10.1016/j.cell.2015.09.038

The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA
journal, April 2016

  • Fonfara, Ines; Richter, Hagen; Bratovič, Majda
  • Nature, Vol. 532, Issue 7600
  • DOI: 10.1038/nature17945

Targeted mutagenesis in mice by electroporation of Cpf1 ribonucleoproteins
journal, June 2016

  • Hur, Junho K.; Kim, Kyoungmi; Been, Kyung Wook
  • Nature Biotechnology, Vol. 34, Issue 8
  • DOI: 10.1038/nbt.3596

Generation of knockout mice by Cpf1-mediated gene targeting
journal, June 2016

  • Kim, Yongsub; Cheong, Seung-A; Lee, Jong Geol
  • Nature Biotechnology, Vol. 34, Issue 8
  • DOI: 10.1038/nbt.3614

Diversity and evolution of class 2 CRISPR–Cas systems
journal, January 2017

  • Shmakov, Sergey; Smargon, Aaron; Scott, David
  • Nature Reviews Microbiology, Vol. 15, Issue 3
  • DOI: 10.1038/nrmicro.2016.184

Multiplex gene editing by CRISPR–Cpf1 using a single crRNA array
journal, December 2016

  • Zetsche, Bernd; Heidenreich, Matthias; Mohanraju, Prarthana
  • Nature Biotechnology, Vol. 35, Issue 1
  • DOI: 10.1038/nbt.3737

Crystal Structure of Cpf1 in Complex with Guide RNA and Target DNA
journal, May 2016


Rationally engineered Cas9 nucleases with improved specificity
journal, December 2015


High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects
journal, January 2016

  • Kleinstiver, Benjamin P.; Pattanayak, Vikram; Prew, Michelle S.
  • Nature, Vol. 529, Issue 7587
  • DOI: 10.1038/nature16526

Engineered Cpf1 variants with altered PAM specificities
journal, June 2017

  • Gao, Linyi; Cox, David B. T.; Yan, Winston X.
  • Nature Biotechnology, Vol. 35, Issue 8
  • DOI: 10.1038/nbt.3900

Improving CRISPR-Cas nuclease specificity using truncated guide RNAs
journal, January 2014

  • Fu, Yanfang; Sander, Jeffry D.; Reyon, Deepak
  • Nature Biotechnology, Vol. 32, Issue 3
  • DOI: 10.1038/nbt.2808

Structures of Cas9 Endonucleases Reveal RNA-Mediated Conformational Activation
journal, February 2014


Structure-guided SCHEMA recombination of distantly related β-lactamases
journal, November 2006

  • Meyer, Michelle M.; Hochrein, Lisa; Arnold, Frances H.
  • Protein Engineering, Design and Selection, Vol. 19, Issue 12
  • DOI: 10.1093/protein/gzl045

Computational method to reduce the search space for directed protein evolution
journal, March 2001

  • Voigt, C. A.; Mayo, S. L.; Arnold, F. H.
  • Proceedings of the National Academy of Sciences, Vol. 98, Issue 7
  • DOI: 10.1073/pnas.051614498

Rational design of highly active sgRNAs for CRISPR-Cas9–mediated gene inactivation
journal, September 2014

  • Doench, John G.; Hartenian, Ella; Graham, Daniel B.
  • Nature Biotechnology, Vol. 32, Issue 12
  • DOI: 10.1038/nbt.3026

Sequence determinants of improved CRISPR sgRNA design
journal, June 2015


Internal guide RNA interactions interfere with Cas9-mediated cleavage
journal, June 2016

  • Thyme, Summer B.; Akhmetova, Laila; Montague, Tessa G.
  • Nature Communications, Vol. 7, Issue 1
  • DOI: 10.1038/ncomms11750

Rapid and Efficient One-Step Metabolic Pathway Integration in E. coli
journal, April 2016

  • Bassalo, Marcelo C.; Garst, Andrew D.; Halweg-Edwards, Andrea L.
  • ACS Synthetic Biology, Vol. 5, Issue 7
  • DOI: 10.1021/acssynbio.5b00187

A Biophysical Model of CRISPR/Cas9 Activity for Rational Design of Genome Editing and Gene Regulation
journal, January 2016


Nitroreductase (GlNR1) increases susceptibility of Giardia lamblia and Escherichia coli to nitro drugs
journal, February 2011

  • Nillius, D.; Muller, J.; Muller, N.
  • Journal of Antimicrobial Chemotherapy, Vol. 66, Issue 5
  • DOI: 10.1093/jac/dkr029

RNA-guided editing of bacterial genomes using CRISPR-Cas systems
journal, January 2013

  • Jiang, Wenyan; Bikard, David; Cox, David
  • Nature Biotechnology, Vol. 31, Issue 3, p. 233-239
  • DOI: 10.1038/nbt.2508

Rapid characterization of CRISPR-Cas9 protospacer adjacent motif sequence elements
journal, November 2015


High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity
journal, August 2013

  • Pattanayak, Vikram; Lin, Steven; Guilinger, John P.
  • Nature Biotechnology, Vol. 31, Issue 9
  • DOI: 10.1038/nbt.2673

Programmable Removal of Bacterial Strains by Use of Genome-Targeting CRISPR-Cas Systems
journal, January 2014


Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells
journal, June 2016

  • Kleinstiver, Benjamin P.; Tsai, Shengdar Q.; Prew, Michelle S.
  • Nature Biotechnology, Vol. 34, Issue 8
  • DOI: 10.1038/nbt.3620

High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells
journal, June 2013

  • Fu, Yanfang; Foden, Jennifer A.; Khayter, Cyd
  • Nature Biotechnology, Vol. 31, Issue 9
  • DOI: 10.1038/nbt.2623

Enhanced proofreading governs CRISPR–Cas9 targeting accuracy
journal, September 2017

  • Chen, Janice S.; Dagdas, Yavuz S.; Kleinstiver, Benjamin P.
  • Nature, Vol. 550, Issue 7676
  • DOI: 10.1038/nature24268

Engineered CRISPR-Cas9 nucleases with altered PAM specificities
journal, June 2015

  • Kleinstiver, Benjamin P.; Prew, Michelle S.; Tsai, Shengdar Q.
  • Nature, Vol. 523, Issue 7561
  • DOI: 10.1038/nature14592

Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems
journal, March 2013

  • DiCarlo, James E.; Norville, Julie E.; Mali, Prashant
  • Nucleic Acids Research, Vol. 41, Issue 7, p. 4336-4343
  • DOI: 10.1093/nar/gkt135

Methods for the directed evolution of proteins
journal, June 2015

  • Packer, Michael S.; Liu, David R.
  • Nature Reviews Genetics, Vol. 16, Issue 7
  • DOI: 10.1038/nrg3927

Quick and easy yeast transformation using the LiAc/SS carrier DNA/PEG method
journal, January 2007