SLiCE: a novel bacterial cell extract-based DNA cloning method

We describe a novel cloning method termed SLiCE (Seamless Ligation Cloning Extract) that utilizes easy to generate bacterial cell extracts to assemble multiple DNA fragments into recombinant DNA molecules in a single in vitro recombination reaction. SLiCE overcomes the sequence limitations of traditional cloning methods, facilitates seamless cloning by recombining short end homologies (≥15 bp) with or without flanking heterologous sequences and provides an effective strategy for directional subcloning of DNA fragments from Bacteria Artificial Chromosomes (BACs) or other sources. SLiCE is highly cost effective as a number of standard laboratory bacterial strains can serve as sources for SLiCE extract. In addition, the cloning efficiencies and capabilities of these strains can be greatly improved by simple genetic modifications. As an example, we modified the DH10B Escherichia coli strain to express an optimized λ prophage Red recombination system. This strain, termed PPY, facilitates SLiCE with very high efficiencies and demonstrates the versatility of the method.

[1]  Yongping Huang,et al.  The CRISPR/Cas System mediates efficient genome engineering in Bombyx mori , 2013, Cell Research.

[2]  A. Stewart,et al.  Rapid modification of bacterial artificial chromosomes by ET-recombination. , 1999, Nucleic acids research.

[3]  G. Freeman,et al.  In-fusion assembly: seamless engineering of multidomain fusion proteins, modular vectors, and mutations. , 2007, BioTechniques.

[4]  Christopher M. Vockley,et al.  RNA-guided gene activation by CRISPR-Cas9-based transcription factors , 2013, Nature Methods.

[5]  B. González,et al.  Modular system for the construction of zinc-finger libraries and proteins , 2010, Nature Protocols.

[6]  U. Bonas,et al.  Xanthomonas AvrBs3 family-type III effectors: discovery and function. , 2010, Annual review of phytopathology.

[7]  Randall J. Platt,et al.  Optical Control of Mammalian Endogenous Transcription and Epigenetic States , 2013, Nature.

[8]  Rudolf Jaenisch,et al.  One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering , 2013, Cell.

[9]  Eli J. Fine,et al.  DNA targeting specificity of RNA-guided Cas9 nucleases , 2013, Nature Biotechnology.

[10]  Jeffry D Sander,et al.  FLAsH assembly of TALeNs for high-throughput genome editing , 2022 .

[11]  S. Lovett,et al.  Mechanisms of recombination: lessons from E. coli. , 2008, Critical reviews in biochemistry and molecular biology.

[12]  Detlef Weigel,et al.  Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease , 2013, Nature Biotechnology.

[13]  Dana Carroll,et al.  Heritable Gene Knockout in Caenorhabditis elegans by Direct Injection of Cas9–sgRNA Ribonucleoproteins , 2013, Genetics.

[14]  Jeffry D Sander,et al.  Targeted DNA demethylation and activation of endogenous genes using programmable TALE-TET1 fusion proteins , 2013, Nature Biotechnology.

[15]  G. Church,et al.  CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering , 2013, Nature Biotechnology.

[16]  Bing Yang,et al.  Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice , 2013, Nucleic acids research.

[17]  Daniel H. Haft,et al.  A Guild of 45 CRISPR-Associated (Cas) Protein Families and Multiple CRISPR/Cas Subtypes Exist in Prokaryotic Genomes , 2005, PLoS Comput. Biol..

[18]  Jianglin Fan,et al.  Effective gene targeting in rabbits using RNA-guided Cas9 nucleases. , 2014, Journal of molecular cell biology.

[19]  Gary D. Stormo,et al.  An optimized two-finger archive for ZFN-mediated gene targeting , 2012, Nature Methods.

[20]  P. Duchateau,et al.  A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences , 2006, Nucleic acids research.

[21]  Emmanuelle Charpentier,et al.  The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems , 2013, RNA biology.

[22]  Drena Dobbs,et al.  Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly , 2006, Nature Protocols.

[23]  Melissa M. Harrison,et al.  Genome Engineering of Drosophila with the CRISPR RNA-Guided Cas9 Nuclease , 2013, Genetics.

[24]  Qi Zhou,et al.  Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems , 2013, Nature Biotechnology.

[25]  Yongxiang Zhao,et al.  Heritable gene targeting in the mouse and rat using a CRISPR-Cas system , 2013, Nature Biotechnology.

[26]  M. Weiner,et al.  Polishing with T4 or Pfu polymerase increases the efficiency of cloning of PCR fragments. , 1994, Nucleic acids research.

[27]  J. Clark,et al.  Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases. , 1988, Nucleic acids research.

[28]  David A. Scott,et al.  Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity , 2013, Cell.

[29]  Wei Zhang,et al.  Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System , 2014, Cell.

[30]  A. J. Mixson,et al.  Alteration in the IL‐2 signal peptide affects secretion of proteins in vitro and in vivo , 2005, The journal of gene medicine.

[31]  John A. Calarco,et al.  Heritable Custom Genomic Modifications in Caenorhabditis elegans via a CRISPR–Cas9 System , 2013, Genetics.

[32]  B A Neilan,et al.  Enzyme-free cloning: a rapid method to clone PCR products independent of vector restriction enzyme sites. , 1999, Nucleic acids research.

[33]  Matthew J. Moscou,et al.  A Simple Cipher Governs DNA Recognition by TAL Effectors , 2009, Science.

[34]  J. W. Little [34a] An exonuclease induced by bacteriophage lambda , 1967 .

[35]  Jeffry D. Sander,et al.  Heritable and Precise Zebrafish Genome Editing Using a CRISPR-Cas System , 2013, PloS one.

[36]  Hamilton O. Smith,et al.  A restriction enzyme from Hemophilus influenzae: II. Base sequence of the recognition site , 1970 .

[37]  Yarden Katz,et al.  Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system , 2013, Cell Research.

[38]  A. Kuzminov Recombinational repair of DNA damage in Escherichia coli and bacteriophage lambda. , 1999, Microbiology and molecular biology reviews : MMBR.

[39]  H. Smith,et al.  A restriction enzyme from Hemophilus influenzae. I. Purification and general properties. , 1970, Journal of molecular biology.

[40]  Dana Carroll,et al.  Design, construction and in vitro testing of zinc finger nucleases , 2006, Nature Protocols.

[41]  Steven Lin,et al.  Precise and Heritable Genome Editing in Evolutionarily Diverse Nematodes Using TALENs and CRISPR/Cas9 to Engineer Insertions and Deletions , 2013, Genetics.

[42]  Bo Zhang,et al.  Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish , 2013, Nucleic acids research.

[43]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[44]  Youming Zhang,et al.  RecE/RecT and Redα/Redβ initiate double-stranded break repair by specifically interacting with their respective partners , 2000, Genes & Development.

[45]  J. Joung,et al.  Locus-specific editing of histone modifications at endogenous enhancers using programmable TALE-LSD1 fusions , 2013, Nature Biotechnology.

[46]  Gang Bao,et al.  CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity , 2013, Nucleic acids research.

[47]  David A. Mead,et al.  A Universal Method for the Direct Cloning of PCR Amplified Nucleic Acid , 1991, Bio/Technology.

[48]  R. Tuli,et al.  RNA-Guided Genome Editing for Target Gene Mutations in Wheat , 2013, G3: Genes, Genomes, Genetics.

[49]  E. Lander,et al.  Genetic Screens in Human Cells Using the CRISPR-Cas9 System , 2013, Science.

[50]  K. Hsiao,et al.  Exonuclease III induced ligase-free directional subcloning of PCR products. , 1993, Nucleic acids research.

[51]  Marilyn Fisher,et al.  Simple and efficient CRISPR/Cas9‐mediated targeted mutagenesis in Xenopus tropicalis , 2013, Genesis.

[52]  A. C. Chang,et al.  Construction of biologically functional bacterial plasmids in vitro. , 1973, Proceedings of the National Academy of Sciences of the United States of America.

[53]  Bo Zhang,et al.  Highly Efficient Genome Modifications Mediated by CRISPR/Cas9 in Drosophila , 2013, Genetics.

[54]  C. Ostermeier,et al.  An improved method for fast, robust, and seamless integration of DNA fragments into multiple plasmids. , 2006, Protein expression and purification.

[55]  S. Elledge,et al.  Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC , 2007, Nature Methods.

[56]  J. Keith Joung,et al.  High frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells , 2013, Nature Biotechnology.

[57]  C. Radding,et al.  The role of exonuclease and beta protein of phage lambda in genetic recombination. 3. Binding to deoxyribonucleic acid. , 1971, The Journal of biological chemistry.

[58]  D. Belin,et al.  Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter , 1995, Journal of bacteriology.

[59]  Feng Zhang,et al.  Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system , 2013, Nucleic acids research.

[60]  G. Freeman,et al.  The TIM gene family: emerging roles in immunity and disease , 2003, Nature Reviews Immunology.

[61]  G. Hu,et al.  DNA Polymerase-Catalyzed Addition of Nontemplated Extra Nucleotides to the 3′ of a DNA Fragment , 1993 .

[62]  C. Radding,et al.  The Role of Exonuclease and β Protein of Phage λ in Genetic Recombination III. BINDING TO DEOXYRIBONUCLEIC ACID , 1971 .

[63]  Timothy B. Stockwell,et al.  Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome , 2008, Science.

[64]  Luke A. Gilbert,et al.  Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression , 2013, Cell.

[65]  Tamás Fehér,et al.  Scarless engineering of the Escherichia coli genome. , 2008, Methods in molecular biology.

[66]  V. Jung,et al.  Cloning of polymerase chain reaction-generated DNA containing terminal restriction endonuclease recognition sites. , 1993, Methods in enzymology.

[67]  Yoshio Koyanagi,et al.  Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus , 2013, Scientific Reports.

[68]  M. A. Smith,et al.  Improved cloning efficiency of polymerase chain reaction (PCR) products after proteinase K digestion , 1991, Nucleic Acids Res..

[69]  Albert J R Heck,et al.  RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions , 2011, Proceedings of the National Academy of Sciences.

[70]  P. D. de Jong,et al.  Ligation-independent cloning of PCR products (LIC-PCR). , 1990, Nucleic acids research.

[71]  M. Kozak,et al.  Regulation of translation via mRNA structure in prokaryotes and eukaryotes. , 2005, Gene.

[72]  F. Collins,et al.  Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR products. , 1991, Nucleic acids research.

[73]  Chad A. Cowan,et al.  Enhanced efficiency of human pluripotent stem cell genome editing through replacing TALENs with CRISPRs. , 2013, Cell stem cell.

[74]  A. Stewart,et al.  RecE/RecT and Redalpha/Redbeta initiate double-stranded break repair by specifically interacting with their respective partners. , 2000, Genes & development.

[75]  A. Kuzminov Recombinational Repair of DNA Damage inEscherichia coli and Bacteriophage λ , 1999, Microbiology and Molecular Biology Reviews.

[76]  Claudio Mussolino,et al.  Engineered zinc finger nickases induce homology-directed repair with reduced mutagenic effects , 2012, Nucleic acids research.

[77]  A. Kuzminov Single-strand interruptions in replicating chromosomes cause double-strand breaks , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[78]  Susan R. Wente,et al.  Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system , 2013, Proceedings of the National Academy of Sciences.

[79]  T. Holton,et al.  A simple and efficient method for direct cloning of PCR products using ddT-tailed vectors. , 1991, Nucleic acids research.

[80]  A. Aguilera,et al.  Double‐strand breaks arising by replication through a nick are repaired by cohesin‐dependent sister‐chromatid exchange , 2006, EMBO reports.

[81]  S. Lovett,et al.  RecA-independent recombination is efficient but limited by exonucleases , 2007, Proceedings of the National Academy of Sciences.

[82]  S. Lovett,et al.  Crossing over between regions of limited homology in Escherichia coli. RecA-dependent and RecA-independent pathways. , 2002, Genetics.

[83]  C. Gomez-Sanchez,et al.  Universal cloning method by TA strategy. , 1995, BioTechniques.

[84]  G. Church,et al.  Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. , 2011, Nature biotechnology.

[85]  Morgan L. Maeder,et al.  CRISPR RNA-guided activation of endogenous human genes , 2013, Nature Methods.

[86]  C. Radding,et al.  The Role of Exonuclease and β Protein of Phage λ in Genetic Recombination: II. SUBSTRATE SPECIFICITY AND THE MODE OF ACTION OF λ EXONUCLEASE , 1971 .

[87]  Jun Li,et al.  Targeted genome modification of crop plants using a CRISPR-Cas system , 2013, Nature Biotechnology.

[88]  Y. Doyon,et al.  Targeted gene addition to a predetermined site in the human genome using a ZFN-based nicking enzyme , 2012, Genome research.

[89]  P. Tucker,et al.  Construction of recombinant DNA by exonuclease recession. , 1993, Nucleic acids research.

[90]  Jun Zhang,et al.  Generation of gene-modified mice via Cas9/RNA-mediated gene targeting , 2013, Cell Research.

[91]  J. Keith Joung,et al.  Improving CRISPR-Cas nuclease specificity using truncated guide RNAs , 2014, Nature Biotechnology.

[92]  C. Pabo,et al.  DNA recognition by Cys2His2 zinc finger proteins. , 2000, Annual review of biophysics and biomolecular structure.

[93]  Feng Zhang,et al.  Selection-Free Zinc-Finger Nuclease Engineering by Context-Dependent Assembly (CoDA) , 2010, Nature Methods.

[94]  Xiaojun Zhu,et al.  Genome editing with RNA-guided Cas9 nuclease in Zebrafish embryos , 2013, Cell Research.

[95]  Elo Leung,et al.  A TALE nuclease architecture for efficient genome editing , 2011, Nature Biotechnology.

[96]  C. Radding,et al.  The role of exonuclease and beta protein of phage lambda in genetic recombination. II. Substrate specificity and the mode of action of lambda exonuclease. , 1971, The Journal of biological chemistry.

[97]  I. Katic,et al.  Targeted Heritable Mutation and Gene Conversion by Cas9-CRISPR in Caenorhabditis elegans , 2013, Genetics.

[98]  Frank Buchholz,et al.  A new logic for DNA engineering using recombination in Escherichia coli , 1998, Nature Genetics.

[99]  P. Duchateau,et al.  Meganucleases and Other Tools for Targeted Genome Engineering: Perspectives and Challenges for Gene Therapy , 2011, Current gene therapy.

[100]  T. Ochiya,et al.  Genome engineering of mammalian haploid embryonic stem cells using the Cas9/RNA system , 2013, PeerJ.

[101]  J. Keith Joung,et al.  TALENs: a widely applicable technology for targeted genome editing , 2012, Nature Reviews Molecular Cell Biology.

[102]  Neville E. Sanjana,et al.  Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells , 2014, Science.

[103]  Jin-Soo Kim,et al.  Preassembled zinc-finger arrays for rapid construction of ZFNs , 2011 .

[104]  Farshid Guilak,et al.  Synergistic and tunable human gene activation by combinations of synthetic transcription factors , 2013, Nature Methods.

[105]  A. Rashtchian,et al.  Novel methods for cloning and engineering genes using the polymerase chain reaction. , 1995, Current opinion in biotechnology.

[106]  Mike Boxem,et al.  CRISPR/Cas9-Targeted Mutagenesis in Caenorhabditis elegans , 2013, Genetics.

[107]  D. Nathans,et al.  Specific cleavage of simian virus 40 DNA by restriction endonuclease of Hemophilus influenzae. , 1971, Proceedings of the National Academy of Sciences of the United States of America.

[108]  Konstantin Severinov,et al.  Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence , 2011, Proceedings of the National Academy of Sciences.

[109]  J. LaBaer,et al.  Many paths to many clones: a comparative look at high-throughput cloning methods. , 2004, Genome research.

[110]  B. W. Lee,et al.  High-level expression of a codon optimized recombinant dust mite allergen, Blo t 5, in Chinese hamster ovary cells. , 2004, Biochemical and biophysical research communications.

[111]  D. Court,et al.  An efficient recombination system for chromosome engineering in Escherichia coli. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[112]  P. Sternberg,et al.  Transgene-Free Genome Editing in Caenorhabditis elegans Using CRISPR-Cas , 2013, Genetics.

[113]  Yilong Li,et al.  Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library , 2013, Nature Biotechnology.

[114]  Luke A. Gilbert,et al.  CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes , 2013, Cell.

[115]  D. Court,et al.  A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. , 2001, Genomics.

[116]  E. Rebar,et al.  Genome editing with engineered zinc finger nucleases , 2010, Nature Reviews Genetics.

[117]  Chris P. Ponting,et al.  Highly Efficient Targeted Mutagenesis of Drosophila with the CRISPR/Cas9 System , 2013, Cell reports.

[118]  K. Murphy,et al.  Use of Bacteriophage λ Recombination Functions To Promote Gene Replacement in Escherichia coli , 1998, Journal of bacteriology.

[119]  Ronnie J Winfrey,et al.  Rapid "open-source" engineering of customized zinc-finger nucleases for highly efficient gene modification. , 2008, Molecular cell.

[120]  Kabin Xie,et al.  RNA-guided genome editing in plants using a CRISPR-Cas system. , 2013, Molecular plant.

[121]  D. Hoover,et al.  DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis. , 2002, Nucleic acids research.

[122]  Philippe Horvath,et al.  The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli , 2011, Nucleic acids research.

[123]  R. Buller,et al.  Duplex strand joining reactions catalyzed by vaccinia virus DNA polymerase , 2006, Nucleic acids research.

[124]  N. Grishin,et al.  A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action , 2006, Biology Direct.

[125]  J. Keith Joung,et al.  Robust, synergistic regulation of human gene expression using TALE activators , 2013, Nature Methods.

[126]  R. Kolodner Genetic recombination of bacterial plasmid DNA: electron microscopic analysis of in vitro intramolecular recombination. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[127]  J. W. Little An exonuclease induced by bacteriophage lambda. II. Nature of the enzymatic reaction. , 1967, The Journal of biological chemistry.

[128]  George M. Church,et al.  Heritable genome editing in C. elegans via a CRISPR-Cas9 system , 2013, Nature Methods.

[129]  Jin-Soo Kim,et al.  Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases , 2014, Genome research.

[130]  Eunji Kim,et al.  Precision genome engineering with programmable DNA-nicking enzymes , 2012, Genome research.

[131]  C. Uyttenhove,et al.  Tumor rejection requires a CTLA4 ligand provided by the host or expressed on the tumor: superiority of B7-1 over B7-2 for active tumor immunization. , 1996, Journal of immunology.

[132]  Yoshio Kato,et al.  targeted gene knockout by direct delivery of zinc-finger nuclease proteins , 2012 .