Improved CRISPR‐Cas12a‐assisted one‐pot DNA editing method enables seamless DNA editing

As the clustered regularly interspaced short palindromic repeats (CRISPR)–Cas12a (previously known as Cpf1) system cleaves double‐stranded DNA and produces a sticky end, it could serve as a useful tool for DNA assembly/editing. To broaden its application, a variety of engineered FnCas12a proteins are generated with expanded protospacer adjacent motif (PAM) requirements. Two variants (FnCas12a‐EP15 and EP16) increased the targeting range of FnCas12a by approximately fourfold. They can efficiently recognize a broad range of PAM sequences including YN (Y = C or T), TAC and CAA. Meanwhile, based on our demonstration that FnCas12a is active from 16 to 60°C, we developed an "improved CRISPR‐Cas12a‐assisted one‐pot DNA editing" (iCOPE) method to facilitate DNA editing by combining the crRNA transcription, digestion, and ligation in one pot. By applying iCOPE, the editing efficiency reached 72–100% for two DNA fragment assemblies, and for the 21 kb large DNA construct modification, the editing efficiency can reach 100%. Thanks to the advantages of Cas12a, iCOPE with only one digestion enzyme could replace current a variety of restriction enzymes to perform the cloning in one pot with almost no sequence constraints. Taken together, this study offers an expanded DNA targeting scope of CRISPR systems and could serve as an efficient seamless one‐pot DNA editing tool.

[1]  D. Patel,et al.  Type V CRISPR-Cas Cpf1 endonuclease employs a unique mechanism for crRNA-mediated target DNA recognition , 2016, Cell Research.

[2]  J. Nielsen,et al.  Advancing biotechnology with CRISPR/Cas9: recent applications and patent landscape , 2018, Journal of Industrial Microbiology & Biotechnology.

[3]  Shiyuan Li,et al.  C-Brick: A New Standard for Assembly of Biological Parts Using Cpf1. , 2016, ACS synthetic biology.

[4]  Jeffry D. Sander,et al.  CRISPR-Cas systems for editing, regulating and targeting genomes , 2014, Nature Biotechnology.

[5]  Sungroh Yoon,et al.  Deep learning improves prediction of CRISPR–Cpf1 guide RNA activity , 2018, Nature Biotechnology.

[6]  Zengyi Shao,et al.  DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways , 2008, Nucleic acids research.

[7]  Guoping Zhao,et al.  Tandem assembly of the epothilone biosynthetic gene cluster by in vitro site-specific recombination , 2011, Scientific reports.

[8]  Qiang Wu,et al.  Precise and Predictable CRISPR Chromosomal Rearrangements Reveal Principles of Cas9-Mediated Nucleotide Insertion. , 2018, Molecular cell.

[9]  Bo Salomonsen,et al.  USER-derived cloning methods and their primer design. , 2014, Methods in molecular biology.

[10]  Carola Engler,et al.  A One Pot, One Step, Precision Cloning Method with High Throughput Capability , 2008, PloS one.

[11]  Feng Zhang,et al.  Engineered Cpf1 variants with altered PAM specificities increase genome targeting range , 2017, Nature Biotechnology.

[12]  J. Keasling,et al.  Rapid metabolic pathway assembly and modification using serine integrase site-specific recombination , 2013, Nucleic acids research.

[13]  A. Regev,et al.  Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System , 2015, Cell.

[14]  Hadi Bayat,et al.  The Conspicuity of CRISPR-Cpf1 System as a Significant Breakthrough in Genome Editing , 2017, Current Microbiology.

[15]  Xingxu Huang,et al.  sgRNAcas9: A Software Package for Designing CRISPR sgRNA and Evaluating Potential Off-Target Cleavage Sites , 2014, PloS one.

[16]  A. Stewart,et al.  ExoCET: exonuclease in vitro assembly combined with RecET recombination for highly efficient direct DNA cloning from complex genomes , 2017, Nucleic acids research.

[17]  Kornel Labun,et al.  CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering , 2016, Nucleic Acids Res..

[18]  Sylvestre Marillonnet,et al.  Fast track assembly of multigene constructs using Golden Gate cloning and the MoClo system , 2012 .

[19]  Wenjun Jiang,et al.  Cas9-Assisted Targeting of CHromosome segments CATCH enables one-step targeted cloning of large gene clusters , 2015, Nature Communications.

[20]  Tao Zhang,et al.  A CRISPR–Cpf1 system for efficient genome editing and transcriptional repression in plants , 2017, Nature Plants.

[21]  Charles E. Vejnar,et al.  CRISPRscan: designing highly efficient sgRNAs for CRISPR/Cas9 targeting in vivo , 2015, Nature Methods.

[22]  S. Brady,et al.  Multiplexed CRISPR/Cas9- and TAR-Mediated Promoter Engineering of Natural Product Biosynthetic Gene Clusters in Yeast. , 2016, ACS synthetic biology.

[23]  Carola Engler,et al.  Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes , 2009, PloS one.

[24]  D. G. Gibson,et al.  Enzymatic assembly of DNA molecules up to several hundred kilobases , 2009, Nature Methods.

[25]  S. Elledge,et al.  SLIC: a method for sequence- and ligation-independent cloning. , 2012, Methods in molecular biology.

[26]  Junhao Fu,et al.  A ‘new lease of life’: FnCpf1 possesses DNA cleavage activity for genome editing in human cells , 2017, Nucleic acids research.

[27]  W. Wang,et al.  Two-stage PCR protocol allowing introduction of multiple mutations, deletions and insertions using QuikChange Site-Directed Mutagenesis. , 1999, BioTechniques.

[28]  M. Jinek,et al.  Structural Plasticity of PAM Recognition by Engineered Variants of the RNA-Guided Endonuclease Cas9. , 2016, Molecular cell.

[29]  Yunkun Liu,et al.  In Vitro CRISPR/Cas9 System for Efficient Targeted DNA Editing , 2015, mBio.

[30]  Ines Fonfara,et al.  The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA , 2016, Nature.

[31]  Osamu Nureki,et al.  Structural Basis for the Altered PAM Specificities of Engineered CRISPR-Cas9. , 2016, Molecular cell.

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

[33]  Sheng Yang,et al.  CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum , 2017, Nature Communications.

[34]  Chase L. Beisel,et al.  Identifying and Visualizing Functional PAM Diversity across CRISPR-Cas Systems. , 2016, Molecular cell.

[35]  H. Nishimasu,et al.  Structural Basis for the Altered PAM Recognition by Engineered CRISPR-Cpf1. , 2017, Molecular cell.

[36]  Emmanuelle Charpentier,et al.  The Biology of CRISPR-Cas: Backward and Forward , 2018, Cell.

[37]  Huimin Zhao,et al.  Programmable DNA-Guided Artificial Restriction Enzymes. , 2017, ACS synthetic biology.

[38]  David R. Liu,et al.  Evolved Cas9 variants with broad PAM compatibility and high DNA specificity , 2018, Nature.