Development and applications of programmable DNA-guided Argonaute-based artificial restriction enzymes

Enghiad, Behnam

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https://hdl.handle.net/2142/113234 Copy

Description

Title

Development and applications of programmable DNA-guided Argonaute-based artificial restriction enzymes

Author(s)

Enghiad, Behnam

Issue Date

2020-12-03

Director of Research (if dissertation) or Advisor (if thesis)

Zhao, Huimin

Doctoral Committee Chair(s)

Zhao, Huimin

Committee Member(s)

• Metcalf, William W
• Rao, Christopher V
• Shukla, Diwakar

Department of Study

Chemical & Biomolecular Engr

Discipline

Chemical Engineering

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Argonaute
• Artificial Restriction Enzymes
• DNA Assembly
• Natural Products

Abstract

Restriction enzymes or formally known as restriction endonucleases are a class of nuclease enzymes which recognize short DNA sequences and cleave DNA molecules at or near their recognition site. Type II restriction enzymes are capable of cleavage of DNA at a fixed location with respect to their recognition sequence and some type II restriction enzymes are able to generate defined cohesive ends (a.k.a sticky ends) on DNA molecules after cleavage. Because of these two remarkable features, upon their discovery, type II restriction enzymes revolutionized molecular biology and helped give rise to the field of modern biotechnology. To date, type II restriction enzymes still play a major role in biological research with more than 600 enzymes with >235 distinct sequence specificities commercially available. While type II restriction enzymes are able to cleave DNA molecules specifically, they only recognize short DNA sequences (4-8 base pairs) which limits some of their applications. To address this challenge, artificial restriction enzymes (AREs) such as ZFNs, TALENs, or CRISPR-Cas nucleases were developed. While these AREs have longer recognition sequences compared to type II restriction enzymes, they are not able to produce defined sticky ends on DNA molecules or target all desired DNA sequences which significantly constrains their applications in vitro. In this dissertation, I describe the development and applications of a new class of artificial restriction enzymes capable of targeting virtually any DNA sequences with high specificities and generating defined sticky ends of varying length. Argonaute proteins are a family of nucleic acid guide-dependent proteins which can be found in all domains of life. Some prokaryotic Argonaute proteins (pAgos) are able to use short single-stranded DNA molecules as guides to target complementary DNA sequences. I first utilized this capability of pAgos and developed a Pyrococcus furiosus Argonaute (PfAgo) based platform for generation of programmable DNA-guided artificial restriction enzymes. This platform was used to generate 18 AREs for DNA fingerprinting and molecular cloning of PCR-amplified or genomic DNAs. Next, I studied the potential of other pAgos for use as AREs. Through these studies, I was able to create an engineered version of PfAgo enzyme which demonstrates lower non-guided nuclease activity as well as higher specificity for cleavage of high GC-content DNA sequences compared to the wild-type PfAgo enzyme. To demonstrate some of the applications of the newly developed AREs, I first created a method for rapid and highly accurate assembly of linear DNA molecules by PfAgo-based AREs. Using this method, plasmid DNA molecules up to 27 kb in size can be assembled from up to 10 DNA fragments with high efficiencies. This method also exhibits extremely low error rates and is able to assemble DNA molecules containing sequence repeats as well as DNA molecules with high GC-content. Next, I evaluated the capability and limitations of PfAgo-based AREs in direct cloning of microbial biosynthetic gene clusters (BGCs). Using PfAgo-based AREs, I was able to clone microbial BGCs ranging from 13-42 kb in size from both Bacillus and Streptomyces species with high efficiencies. However, PfAgo-based AREs did not exhibit 100% success rate for this application. As a result, I developed an alternative method for cloning microbial BGCs named Cas12a assisted precise targeted cloning using in vivo Cre-lox recombination (CAPTURE). This method which consists of Cas12a digestion, a newly developed DNA assembly approach termed T4 polymerase exo + fill-in DNA assembly, and Cre-lox in vivo DNA circularization, is capable of cloning microbial natural product BGCs ranging from 10-113 kb in size regardless of their GC-content or repetitive DNA sequence with ~100% cloning efficiency and success rate.

Graduation Semester

2020-12

Type of Resource

Thesis

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http://hdl.handle.net/2142/113234

Copyright and License Information

Copyright 2020 Behnam Enghiad

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