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Modern Methods for Laboratory Diversification of Biomolecules Bratulic and Badran 51 Available online at www.sciencedirect.com ScienceDirect Modern methods for laboratory diversification of biomolecules Sinisa Bratulic and Ahmed H Badran Genetic variation fuels Darwinian evolution, yet spontaneous to the success of a directed evolution campaign, as the mutation rates are maintained at low levels to ensure cellular sequence landscape of a standard protein or biopolymer is viability. Low mutation rates preclude the exhaustive typically too vast to be exhaustively searched [1]. Where a exploration of sequence space for protein evolution and priori information is limited, unbiased and random in vitro genome engineering applications, prompting scientists to [2,3] or in vivo [4–7] mutagenesis methods have success- develop methods for efficient and targeted diversification of fully generated libraries of variants with improved or nucleic acid sequences. Directed evolution of biomolecules novel functionalities. Alternatively, bioinformatics, struc- relies upon the generation of unbiased genetic diversity to tural, or biochemical information can be leveraged to discover variants with desirable properties, whereas genome- comprehensively explore a portion of the variant land- engineering applications require selective modifications on a scape by focusing mutagenesis on functionally relevant genomic scale with minimal off-targets. Here, we review the sites [8,9]. Finally, newly identified beneficial mutations current toolkit of mutagenesis strategies employed in directed can be isolated or integrated into single sequences using evolution and genome engineering. These state-of-the-art in vitro [10,11] or in vivo [12 ,13] recombination methods. methods enable facile modifications and improvements of Recently, laboratory evolution has shifted to techniques single genes, multicomponent pathways, and whole genomes that directly couple the diversification and assessment for basic and applied research, while simultaneously paving the steps, providing the basis for continuous in vivo evolution way for genome editing therapeutic interventions. strategies [14] that streamline previously lengthy experi- ments and minimize the need for human intervention. Address Whereas these methods have proven crucial for studying Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA structure-function relationships of single macromolecules Corresponding author: Badran, Ahmed H ([email protected]) [15], functional genomic screening and genome engineer- ing applications typically require unbiased methods for in situ genome modification. Early methods to produce Current Opinion in Chemical Biology 2017, 41:50–60 strain libraries relied on treatments with chemical muta- This review comes from a themed issue on Mechanistic biology gens/stressors [16] or transposon mutagenesis [17], and Edited by Gregory A. Weiss later integrated targeted methods employing homologous For a complete overview see the Issue and the Editorial recombination capabilities, or recombineering (recombina- Available online 2nd November 2017 tion-mediated genetic engineering) [18]. These http://dx.doi.org/10.1016/j.cbpa.2017.10.010 approaches have been applied in both eukaryotes [19] and prokaryotes [20], and extended to enable in vivo ã 1367-5931/ 2017 Elsevier Ltd. All rights reserved. continuous genome engineering [21 ,22]. The recent discovery of CRISPR-Cas9 systems has reshaped this field, owing to their effectiveness as programmable nucleases or DNA-binding domains, and enabling novel, comparatively facile strategies for targeted diversification Introduction in cells [23]. Naturally occurring biomolecules are products of evolu- tion in service of the survival of an organism, but they In this review we focus on novel approaches for the often lack the catalytic efficiency, specificity, or stability diversification of biomolecules and generation of variant necessary for industrial or therapeutic applications. To libraries, which underlie the application of evolutionary improve these properties, proteins and other biopolymers principles in molecular biology research and engineering. are subjected to rounds of directed evolution, a powerful We first discuss untargeted mutagenesis methods that are and flexible scheme to systematically endow biomole- commonly applied in generating diversity for directed cules with desirable traits. This approach can employ a protein evolution, and highlight novel methods that have variety of mutagenesis strategies to modulate the fre- been developed over the past decade. We transition to quency, distribution, and spectrum of mutations to more targeted mechanisms of creating diversity, and explore biomolecule sequence landscapes, and applies address recent advances in targeted genome modifica- selections or screens to identify and assess improved tions with an emphasis on the latest developments in variants. The choice of diversification strategy is critical CRISPR-based systems. Current Opinion in Chemical Biology 2017, 41:50–60 www.sciencedirect.com Modern methods for laboratory diversification of biomolecules Bratulic and Badran 51 Diversification methods for directed protein through Mu transposon mutagenesis [33], where the gene evolution and functional studies of interest bridges a TAT periplasm-directing signal and Random mutagenesis TEM-1 b-lactamase, ensuring that only an in-frame À9 Natural mutation rates are low (10 mutations/nucleo- transposition event creates a functional TAT-b-lactamase tide/generation [24]) and, therefore, inappropriate for product. diversification of nucleic acid sequences in a laboratory setting. Laboratory evolution of biomolecules critically Conversely, approaches that do not rely on PCR can depends on elevated mutation rates for the discovery of simplify library generation by minimizing researcher improved or novel activities, mirroring biological princi- intervention. In error-prone rolling circle amplification ples that exist naturally (e.g. somatic hypermutation is (epRCA) [34], isothermal amplification and mutagenesis employed to generate substantial antibody diversity [25]). are coupled, and RCA-generated libraries can be directly Early protein evolution efforts catalyzed the develop- transformed into Escherichia coli without further proces- ment of approaches to increase mutation rates in a sing by restriction and ligation reactions (Table 1). RCA sequence-independent fashion to facilitate the unbiased can also be combined with Kunkel mutagenesis [35] in construction of large and diverse gene libraries, spear- selective RCA (sRCA) mutagenesis [36]. Plasmids are À À headed by techniques like error-prone PCR (epPCR). first produced from an ung dut E. coli strain to undergo PCR protocols can be modified to reduce the fidelity of non-specific uridylation (dT!dU), and subsequently the reaction by modulating buffer composition [26] and amplified by PCR using mutagenic primers. Treatment dNTP ratios [3], introducing nucleoside analogues [2], with uracil-DNA glycosylase (UDG) creates abasic sites using proofreading-deficient polymerases [27,28], or in the uracil-containing template, leaving only the muta- treating oligonucleotides with chemical mutagens [29]. genized product for amplification by RCA. The sRCA While these various approaches increase overall mutation approach increases effective library sizes and improves rates, the distribution of specific base changes that are the mutagenesis efficiency by eliminating the non- generated can limit the chemical diversity of the resultant mutated background sequences (Table 1). libraries. This distribution, called the mutational spec- trum, cumulatively describes the efficiency and bias of Compared to in vitro mechanisms that require discreet sequence space exploration by a mutagenesis method. manipulations to achieve the desired mutagenesis, in vivo Despite widespread implementation, epPCR suffers mutagenesis approaches take inspiration from naturally from a bias in mutational spectrum (Table 1) to predomi- occurring cellular, error-prone replication machinery. nately incorporate transitions (A$G or T$C), yielding Early attempts at in vivo mutagenesis were inspired by libraries enriched in synonymous mutations or conserva- SOS response and relied on its components [25], where tive nonsynonymous mutations given the redundancy and mutator strains [4] enabled elevated mutagenesis in vivo, assignments of the 64 natural codons. The sequence but to date these approaches have lacked mechanisms to saturation mutagenesis (SeSaM) [30 ] method was devel- control the resultant mutation rates and spectra (Table 1). oped to specifically address this bias (Table 1), where the In instances where unbiased, whole organism mutagene- promiscuous base-pairing nucleotide inosine is enzymat- sis is desirable (e.g. for genome, plasmid, and viral evo- ically incorporated in the variant library and later replaced lution), a mutagenesis plasmid (MP) system encoding with canonical nucleotides through standard PCR. A inducible dominant mutator alleles can be employed recent improvement, SeSam-Tv-II [31 ], increases the across variable E. coli strains. This in vivo mutagenesis likelihood of consecutive mutations, especially double approach was developed to enable control over a broad transversions (A/G$C/T), thereby improving library dynamic range of mutation rates concomitant with an quality and generating variants that are typically inacces- unbiased mutational spectrum (Figure 1a) [6]. sible by conventional epPCR [31 ]. Traditional in vivo mutagenesis approaches can also While epPCR-based methods
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