This work was supported by the grants from the Ministry of Science and Higher Education (0295/B/PO1/2008/34 to MB and N301 100 31/3043 to KS), from the Polish National Science Centre (NCN) (UMO-2011/02/A/NZ1/00052, UMO-2014/13/B/NZ1/03991 and UMO-2014/14/M/NZ5/00558 to MB) and by short term EMBO fellowship to KS (ATSF 277.00-05).

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redesign+of+extensive+protein-DNA+interfaces+of+meganucleases+using+iterative+cycles+of+in+vitro+compartmentalization.">Redesign of extensive protein-DNA interfaces of meganucleases using iterative cycles of in vitro compartmentalization.</a> Proceedings of the National Academy of Science U.S.A. 111 (11), 4061-4066 (2014).</li><li>Howland, J. L., Ausubel, F., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., Struhl, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Short Protocols in Molecular Biology. , (1995).</li><li>Wilson, D. S., Keefe, A. 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The authors have nothing to disclose.

The selection protocol described here was tested for NlaIV8, a dimeric PD-(D/E)XK fold recognition sequence that recognizes a palindromic target site with central NN bases and catalyzes a blunt end cut between the NN bases. NlaIV was picked because cleavage between the NN bases suggests that these bases are close to the protein in the complex. In principle, the protocol could be used for any sequence specific restriction endonuclease, monomeric or dimeric, of any fold group, catalyzing double strand breaks of any stagger, irrespective of whether catalytic and specificity domains coincide (as in the NlaIV example) or are separate (e.g., FokI). Moreover, the protocol in principle is useful not only for the generation of new, more narrow enzyme specificity, but could also be used to eliminate star activities, or to create high fidelity endonucleases. However, all this has not been tested yet. In particular, targeted elimination of star activity may be complicated, because the same amino acid residues could be involved in binding to the desired and undesired bases. The in vitro steps described in this protocol are not limited to the selection of narrowed down specificities but could also be used to select otherwise altered specificities. However, there is then a problem with variant endonucleases: if the spectrum of substrates includes novel targets not cleaved by the parental endonuclease, there is in general no good way to protect cells from the harmful effects of this activity. In contrast, if endonuclease specificity is only narrowed down, the targets are a subset of the wild type targets, and hence the already available cognate methyltransferase should be fully protective.
Our protocol differs in several respects from many directed evolution protocols. Open reading frame diversity is generated once at the beginning of the experiment, not in every iteration. Moreover, it is created by split-and-mix synthesis, rather than by EP-PCR. For NNS substitutions of codons, as used in this work, there are (4 x 4 x 2)6 ~ 1.07 x 109 combinations for six positions. Therefore, any given variant is present on average once in 1.7 fmoles of ESC. This capacity can be increased to seven positions by using synthesis with a mixture of 20 trinucleotide precursors that is offered by Glen Research or by decreasing mutation frequency in less promising positions with split-and-mix oligonucleotide synthesis. If possible, it is recommended to limit the extent of variation to six positions. Obviously, such mutagenesis targeting requires some preexisting knowledge about at least the regions of the REase involved in substrate binding. The split-and-mix protocol to generate diversity has clear advantages in comparison to EP-PCR. Using EP-PCR, we obtained unchanged variants and sequences carrying eight substitutions for NlaIV ESCs in the same EP-PCR (Table 4). The library from EP-PCR contains a substantial fraction of clones that should be avoided (wild type sequences, multiple substitutions, frameshift and nonsense mutations, and mutations in places unlikely to affect sequence specificity).
Our protocol also differs from many other directed evolution protocols by the presence of two sequential selection steps. Positive selection makes sure that the desired activity is retained, otherwise the biotin tag is not removed, and the coding sequence can be removed by pull-down. It is technically possible that the fortuitous emergence of a novel, non-overlapping specificity (e.g., GCATGC) could lead to severing of the biotin tag as well, if a suitable cleavage site is present near the desired cleavage, but not elsewhere. However, this should be highly unlikely. Negative selection removes open reading frames that code for enzymes that still have the undesired activity. This step is not strictly mandatory, because the protocol will still enrich the output library with variants that are able to cleave the selection sequence but not able to cleave elsewhere in the ESC, therefore rendering it unsuitable for PCR amplification. However, selection effectiveness is expected to be lower because enzymes with the original sequence specificity will not be removed from the output and will outcompete promising variants with altered specificity but also decreased enzymatic activity. Note that at the population level, both desired and undesired target sequences can, but need not be, degenerate. In the NlaIV example, the anti-target was degenerate and the target non-degenerate. Even when there is degeneracy at the population level, in a single droplet only one (non-degenerate) target or anti-target is present. In our protocol, target and anti-target sequences are reintroduced at every repetition of the selection steps. Therefore, an open reading frame must encode an enzyme capable of cleaving all possible targets, and unable to cleave any of the anti-targets, to survive multiple selection rounds. Notice that the need to reintroduce the antiselection target at each iteration of the protocol enforces two sequential PCRs. The first PCR uses a primer that anneals outside the anti-target, so that cleavage of the anti-target prevents the PCR reaction. The second PCR requires a primer that reaches beyond the anti-target, and reintroduces anti-target, to make sure that during multiple rounds of selection, each open reading frame is tested against all variants of the anti-target.
For enzymes that generate sticky ends, a related alternative protocol based on a previously described method for isolation of REase ORF10 can be used. The depletion of inactive variants by biotin capture that is used in our experiments is replaced in the alternative protocol by ligation of the compatible adapter with a sequence that is used as a primer binding site in a selective PCR (Figure 9). Only ESCs that produce enzymes with the selected specificity generate ligation-capable ends and will therefore be selected. The sequence of the sticky end of the counter eselected sequence must be designed in such a way that it cannot participate in ligation with adapters. Iteration of the selection process can be easily achieved by switching between two different adapters and consequently two different reverse primers in selective PCR.
Even with new protocols, the task of engineering novel specificities in vitro is still very challenging. For typical type II REases, sequence specificity and endonucleolytic activity depend on the same protein regions. It is therefore difficult to alter one without affecting the other. Success is made more likely by a strategy that takes into account the footprint of the enzyme, respects the symmetry of protein-DNA interactions, and builds on preexisting enzymatic preferences, which should be determined upfront in biochemical experiments, as was done for the NlaIV example8.

Sequence specificity engineering is extremely challenging for class II REases. In this class of endonucleases, sequence recognition and catalysis are closely intertwined, most probably as an evolutionary safeguard against creation of an endonuclease of broader specificity than its cognate methyltransferase, which would damage host DNA. Directed evolution of new specificities in cells is further complicated by the need to protect host DNA against the newly engineered endonuclease activity. Therefore, there are only a few successful attempts of REase engineering reported and all of them exploit the unique features of a particular enzyme1,2,3,4,5,6,7.
Here we provide a detailed protocol for specificity engineering that can be used to generate endonuclease variants that have narrower specificity than a parental enzyme that is based on our successful engineering of a NlaIV endonuclease8. For any such enzyme with an arbitrary recognition sequence, extra specificity can be introduced for bases in the flanks. For parental enzymes that recognize partially degenerate sequences (such as NlaIV with its GGNNCC target), additional specificity can also be introduced within the recognition sequence. As extra specificity will likely require protein-DNA contacts, the newly recognized bases should lie within the footprint of the parental endonuclease on DNA. In principle, selection schemes can be set up for any desired specialization of the recognition sequence. However, most REases that recognize palindromic and nearly palindromic target sequences are functional dimers that recognize only a half-site of the palindrome. Hence, selection of new specificities that violate the symmetry of protein nucleic interactions is unlikely to work. For the dimeric NlaIV, for example, the GGNNCC sequence can theoretically be narrowed down to GGATCC but narrowing the specificity down to GGAACC is expected to be more difficult. Our scheme involves both positive and negative selection.
The process is more efficient when negative selection is also used to remove the specificities able to cleave all sequences other than the preferred narrower specificity. For example, selection for GGATCC could be combined with antiselection against GGBVCC (where B is any base other than A, and V is any base other than T). When some of the possible target sequences are not covered, the outcome of the selection experiment depends on the effectiveness of positive and negative selection. In our NlaIV work, we selected for GGATCC, and against GGSSCC (where S is G or C), and obtained a specificity that, ignoring symmetry breaking targets, could be described as GGWWCC (where W is A or T), suggesting that in this particular case, negative selection was more important than positive selection.
Our approach starts with the creation of an expression selection cassette (ESC). The ESC is structured in sections. On the inside core section, there are variants of the open reading frame (ORF) of the REase, under T7 promoter control. This core section of the ESC cannot contain any cognate site for the engineered REase. The core is sandwiched between two cognate sites for wild type REase: a cleavage site for the undesired activity (counter selected sequence, GGSSCC in this example) and a cleavage site for the desired activity (selected sequence, GGATCC in the example). The final step of the preparation of the ESC in PCR adds biotin close to the desired activity at the 5&#39; end and creates a variety of counter selected sequences (GGSSCC in the example). The selection strategy relies on the use of carefully designed primers at the ESC reamplification protocol after an in vitro transcription/translation/selection protocol (Figure 1A). The ESC library is expressed in an in vitro compartmentalized transcription translation water-in-oil emulsion9,10,11. Within each droplet, the specificity of the expressed enzyme affects the state of the ESC (Figure 1B, step I). For the described arrangement, the desired cleavage activity of the translated protein removes the DNA&#39;s biotin tag but does not affect the other ESC end with the counter selected sequence. When the emulsion is broken, biotinylated fragments are removed by streptavidin affinity pulldown, so that only fragments from droplets with the desired activity remain (Figure 1B, step II). This step removes inactive REase variants. The supernatant fraction of the pull-down step is then amplified by PCR. In the first PCR reaction primers F2 and R1 are used (Figure 1A,B, step III). Primer F2 binds to the ESC section between the counter selected sequence and the molecule end. Therefore, ESCs expressing variants that are capable of cleaving the counter selected sequence (and, therefore, separate the binding sites for primers F2 and R1 into two different DNA molecules) are not amplified and are thus removed from the library. The primer R1 binds between the selected site and the core of the ESC so that it is not affected by the cleavage status of the selected site and restores the cleavage site for the desired activity (GGATCC). The cycle is closed by a second PCR (with primers F1 and R2) that adds biotin at the 5&#39; end close to the selected site and restores designed variation at the counter selected site close to the opposite end of the ESC (Figure 1B, step IV). The resulting DNA mixture is ready for another round of selection.
The success of the selection protocol depends strongly on the proper choice of the new, more stringent target recognition sequence and on careful design of the mutagenesis strategy and its effective implementation. Because it is much easier to improve upon slight preexisting preferences of the REase than to overcome them, we recommend starting with a kinetic study of any preexisting preferences. The necessity of careful mutagenesis design results from the limited size of a mutant library that can be processed by the presented protocol (109 clones in a single experiment). Therefore all 20 possible amino acid substitutions can be effectively tested in only a few positions (see Discussion). Random mutagenesis, such as error-prone PCR (EP-PCR) presented as an alternative method, will lead to profound undersampling of existing complexity. If any information concerning potential amino acid positions involved in contacts with DNA (or even located in a close proximity to the degenerate nucleotides in a cognate sequence) is available, it certainly should be used to select a few amino acids for oligonucleotide guided saturation mutagenesis (protocol steps 1.6-3.10).

<table><tbody><tr><td>1000&amp;Aring; CPG Support (dA, dT, dC, dG)</td><td>Biosset</td><td>45-1000-050</td><td>Other vendors can be used as well</td></tr><tr><td>ASM-800 DNA/RNA</td><td>Biosset</td><td>800-001-000</td><td></td></tr><tr><td>GeneJET Gel Extraction Kit</td><td>Thermo Scientific</td><td>K0691</td><td>Any other kit can be used</td></tr><tr><td>Glen-Pak DNA purification cartridge</td><td>Glen Research</td><td>60-5200</td><td></td></tr><tr><td>HIS-Select Nickel Affinity Gel</td><td>Sigma</td><td>P6611</td><td></td></tr><tr><td>pET 28a vector</td><td></td><td></td><td>Any other vector with T7 promoter upstream of plycloning site can be used instead</td></tr><tr><td>Phusion High-Fidelity DNA Polymerase</td><td>Thermo Scientific</td><td>F530S</td><td>Any other high fidelity and highly processive thermophilic polymearse can be used instead</td></tr><tr><td>Porous steel foil</td><td>Biosset</td><td>40-063</td><td></td></tr><tr><td>Rapid Translation System&lt;br/&gt; RTS 100, E.coli HY Kit</td><td>Roche</td><td>3 186 148</td><td></td></tr><tr><td>Restriction endonucleases</td><td>Thermo Scientific</td><td></td><td>Obviously other vendors, enzymes can be used</td></tr><tr><td>Streptavidin Magnetic Beads</td><td>New England Biolabs</td><td>S1420S</td><td>Other vendors can be used as well. We have positively tested beds form Sigma</td></tr><tr><td>Synthesis chemicals including phosphoramidities</td><td>Carl Roth</td><td></td><td>Other vendors can be used as well</td></tr><tr><td>Synthesis columns (different sizes)</td><td>Biosset</td><td></td><td></td></tr><tr><td>T4 DNA ligase</td><td>Thermo Scientific</td><td>EL0011</td><td>Any other ligase can be used</td></tr></tbody></table>

in vitro directed evolution of a restriction endonuclease with more stringent specificity

Restriction endonuclease (REase) specificity engineering is extremely difficult. Here we describe a multistep protocol that helps to produce REase variants that have more stringent specificity than the parental enzyme. The protocol requires the creation of a library of expression selection cassettes (ESCs) for variants of the REase, ideally with variability in positions likely to affect DNA binding. The ESC is flanked on one side by a sequence for the restriction site activity desired and a biotin tag and on the other side by a restriction site for the undesired activity and a primer annealing site. The ESCs are transcribed and translated in a water-in-oil emulsion, in conditions that make the presence of more than one DNA molecule per droplet unlikely. Therefore, the DNA in each cassette molecule is subjected only to the activity of the translated, encoded enzyme. REase variants of the desired specificity remove the biotin tag but not the primer annealing site. After breaking the emulsion, the DNA molecules are subjected to a biotin pulldown, and only those in the supernatant are retained. This step assures that only ESCs for variants that have not lost the desired activity are retained. These DNA molecules are then subjected to a first PCR reaction. Cleavage in the undesired sequence cuts off the primer binding site for one of the primers. Therefore, PCR amplifies only ESCs from droplets without the undesired activity. A second PCR reaction is then carried out to reintroduce the restriction site for the desired specificity and the biotin tag, so that the selection step can be reiterated. Selected open reading frames can be overexpressed in bacterial cells that also express the cognate methyltransferase of the parental REase, because the newly evolved REase targets only a subset of the methyltransferase target sites.

This protocol is just a tool to increase the frequency of desired variants of an engineered REase by depleting (but not eliminating) two unwanted classes: inactive enzymes and endonucleases with unchanged wild type sequence specificity. On the other hand, because changing REase specificity is extremely difficult, finding even one such variant producing a cleavage pattern that is different from the wild type enzyme in a single screening of 24 clones should be considered a success. In our hands the best screens could identify up to 20% of promising variants (Figure 8A).
The positive outcome strongly depends on a library quality (i.e., limited frequency of substitutions and their random distribution) and efficient capture of the biotinylated population of library members (steps 3.6&#8211;3.7). Both problems can be detected. The library quality should be checked prior to the selection by sequencing as many clones as possible (&#62;15) or by direct sequencing of the library by high throughput sequencing (step 3.10, Table 3). If a majority of the selected clones are not active, this is a clear indication of failure of the streptavidin capture selection. A similar effect is observed in the case of libraries that undergo many selection cycles, because such libraries are most probably dominated by inactive variants that escaped the streptavidin capture selection step (Figure 8B). Therefore, it is advisable to run screening after every selection cycle and further develop manually selected promising variants rather than to depend on selection iteration.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60807/60807fig1.jpg" /> 
Figure 1: In vitro selection of a new sequence specificity based on NlaIV engineering. (A) The organization of the expression/selection cassette (ESC) includes two recognition sites for REase, 1) the selected sequence (GGATCC) close to the right end and 2) the counter selected sequence (GGSSCC) close to the left end, as well as the T7p and T7t-T7 promoter and T7 terminator. The primer binding sites are shown below. Cleavage by wild type and selected NlaIV variants are shown as red and green triangles respectively. (B) Selection cycle steps: I) Emulsification of transcription-translation-cleavage reaction mixes with the ESC library; II) All biotinylated DNA is captured on magnetic particles coated with streptavidin and removed, thus removing encoding inactive variants; III) ESCs encoding REases with wild type activity (i.e., those able to cleave the GGSSCC sequence) are eliminated because cleavage of the sequence separates the binding sites for the forward and reverse primers. Therefore, no amplification of these ESCs occurs; IV) Input for the next selection round is created by addition of biotin on the right end and reintroducing variation of the counter selected sequences on the left end. Reprinted from Czapinska et al.8 with permission from Elsevier. <a href="https://www.jove.com/files/ftp_upload/60807/60807fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60807/60807fig2.jpg" /> 
Figure 2: Preparation of ESC. Fragment derived from the original construct in an expression vector containing NlaIV ORF under control of the T7 promoter was modified to be suitable for expression/selection. The NlaIV site downstream from the NlaIV ORF was removed and unique sites (SalI, EcoRI and Eco52I) that were used to mutagenize selected positions were introduced in the NlaIV ORF as silent mutations. The final construct was amplified with flanking primers that introduced two flanking NlaIV sites: The counter selected sequence (GGSSCC) on the left and selected sequence (GGATCC) on the right. The reverse primer also introduced biotin. Primers used in creation of mutated ECS are shown as blue arrows and labeled below (see Table 1B,C). <a href="https://www.jove.com/files/ftp_upload/60807/60807fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60807/60807fig3.jpg" /> 
Figure 3: Scheme of split and mix synthesis. The example refers to MutB primer synthesis where an NNS sequence was introduced at 0.8 frequency at four positions (see also Table 3). Note that chemical synthesis is carried out from 3&#39; to 5&#39; but all sequences are shown in canonical 5&#39;-3&#39; orientation (i.e., it proceeds from right to left in this scheme). Wild type sequences at mutagenized positions are shown in green while NNS mutagenic sequences are in red. The SalI recognition site that is later used to introduce mutations in ESCs is underlined. Points of mixing and splitting steps (2 and 4) are indicated. <a href="https://www.jove.com/files/ftp_upload/60807/60807fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60807/60807fig4.jpg" /> 
Figure 4: Use of unique restriction enzyme sites in oligonucleotide targeted mutagenesis. The strategy of mutation introduction is shown on an example of the construction of libraries A-C (see steps 3.1&#8211;3.7). Reprinted from Czapinska et al.8 with permission from Elsevier. <a href="https://www.jove.com/files/ftp_upload/60807/60807fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60807/60807fig5.jpg" /> 
Figure 5: Endonucleolytic cleavage in in vitro transcription-translation. (A) Cleavage of a test substrate in optimal REase buffer: 1) Substrate, 612 bp PCR product with a single NlaIV recognition site; 2) Cleavage products, 355 bp and 257 bp. (B) Cleavage in an in vitro transcription-translation reaction (containing 0.5 &#181;g of ESC): 1&#8211;2) 15 &#181;L aliquots of in vitro transcription translation without substrate (line 2: reaction supplemented with 1.5 mM MgCl2); 3&#8211;4) 15 &#181;L aliquots of in vitro transcription-translation with 1 &#181;g of test substrate; (line 4: reaction supplemented with 1.5 mM&#160;MgCl2). S-DNA size marker (pBR322 digested with MspI). Samples were resolved in 6% native PAGE. DNA was stained with ethidium bromide. <a href="https://www.jove.com/files/ftp_upload/60807/60807fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/60807/60807fig6.jpg" /> 
Figure 6: Products of the first PCR in the selection cycle. See Figure 1B, step III; protocol step 5.10. Column sets 1 and 2 are aliquots of two different libraries loaded in triplicate. S-DNA size standard (lambda DNA digested with HindIII and EcoRI). Arrow indicates position of the full-length ESC (1,050 bp). <a href="https://www.jove.com/files/ftp_upload/60807/60807fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/60807/60807fig7.jpg" /> 
Figure 7: NlaIV variants purified for further screening in mini scale. See step 6.3.11. Each line contains a 10 &#181;L aliquot of a different variant. S-protein molecular weight standard. Molecular mass of NaIV REase subunit is 29.9 kDa. <a href="https://www.jove.com/files/ftp_upload/60807/60807fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/60807/60807fig8.jpg" /> 
Figure 8: Examples of screening of NlaIV variants for sequence specificity alteration. See step 6.4.2. (A) Successful screening with high frequency of promising variants. S = DNA size marker, lambda DNA cleaved with HindIII and EcoRI; wild type (wt) = lambda DNA cleaved with wild type NlaIV; &#955; = lambda DNA substrate, not cleaved; other columns=variants with very low activity. Variants are labeled ! = promising variants that produce a cleavage pattern distinct from the wild type enzyme; ? = variants that also might have altered sequence preference. (B) Unsuccessful screening, with a majority of variants inactive and one variant with apparently unaltered cleavage pattern. <a href="https://www.jove.com/files/ftp_upload/60807/60807fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/60807/60807fig9.jpg" /> 
Figure 9: Alternative selection by ligation. This alternative can be used for all REases generating sticky ends. Here we present an example protocol for a selection scheme for MwoI enzyme (unpublished). I) Selected sequence (located at the right end of the ESC) with defined residues shown in red and selected variation of the cognate sequence shown in blue. In parentheses below the counter selected sequence to be placed at the left end of the ESC is shown; II) Product of MwoI cleavage; III) After terminating in vitro transcription/translation, products are purified and ligation is performed with excess adapter. Only the cleavage products that were cleaved in the selected sequence can participate in ligation. Therefore, inactive variants are eliminated, and the pulldown step is unnecessary. The cleavage product in the counter selected sequence (on left end of the ESC, not shown) cannot participate in this ligation because the protruding end of the adaptor is not complementary to the counter selected sequence; IV) Selective PCR uses the same strategy as in the main protocol to eliminate variants with the wild type degenerate sequence specificity (F1 primer binding distal to the counter selected site) whereas inactive variants are eliminated by the selective reverse primer that cannot bind to the uncleaved (and therefore not modified by adapter ligation) right end. In the next cycle the process can be iterated by using adaptor that is identical to the cleavage product of the preceding step (i.e., the &#34;cleaved cassette&#34; in panel III), and an appropriate selective reverse primer. <a href="https://www.jove.com/files/ftp_upload/60807/60807fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Primers used in NlaIV engineering. Sequences of the restriction sites mentioned in the comments are underlined. Small letters indicate sequences that do not have complements in the DNA templates. <a href="https://www.jove.com/files/ftp_upload/60807/table1.xlsx" target="_blank">Please click here to view this table (Right click to download).</a>
Table 2: Conditions of PCR reactions to be used in the protocol. Tm = primer melting temperature (if Tm is different for the primers, the lower Tm should be used). <a href="https://www.jove.com/files/ftp_upload/60807/table2.xlsx" target="_blank">Please click here to view this table (Right click to download).</a>
Table 3: Results of quality check of two mutagenic primers synthesized with split-and-mix strategy. Mutagenized codons are indicated with [XXX]. A lower index number indicates the position of an encoded amino acid. Adapted from Czapinska et al.8 with permission from Elsevier. <a href="https://www.jove.com/files/ftp_upload/60807/table3.xlsx" target="_blank">Please click here to view this table (Right click to download).</a>
Table 4: Results of EP-PCR. Main parameters derived from sequence analysis of 22 clones of ECS. <a href="https://www.jove.com/files/ftp_upload/60807/table4.xlsx" target="_blank">Please click here to view this table (Right click to download).</a>

Watch this Scientific Journal Video about In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity at JoVE.com

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

Restriction endonucleases with new sequence specificity can be developed from enzymes recognizing a partially degenerate sequence. Here we provide a detailed protocol that we successfully used to alter the sequence specificity of NlaIV enzyme. Key ingredients of the protocol are the in vitro compartmentalization of the transcription/translation reaction and selection of variants with new sequence specificities.

Restriction endonuclease (REase) specificity engineering is extremely difficult. Here we describe a multistep protocol that helps to produce REase ...

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Research

JoVE Journal

Genetics

7.2K Views.  International Institute of Molecular and Cell Biology, Warsaw (IIMCB). Ons protocol maakt het mogelijk om restrictieenzymen te maken met gewijzigde, strengere sequentiespecificiteiten met behulp van in vitro compartimentering en een unieke selectiestrategie in gerichte evolutie. Potentieel is het vrij universeel, aangezien het op om het even welke beperking van toepassing zou moeten zijn endonuclease en de selectie naar om het even welke striktere versie van een originele cognateopeenvolging kan worden geleid. Als nieuw gecreëerde varianten worden uitgedrukt do...

Video: In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution

The development of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) into therapeutic modalities requires the avoidance of its potentially deleterious off-target effects. Several methods have been devised to reduce such effects. Here, we present an Escherichia coli-based directed evolution method called Sniper-screen to obtain a Cas9 variant with optimized specificity and retained on-target activity, called Sniper-Cas9. Using Sniper-screen, positive and negative selection can be performed simultaneously. The screen can also be repeated with other single-guide RNA (sgRNA) sequences to enrich for the true positive hits. By using the CMV-PltetO1 dual promoter to express Cas9 variants, the performance of the pooled library can be quickly checked in mammalian cells. Methods to increase the specificity of Sniper-Cas9 are also described. First, the use of truncated sgRNAs has previously been shown to increase Cas9 specificity. Unlike other engineered Cas9s, Sniper-Cas9 retains a wild-type (WT) level of on-target activity when combined with truncated sgRNAs. Second, the delivery of Sniper-Cas9 in a ribonucleoprotein (RNP) format instead of a plasmid format is possible without affecting its on-target activity.

Here, we present a protocol to optimize CRISPR-Cas9 to achieve a higher specificity without the loss of on-target activity. We use a directed evolution approach called Sniper-screen to find a mutant Cas9 with the desired characteristics. Sniper-Cas9 is compatible with truncated single-guide RNAs and delivery in a ribonucleoprotein format, well-known strategies for achieving higher specificities.

The development of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) into therapeutic modalities requires the ...

A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes

Directed evolution is defined as a method to harness natural selection in order to engineer proteins to acquire particular properties that are not associated with the protein in nature. Literature has provided numerous examples regarding the implementation of directed evolution to successfully alter molecular specificity and catalysis1. The primary advantage of utilizing directed evolution instead of more rational-based approaches for molecular engineering relates to the volume and diversity of variants that can be screened2. One possible application of directed evolution involves improving structural stability of bacteriolytic enzymes, such as endolysins. Bacteriophage encode and express endolysins to hydrolyze a critical covalent bond in the peptidoglycan (i.e. cell wall) of bacteria, resulting in host cell lysis and liberation of progeny virions. Notably, these enzymes possess the ability to extrinsically induce lysis to susceptible bacteria in the absence of phage and furthermore have been validated both in vitro and in vivo for their therapeutic potential3-5. The subject of our directed evolution study involves the PlyC endolysin, which is composed of PlyCA and PlyCB subunits6. When purified and added extrinsically, the PlyC holoenzyme lyses group A streptococci (GAS) as well as other streptococcal groups in a matter of seconds and furthermore has been validated in vivo against GAS7. Significantly, monitoring residual enzyme kinetics after elevated temperature incubation provides distinct evidence that PlyC loses lytic activity abruptly at 45 &deg;C, suggesting a short therapeutic shelf life, which may limit additional development of this enzyme. Further studies reveal the lack of thermal stability is only observed for the PlyCA subunit, whereas the PlyCB subunit is stable up to ~90 &deg;C (unpublished observation). In addition to PlyC, there are several examples in literature that describe the thermolabile nature of endolysins. For example, the Staphylococcus aureus endolysin LysK and Streptococcus pneumoniae endolysins Cpl-1 and Pal lose activity spontaneously at 42 &deg;C, 43.5 &deg;C and 50.2 &deg;C, respectively8-10. According to the Arrhenius equation, which relates the rate of a chemical reaction to the temperature present in the particular system, an increase in thermostability will correlate with an increase in shelf life expectancy11. Toward this end, directed evolution has been shown to be a useful tool for altering the thermal activity of various molecules in nature, but never has this particular technology been exploited successfully for the study of bacteriolytic enzymes. Likewise, successful accounts of progressing the structural stability of this particular class of antimicrobials altogether are nonexistent. In this video, we employ a novel methodology that uses an error-prone DNA polymerase followed by an optimized screening process using a 96 well microtiter plate format to identify mutations to the PlyCA subunit of the PlyC streptococcal endolysin that correlate to an increase in enzyme kinetic stability (Figure 1). Results after just one round of random mutagenesis suggest the methodology is generating PlyC variants that retain more than twice the residual activity when compared to wild-type (WT) PlyC after elevated temperature treatment.

A novel directed evolution method specific to the field of thermostability engineering was developed and consequently validated for bacteriolytic enzymes. After only one round of random mutagenesis, an evolved bacteriolytic enzyme, PlyC 29C3, displayed greater than twice the residual activity when compared to the wild-type protein after elevated temperature incubation.

Directed evolution is defined as a method to harness natural selection in order to engineer proteins to acquire particular properties that are not ...

Immunology and Infection

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic acid (sRNA) molecules and target messenger RNAs (mRNAs). In addition, site-directed mutagenesis is used to map specific protein binding sites to RNA. A 2-step and 3-step PCR based introduction of mutations is described. The approach is relevant to all protein-RNA and RNA-RNA interaction studies. In short, the technique relies on designing primers with the desired mutation(s), and through 2 or 3 steps of PCR synthesizing a PCR product with the mutation. The PCR product is then used for cloning. Here, we describe how to perform site-directed mutagenesis with both the 2- and 3-step approach to introduce mutations to the sRNA, McaS, and the mRNA, csgD, to investigate RNA-RNA and RNA-protein interactions. We apply this technique to investigate RNA interactions; however, the technique is applicable to all mutagenesis studies (e.g., DNA-protein interactions, amino-acid substitution/deletion/addition). It is possible to introduce any kind of mutation except for non-natural bases but the technique is only applicable if a PCR product can be used for downstream application (e.g., cloning and template for further PCR).

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in deoxyribonucleic acid (DNA). This protocol describes how to do site-directed mutagenesis with a 2-step and 3-step polymerase chain reaction (PCR) based approach, which is applicable to any DNA fragment of interest.

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic ...

Biochemistry

Reverse Genetics to Engineer Positive-Sense RNA Virus Variants

The lack of a convenient method for the iterative generation of diverse full-length viral variants has impeded the study of directed evolution in RNA viruses. By integrating a full RNA genome error-prone PCR and reverse genetics, random genome-wide substitution mutagenesis can be induced. We have developed a method using this technique to synthesize diverse libraries to identify viral mutants with phenotypes of interest. This method, called full-length mutant RNA synthesis (FL-MRS), offers the following advantages: (i) the ability to create a large library via a highly efficient one-step error-prone PCR; (ii) the ability to create groups of libraries with varying levels of genetic diversity by manipulating the fidelity of DNA polymerase; (iii) the creation of a full-length PCR product that can directly serve as a template for mutant RNA synthesis; and (iv) the ability to create RNA that can be delivered into host cells as a non-selected input pool to screen for viral mutants of the desired phenotype. We have found, using a reverse genetics approach, that FL-MRS is a reliable tool to study viral-directed evolution at all stages in the life cycle of the hepatitis C virus, JFH1 isolate. This technique appears to be an invaluable tool to employ directed evolution to understand adaptation, replication, and the role of viral genes in pathogenesis and antiviral resistance in positive-sense RNA viruses.

The protocol describes a method for introducing controllable genetic diversity in the hepatitis C virus genome by combining full-length mutant RNA synthesis using error-prone PCR and reverse genetics. The method provides a model for phenotype selection and can be used for 10 kb long positive-sense RNA virus genomes.

The lack of a convenient method for the iterative generation of diverse full-length viral variants has impeded the study of directed evolution in RNA ...

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

Biology

Substrate Generation for Endonucleases of CRISPR/Cas Systems

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) 1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among traditional technologies, RNA interference suffers from partial target abrogation and the requirement for life-long treatments. In contrast, artificial nucleases can impose stable gene inactivation through induction of a DNA double strand break (DSB), but recent studies are questioning the safety of this approach. Targeted epigenetic editing via engineered transcriptional repressors (ETRs) may represent a solution, as a single administration of specific ETR combinations can lead to durable silencing without inducing DNA breaks.
ETRs are proteins containing a programmable DNA-binding domain (DBD) and effectors from naturally occurring transcriptional repressors. Specifically, a combination of three ETRs equipped with the KRAB domain of human ZNF10, the catalytic domain of human DNMT3A and human DNMT3L, was shown to induce heritable repressive epigenetic states on the ETR-target gene. The hit-and-run nature of this platform, the lack of impact on the DNA sequence of the target, and the possibility to revert to the repressive state by DNA demethylation on demand, make epigenetic silencing a game-changing tool. A critical step is the identification of the proper ETRs&#39; position on the target gene to maximize on-target and minimize off-target silencing. Performing this step in the final ex vivo or in vivo preclinical setting can be cumbersome.
Taking the CRISPR/catalytically dead Cas9 system as a paradigmatic DBD for ETRs, this paper describes a protocol consisting of the in vitro screen of guide RNAs (gRNAs) coupled to the triple-ETR combination for efficient on-target silencing, followed by evaluation of the genome-wide specificity profile of top hits. This allows for reduction of the initial repertoire of candidate gRNAs to a short list of promising ones, whose complexity is suitable for their final&#160;evaluation in the therapeutically relevant setting of interest.

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Here, we present a protocol for the in vitro selection of engineered transcriptional repressors (ETRs) with high, long-term, stable, on-target silencing efficiency and low genome-wide, off-target activity. This workflow allows for reducing an initial, complex repertoire of candidate ETRs to a short list, suitable for further evaluation in therapeutically relevant settings.

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among ...

Bioengineering

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that involves the construction, cloning and expression of mutant libraries, coupled to high frequency homologous DNA recombination in vivo. Here, we present a protocol to create and screen mutant libraries in yeast based on the example of a fungal aryl-alcohol oxidase (AAO) to enhance its total activity. Two protein segments were subjected to focused-directed evolution by random mutagenesis and in vivo DNA recombination. Overhangs of ~50 bp flanking each segment allowed the correct reassembly of the AAO-fusion gene in a linearized vector giving rise to a full autonomously replicating plasmid. Mutant libraries enriched with functional AAO variants were screened in S. cerevisiae supernatants with a sensitive high-throughput assay based on the Fenton reaction. The general process of library construction in S. cerevisiae described here can be readily applied to evolve many other eukaryotic genes, avoiding extra PCR reactions, in vitro DNA recombination and ligation steps.

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

We present a detailed protocol to construct and screen mutant libraries for directed evolution campaigns in Saccharomyces cerevisiae.

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that ...

Conservative Site-specific Recombination and Phase Variation

Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP sites are around 34 basepairs in length. LoxP sites contain 13 bp palindromic sequences, meaning that the nucleotide sequence reads the same in both 5&#8217; to 3&#8217; and 3&#8217; to 5&#8217; directions. Site-specific recombination mediated by Cre recombinases is one of the most popular methods used in the creation of transgenic mice with acquired mutations. Using thermostable variants of Cre recombinase with tissue specific promoters allows for spatial control over Cre recombinase&#39;s expression and action. For instance, placing a kidney-specific Cadherin promoter upstream of the Cre gene allows the enzyme to be expressed only in renal tissues. For temporal control of Cre recombinase activity, the enzyme gene is fused with a ligand binding domain so that the enzyme is expressed only in the specific ligand&#8217;s presence.
A major limitation in using site-specific recombination as a genome editing tool is that the recombination target site or sites must be first inserted or must be present by chance. If a genomic site congruent with the enzyme recognition site can be preselected, the recombinases can be used with &#8220;made-to-order&#8221; target specificity. Recent studies have used mutagenesis and gene shuffling to design Flp variants that can functionally recognize sites with combinatorial mutations. The results are promising for future iterations of gene shuffling that can yield more specific Flp variants and can be used commercially as a molecular tool for engineering large genomes.

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

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