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).

<ol>
	<li>Sch&#246;ttler, S., Wenz, C., Lanio, T., Jeltsch, A., Pingoud, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+engineering+of+the+restriction+endonuclease+EcoRV--structure-guided+design+of+enzyme+variants+that+recognize+the+base+pairs+flanking+the+recognition+site.">Protein engineering of the restriction endonuclease EcoRV--structure-guided design of enzyme variants that recognize the base pairs flanking the recognition site.</a> European Journal of Biochemistry. 258 (1), 184-191 (1998).</li><li>Wenz, C., Hahn, M., Pingoud, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+variants+of+the+restriction+endonuclease+EcoRV+that+depend+in+their+cleavage+activity+on+the+flexibility+of+sequences+flanking+the+recognition+site.">Engineering of variants of the restriction endonuclease EcoRV that depend in their cleavage activity on the flexibility of sequences flanking the recognition site.</a> Biochemistry. 37 (8), 2234-2242 (1998).</li><li>Samuelson, J. C., Xu, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+evolution+of+restriction+endonuclease+BstYI+to+achieve+increased+substrate+specificity.">Directed evolution of restriction endonuclease BstYI to achieve increased substrate specificity.</a> Journal of Molecular Biology. 319 (3), 673-683 (2002).</li><li>Samuelson, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+a+rare-cutting+restriction+enzyme:+genetic+screening+and+selection+of+NotI+variants.">Engineering a rare-cutting restriction enzyme: genetic screening and selection of NotI variants.</a> Nucleic Acids Research. 34 (3), 796-805 (2006).</li><li>Rimseliene, R., Maneliene, Z., Lubys, A., Janulaitis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+restriction+endonucleases:+using+methylation+activity+of+the+bifunctional+endonuclease+Eco57I+to+select+the+mutant+with+a+novel+sequence+specificity.">Engineering of restriction endonucleases: using methylation activity of the bifunctional endonuclease Eco57I to select the mutant with a novel sequence specificity.</a> Journal of Molecular Biology. 327 (2), 383-391 (2003).</li><li>Morgan, R. D., Luyten, Y. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+engineering+of+type+II+restriction+endonuclease+DNA+binding+and+cleavage+specificity.">Rational engineering of type II restriction endonuclease DNA binding and cleavage specificity.</a> Nucleic Acids Research. 37 (15), 5222-5233 (2009).</li><li>Skowronek, K., Boniecki, M. J., Kluge, B., Bujnicki, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+engineering+of+sequence+specificity+in+R.MwoI+restriction+endonuclease.">Rational engineering of sequence specificity in R.MwoI restriction endonuclease.</a> Nucleic Acids Research. 40 (17), 8579-8592 (2012).</li><li>Czapinska, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+Structure+and+Directed+Evolution+of+Specificity+of+NlaIV+Restriction+Endonuclease.">Crystal Structure and Directed Evolution of Specificity of NlaIV Restriction Endonuclease.</a> Journal of Molecular Biology. 431 (11), 2082-2094 (2019).</li><li>Miller, O. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+evolution+by+in+vitro+compartmentalization.">Directed evolution by in vitro compartmentalization.</a> Nature Methods. 3 (7), 561-570 (2006).</li><li>Zheng, Y., Roberts, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+restriction+endonucleases+using+artificial+cells.">Selection of restriction endonucleases using artificial cells.</a> Nucleic Acids Research. 35 (11), e83 (2007).</li><li>Takeuchi, R., Choi, M., Stoddard, B. 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. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+8+Unit+8.3:+Random+mutagenesis+by+PCR.">Chapter 8 Unit 8.3: Random mutagenesis by PCR.</a> Current Protocols in Molecular Biology. , (2001).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1000&#197; 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 
			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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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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Nanyang Technological University

Research

JoVE Journal

Genetics

7.2K Views.  International Institute of Molecular and Cell Biology, Warsaw (IIMCB). 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.

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

Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection

We describe a low cost, configurable morbidostat for characterizing the evolutionary pathway of antibiotic resistance. The morbidostat is a bacterial culture device that continuously monitors bacterial growth and dynamically adjusts the drug concentration to constantly challenge the bacteria as they evolve to acquire drug resistance. The device features a working volume of ~10 ml and is fully automated and equipped with optical density measurement and micro-pumps for medium and drug delivery. To validate the platform, we measured the stepwise acquisition of trimethoprim resistance in Escherichia coli MG 1655, and integrated the device with a multiplexed microfluidic platform to investigate cell morphology and antibiotic susceptibility. The approach can be up-scaled to laboratory studies of antibiotic drug resistance, and is extendible to adaptive evolution for strain improvements in metabolic engineering and other bacterial culture experiments.

We describe a low cost, configurable morbidostat that enables the characterization of antibiotic drug resistance by dynamically adjusting the drug concentration. The device can be integrated with a multiplexed microfluidic platform. The approach can be scaled up for laboratory antibiotic drug resistance studies.

We describe a low cost, configurable morbidostat for characterizing the evolutionary pathway of antibiotic resistance. The morbidostat is a bacterial ...

Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) refers to the experimental situation in which evolution is observed using living organisms under controlled conditions and stressors; organisms are thereby artificially forced to make evolutionary changes. Microorganisms are subject to a variety of stressors in the environment and are capable of regulating certain stress-inducible proteins to increase their chances of survival. Naturally occurring spontaneous mutations bring about changes in a microorganism's genome that affect its chances of survival. Long-term exposure to chemostat culture provokes an accumulation of spontaneous mutations and renders the most adaptable strain dominant. Compared to the colony transfer and serial transfer methods, chemostat culture entails the highest number of cell divisions and, therefore, the highest number of diverse populations. Although chemostat culture for ALE requires more complicated culture devices, it is less labor intensive once the operation begins. Comparative genomic and transcriptome analyses of the adapted strain provide evolutionary clues as to how the stressors contribute to mutations that overcome the stress. The goal of the current paper is to bring about accelerated evolution of microorganisms under controlled laboratory conditions.

Here, we present a protocol to obtain adaptive laboratory evolution of microorganisms under conditions using chemostat culture. Also, genomic analysis of the evolved strain is discussed.

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) ...

Genome Editing and Directed Differentiation of hPSCs for Interrogating Lineage Determinants in Human Pancreatic Development

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding human development and dissecting disease mechanisms in a dish. To capitalize on this potential application requires efficient genome-editing tools to generate hPSC mutants in disease-associated genes, as well as in vitro hPSC differentiation protocols to produce disease-relevant cell types that closely recapitulate their in vivo counterparts. An efficient genome-editing platform for hPSCs named iCRISPR has been developed through the TALEN-mediated targeting of a Cas9 expression cassette in the AAVS1 locus. Here, the protocols for the generation of inducible Cas9 hPSC lines using cells cultured in a chemically defined medium and a feeder-free condition are described. Detailed procedures for using the iCRISPR system for gene knockout or precise genetic alterations in hPSCs, either through non-homologous end joining (NHEJ) or via precise nucleotide alterations using a homology-directed repair (HDR) template, respectively, are included. These technical procedures include descriptions of the design, production, and transfection of CRISPR guide RNAs (gRNAs); the measurement of the CRISPR mutation rate by T7E1 or RFLP assays; and the establishment and validation of clonal mutant lines. Finally, we chronicle procedures for hPSC differentiation into glucose-responsive pancreatic &#946;-like cells by mimicking in vivo pancreatic embryonic development. Combining iCRISPR technology with directed hPSC differentiation enables the systematic examination of gene function to further our understanding of pancreatic development and diabetes disease mechanisms.

Protocols to generate hPSC mutant lines using the iCRISPR platform and to differentiate hPSCs into glucose-responsive &#946;-like cells are described. Combining genome editing technology with hPSC-directed differentiation provides a powerful platform for the systematic analysis of the role of lineage determinants in human development and disease progression.

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding ...

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in which the gene of interest has been disrupted. A gene-disruption DNA construct containing a suitable antibiotic resistance marker gene is useful for the generation of gene-disrupted strains in bacteria. However, conventional construction methods, which require gene cloning steps, involve complex and time-consuming protocols. Here, a relatively facile, rapid, and cost-effective method for targeted gene disruption in Streptococcus mutans is described. The method utilizes a 2-step fusion polymerase chain reaction (PCR) to generate the disruption construct and electroporation for genetic transformation. This method does not require an enzymatic reaction, other than PCR, and additionally offers greater flexibility in terms of the design of the disruption construct. Employment of electroporation facilitates the preparation of competent cells and improves the transformation efficiency. The present method may be adapted for the generation of gene-disrupted strains of various species.

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

We describe a facile, rapid, and relatively inexpensive method for the generation of gene-disrupted Streptococcus mutans strains; this technique may be adapted for the generation of gene-disrupted strains of various species.

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in ...

Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the increase in gene copy number per cell provided by multicopy plasmids could accelerate the evolution of plasmid-encoded genes. In this work, we present an experimental system to test the ability of multicopy plasmids to promote gene evolution. Using simple molecular biology methods, we constructed a model system where an antibiotic resistance gene can be inserted into Escherichia coli MG1655, either in the chromosome or on a multicopy plasmid. We use an experimental evolution approach to propagate the different strains under increasing concentrations of antibiotics and we measure survival of bacterial populations over time. The choice of the antibiotic molecule and the resistance gene is so that the gene can only confer resistance through the acquisition of mutations. This &#34;evolutionary rescue&#34; approach provides a simple method to test the potential of multicopy plasmids to promote the acquisition of antibiotic resistance. In the next step of the experimental system, the molecular bases of antibiotic resistance are characterized. To identify mutations responsible for the acquisition of antibiotic resistance we use deep DNA sequencing of samples obtained from whole populations and clones. Finally, to confirm the role of the mutations in the gene under study, we reconstruct them in the parental background and test the resistance phenotype of the resulting strains.

Here we present an experimental method to test the role of multicopy plasmids in the evolution of antibiotic resistance.

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the ...

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective tag single nucleotide polymorphism (SNP) approach using a multiplexed genotyping assay with mass spectrometry, to investigate gene pathway associations in clinical cohorts. We investigate the food allergy candidate locus Interleukin13 (IL13) as an example. This method efficiently maximizes the coverage by taking advantage of shared linkage disequilibrium (LD) within a region. Selected LD SNPs are then designed into a multiplexed assay, enabling up to 40 different SNPs to be analyzed simultaneously, boosting cost-effectiveness. Polymerase chain reaction (PCR) is used to amplify the target loci, followed by single nucleotide extension, and the amplicons are then measured using matrix-assisted laser desorption/ionization-time of flight(MALDI-TOF) mass spectrometry. The raw output is analyzed with the genotype calling software, using stringent quality control definitions and cut-offs, and high probability genotypes are determined and output for data analysis.

Identification of genetic variants contributing to complex human disease allows us to identify novel mechanisms. Here, we demonstrate a multiplex genotyping approach to candidate genes or gene pathway analysis that maximizes the coverage at low cost and is amenable to cohort-based studies.

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective ...

Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that control the fidelity of transcription. To fill this gap in scientific knowledge, we recently optimized the circle-sequencing assay to detect transcription errors throughout the transcriptome of Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans. This protocol will provide researchers with a powerful new tool to map the landscape of transcription errors in eukaryotic cells so that the mechanisms that control the fidelity of transcription can be elucidated in unprecedented detail.

This protocol provides researchers with a new tool to monitor the fidelity of transcription in multiple model organisms.

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that ...

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

A first approach to study the function of an unknown gene in bacteria is to create a knock-out of this gene. Here, we describe a robust and fast protocol for transferring gene deletion mutations from one Escherichia coli strain to another by using generalized transduction with the bacteriophage P1. This method requires that the mutation be selectable (e.g., based on gene disruptions using antibiotic cassette insertions). Such antibiotic cassettes can be mobilized from a donor strain and introduced into a recipient strain of interest to quickly and easily generate a gene deletion mutant. The antibiotic cassette can be designed to include flippase recognition sites that allow the excision of the cassette by a site-specific recombinase to produce a clean knock-out with only a ~100-base-pair-long scar sequence in the genome. We demonstrate the protocol by knocking out the tamA gene encoding an assembly factor involved in autotransporter biogenesis and test the effect of this knock-out on the biogenesis and function of two trimeric autotransporter adhesins. Though gene deletion by P1 transduction has its limitations, the ease and speed of its implementation make it an attractive alternative to other methods of gene deletion.

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

Here we present a protocol for the use of pre-existing antibiotic resistance-cassette deletion constructs as a basis for making deletion mutants in other E. coli strains. Such deletion mutations can be mobilized and inserted into the corresponding locus of a recipient strain using P1 bacteriophage transduction.

A first approach to study the function of an unknown gene in bacteria is to create a knock-out of this gene. Here, we describe a robust and fast protocol ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences is desired. We herein provide a detailed in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) protocol to study the RNA structural change in the presence of an experimental drug for spinal muscular atrophy (SMA), survival of motor neuron (SMN)-C2, and in exon 7 of the pre-mRNA of the SMN2 gene. In in vitro SHAPE, an RNA sequence of 140 nucleotides containing SMN2 exon 7 is transcribed by T7 RNA polymerase, folded in the presence of SMN-C2, and subsequently modified by a mild 2&#39;-OH acylation reagent, 2-methylnicotinic acid imidazolide (NAI). This 2&#39;-OH-NAI adduct is further probed by a 32P-labeled primer extension and resolved by polyacrylamide gel electrophoresis (PAGE). Conversely, 2&#39;-OH acylation in in-cell SHAPE takes place in situ with SMN-C2 bound cellular RNA in living cells. The pre-mRNA sequence of exon 7 in the SMN2 gene, along with SHAPE-induced mutations in the primer extension, was then amplified by PCR and subject to next-generation sequencing. Comparing the two methodologies, in vitro SHAPE is a more cost-effective method and does not require computational power to visualize results. However, the in vitro SHAPE-derived RNA model sometimes deviates from the secondary structure in a cellular context, likely due to the loss of all interactions with RNA-binding proteins. In-cell SHAPE does not need a radioactive material workplace and yields a more accurate RNA secondary structure in the cellular context. Furthermore, in-cell SHAPE is usually applicable for a larger range of RNA sequences (~1,000 nucleotides) by utilizing next-generation sequencing, compared to in vitro SHAPE (~200 nucleotides) that usually relies on PAGE analysis. In case of exon 7 in SMN2 pre-mRNA, the in vitro and in-cell SHAPE derived RNA models are similar to each other.

Detailed protocols for both in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) experiments to determine the secondary structure of pre-mRNA sequences of interest in the presence of an RNA-targeting small molecule are presented in this article.

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

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 ...

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

Summary

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Chapters in this video

Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection

Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

Genome Editing and Directed Differentiation of hPSCs for Interrogating Lineage Determinants in Human Pancreatic Development

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

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