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Recombination-independent genome editing through CRISPR/Cas9-enhanced targetron delivery Elena Velazquez, Victor de Lorenzo, and Yamal Al-Ramahi ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.9b00293 • Publication Date (Web): 16 Aug 2019 Downloaded from pubs.acs.org on August 17, 2019

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ACS Synthetic Biology

1

Recombination-independent genome editing through CRISPR/Cas9-enhanced TargeTron delivery

by

Elena Velázquez, Víctor de Lorenzo* and Yamal Al-Ramahi

Systems Biology Program, Centro Nacional de Biotecnología-CSIC, Campus de Cantoblanco, Madrid 28049, Spain.

____________________________________________________________________________ * Correspondence to:

Víctor de Lorenzo Centro Nacional de Biotecnología-CSIC Campus de Cantoblanco, Madrid 28049, Spain Tel.: 34- 91 585 45 36; Fax: 34- 91 585 45 06 E-mail: [email protected]

____________________________________________________________________________

Abbreviations: DIV, domain IV; IEP, Intron-Encoded Protein; Sm, streptomycin; Km, kanamycin; Cm, chloramphenicol; IPTG, Isopropyl ß-D-1-thiogalactopyranoside; X-gal, 5-bromo-4-chloro-3indolyl-β-D-galactopyranoside; CRISPR, clustered regularly interspaced short palindromic repeats; PAM, Protospacer adjacent motif; wt, wild-type; ORF, Open reading frame; PCR, polymerase chain reaction; RAM, retrotransposition-activated selectable marker; Cas9, CRISPRassociated protein 9.

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2 ABSTRACT. Group II introns were developed time ago as tools for the construction of knockout mutants in a wide range of organisms, ranging from Gram-positive and Gram-negative bacteria to human cells. Utilizing these introns is advantageous because they are independent of the host's DNA recombination machinery, they can carry heterologous sequences (and thus be used as vehicles for gene delivery), and they can be easily retargeted for subsequent insertions of additional genes at the user's will. Alas, the use of this platform has been limited, as insertion efficiencies greatly change depending on the target sites and cannot be predicted a priori. Moreover, the ability of introns to perform their own splicing and integration is compromised when they carry foreign sequences. To overcome these limitations, we merged the group II intron-based TargeTron system with CRISPR/Cas9 counterselection. To this end, we first engineered a new group-II intron by replacing the retrotransposition-activated selectable marker (RAM) with ura3 and retargeting it to a new site in the lacZ gene of E. coli. Then, we showed proved that directing CRISPR/Cas9 towards the wild-type sequences dramatically increased the chances of finding clones that integrated the retrointron into the target lacZ sequence. The CRISPR-Cas9 counterselection strategy presented herein thus overcomes a major limitation that has prevented the use of group II introns as devices for gene delivery and genome editing at large in a recombination-independent fashion.

KEYWORDS: CRISPR/Cas9, targetron, group II intron, genome editing, retroelements

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Group II introns are a class of retroelements widely found in the genomes of different bacteria and organelles. Although sequence homology has not been found between them, when these introns are in their RNA form, they fold into a very conserved secondary structure. This structure is composed of six domains that spread from a central wheel (reviewed in1,2). While the rest of these domains are similar between group II introns, domain IV (DIV) tends to diverge in length, as there are cases in which an ORF is carried inside of it. This ORF is translated into a protein known as intron-encoded protein (IEP), which is indispensable for the mobility of group II introns3-5. When the locus where the intron is inserted is transcribed into RNA, it is spliced from the rest of the sequence. Afterward, the intron and the IEP start to scan DNA molecules looking for a correct target sequence to insert. When this location is found, the intron RNA is integrated and retrotranscribed into DNA by the IEP. This


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3 process is called retrohoming, as these molecules tend to invade intronless alleles instead of new genomic locations (reviewed in6). Thus, group II introns are mobile elements with a specific target sequence7.

The first biotechnological application of group II introns came when they were modified to insert into places that were different from their original, designated places. The first intron to be used like this was Ll.LtrB from Lactococcus lactis, as it was the best known intron of this class8. On the one hand, since recognition of the target sequence is mostly driven by base complementarity, it was possible to change specific regions inside this intron to make it recognize different loci. Nevertheless, because this identification is not only dependent upon base-pairing, algorithms to predict better insertion points were developed9,10. Therefore, in a given sequence, these algorithms return a list of compatible loci for the retargeting of the intron as well as primers to modify these molecules through a simple PCR step. This technology was exploited as a disruption tool for the generation of knockout mutants and received the name of TargeTron9.

In addition to their use as mutagenic tools, some attempts to employ these introns as delivery systems were made11,12. There are several reasons why Ll.LtrB is a good candidate for this. First, it has high sequence recognition specificity13,14 as well as the potential to modify this specificity towards a different region9,15,16. Second, Ll.LtrB has been proven to work in recA- strains17, which do not require a functional homologous recombination system. Third, this intron has been employed in a wide range of organisms, from Gram-negative to Gram-positive bacteria as well as human cells10,13,17,18. Finally, as has been observed, the IEP gene can be expressed in trans without affecting the splicing and integration efficiency of the intron. This ORF in the DIV can be substituted by an exogenous sequence to be transported into the desired genomic location. On the other hand, there are some limitations that have impeded these attempts. The most important one is the changing integration efficiency of retargeted introns, which can make identification of expected mutants laborious. This was partially solved by inserting a retrotransposition-activated selectable marker (RAM) in DIV15,19. This consists of an antibiotic resistance gene interrupted by a group I intron. The construct is arranged so that if only the intron is integrated, then the group I intron is lost, and the resistance gene is activated. This approach improved the screening of mutants but hindered



4 the possibility of introducing different cargos inside DIV. This highlights the second restriction of group II introns, which lies in the existence of a limit in the length of sequences DIV can harbor11.

In the present work, we have outmaneuvered these limitations by combining group II introns with CRISPR technology. This machinery is found in Eubacteria and Archaea and was reported to work as an immune system against bacteriophages20,21. It is basically based on CRISPR-associated proteins (Cas) and specific sequences called spacers22,23. Among the Cas proteins, the most characteristic is Cas9, a double-stranded DNA endonuclease. Regarding the spacers, they are placed between identical repeats, which received the name of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). When they are transcribed and processed, the spacers are complexed with Cas9 and, if their target is near a sequence called protospacer-adjacent motif (PAM24), the endonuclease will cleave the genomic locus. The discovery that Cas9 could be directed to specific sites by changing these spacers triggered the development of CRISPR systems as gene editing tools25. The CRISPR system from Streptococcus pyogenes was the first one adapted for this purpose and different variants have emerged since then. One of them is the use of CRISPR-Cas9 as a counterselection to remove wild type bacteria after a mutagenesis procedure26-31.

In the case presented here, we have adapted this technology as an external counterselection system of group II introns mutants. By choosing intron insertion sites near a PAM sequence (5’-NGG-3’ in the case of S. pyogenes-CRISPR system), we can design CRISPR spacers that specifically recognize this region. Therefore, if the intron retrohomes in the selected site, then the CRISPR-Cas9 system is expected to no longer recognize this locus, and the mutated bacteria will survive. In contrast, those bacteria that do not incorporate the intron will be killed. The experiments reported below document the potential of group II introns for genome editing in Escherichia coli and lead the way to its adaptation to other organisms with fewer available tools.

RESULTS AND DISCUSSION

Efficiency of crRNA-guided genome cleavage with the Cas9 RNP complex. To test the quality of crRNA-635sLacZ in crRNA-guided cleavage of DNA with Cas9-RNP complexes, the E. coli strain BL21/DE3 was transformed with pSEVA421-Cas9tr (Figure 1). Afterwards, the resulting strain was


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5 transformed with either the spacer-free vector, pSEVA231-CRISPR, or with the plasmid, pSEVA231CRISPR-635s, which produces what we named crRNA-635sLacZ, a crRNA targeted to the vicinity of the position 635 in the lacZ sequence (Figure 2).

Figure 1. The main plasmids used in this study. The plasmids above encode all the parts necessary to carry out the CRISPR-Cas9 counterselection31. Briefly, pSEVA421-Cas9tr enables the expression of Cas9 endonuclease, with the capability to cleave the genome, and the expression of tracrRNA, which allows the attachment between Cas9 and the crRNA form. The plasmid pSEVA231-CRISPR is used for CRISPR spacer expression. After being digested with BsaI enzyme and synthesized, specific spacers can then be ligated into it. The plasmid pACDura3 harbors both the group II intron and IEP (LtrA protein). Their expression is controlled by a T7 promoter; therefore, it is necessary to express T7 polymerase first to induce their production. This group II intron was retargeted to insert into the 635s locus of the lacZ gene, and the resulting plasmid was called pACDura3-635sLacZ.

In this context, we expected the following. Provided that crRNA-635LacZ is absent, Cas9 and tracrRNA form a guideless complex that is unable/barely able to bind and cut the genomic DNA; therefore, it has no important effect on cell viability. In contrast, if Cas9, tracrRNA and crRNA635sLacZ are together in the cell, they form a Cas9-RNP complex that examines the genome using the crRNA-635LacZ as a guide to find its specific target by sequence complementarity (Figure 2B). If the complex finds a target in the genome that is adjacent to a PAM motif, Cas9 cleaves both DNA strands, and the cell dies. Reports show that the effectiveness of interference varies with different crRNA guides26,31. For this reason, as was the case in other contexts31, it is advisable to estimate in



6 practice to what extent a new crRNA guide works well in combination with Cas9 and tracrRNA in the elimination of cells with an intact lacZ sequence.

Figure 2. Designing CRISPR/Cas9 counterselection. (A) Interference of the Cas9 complex with the target DNA in lacZ. The Cas9-RNP complex scans the genomic DNA, interacts with the PAM motif and binds to the opposite strand of the protospacer (orange), to a sequence that is complementary to the crRNA. Then, Cas9 cleaves both genomic DNA strands, and the cell dies. As part of our gene delivery system, the group II intron was retargeted to an insertion site within the selected protospacer (orange). Thus, successful insertion of the intron disrupts the original configuration of this sequence and prevents interference by the Cas9-RNP complex, thus enabling cell survival. (B) Group II intron insertion site and CRISPR interference in lacZ. Retargeted group II intron insertion site at 3 nts of a PAM (red, bold letters) and the region recognized by crRNA-635sLacZ (underlined text) are indicated. This spacer (green) is constructed to match the 30 nts next to the PAM. After the maturing process, only the first 20 nts (in green, bold letters) are attached to Cas9 protein to perform the counterselection. (C) Synthesis of the CRISPR spacer. To clone the designed spacer into the pSEVA231-CRISPR plasmid for its expression, two single-stranded oligonucleotides were synthesized that included additional sequences (blue) to fit into the pSEVA231-CRISPR plasmid after it was digested with the BsaI enzyme.

Our results in the interference experiment (Figure 3 and Supplementary Figure S1A) confirm the reasoning we have just stated. E. coli BL21/DE3 cells (pSEVA421-Cas9tr, pSEVA231-CRISPR635s), which produce Cas9, tracrRNA and crRNA-635sLacZ, result in a colony count at least two orders of magnitude lower than that from E. coli BL21/DE3 (pSEVA421-Cas9tr, pSEVA231CRISPR), which produces Cas9 and tracrRNA but not crRNA-635sLacZ. This decrease in colony counts is in agreement with what could be expected when the Cas9-RNP complex is properly guided


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7 to a specific target and performs the lethal cut of the genome efficiently. In brief, our interference experiment results show that the crRNA-635sLacZ guide, in combination with Cas9 and tracrRNA, is effective in eliminating cells that have an intact/wild type lacZ gene, thus proving that it is a suitable choice for the counterselection experiment discussed later in this article.

1,00E+09

Transformation efficiency (KmR+SmR/SmR CFU per 109)

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1,00E+08

1,00E+07

1,00E+06

1,00E+05

1,00E+04

1,00E+03

Figure 3. Interference assay. Testing the spacer effectiveness was performed by transformation of pSEVA231-CRISPR (green) or pSEVA231-CRISPR-635s (blue) in E. coli BL21/DE3 (pSEVA421-Cas9tr). Then, this effectiveness was estimated as the ratio of escapers (cells growing on LB/Sm/Km plates) to the total viable cells (cells growing on LB/Sm plates) and normalized to 109 cells. The average and standard deviation of the three different biological assays were plotted.

We also performed two more replicates of this experiment but supplementing the plates with X-gal to determine the number of white escapers. As these mutants display the same phenotype as cells with the intron inserted inside of the lacZ gene, this experiment allowed us to know the extent of false positive colonies that we might expect when carrying out the counterselection with CRISPR. The assay revealed a proportion of ~4% of escapers showing a white phenotype of the total survival when pSEVA231-CRISPR-635s was electroporated (Supplementary Figure S1B). In the case, when pSEVA231-CRISPR was transformed, no white colonies were spotted. This was expected, as spontaneous mutations in lacZ gene are being selected for when crRNA-635sLacZ is present. Moreover, cells might include mutations after successfully repairing the double-strand break caused by Cas9 endonuclease in this region, leading to loss of function in this gene. These two scenarios



8 explain the increased ratio of white escapers in comparison to the control assay, i.e., when pSEVA231-CRISPR was delivered into the cells.

Native integration efficiency of the engineered group II intron in pACDura3_635sLacZ without counterselection. After proving that crRNA-635sLacZ is a good spacer to perform CRISPR-Cas9 counterselection, we modified the Ll.LtrB intron to employ it in combination with this system. The group II intron used in this work came from pACD4K-C (TargeTron gene knockout system, SigmaAldrich). This intron carries a RAM, which serves as a method of counterselection of clones where the intron successfully retrohomed. This RAM consists of a Km resistance gene interrupted by a group I intron such that if only the intron is successfully spliced and integrated in a new genetic location, then this group I intron is also spliced, and the resistance gene is restored19. First, we replaced this RAM with the gene ura3 (Figure 1). Removing RAM prevented interference with pSEVA231-CRISPR and pSEVA231-CRISPR-635s, which also confers resistance to Km and enabled the use of any of these two plasmids in combination with the retrotransposon in subsequent experiments (next section). Having the engineered group II intron with no counterselection genes allowed us to estimate its native efficiency of integration as a reference. Finally, as we wanted to test the retrointron as a device to deliver genes, we introduced ura3 to fulfill the function as cargo. As such, we placed the sequence of interest to be delivered and made the plasmid with an intron compatible with the CRISPR-Cas9 method of counterselection. We selected the ura3 gene as candidate because it has a length that is similar to the RAM of approximately 1 kb (the gene along with one constitutive promoter), and fragments of this size have been used before in similar experiments11 12. Although good sites to accommodate cargo sequences are usually located in the DIV loops, they are not present along the entire region. It must be noted that it is important to keep the LtrA binding region within DIV intact to ensure that the intron retains its LtrA-binding properties. In this sense, replacing RAM with the ura3 gene, as we did in the MluI site, locates the cargo at domain IVb of Ll.LtrB, which has been described to be the optimum region of group II introns to harbor foreign sequences11.

Next, modifications were aimed at retargeting the retrointron. Although the Ll.LtrB intron in pACD4KC was targeted to insert into locus 1063a in lacZ, this insertion site is not close to a PAM sequence, which is indispensable for Cas9 to cleave both DNA strands. To choose an adequate insertion site,


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9 we considered target sites compatible with both: TargeTron insertion into lacZ and with crRNAguided genome cleavage by Cas9. First, we used the Clostron website to search for possible insertion sites in lacZ.

Figure 4. Retargeting the group II intron for delivery into a new location. (A) Retargeting of the group II intron to insert into the 635s locus of lacZ gene. Primers for pIBS, pEBS2, pEBS1d and pEBS universal were designed with the Clostron algorithm and used in a SOEing PCR to modify the three regions that determine the intron integration target. Finally, the resulting fragment was ligated into the intron expression vector, pACDura3, so that the intron was retargeted and ready to insert into the new specified gene. (B) Base-pairing between the intronic RNA and the DNA locus where the intron will insert into. In this case, locus 635s of the lacZ gene was chosen so that the intron would insert between nucleotides 635 and 636 in the sense strand (black triangle). EBS2: Exon Binding Site 2; EBS1: Exon Binding Site 1; IBS2: Intron Binding Site 2; IBS1: Intron Binding Site 1.

This website uses an algorithm9 to scan the given sequence and returns a list of potential insertion regions ordered by a score corresponding to the predicted intron insertion efficiency from higher to lower. Nevertheless, due to its probabilistic nature, the algorithm does not always reliably predict relative integration efficiency, as shown in a study where two retrointrons received high scores by the algorithm, and one of them showed a much lower than expected integration frequency compared with the other18. Therefore, although serving as a guide, these predictions should be taken with caution, as empirical verification is more representative. Among the predicted sequences in the Clostron output, we chose 635s, which scored high (10.063) and included a PAM motif at only 3 nucleotides from the intron insertion site (Figure 2B). Finally, we obtained the vector,



10 pACDura3_635sLacZ, which carries a group II intron with the ura3 gene inserted in its domain IVb and was redirected to insert into locus 635s of lacZ (Figure 4), satisfying the compatibility requisites stated above.

Once we had successfully modified the Ll.LtrB intron, we studied its native integration efficiency. This is important so that an estimation of the basal capacity to insert sequences without a counterselection system can be made. Therefore, after inducing intron expression with IPTG, we plated several dilutions to observe the efficiency. As the intron was retargeted to insert into the lacZ gene, we used blue/white screening to easily find potential disruption mutants. Those bacteria that incorporated the intron at the correct locus would display a white phenotype in the presence of the reagent X-gal. On the other hand, if the intron did not insert, the lacZ gene would be functional and the colonies arising would be blue (Figure 5A). After incubation, two white colonies were observed among 831 blue ones in a dilution of 10-5 (Figure 6A, Supplementary Table S1). This resulted in a frequency of 0.24%. This percentage is significantly lower than that previously reported for the same intron targeted to the same locus, where frequencies up to 10% were found18. This discrepancy could be due to the presence of the ura3 gene in the intron (in our work) considering that cargo sequences of a certain length can hinder the transposition of the retrointron. This hypothesis is in agreement with previous reports that also suggest that the presence of large DNA inserts within the Ll.LtrB intron can hinder its integration capability. As an example, a study showed that the insertion of a gfp gene (which is shorter than the ura3 that we used in our experiments) as cargo in the Ll.ltrB retrointron reduced the invasion frequency by almost half compared with that of the Ll.ltrB alone12. Another study showed that insertion of foreign sequences shorter than 100 bp as cargo in the Ll.ltrB transposon almost did not affect mobility. In contrast, the mobility of Ll.ltrB variants carrying cargos of more than 1 Kb was significantly reduced and, in some cases, even not detected. In general, hindrance of Ll.ltrB mobility was not due to cargo gene expression, toxicity or sequence specificity but was mostly size related 11. Accordingly, we may expect even a greater hindrance if placing longer cargo sequences inside group II introns. Moreover, this rate will differ for other integration sites18 and could produce even lower frequencies. Taken together, the cargo size and the quality of the insertion site could make generation of mutants difficult without the adoption of a counterselection method.


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11 To confirm our results with the native integration frequency, the presence of the intron in the lacZ gene was verified through two PCRs (Figure 6D). By using a primer annealing inside the intron paired with a lacZ specific primer, we could confirm the presence of this molecule at the expected location, as an amplicon of 2 kb is produced only if the intron is placed in the specified insertion site (Supplementary Figure S2).

Figure 5. Mechanism of action of the TargeTron and counterselection strategy. A) Group II intron expression and integration mechanism. Group II intron expression is controlled by a T7 promoter and, simultaneously, the T7 polymerase is expressed from the genome of E. coli BL21/DE3 after the addition of IPTG. Once the group II intron and ltrA gene are transcribed into RNA, the LtrA protein is translated, and the splicing of the group II intron takes place. These two molecules remain joined and start to scan DNA molecules until they find their target sequence. In this case, the Group II intron is retargeted to identify locus 635s in the lacZ gene. After recognition, reverse splicing occurs, and the intron is inserted into lacZ at a certain efficiency, thus disrupting its ORF. This process leads to two possible genotypes: wild type lacZ gene (WT, without the intron) and the mutated lacZ gene (Mut, with the intron). B) Counterselection strategy based on CRISPR-Cas9 technology. Part of the induced bacteria are not able to incorporate the intron at the correct locus, remaining as wild type genotypes. To reduce the amount of this WT population, a CRISPR spacer is designed to base-pair with the recognition region of the intron. If there is a PAM near the insertion site and the intron did not reverse splice into the 635s locus, then the Cas9-RNP complex, guided by crRNA-635sLacZ, will interact with this region and will cleave the bacterial genome of the WT cells. If the intron is present, then crRNA-635sLacZ cannot base-pair with this region, and the Cas9 endonuclease will not act.

With the second PCR, by using primers annealing at both sides of the insertion site, we could verify the presence of the intron and the purity of the population conforming each colony (Supplementary Figure S2). The amplification of a fragment with the size of the WT genotype (1.5 kb) in white



12 colonies or even the presence of two bands in this PCR revealed a mixture of WT and disrupted genotypes (3.5 kb). As previously described, it is possible to find these mixed populations because there is a proportion of late integrations that occur during colony growth after plating18. Additionally, thanks to the blue/white screening, we could visually witness this effect in some of the identified colonies (Figure 6B). In this case, the arrow points out a sector composed by WT cells inside of a big, white colony with mostly mutated bacteria. This finding supports the existence of these mixed colonies where there are non-mutated cells along with disrupted ones. On the other hand, the reason why we could not see this mosaic in the two analyzed colonies may be that this WT population is so low that a blue phenotype cannot be observed. This could also explain why the bands are so weak if we compare them with amplifications from blue-looking colonies (Supplementary Figure S2). Nevertheless, this problem could be easily solved by restreaking to isolate a pure, mutated strain.

Delivery of a group II intron in combination with the CRISPR/Cas9 counterselection method. To boost the chances of finding clones that integrated the retrointron into lacZ, we used our plasmid for group II intron delivery in combination with the CRISPR/Cas9 system to kill the cells that did not incorporate the retroelement in the specified target site (Figure 5B). To do so, E. coli BL21/DE3 (pACDura3_635sLacZ, pSEVA421-Cas9tr) cells were induced with IPTG to produce T7 RNA polymerase and, consequently, to express the engineered retrointron, which is under control of the T7 promoter (Figure 5A). In this context, this retargeted group II intron has the chance to integrate, by retrohoming, into the specified insertion site inside lacZ. Next, we tested two conditions. On the one hand, these induced cells were provided with the spacer-free pSEVA231-CRISPR as a negative control (absence of crRNA) and on the other hand, with pSEVA-CRISPR-635s for production of crRNA-635s. As we have already seen (Figure 6A), the retrohoming process does not occur frequently; therefore, the probability of finding colonies that have the retroelement integrated into the genome (white colonies in X-gal) is very low. It is here that the CRISPR/Cas9 counterselection makes it easier to find white colonies, as it helps to clear up “WT cells” (blue colonies in X-gal) that did not integrate the retrointron into lacZ. For our purpose, as already discussed (Figure 5B), the Cas9-RNP complex is guided by crRNA-635s; when it finds the proper sequence adjacent to a PAM in lacZ, Cas9 then cuts both DNA strands, and the cell dies. This combination of the PAM and the adjacent target sequence is present in cells with the WT lacZ; therefore, they will be killed after Cas9 cuts the DNA double strand. Conversely, the integration of the retrointron into lacZ disrupts the


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13 original configuration of the sequence in the gene. As such, the PAM (5´-NGG-3´) and the sequence complementary to crRNA-635s are separated enough to prevent interference by the Cas9-RNP complex and thus, enabling these cells to survive. Consequently, the resulting culture (Figure 6C) presents an enrichment in white colonies compared with the control experiment (Figure 6A).

Following this approach, we plated dilutions in different conditions, and the results (Figure 6C, Supplementary Table S1) show that in plates with and without Km, the colony counts after cells are endowed with pSEVA231-CRISPR agree with a situation where there is no counterselection. These results can be explained in the case at hand, as cells with pSEVA231-CRISPR are spacer-free, and although the Cas9 and tracRNA are present, they form a guideless Cas9-RNP complex that does not cut the DNA. All the colonies found after plating in Sm/Cm were blue (no white colony was found), which is in agreement with the very low retrohoming efficiency of the group II intron, entailing low probabilities of finding a white colony. In contrast, the proportion of white colonies considerably increased (from 0.24% to 88%) after the electroporation of pSEVA231-CRISPR-635s (Figure 6C, right plates). This outcome is totally opposed to the result obtained without CRISPR counterselection, where most of the observed colonies were blue (Figure 6A). As before, the correct integration of the testing intron was also confirmed through two PCRs (Figure 6D). In this case, the specific PCR for detecting the intron was the same but, in the case where two primers annealing at both sides of the insertion point were used, we changed to primers pLacZ-F2 and pLacZ-R2, as these primers produced smaller amplifications and displayed better performance, generating fragments that were easier to detect after the PCR (Figure 6D). A total of 16 colonies (2 blue and 14 white colonies) were tested and we observed the presence of three white colonies that did not incorporate the intron, despite displaying a white phenotype. This agrees with the results obtained in the interference assay where we found white escapers after repeating the experiment supplementing the plates with X-gal (Supplementary Figure S1B). Taking this into account, this result leads to a final estimated efficiency of the method as a 78% (11 confirmed mutated colonies/14 total white screened colonies) probability of finding the desired insertion after combining with CRISPR counterselection. Thereby, this is still an improvement of the technique, as in most of the cases, a visual screening is not possible. This increase in the mutated/non-mutated bacteria ratio can support finding lowerfrequency integration events in an easier way18. Therefore, even if the total number of mutated



14 bacteria is lower than the genuine integration yield of group II introns, the chance to find a disruption is much higher with the use of CRISPR counterselection.

Figure 6. Efficiency of counterselection. (A) Plate with E. coli BL21/DE3 (pACDura3_635sLacZ,

pSEVA421-Cas9tr) cells exhibiting the native insertion efficiency of the engineered group II intron into 635s-lacZ locus. White colonies (arrows) found in the 10-5 dilution are possible mutants with the intron inserted at the correct locus. (B) First picture: Detail of a bacterial colony with two different phenotypes. A small population seems to be wild type (blue sector) while the rest of it appears as mutated (white sector). Second and third pictures: Detail of white colonies found in plate A. (C) Efficiency test of CRISPR counterselection in combination with targetron. After induction of group II intron, pSEVA231-CRISPR-635s spacer plasmid (top plates) or spacer-free pSEVA231-CRISPR plasmid (bottom plates) were electroporated into the cells. The viability of cells was tested by plating in LB medium without Km (plates to the left) while the efficiency of counterselection was measured by plating in presence of this antibiotic (plates to the right). (D) Colony PCR Check: Two different reactions were set up to determine the presence of group II intron at the correct genomic place. The first PCR is specific of the intron since one primer anneals inside the ura3 gene and the second primer, in the lacZ gene. Only if the intron is correctly inserted, an amplification product of 2 kb will be generated (top gel). The second PCR used primers base-pairing at both sides of the insertion site of the intron in the lacZ gene. If the intron is correctly inserted, the amplification product should be 2.5 kb long, while if it is absent, the product is 0.5 kb long (bottom gel). The PRC-tested colonies come from the CRISPR-Cas9 counterselection assay (C, pSEVA231-CRISPR-635 spacer). WC: White colony; BC: Blue colony; H2O: control reaction without DNA.


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15 This improved probability allows for the selection of integration loci that are expected to be less amenable to the procedure. Moreover, the use of CRISPR-Cas9 enables the maximization of the load capacity of the TargeTron because it has been released from the need to include a specific sequence for counterselection as part of the cargo. Thus, it is possible to introduce longer sequences in the DIV of the Ll.LtrB.

Delivery of a group II intron alone or in combination with CRISPR/Cas9 counterselection in E. coli BL21/DE3 ΔrecA. As it was previously stated in the text, one of the advantages that group II introns have in comparison with other delivery systems is their capability to function independently of homologous recombination17. Therefore, to prove the correct performance of the system in a ΔrecA strain, we deleted the recA gene from E. coli BL21/DE3 (pACDura3_635sLacZ, pSEVA421-Cas9tr), as explained in the Supplementary Methods, and repeated the same experiments explained in the previous sections; however, this time we used BL21/DE3 ΔrecA as the background E. coli strain. Next, we studied both the native integration efficiency of our group II intron and the counterselection performance of CRISPR/Cas9 in this new strain. As shown in Supplementary Figure S3 and Supplementary Table S6, we observed similar results to those already presented for E. coli BL21/DE3 (Figure 6C), confirming the performance of the introduced methodology in a recombination deficient strain.

CONCLUSION

Even though new systems are being developed with the capability of inserting exogenous sequences in bacterial genomes without requiring the recombination machinery of the cell34, there are currently not many available tools that can be used in this scenario. Our approach broadens the number of such genetic devices and allows the exploitation of group II introns as effective systems for genome editing in bacteria with hindered or absent recombination skills or in bacteria that are required to be recombination-deficient to comply with safety regulations. Despite their independence of recombination, the use of RNA-based retroelements for introducing a suite of genomic edits in a large variety of microorganisms has been limited thus far by the low frequency of occurrences of the pursued changes. The association of insertions or changes otherwise to selectable markers can indeed improve the system, although the resulting strains keep the marker gene (which might be



16 removed later) and the same platform cannot be reused for implementing additional genomic alterations. By using CRISPR/Cas9 as a counterselection device against wild type genomic sequences, we have shown above that directed and seamless modifications can be introduced in the genome of E. coli with the TargeTron system, which can subsequently be reused for further rounds of changes. As the parts included in the insertion/counterselection events are virtually orthogonal with respect to the host, in particular regarding the endogenous DNA recombination system, the described strategy is amenable to many types of organisms, both prokaryotic and eukaryotic. We thus advocate for this technique in cases in which the same DNA sequence is to be entered in diverse strains with different purposes, such as performing comparative gene expression studies.

METHODS

Bacterial strains and media. E. coli strain CC118 [Δ (ara-leu), araD139, Δ lacX74, galE, galK phoA20, thi-1, rpsE, rpoB, argE (Am), recA1, OmpC+, OmpF+] was used for plasmid cloning and propagation. BL21/DE3 [fhuA2, [lon], ompT, gal, (λ DE3), [dcm], ∆hsdS; (λ DE3) = λ sBamHIo ∆EcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 ∆nin5] and (BL21)DE3 ΔrecA (Supplementary Methods) strains were used for the TargeTron and counterselection assay32. Luria-Bertani (LB) medium was used for growth and was supplemented when needed with kanamycin (Km; 50 µg/mL), chloramphenicol (Cm; 30 µg/mL) and/or streptomycin (Sm; 50 µg/mL). Isopropyl-β-D-1tiogalactopiranoside (IPTG) was used to induce the expression of T7 polymerase in the BL21/DE3 strain at 0.5 mM. For solid culture, LB-agar (1,5 %) was used, and X-gal (5-Bromo-4-chloro-3-indolylβ-D-galactopyranoside) was added when needed at a final concentration of 30 µg/mL.

Plasmid construction. To build the pACDura3 plasmid, the retro-transposition-activated selectable marker (RAM) in pACD4K-C (TargeTron gene knockout system, Sigma-Aldrich) was excised by digestion with the MluI enzyme. Later, the ura3 gene was amplified by PCR with Q5 high-fidelity DNA polymerase (New England Biolabs) and primers ura3.mluI_F and ura3.mluI_R (Supplementary Table S2). The template DNA used was pSEVA421-pEM7-URA3 (Supplementary Table S3) and the PCRs were performed following the manufacturer’s instructions and are listed in Supplementary Table S4. The resulting amplicon was digested with MluI and cloned into digested pACD4K-C. Finally, the insertion of ura3 in the sense orientation in the new plasmid was confirmed by


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17 sequencing. To retarget pACDura3, primers pIBS, pEBS1d, pEBS2 and pEBSuniversal (Supplementary Table S2) were designed using ClosTron platform (http://clostron.com) with lacZ as query sequence. From the output list, primer sequences redirecting to 635s locus were selected. Oligonucleotides synthesized accordingly were used in a SOEing PCR (Supplementary Table S4) with pACDura3 as template to yield a 350 bp amplicon, which was later digested with BsrGI/HindIII and used to replace the corresponding segment in pACDura3, following the TargeTron protocol from Sigma-Aldrich. The obtained plasmid was named pACDura3_635sLacZ. Plasmids pSEVA421Cas9tr and pSEVA231-CRISPR are described elsewhere31. The spacer for the counterselection assay was designed and cloned into the pSEVA231-CRISPR as explained in the same study. Briefly, the 635s spacer was assembled by hybridization of two complementary oligonucleotides with BsaI sticky ends and 5’-phosphorylation (635s_spacer_S and 635s_spacer_AS). The assembled spacer was directly ligated into BsaI-digested pSEVA231-CRISPR. The resulting plasmid was named pSEVA231-CRISPR-635s.

Interference assay of selected spacers. To test the cleavage efficiency of the chosen spacers, E. coli BL21/DE3 harboring pSEVA421-Cas9tr were grown in LB/Sm/MgSO4 (2 mM)/glucose (20 mM) until their OD600 reached 0.4. Then, the cells were collected and washed four times with cold MilliQ water to produce competent cells33. One hundred nanograms of pSEVA231-CRISPR-635s and spacer-free pSEVA231-CRISPR (control) were electroporated into two aliquots of prepared competent cells. Transformed bacteria were grown in LB/Sm for 1 h at 37°C and were afterwards plated on LB/Sm to test viability and LB/Sm/Km to test the efficiency of cleavage. Two more replicates of this experiment were performed in the same way, but X-gal was added to the plates in order to calculate the proportion of white escapers that appeared (Supplementary Figure S1).

Insertion assay. E. coli BL21/DE3 or E. coli BL21/DE3 ΔrecA (Supplementary Methods) bearing pSEVA421-Cas9tr and pACDura3_635sLacZ were grown at 37°C in LB/Sm/Cm plus 1% glucose until the OD600=0.2. Then, IPTG was added to the culture, which was further incubated for 30 minutes at 30°C in a 8 mL total volume. Next, the samples were centrifuged at 8,000 g for 3 minutes, and then, the cells were washed with LB medium to eliminate the rest of the inducer. After this, 1 mL of LB/Sm was added to the pellet and was incubated for 1 h at 30°C. Serial dilutions of this culture



18 were plated on LB/agar plus X-gal to see the genuine insertion efficiency of the engineered group II intron.

Counterselection assay. An adaptation of the same procedure employed in the interference assay was used to turn the rest of the induced culture into electrocompetent cells33. Briefly, the induced culture was added to fresh LB/Sm/Cm medium plus MgSO4 (2 mM) and glucose (20 mM) at a final OD600=0.2 and was grown at 37°C until the OD600 reached 0.4. Then, 12 mL of this culture were placed at 4°C, washed with cold MilliQ H2O four times, and finally resuspended in 150 µL of cold MilliQ H2O. One hundred nanograms of pSEVA231-CRISPR-635s or pSEVA231-CRISPR (control) were electroporated into 75 µL of competent cells and then cultured in 1.5 mL of LB/Sm for 1 h at 37°C. Serial dilutions of both conditions were plated on LB-agar plus Sm, Cm and X-gal to analyze viability. Counterselection was studied by plating 150 µL or the rest of the culture on LB-agar plates plus Sm, km and X-gal. All plates were grown at 37°C overnight. Analysis of integration by colony PCR. Two different PCRs were prepared to determine the presence or absence of the group II intron in the correct position of the lacZ gene (Supplementary Table S4). One of the reactions used primers flanking the insertion region: pLacZ-F and pLacZ-R or pLacZ-F2 and pLacZ-R2 (Supplementary Table S2). The second one used primer pLacZ-R and one primer annealing inside the ura3 gene, pURA3-F (Supplementary Table S2). Both PCRs were checked by electrophoresis on a 0.8% agarose gel and 1x TAE (Tris-Acetate-EDTA). EZ Load 500 bp Molecular Ruler (Bio-Rad) was used as the DNA ladder. Supporting Information Supplementary Table S1:

Colony counts in targetron and CRISPR/Cas9 counterselection assay in E. coli

Supplementary Table S2:

List of oligonucleotides used in this work


List of plasmids used in this work


PCR conditions


Colony counts and normalized data from interference assay in E. coli BL21/DE3


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19 Supplementary Table S6:

Colony counts after targetron insertion and CRISPR/Cas9 counterselection assay in E. coli BL21/DE3 ΔrecA.

Supplementary Methods:

Deletion of recA gene in E. coli BL21/DE3 (pACDura3_635sLacZ, pSEVA421-Cas9tr). Delivery of group II intron alone or in combination with CRISPR/Cas9 counterselection in E. coli BL21DE3 ΔrecA.

Supplementary Figure S1:

Colony counts of escapers


Colony PCR check of insertions.


Efficiency of counterselection in E. coli BL21/DE3 ΔrecA

Funding Sources. This work was funded by the SETH Project of the Spanish Ministry of Science RTI 2018-095584-B-C42, MADONNA (H2020-FET-OPEN-RIA-2017-1-766975),

BioRoboost

(H2020-NMBP-BIO-CSA-2018), and SYNBIO4FLAV (H2020-NMBP/0500) Contracts of the European Union and the S2017/BMD-3691 InGEMICS-CM funded by the Comunidad de Madrid (European Structural and Investment Funds). EV was the recipient of a Fellowship from the Education Ministry, Madrid, Spanish Government (FPU15/04315).

Author Contributions. YA and VdL planned the experiments and EV did the experimenal work. All Authors analyzed and discussed the data and contributed to the writing of the article.

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23 For Table of Contents Only

Tragetron technology enables genomic edits (eg insertions) through a molecular mechanism alien to recombination and which can be made very efficient through CRISPR/Cas9 counterselection



Tragetron technology enables genomic edits (eg insertions) through a molecular mechanism alien to recombination and which can be made very efficient through CRISPR/Cas9 counterselection 254x190mm (72 x 72 DPI)


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Cas9

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