No-SCAR genome editing is an editing method that is able to manipulate the Escherichia coli (E. coli) genome. The system relies on recombineering whereby DNA sequences are combined and manipulated through homologous recombination. No-SCAR is able to manipulate the E. coli genome without the use of the chromosomal markers detailed in previous recombineering methods. Instead, the λ-Red recombination system facilitates donor DNA integration while Cas9 cleaves double-stranded DNA to counter-select against wild-type cells. Although λ-Red and Cas9 genome editing are widely used technologies, the no-SCAR method is novel in combining the two functions; this technique is able to establish point mutations, gene deletions, and short sequence insertions in several genomic loci with increased efficiency and time sensitivity.
Background and overview
λ-red recombineering system The λ-red recombineering system was published in 1998 and allows for insertion, deletion, or mutations to E. coli genes. In this system, the red operon from bacteriophage λ is transfected into E. coli cells to facilitate incorporation of linear target DNA into the E. coli genome. The bacteriophage λ-red operon consists of the exo, bet, and gam genes which, together, are responsible for recombineering. Phage λ exonuclease (exo) degrades transfected linear target DNA from the 5' end. Beta binds to the resulting single stranded 3' end and incorporates it into the target DNA to form the recombinant DNA. Phage λ gamma is necessary to inhibit E. coli nuclease activity and protect the transformed linear DNA in vivo.
Following λ-red operon activity induction, a linear, double-stranded cassette encoding a selectable marker, such as antibiotic resistance, is transformed into the cells in place of the target gene and incorporated into the DNA behind a specific inducible promoter. This allows for growth selection of the recombinant cells with proper insertion location verified using polymerase chain reaction (PCR). Specific incorporation can be achieved by including flanking PCR primers around the inserted linear DNA that are complement to the targeted insertion site. After selection of the recombinants, a second transformation is needed to remove the selective marker. A plasmid expressing flippase (FLP) can be transformed into the recombined cells, which can specifically cleave FLP recognition target sites (FRTs) flanking the antibiotic resistance gene. While this successfully removes the selective marker from the genome, it leaves FRT scars in place of the target gene.
The λ-red system has also been optimized for scarless recombination; however, this is a two-step system consisting of selection and counterselection. In this case, a gene cassette with a dual selectable marker can be incorporated into the DNA at the specific location of mutagenesis. After selection of recombinants, a subsequent transformation to transfect linear DNA with the desired mutation is performed, which will then be homologously recombined into the cellular DNA in place of the marker. Therefore, counterselection against the cells containing the marker needs to be performed in order to identify the cells that have successfully incorporated the linear DNA into the target sequence. This can be verified using PCR screening. The method of recombination detailed above is advantageous as it provides an alternative to the low-efficiency, laborious, and multi-step recombination processes using endonucleases and ligases. Therefore, λ-red recombination is more specific in terms of possible genomic alterations that are not governed by locations of restriction enzyme recognition sites. However, it also has many limitations. Multiple rounds of transformations can increase the risk of error and increase recombination time. Therefore, efficiency can be low (0.1–10% for point mutations; 10−5–10−6 for insertions, deletions, or replacements) and requires long growth into workable colonies between transformations and recombinant selections. All together, this contributes to a lengthy and inefficient mutagenesis procedure for even single mutations. This technique can also leave scars that can contribute to destabilization of the chromosome and impact the success of the manipulation.
CRISPR/Cas9 recombination
A more recent method for genome editing uses CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) sequences and the endonuclease Cas9 (CRISPR-associated protein 9). These components are an integral part of the immune response for some bacteria. and have been repurposed for genome engineering. In this system, sequences matching foreign bacteriophage or plasmid DNA are incorporated as "spacer" sequences into the bacterial genome located between repeating CRISPR loci. Cas endonucleases are able to initiate double strand breaks within these foreign DNAs that are complement to the transcribed CRISPR RNAs (crRNA, or "protospacers"), thus degrading them. A conserved protospacer-adjacent motif (PAM, sequence 5'-NGG-3') located immediately downstream of the protospacer in the cellular genome is necessary for Cas9 cleavage. Together, this system allows for adaptive immunity to the dynamic viral genetic material.
In 2013, methods for harnessing this system for use in editing mutations or insertions of specific E. coli sequences were published. This method includes constructing a plasmid consisting of the cas9 gene and CRISPR loci containing the matching target DNA, called single guide RNA (sgRNA). After expression is induced, Cas9 is able to identify the target DNA sequence of the cellular genome by finding the sgRNA complement and initiate strand breaks in the E. coli genome. This allows a transfected, linear DNA sequence to be incorporated into the genome, relying on the cellular machinery to use the linear DNA flanked by homologous regions specific to the cleaved location as a template to rebuild using homology directed repair.
This method is advantageous in that it allows multiple mutations to be introduced into the genome in one experiment. It also has vastly improved efficiency over previous technologies and allows for almost any kind of mutation with ease. These advantages make the CRISPR/Cas9 recombination technique the most promising for application in human health. However, major drawbacks exist, including, most prominently, off-target cleavage that can result in unintended genome disruptions.
Integrating λ-red and Cas9 recombination Reisch and Prather pioneered a technique that combines both the λ-red and CRISPR/Cas9 recombination systems to form a novel methodology called no-SCAR (Scarless Cas9 Assisted Recombineering) for E. coli genome modifications. In this method, a plasmid containing the gene for Cas9 expression (cas9) is first transformed into E. coli cells. After selecting for the transformants using antibiotic resistance, another plasmid containing the targeted gene of interest in the form of sgRNA and the λ-red operon is transformed. After induced expression of the λ-red recombineering system, linear DNA to be incorporated into the E. coli genome is transformed into the cells. The expression of Cas9 and the sgRNA are then induced, which results in the Cas9 locating the E. coli target DNA based on the sgRNA complement. Cas9 is able to initiate a double strand break and the λ-red system is able to bring the linear DNA to E. coli genome for homologous recombination. The cells are then cured of the plasmid containing the specific sgRNA and then the next plasmid containing the specific sgRNA target sequence can be transformed and the process is repeated.
This system is able to modify numerous genome locations rapidly. In instances when a small number of mutations are introduced into the genome, the time efficiency of no-SCAR is comparable to other methods. However, with a large number of alterations, this technique is superior.
Methodology
Plasmids SCAR-less editing uses plasmids for genome modification created using circular polymerase extension cloning. Briefly, in a one-step reaction, this process can assemble and clone multiple inserts into any vector and requires no restriction enzyme digestion, ligation, or homologous recombination. Therefore, this method contributes to the cost-effectiveness and throughput of the no-SCAR system.
The no-SCAR technique uses a two-plasmid system; this is because co-transformation of both cas9 containing plasmid and the sgRNA plasmid results in cell death. More specifically, the cell lethality is a consequence of Cas9 cleaving the E. coli DNA that matches the sgRNA. In order to circumvent this issue, multiple plasmids are used in order to maintain expressional control of both cas9 and sgRNA.
The PTET promoter plays an integral role in the expression of both plasmids. It drives transcriptional expression of both the sgRNA of interest and cas9 in the host cell upon induction with anhydrotetracycline (aTc). In the absence of the inducer, the PTET promoter is repressed by the constitutively expressed tetracycline repressor protein (TetR). Therefore, the presence of TetR in the host cell prior to the introduction of both sgRNA and cas9 is a measure to avoid cell lethality.
The first plasmid used in the pioneering work of Reisch and Prather is composed of: the cas9 gene under the control of the PTET promoter; the tetR gene, which codes for tetracycline repressor protein, under control of a constitutive promoter; and the cmr gene for chloramphenicol resistance. It was observed that leaky expression of Cas9 occurred even without induction of the PTET promoter. Therefore, to avoid cell death, a transfer messenger RNA (ssrA) tag was included in the plasmid downstream of the cas9 gene. In the event of leaky Cas9 expression, the C-terminal ssrA tag would be recognized by ClpP protease and degrade Cas9 to allow for better expressional control of the protein. Together, these components make up the pCas9cr4 plasmid and allow targeting of the host cell chromosome.
The second plasmid used in the no-SCAR method consists of: the sgRNA of interest expressed under the control of the PTET promoter; the three genes that make up the λ-red system (exo, bet, and gam) under the control of the ParaB promoter, which is induced by arabinose; and the gene conferring resistance to aminoglycosides, such as spectinomycin and streptomycin. These components make up the pKDsg-XXX plasmid to facilitate λ-red mediated alterations of the E. coli genome, where -XXX denotes the targeted sequence to modify.
In summary, theoretically, the plasmids for use in this method can be constructed to allow for absolute customization of the protocol. One stipulation is that each of the two constructed plasmids should have distinct selectable markers, such as genes conferring resistance to two different antibiotics, to allow for targeted selection and counterselection. Furthermore, the pCas9cr4 plasmid can be purchased from Addgene (ID 62655) for direct implementation into a no-SCAR recombination experiment, and the pKDsgRNA-p15 (ID 62656) and pKDsgRNA-ack (ID 62654) plasmids can also be purchased from Addgene.
Transformations The next step in the no-SCAR protocol is to transform the pCas9cr4 and pKDsg-XXX plasmids and linear oligonucleotides into the E. coli cells themselves. In order to achieve this, the cells are made to be electrocompetent; one such method, as used by Reisch and Pather, is using a glycerol/mannitol density step gradient, which is fast and simple. This allows for transformation through electroporation and introduction of the plasmids and DNA into the cells.