Guest post by Thomas Clements
Gene editing technology is continuously evolving. CRISPR-Cas9 mutagenesis hit the world by storm in 2013 and has since been extensively used as a tool to knockout specific genes of interest in vitro, in vivo, and recently even in humans. There have been many improvements to the system, but up until recently no one has described a mechanism to insert novel DNA into specific locations without creating potentially toxic double-strand breaks in DNA. Here, I describe two reports of RNA-guided DNA integration with CRISPR-associated transposases.
The Current Technology
The clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated (Cas) endonuclease system is present in roughly 40% of sequenced bacteria and 90% of sequenced archaea (Antonova et al., 2018). Scientists have leveraged this natural tool to edit DNA. Editing is initiated by inducing novel double-strand breaks (DSBs) in the DNA itself through the use of the Cas9 endonuclease directed by a single-guide RNA (sgRNA).
Once a DSB break occurs, cells respond through two competing mechanisms: the dominant non-homologous end-joining (NHEJ) and homologous recombination (HR) (Huertas et al., 2008). This dominant NHEJ process is error-prone and leads to insertions and/or deletions (indels) of nucleotides at the break site, but only produces indels on the order 10 bps on average (Carrington et al., 2015). If scientists want to insert larger chunks of DNA at precise locations, they must rely on HR, which requires the exchange of similar or analogous DNA to DSBs. However, while CRISPR-based integration has had some success in vitro (Antonova et al., 2018), it has been increasingly problematic in vivo (Hruscha et al., 2013)with incorporation rates sometimes on the order of less than 1%.
There is a great need in molecular biology to insert specific large chunks of DNA in discrete locations. This technology could easily insert novel constructs, such as GFP, into gene-specific locations to visually measure gene activity, rescue disease specific genetic mutations through gene therapy, and even improve crop yield by allowing gene specific alterations to be visible in fewer generations among others. In order to find new ways to accomplish this, scientists have focused their attention back to how CRISPR works in the natural population. In bacteria and archaea, CRISPRs serve as an adaptive immune system. When a foreign nucleic acid invades a host cell and it is degraded, a small portion of it is incorporated into the CRISPR array in the form of a small spacer. This locus can then produce small RNAs that bind and guide Cas endonucleases to cleave previously degraded foreign nucleic acids (Wei et al., 2013).
In its natural role, CRISPR has two big functions, it can quickly target and degrade foreign DNA, but it also incorporates a memory of this foreign invasion into its own genome to prevent further attacks. In recent publications, scientists have increasingly focused on the two discrete functions: it not only knocks out (destroys via direct offensive attack) foreign viral invaders, but also incorporates a novel DNA spacer into its own genome to serve as a “memory” of this attack to prevent further viral attacks in the future by more quickly recognizing this foreign invader (a more defensive mechanism).There are two classes of CRISPR: class 1 and class 2. Within class 1, there are types I, III and IV. Within class 2, there are types II, V and VI. The three main types are type I, II and III, and they’re distinguished by unique Cas proteins. Proteins Cas 1, Cas 2, and Cas4 play key roles in spacer acquisition, whereas Cas proteins 3 and 5-10 play significant roles in target binding and cleavage (Hatoum-Aslan et al., 2013; Jiang et al., 2013). In summary, CRISPR is known to have a role in both targeting and transposition. In most publications, scientists have focused most of their attention CRISPR’s role in targeting via the type II protein Cas9 because of its simplicity (it’s the only protein involved in targeting binding).
In new papers out in Science and Nature, researchers shifted their focus to the Cas proteins involved spacer incorporation and their role in transposition (Klompe et al., 2019) and (Strecker et al., 2019) respectively.
So what exactly are transposons and how have scientists utilized them before? Transposons are mobile DNA elements that can jump locations within the genome and were first described by Barbara McClintock, which earned her a Nobel Prize in 1983. There are two types of transposons: retrotransposons which rely on reverse transcriptase for integration and DNA transposons, which encode for an enzyme called transposase that acts in a “cut and paste” mechanism to excise genetic elements and insert themselves in that place. They are selfish genetic elements in that they often enhance their own transcription even at the expense of organismal fitness. In research settings, many transposons are used to rescue loss of function phenotypes by expressing the fully functional gene. One specific example, (Luft, 2010) successfully used the transposon Sleeping Beauty to insert sequences into mice with sickle cell anemia so they can produce the enzymes need to counteract this disease. Other applications include gene traps among others. Here, transposons like Tol2 are used to mark genes with labels like GFP (Kawakami, 2007). Nonetheless, the use transposons are not specific and the DNA can be inserted with the genome in essentially random locations.
It is hypothesized that CRISPR-associated targeting complexes lead transposons to discrete DNA locations sites via a specific sgRNA. This hypothesis is based on the fact that Cas9 binding to DNA results in an R-loop structure (Figure 2) and transposons like Tn7 is suggested to have hijacked CRISPR effectors to generate specific R-loops. Thus, enhancing the spread of transposons via plasmids and phage infection (Kawakami, 2007).
Klompe et al 2019 Nature
In order to test this hypothesis, (Klompe et al., 2019) used a transposon from Vibrio cholerae strain HE-45, Tn6677, which encodes a variant Type I-F CRISPR–Cas system. This system is known as Cascade and does not have the cas1 and cas2 genes responsible for spacer insertions as well as the cas3 gene responsible for target DNA degradation. Because of this, this system is designed to produce sgRNAs that target 48-50 base pairs downstream the target site. Cascade’s association with tniQ accomplishes integration. tniQ, which is a homolog of tnsD, specifies DNA integration sites and is a key component of the well-known transposon Tn7. All together, this allows for DNA integration in a replicative transposition mechanism with a 5 base pair duplication signature at both ends of the insertion at sites 48-50 base pairs downstream the sgRNA target site.Klompe and colleagues have termed this new tool “Insertion of transposable elements by guide RNA-assisted targeting” or INTEGRATE.
Strecker et al 2019 Science
In similar fashion, (Strecker et al., 2019) studied the CRISPR-associated transposase (CAST) from S. hofmanni (ShCAST). This complex consists of Cas12k, which is a natural cas protein (also known as Cpf1) that can bind to but not cleave DNA (similar to dead Cas9). This is due to a natural mutation and its association with the tnsB/tnsC/tniQcomplex (tnsB is involved in excision as well as integration and tnsC is an ATPase). The researchers selected 48 targets in E. coli and detected novel insertions by PCR at 29 out of the 48 sites (60.4%). Here, ShCAST produces unidirectional insertions 60-66 base pairs downstream of the target and prevents repeated insertions into a single target site.
Thisbreakthrough is significant because the sgRNAs produced from Cascade and ShCAST in these mechanisms are easily manipulated and thus can lead site-specific insertions without creating the lethal DSB and without utilizing the rare HR pathway. In previous studies, transposons can be use to insert sequences into organisms, but the integration location was either completely random or restricted to a specific site. Now we can direct site-specific insertion of DNA segments to any location! This technology could easily insert novel constructs, such as GFP, into gene-specific locations to visually measure gene activity, even more efficiently rescue disease specific genetic mutations, and play key roles in gene therapy as well as crop enhancement. I’m curious as to the first individuals who publish the use of CRISPR-associated transposases in mice, zebrafish, and potentially even humans!
Thomas Clements, Ph.D. is a Senior Lecturer in the Biological Sciences Department at Vanderbilt University. He graduated with his Ph.D. in Biochemistry and Cell Biology from Rice University in May 2018. His research centers on improving CRISPR-Cas9 gene editing in zebrafish and is continuing this work in a Discovery-Based Laboratory Course at Vanderbilt. He is also a member of the Early Career Scientist Policy and the Education Committee at the Genetics Society of America. He is passionate about science literacy and inspiring the next generation of scientists to pursue careers that align with their passions.
Antonova, E., Glazova, O., Gaponova, A., Eremyan, A., Zvereva, S., Grebenkina, N., Volkova, N., and Volchkov, P. (2018). Successful CRISPR/Cas9 mediated homologous recombination in a chicken cell line. F1000Research 7.
Carrington, B., Varshney, G.K., Burgess, S.M., and Sood, R. (2015). CRISPR-STAT: an easy and reliable PCR-based method to evaluate target-specific sgRNA activity. Nucleic Acids Res. gkv802.
Hatoum-Aslan, A., Samai, P., Maniv, I., Jiang, W., and Marraffini, L.A. (2013). A ruler protein in a complex for antiviral defense determines the length of small interfering CRISPR RNAs. J. Biol. Chem. jbc.M113.499244.
Hruscha, A., Krawitz, P., Rechenberg, A., Heinrich, V., Hecht, J., Haass, C., and Schmid, B. (2013). Efficient CRISPR/Cas9 genome editing with low off-target effects in zebrafish. Dev. Camb. Engl.
Huertas, P., Cortés-Ledesma, F., Sartori, A.A., Aguilera, A., and Jackson, S.P. (2008). CDK targets Sae2 to control DNA-end resection and homologous recombination. Nature 455, 689–692.
Jiang, W., Maniv, I., Arain, F., Wang, Y., Levin, B.R., and Marraffini, L.A. (2013). Dealing with the Evolutionary Downside of CRISPR Immunity: Bacteria and Beneficial Plasmids. PLoS Genet 9, e1003844.
Kawakami, K. (2007). Tol2: a versatile gene transfer vector in vertebrates. Genome Biol. 8, S7.
Klompe, S.E., Vo, P.L.H., Halpin-Healy, T.S., and Sternberg, S.H. (2019). Transposon-encoded CRISPR–Cas systems direct RNA-guided DNA integration. Nature 1.
Luft, F.C. (2010). Sleeping Beauty jumps to new heights. J. Mol. Med. 88, 641–643.
Peters, J.E., Makarova, K.S., Shmakov, S., and Koonin, E.V. (2017).Recruitment of CRISPR-Cas systems by Tn7-like transposons. Proc. Natl. Acad. Sci. 114, E7358–E7366.
Strecker, J., Ladha, A., Gardner, Z., Schmid-Burgk, J.L., Makarova, K.S., Koonin, E.V., and Zhang, F. (2019). RNA-guided DNA insertion with CRISPR-associated transposases. Science eaax9181.
Wei, C., Liu, J., Yu, Z., Zhang, B., Gao, G., and Jiao, R. (2013). TALEN or Cas9 – Rapid, Efficient and Specific Choices for Genome Modifications. J. Genet. Genomics 40, 281–289.