PASTE: The Way Forward for Large DNA Insertions.

Abstract

The CRISPR JournalVol. 6, No. 1 First CutFree AccessPASTE: The Way Forward for Large DNA InsertionsMuhammad Arslan Mahmood and Shahid MansoorMuhammad Arslan MahmoodAgricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE) College Pakistan Institute of Engineering and Applied Sciences, Faisalabad, Pakistan.Department of Biological Sciences, University of Sialkot, Sialkot, Pakistan.Search for more papers by this author and Shahid Mansoor*Address correspondence to: Shahid Mansoor, Jamil-ur-Rahman Center for Genome Research, International Center for Chemical and Biological Sciences (ICCBS), University of Karachi, Karachi 75270, Pakistan. E-mail Address: shahidmansoor@iccbs.eduAgricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE) College Pakistan Institute of Engineering and Applied Sciences, Faisalabad, Pakistan.International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan.Search for more papers by this authorPublished Online:9 Feb 2023https://doi.org/10.1089/crispr.2023.0001AboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail A recent report in Nature Biotechnology from the MIT laboratory of Omar Abudayyeh and Jonathan Gootenberg describes a new CRISPR tool (PASTE) that can swap faulty genes with preferred sequences, expanding the scope of gene editing by permitting large multiplex gene insertion beyond the dependence on DNA repair mechanisms.CRISPR-Cas9 genome editing has brought researchers closer to the goal of installing a specific genetic modification at a certain position in the genome with minimal undesirable discrepancy and cellular perturbation. However, the key requirement for the successful correction of faulty genes or the introduction of a new trait is programmable genome insertion. This relies on cellular responses to the double-stranded DNA breaks repair pathways such as nonhomologous end joining,1 homology-independent targeted insertions,2 or homology-directed repair,3 and can cause heterogeneity in editing byproducts as well as triggered aberrant chromosomal translocations and deletions.4Alternative gene editing techniques such as base and prime editing are typically restricted to making only single nucleotide substitutions, minor insertions (less than ∼50 bp), or short deletions (less than ∼80 bp).5A recent report from Anzalone et al at Prime Medicine demonstrated the use of dual prime editing guide RNAs (pegRNAs) with complementary reverse transcription template regions, which can integrate large DNA sequences. Furthermore, this technique utilizes a prime editing protein and two pegRNAs for a programmable replacement or excision of DNA sequences at specific sites in the human genome without requiring double-stranded DNA cuts. When combined with site-specific recombinase, the twin prime editing allowed integration of larger DNA sequences up to 5 kb into safe harbor loci.6 Nonetheless, this tool has low efficiency so far in the 1–5.6 kb range and has yet to demonstrate integration of larger sequences.To overcome these size limitations, Yarnall et al (in the laboratory of Omar Abudayyeh and Jonathan Gootenberg at the MIT McGovern Institute) have devised a new approach, marrying site-specific integrases with programmable CRISPR-based prime editing. It enables efficient integration of large DNA sequences (up to ∼36 kb) at well-defined target regions within human cell lines, nondividing primary human hepatocytes as well as primary T cell, with efficiencies up to 50–60% and 4–5%, respectively.This approach, which the authors have dubbed “programmable addition via site-specific targeting elements” or PASTE, employs a Cas9-nickase attached to reverse transcriptase and a large serine integrase, constructing a blended protein that has the capacity to make 5–50% precise insertions of desired payloads.7Viruses and transposons (mobile genetic elements) encode an enormous diversity of integrative enzymes including recombinases that enable site-specific targeted large DNA insertions without the need for any cellular cofactors or without inducing exposed DNA double-stranded breaks.8 Last year, Durrant et al described the large serine recombinases (LSRs, e.g., Bxb1 and PhiC31) as the DNA integrases that catalyze unidirectional DNA insertion into cognate attachment sites. However, Bxb1 requires the preinstallation of its preferred attachment site (landing pad) in the human genome, whereas PhiC31 can integrate the DNA cargo into pseudo site loci in eukaryotic genomes that resemble its native attachment sites.9By employing Bxb1, a major DNA payload reports successfully of 27 kb integration into the mammalian cells and exhibits low integration efficiency of only ∼10%.10 Furthermore, Durrant et al developed a pipeline to explore the diversity of various LSRs containing microbes that are considered as promising candidates for genome editing in mammalian cell applications and identified a big group of putative functional classes of phage-encoded LSRs including LSRs classes to be utilized for landing pad and single or multitargeting in human genomes. All the LSRs offer the potential of targeting the human genome for both research and clinical applications. However, the two main drawbacks of recombinases are a lack of programmability and low-to-moderate insertion activity.9Yarnall et al formulated a clever solution to these limitations by making a complex of highly active LSRs with an existing CRISPR-based prime editing technique (Fig. 1A) in which LSRs are fused to a prime editor as well as modified pegRNA that encodes the attB site. Using a circular double-stranded DNA template with an attP attachment site, the expressed integrase can directly integrate the DNA cargo at the desired target site with a single delivery process. Hence, the installation of the attachment site can be followed by integrating the donor DNA that has a compatible attP site.FIG. 1. Improved tools for programmable large-scale DNA integration independent of DNA repair pathways. (A) Prime editing technology consists of a fusion of nCas9 with pegRNA and reverse transcriptase. The desired mutations (red pegs) are carried on the pegRNA at the 3′ end of the reverse transcriptase template. The primer binding site binds to the nicked strand of DNA, thereby priming the reverse transcription of a template into the desired DNA sequence. Then, in a precise way, edited nucleotides are inserted into the target site. (B) Bioinformatic mining and experimental screening of potential LSRs revealed new recombinases that are highly effective in combining donor DNA into either a preinstalled landing site or a naturally occurring pseudo site. In addition, LSRs can be used for functional genomics research, in which a donor DNA library that has been amplified by polymerase chain reaction can be placed at a single genomic target site without the requirement for cloning or lentiviral transmission (bottom). (C) PASTE editing employs a combination of a recombinase-prime editor and a pegRNA encoding the attachment site (termed atgRNA) to enable a two-step editing procedure in which attB is inserted first followed by donor DNA integration. The efficacy of this methodology was confirmed with multiple delivery approaches in a variety of cell types including in vivo experiments in mice. LSR, large serine recombinase; pegRNA, prime editing guide RNA.Current research has revealed that prime editing still works if reverse transcriptase and Cas9 nickase are expressed separately. Nonetheless, further experimentation is desirable and likely necessary to determine whether the PASTE fusion layout improves the editing efficiency beyond that achieved with separate constituents. In addition, when PASTE was benchmarked against the other prime editing and integrase-based insertion methods, it was found promisingly successful, driven by the combination of more optimized attB and attP sequences to vintage-enhanced variants of PASTEv2 and v3.7The authors employed PASTEv3 to execute targeted DNA insertions together in primary and nondividing cells of the human genome compared with the other delivery formulations such as adenovirus, adeno-associated virus, and RNA viruses.7 This new approach is straightforwardly exploited for new genes and could be carried by a single dosage of plasmids. This tool also overcomes the problems resulting from dsDNA breaks caused by other CRISPR tools such as chromosomal rearrangement or large-scale chromosome arm deletions. Employing this strategy, complete replacement of genes at their natural sites can be contemplated instead of creating variant-specific treatments.These exciting results7 enhance the versatility of the CRISPR-based gene editing along with the LSRs that provide the opportunity for genome engineering, combinatory screening of bulky DNA libraries aiding to spread these applications for treating the diseases in both animals and plants, which are caused by defective genes. Highly active and novel serine integrases that fit into naturally occurring pseudo sites overcome the requisite of preinstalled landing pads and reliance on safe harbor loci (Fig. 1B).PASTE is a tool not just for correcting individual mutations; it can replace faulty genes with new genes without inducing any dsDNA breaks. Hence this approach comes with a golden opportunity to make desirable DNA insertion and gene replacement of kilobase-scale DNA cargoes. The combination of these novel recombinases with the CRISPR-Cas9 system enables easy reprogramming and insertion system to target specific genome sequences in plants that act as susceptibility genes or loci for several abiotic and biotic stresses.Yet, certain complex reaction setups involved such as flap equilibration and resolution, DNA nicking, reverse transcription, and recombination will be imperative for the comparison of PASTE with other engineering toolkits, including CRISPR-associated transposases and phage-derived single-stranded annealing proteins.11A common theme of this novel approach, PASTE has the capacity for snipping out and replacing the susceptibility genes with new effective genes in an efficient and safer way and becomes a huge value to both basic science and biotechnology studies (Fig. 1C). In addition, we look forward to seeing it further applied to agricultural applications in which it could be utilized to protect crops from heat, drought, salinity, hypoxia, pests, and various diseases such as rust diseases in bread wheat and viral diseases in food and fiber crops with a goal to achieve high-yielding climate-smart crop plants.