{"id":{"repo_id":"texas","oai_identifier":"oai:repositories.lib.utexas.edu:2152/130678"},"canonical_url":"https://search.dev.ndltd.org/etd/texas/oai:repositories.lib.utexas.edu:2152/130678","repository":{"repo_id":"texas","name":"University of Texas","base_url":"https://repositories.lib.utexas.edu/server/oai/request"},"display":{"title":"Discovery of novel systems for genome editing","abstract":"Genome editing techniques allow for targeted modifications to specific genes, enabling the correction of genetic mutations that cause diseases, as well as the functional study of specific genes. The capacity to precisely modify genes at desired loci offers unprecedented opportunities in therapeutic applications, particularly in monogenic disorders, where a single genetic aberration is the main cause. Moreover, genome editing can facilitate the development of transgenic organisms for agricultural advancements, increasing food production and enhancing nutritional quality. Additionally, it can be used for the research and synthesis of biofuels, pharmaceuticals, and other bio-based materials. The increasing global challenges in health and sustainability underscore the necessity of genome editing as a transformative approach to address complex biological and medical questions. The experimental validation of Cas9-based programmable DNA cleavage and its early capacity for targeted genome modification in living eukaryotic cells sparked rapid advancements in CRISPR–Cas genome editing tools. Cas9, when used on its own for genome editing, primarily induces gene knockouts by introducing double-strand breaks and relying on the cell’s error-prone repair mechanisms. Without additional elements, Cas9 lacks the capability for precise sequence insertions or specific nucleotide editing. This limitation restricts its applications to gene knockout. Diverse genome editing challenges necessitate tool kits beyond just Cas9 to offer enhanced specificity, versatility, and the ability to target a broader range of genomic sequences. The one type of genome editing tool depends on site-specific double-strand breaks (DSBs) and homology direct repair. In these systems, cas protein response to induce site-specific DSB at the position needs to be edited, then the cell’s natural repair mechanisms come into play, with external DNA templates, like single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), is provided, precise genetic alterations can be achieved through homology-directed repair (HDR). These methods can be hindered due to challenges in delivering the external templates into cells, and retrons could be the solution for this. Retron’s unique ability to produce programable msDNA as templates within cells could substitute the ssDNA or dsDNA template, but people are still working on Eco1 and Eco2 which were discovered in 1981. There are innumerable returns distributed in bacteria and archaea that might be better than those two waiting for discovery. We have developed a bioinformatic pipeline to identify thousands of novel retron candidates with its msrmsd. We classify them based on their associate open reading frame and pick 100 candidate retron for testing. We developed a rapid screen method for the retron editor in HEK293T cell by the transient reporter assay. We successfully identified multiple novel retron that work much better than Eco1 in genome editing efficiency. Without any optimization, just by transfecting the plasmid containing the cas9 and retron into HEK293T cell can reach 33% editing efficiency. Another type of genome editing technique brought forward innovative tools like prime editors and base editors. These methods capitalize on the Cas nickase’s ability to create a single-strand break, coupled with effector proteins that mediate the desired genetic modifications. Prime editors, for instance, merge the nicking capability of Cas with reverse transcriptase to precisely rewrite DNA sequences. Base editors, on the other hand, employ chemically modified nucleobases to facilitate the direct conversion of one base pair to another without causing double-strand breaks. These methods offer the advantage of reduced indel formation and enhanced precision compared to traditional Cas9 systems. However, they also come with limitations; for example, the editing window is relatively narrow, typically permitting changes to less than 44 base pairs at a time. The CAST (CRISPR-associated transposases) system represents a groundbreaking advancement in the realm of genome editing. Unlike the conventional CRISPR-Cas systems which rely on inducing double-strand breaks and subsequent DNA repair mechanisms, CASTs facilitate direct DNA insertions into specific genomic locations using RNA-guided mechanisms which are fewer independent editing steps, and without the need for extensive optimization. This targeted insertion capability offers a distinct advantage as it circumvents some of the limitations and risks associated with the introduction of double-strand breaks, such as unwanted indels or chromosomal rearrangements. Compared with prime editors and base editors, CASTs potentially allow for the insertion of much larger DNA fragments which could fit many editing scenarios. CRISPR-associated transposons (CASTs) co-opt cas genes for RNA-guided transposition. CASTs are exceedingly rare in genomic databases. We expand the diversity of reported CAST systems via a bioinformatic search of metagenomic databases. We discover new architectures for all known CASTs, including novel arrangements of the Cascade effectors, new target homing modalities, and minimal V-K systems. We also describe new families of CASTs that have co-opted the Type I-C and Type IV CRISPR-Cas systems. Our search for non-Tn7 CASTs identifies putative candidates that include a nuclease dead Cas12. These new systems shed light on how CRISPR systems have co-evolved with transposases and expanded the programmable gene editing toolkit. We show that CASTs instead co-opt defense-associated CRISPR arrays for horizontal transmission. A bioinformatic analysis shows that all CAST sub-types co-occur with defense-associated CRISPR-Cas systems. Using an E. coli quantitative transposition assay, we show that CASTs use CRISPR RNAs (crRNAs) from these defense systems for horizontal gene transfer. A high-resolution structure of the type I-F CAST-Cascade in complex with a type III-B crRNA reveals that Cas6 recognizes direct repeats via sequence-independent π − π interactions. In addition to using heterologous CRISPR arrays, type V CASTs can also transpose via a crRNA-independent unguided mechanism, even when the S15 co-factor is over-expressed. Over-expressing S15 and the trans-activating CRISPR RNA (tracrRNA) or a single guide RNA (sgRNA) reduces, but does not abrogate, off-target integration for type V CASTs. These results provide a possible mechanism for how CASTs horizontally transfer to new hosts. More broadly, this work will guide further efforts to engineer the activity and specificity of CASTs for gene editing applications.","abstract_html":"Genome editing techniques allow for targeted modifications to specific genes, enabling the correction of genetic mutations that cause diseases, as well as the functional study of specific genes. The capacity to precisely modify genes at desired loci offers unprecedented opportunities in therapeutic applications, particularly in monogenic disorders, where a single genetic aberration is the main cause. Moreover, genome editing can facilitate the development of transgenic organisms for agricultural advancements, increasing food production and enhancing nutritional quality. Additionally, it can be used for the research and synthesis of biofuels, pharmaceuticals, and other bio-based materials. The increasing global challenges in health and sustainability underscore the necessity of genome editing as a transformative approach to address complex biological and medical questions. The experimental validation of Cas9-based programmable DNA cleavage and its early capacity for targeted genome modification in living eukaryotic cells sparked rapid advancements in CRISPR–Cas genome editing tools. Cas9, when used on its own for genome editing, primarily induces gene knockouts by introducing double-strand breaks and relying on the cell’s error-prone repair mechanisms. Without additional elements, Cas9 lacks the capability for precise sequence insertions or specific nucleotide editing. This limitation restricts its applications to gene knockout. Diverse genome editing challenges necessitate tool kits beyond just Cas9 to offer enhanced specificity, versatility, and the ability to target a broader range of genomic sequences. The one type of genome editing tool depends on site-specific double-strand breaks (DSBs) and homology direct repair. In these systems, cas protein response to induce site-specific DSB at the position needs to be edited, then the cell’s natural repair mechanisms come into play, with external DNA templates, like single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), is provided, precise genetic alterations can be achieved through homology-directed repair (HDR). These methods can be hindered due to challenges in delivering the external templates into cells, and retrons could be the solution for this. Retron’s unique ability to produce programable msDNA as templates within cells could substitute the ssDNA or dsDNA template, but people are still working on Eco1 and Eco2 which were discovered in 1981. There are innumerable returns distributed in bacteria and archaea that might be better than those two waiting for discovery. We have developed a bioinformatic pipeline to identify thousands of novel retron candidates with its msrmsd. We classify them based on their associate open reading frame and pick 100 candidate retron for testing. We developed a rapid screen method for the retron editor in HEK293T cell by the transient reporter assay. We successfully identified multiple novel retron that work much better than Eco1 in genome editing efficiency. Without any optimization, just by transfecting the plasmid containing the cas9 and retron into HEK293T cell can reach 33% editing efficiency. Another type of genome editing technique brought forward innovative tools like prime editors and base editors. These methods capitalize on the Cas nickase’s ability to create a single-strand break, coupled with effector proteins that mediate the desired genetic modifications. Prime editors, for instance, merge the nicking capability of Cas with reverse transcriptase to precisely rewrite DNA sequences. Base editors, on the other hand, employ chemically modified nucleobases to facilitate the direct conversion of one base pair to another without causing double-strand breaks. These methods offer the advantage of reduced indel formation and enhanced precision compared to traditional Cas9 systems. However, they also come with limitations; for example, the editing window is relatively narrow, typically permitting changes to less than 44 base pairs at a time. The CAST (CRISPR-associated transposases) system represents a groundbreaking advancement in the realm of genome editing. Unlike the conventional CRISPR-Cas systems which rely on inducing double-strand breaks and subsequent DNA repair mechanisms, CASTs facilitate direct DNA insertions into specific genomic locations using RNA-guided mechanisms which are fewer independent editing steps, and without the need for extensive optimization. This targeted insertion capability offers a distinct advantage as it circumvents some of the limitations and risks associated with the introduction of double-strand breaks, such as unwanted indels or chromosomal rearrangements. Compared with prime editors and base editors, CASTs potentially allow for the insertion of much larger DNA fragments which could fit many editing scenarios. CRISPR-associated transposons (CASTs) co-opt cas genes for RNA-guided transposition. CASTs are exceedingly rare in genomic databases. We expand the diversity of reported CAST systems via a bioinformatic search of metagenomic databases. We discover new architectures for all known CASTs, including novel arrangements of the Cascade effectors, new target homing modalities, and minimal V-K systems. We also describe new families of CASTs that have co-opted the Type I-C and Type IV CRISPR-Cas systems. Our search for non-Tn7 CASTs identifies putative candidates that include a nuclease dead Cas12. These new systems shed light on how CRISPR systems have co-evolved with transposases and expanded the programmable gene editing toolkit. We show that CASTs instead co-opt defense-associated CRISPR arrays for horizontal transmission. A bioinformatic analysis shows that all CAST sub-types co-occur with defense-associated CRISPR-Cas systems. Using an E. coli quantitative transposition assay, we show that CASTs use CRISPR RNAs (crRNAs) from these defense systems for horizontal gene transfer. A high-resolution structure of the type I-F CAST-Cascade in complex with a type III-B crRNA reveals that Cas6 recognizes direct repeats via sequence-independent π − π interactions. In addition to using heterologous CRISPR arrays, type V CASTs can also transpose via a crRNA-independent unguided mechanism, even when the S15 co-factor is over-expressed. Over-expressing S15 and the trans-activating CRISPR RNA (tracrRNA) or a single guide RNA (sgRNA) reduces, but does not abrogate, off-target integration for type V CASTs. These results provide a possible mechanism for how CASTs horizontally transfer to new hosts. More broadly, this work will guide further efforts to engineer the activity and specificity of CASTs for gene editing applications.","abstract_has_math":false,"creators":["Hu, Kuang, Ph. D."],"institution":"The University of Texas at Austin","degree_name":"Doctor of Philosophy","degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Finkelstein, Ilya J.","Wilke, C. (Claus)"],"committee_chairs":[],"committee_members":[],"year":2023,"date_issued":"2023-12","date_published":"2023-12","updated_at":"2026-07-24T05:01:06Z","subjects":["Retron","CRISPR","Transposon","Genome editing"],"languages":["en"],"rights":[],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://doi.org/10.26153/tsw/58028"],"render_values":[{"text":"https://doi.org/10.26153/tsw/58028","href":"https://doi.org/10.26153/tsw/58028","code":true}]}]},"links":{"outbound_url":"https://hdl.handle.net/2152/130678","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Finkelstein, Ilya J.","Wilke, C. (Claus)"]},{"key":"dc:creator","label":"Author","values":["Hu, Kuang, Ph. 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The capacity to precisely modify genes at desired loci offers unprecedented opportunities in therapeutic applications, particularly in monogenic disorders, where a single genetic aberration is the main cause. Moreover, genome editing can facilitate the development of transgenic organisms for agricultural advancements, increasing food production and enhancing nutritional quality. Additionally, it can be used for the research and synthesis of biofuels, pharmaceuticals, and other bio-based materials. The increasing global challenges in health and sustainability underscore the necessity of genome editing as a transformative approach to address complex biological and medical questions. The experimental validation of Cas9-based programmable DNA cleavage and its early capacity for targeted genome modification in living eukaryotic cells sparked rapid advancements in CRISPR–Cas genome editing tools. Cas9, when used on its own for genome editing, primarily induces gene knockouts by introducing double-strand breaks and relying on the cell’s error-prone repair mechanisms. Without additional elements, Cas9 lacks the capability for precise sequence insertions or specific nucleotide editing. This limitation restricts its applications to gene knockout. Diverse genome editing challenges necessitate tool kits beyond just Cas9 to offer enhanced specificity, versatility, and the ability to target a broader range of genomic sequences. The one type of genome editing tool depends on site-specific double-strand breaks (DSBs) and homology direct repair. In these systems, cas protein response to induce site-specific DSB at the position needs to be edited, then the cell’s natural repair mechanisms come into play, with external DNA templates, like single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), is provided, precise genetic alterations can be achieved through homology-directed repair (HDR). These methods can be hindered due to challenges in delivering the external templates into cells, and retrons could be the solution for this. Retron’s unique ability to produce programable msDNA as templates within cells could substitute the ssDNA or dsDNA template, but people are still working on Eco1 and Eco2 which were discovered in 1981. There are innumerable returns distributed in bacteria and archaea that might be better than those two waiting for discovery. We have developed a bioinformatic pipeline to identify thousands of novel retron candidates with its msrmsd. We classify them based on their associate open reading frame and pick 100 candidate retron for testing. We developed a rapid screen method for the retron editor in HEK293T cell by the transient reporter assay. We successfully identified multiple novel retron that work much better than Eco1 in genome editing efficiency. Without any optimization, just by transfecting the plasmid containing the cas9 and retron into HEK293T cell can reach 33% editing efficiency. Another type of genome editing technique brought forward innovative tools like prime editors and base editors. These methods capitalize on the Cas nickase’s ability to create a single-strand break, coupled with effector proteins that mediate the desired genetic modifications. Prime editors, for instance, merge the nicking capability of Cas with reverse transcriptase to precisely rewrite DNA sequences. Base editors, on the other hand, employ chemically modified nucleobases to facilitate the direct conversion of one base pair to another without causing double-strand breaks. These methods offer the advantage of reduced indel formation and enhanced precision compared to traditional Cas9 systems. However, they also come with limitations; for example, the editing window is relatively narrow, typically permitting changes to less than 44 base pairs at a time. The CAST (CRISPR-associated transposases) system represents a groundbreaking advancement in the realm of genome editing. Unlike the conventional CRISPR-Cas systems which rely on inducing double-strand breaks and subsequent DNA repair mechanisms, CASTs facilitate direct DNA insertions into specific genomic locations using RNA-guided mechanisms which are fewer independent editing steps, and without the need for extensive optimization. This targeted insertion capability offers a distinct advantage as it circumvents some of the limitations and risks associated with the introduction of double-strand breaks, such as unwanted indels or chromosomal rearrangements. Compared with prime editors and base editors, CASTs potentially allow for the insertion of much larger DNA fragments which could fit many editing scenarios. CRISPR-associated transposons (CASTs) co-opt cas genes for RNA-guided transposition. CASTs are exceedingly rare in genomic databases. We expand the diversity of reported CAST systems via a bioinformatic search of metagenomic databases. We discover new architectures for all known CASTs, including novel arrangements of the Cascade effectors, new target homing modalities, and minimal V-K systems. We also describe new families of CASTs that have co-opted the Type I-C and Type IV CRISPR-Cas systems. Our search for non-Tn7 CASTs identifies putative candidates that include a nuclease dead Cas12. These new systems shed light on how CRISPR systems have co-evolved with transposases and expanded the programmable gene editing toolkit. We show that CASTs instead co-opt defense-associated CRISPR arrays for horizontal transmission. A bioinformatic analysis shows that all CAST sub-types co-occur with defense-associated CRISPR-Cas systems. Using an E. coli quantitative transposition assay, we show that CASTs use CRISPR RNAs (crRNAs) from these defense systems for horizontal gene transfer. A high-resolution structure of the type I-F CAST-Cascade in complex with a type III-B crRNA reveals that Cas6 recognizes direct repeats via sequence-independent π − π interactions. In addition to using heterologous CRISPR arrays, type V CASTs can also transpose via a crRNA-independent unguided mechanism, even when the S15 co-factor is over-expressed. Over-expressing S15 and the trans-activating CRISPR RNA (tracrRNA) or a single guide RNA (sgRNA) reduces, but does not abrogate, off-target integration for type V CASTs. These results provide a possible mechanism for how CASTs horizontally transfer to new hosts. More broadly, this work will guide further efforts to engineer the activity and specificity of CASTs for gene editing applications."]},{"key":"dc:format.mimetype","label":"Dc Format Mimetype","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Discovery of novel systems for genome editing"]}]}],"canonical_facts":{"dc:contributor.advisor":["Finkelstein, Ilya J.","Wilke, C. (Claus)"],"dc:creator":["Hu, Kuang, Ph. D."],"dc:date.accessioned":["2025-01-11T02:40:08Z"],"dc:date.available":["2025-01-11T02:40:08Z"],"dc:date.issued":["2023-12"],"dc:description.abstract":["Genome editing techniques allow for targeted modifications to specific genes, enabling the correction of genetic mutations that cause diseases, as well as the functional study of specific genes. The capacity to precisely modify genes at desired loci offers unprecedented opportunities in therapeutic applications, particularly in monogenic disorders, where a single genetic aberration is the main cause. Moreover, genome editing can facilitate the development of transgenic organisms for agricultural advancements, increasing food production and enhancing nutritional quality. Additionally, it can be used for the research and synthesis of biofuels, pharmaceuticals, and other bio-based materials. The increasing global challenges in health and sustainability underscore the necessity of genome editing as a transformative approach to address complex biological and medical questions. The experimental validation of Cas9-based programmable DNA cleavage and its early capacity for targeted genome modification in living eukaryotic cells sparked rapid advancements in CRISPR–Cas genome editing tools. Cas9, when used on its own for genome editing, primarily induces gene knockouts by introducing double-strand breaks and relying on the cell’s error-prone repair mechanisms. Without additional elements, Cas9 lacks the capability for precise sequence insertions or specific nucleotide editing. This limitation restricts its applications to gene knockout. Diverse genome editing challenges necessitate tool kits beyond just Cas9 to offer enhanced specificity, versatility, and the ability to target a broader range of genomic sequences. The one type of genome editing tool depends on site-specific double-strand breaks (DSBs) and homology direct repair. In these systems, cas protein response to induce site-specific DSB at the position needs to be edited, then the cell’s natural repair mechanisms come into play, with external DNA templates, like single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), is provided, precise genetic alterations can be achieved through homology-directed repair (HDR). These methods can be hindered due to challenges in delivering the external templates into cells, and retrons could be the solution for this. Retron’s unique ability to produce programable msDNA as templates within cells could substitute the ssDNA or dsDNA template, but people are still working on Eco1 and Eco2 which were discovered in 1981. There are innumerable returns distributed in bacteria and archaea that might be better than those two waiting for discovery. We have developed a bioinformatic pipeline to identify thousands of novel retron candidates with its msrmsd. We classify them based on their associate open reading frame and pick 100 candidate retron for testing. We developed a rapid screen method for the retron editor in HEK293T cell by the transient reporter assay. We successfully identified multiple novel retron that work much better than Eco1 in genome editing efficiency. Without any optimization, just by transfecting the plasmid containing the cas9 and retron into HEK293T cell can reach 33% editing efficiency. Another type of genome editing technique brought forward innovative tools like prime editors and base editors. These methods capitalize on the Cas nickase’s ability to create a single-strand break, coupled with effector proteins that mediate the desired genetic modifications. Prime editors, for instance, merge the nicking capability of Cas with reverse transcriptase to precisely rewrite DNA sequences. Base editors, on the other hand, employ chemically modified nucleobases to facilitate the direct conversion of one base pair to another without causing double-strand breaks. These methods offer the advantage of reduced indel formation and enhanced precision compared to traditional Cas9 systems. However, they also come with limitations; for example, the editing window is relatively narrow, typically permitting changes to less than 44 base pairs at a time. The CAST (CRISPR-associated transposases) system represents a groundbreaking advancement in the realm of genome editing. Unlike the conventional CRISPR-Cas systems which rely on inducing double-strand breaks and subsequent DNA repair mechanisms, CASTs facilitate direct DNA insertions into specific genomic locations using RNA-guided mechanisms which are fewer independent editing steps, and without the need for extensive optimization. This targeted insertion capability offers a distinct advantage as it circumvents some of the limitations and risks associated with the introduction of double-strand breaks, such as unwanted indels or chromosomal rearrangements. Compared with prime editors and base editors, CASTs potentially allow for the insertion of much larger DNA fragments which could fit many editing scenarios. CRISPR-associated transposons (CASTs) co-opt cas genes for RNA-guided transposition. CASTs are exceedingly rare in genomic databases. We expand the diversity of reported CAST systems via a bioinformatic search of metagenomic databases. We discover new architectures for all known CASTs, including novel arrangements of the Cascade effectors, new target homing modalities, and minimal V-K systems. We also describe new families of CASTs that have co-opted the Type I-C and Type IV CRISPR-Cas systems. Our search for non-Tn7 CASTs identifies putative candidates that include a nuclease dead Cas12. These new systems shed light on how CRISPR systems have co-evolved with transposases and expanded the programmable gene editing toolkit. We show that CASTs instead co-opt defense-associated CRISPR arrays for horizontal transmission. A bioinformatic analysis shows that all CAST sub-types co-occur with defense-associated CRISPR-Cas systems. Using an E. coli quantitative transposition assay, we show that CASTs use CRISPR RNAs (crRNAs) from these defense systems for horizontal gene transfer. A high-resolution structure of the type I-F CAST-Cascade in complex with a type III-B crRNA reveals that Cas6 recognizes direct repeats via sequence-independent π − π interactions. In addition to using heterologous CRISPR arrays, type V CASTs can also transpose via a crRNA-independent unguided mechanism, even when the S15 co-factor is over-expressed. Over-expressing S15 and the trans-activating CRISPR RNA (tracrRNA) or a single guide RNA (sgRNA) reduces, but does not abrogate, off-target integration for type V CASTs. These results provide a possible mechanism for how CASTs horizontally transfer to new hosts. More broadly, this work will guide further efforts to engineer the activity and specificity of CASTs for gene editing applications."],"dc:format.mimetype":["application/pdf"],"dc:identifier.uri":["https://hdl.handle.net/2152/130678","https://doi.org/10.26153/tsw/58028"],"dc:language.iso":["en"],"dc:subject":["Retron","CRISPR","Transposon","Genome editing"],"dc:title":["Discovery of novel systems for genome editing"],"dc:type":["Thesis"],"thesis:degree_name":["Doctor of Philosophy"],"thesis:institution_name":["The University of Texas at Austin"]},"updated_at":"2026-07-24T05:01:06Z"}