Method for predicting possible off-target occurrences in the process of genome editing using a prime editing system

Abstract

The present application relates to a method for predicting possible off-targets in the process of editing a genome by using a prime editing system.

Description

[Technical Field]

[0001] The present application relates to a method for predicting off-target events in a prime editing system, which is a type of gene editing system. [Background technology]

[0002] Genome editing using the CRISPR / Cas system is an active area of ​​research. Although various studies have been conducted on modified guide RNAs and Cas proteins for gene manipulation, there are still problems with CRISPR / Cas gene editing. The many problems arising from CRISPR / Cas gene manipulation have motivated the development of more sophisticated genome editing techniques. Such motivation has led to the development of a more sophisticated genome editing technique, base editing. However, the scope of application of base editing is still limited.

[0003] David R. Liu and colleagues developed prime-editing, a "search-and-replace" genome editing technique that is effective in inducing insertions, deletions, all 12 base-interchanges, and combinations thereof in the genome.

[0004] A new genome editing platform called "prime editing" was developed by David R. Liu et al., but a method for predicting potential off-target events in genome editing using prime editing has not yet been developed. The development of prime editing as a new genome editing platform necessitates the development of new methods for predicting off-target events suitable for prime editing systems. Summary of the Invention [Problem to be solved by the invention]

[0005] Off-target events occurring in the gene editing process can cause severe side effects. Accordingly, various methods for predicting off-target events have been developed. However, the methods known to date have been developed to target conventional CRISPR / Cas systems, and are therefore difficult to apply to new gene editing systems such as prime editing systems. Therefore, the present application discloses a method or system for predicting off-target events in a prime editing system, which has been developed to target the prime editing system. [Means for solving the problem]

[0006] Some embodiments of the present application provide a method for predicting off-target events that may occur in the process of genome editing using a prime editing system, the method comprising: (a) obtaining an engineered cell; wherein the engineered cell comprises engineered genomic DNA, wherein the engineered genomic DNA comprises a tag sequence, wherein the engineered genomic DNA engages a prime editor protein and a tpegRNA, as follows: (i) contacting genomic DNA with a prime editor protein and a tpegRNA (tagmentation pegRNA), wherein the prime editor comprises a Cas protein and a reverse transcriptase, and the tpegRNA comprises a spacer and an extension region comprising a tag template; (ii) inserting the tag sequence into genomic DNA by a reverse transcription process carried out by reverse transcriptase using the tag template of tpegRNA as a template for reverse transcription; Produced through a process including (b) analyzing the engineered genomic DNA to obtain information about tagmentation; Here, the information about tagmentation includes information about the region of genomic DNA into which the tag sequence is inserted. The present invention provides a method comprising:

[0007] In certain embodiments, the method for predicting off-targets comprises: Obtaining off-target information based on the tagmentation information, wherein the off-target information includes information regarding whether off-target candidates exist, and, if so, information regarding the regions of the off-target candidates; It may further comprise:

[0008] In certain embodiments, the method for predicting off-targets comprises: Verifying the on-target information and comparing the on-target information with the tagmentation information. It may further comprise:

[0009] In certain embodiments, the method for predicting off-targets comprises: determining whether off-target candidates exist by determining the on-target information and comparing the on-target information with the tagmentation information; It may further comprise:

[0010] In certain embodiments, the tag sequence can be inserted into a region of genomic DNA specified by the spacer of the tpegRNA.

[0011] In certain embodiments, the region into which the tag sequence is inserted may be associated with (correspond to) a potential off-target region or an on-target region.

[0012] In certain embodiments, the information about the region into which the tag sequence is inserted may include information about the chromosome in which the tag sequence is located, and information about the region within the chromosome in which the tag sequence is present.

[0013] In certain embodiments, information regarding the region of the off-target candidate may include information regarding the chromosome on which the off-target candidate is located, and the region within the chromosome in which the off-target candidate is located.

[0014] In certain embodiments, the information regarding tagmentation may further include the insertion ratio of the tag sequence by the region into which the tag sequence is inserted.

[0015] In certain embodiments, the information regarding the off-target may further include an off-target prediction score for the off-target candidate.

[0016] In certain embodiments, the information regarding off-targets may further include the number of predicted off-target candidates.

[0017] In certain embodiments, the engineered cell can be obtained by a method comprising contacting a cell with a prime editor protein or a nucleic acid encoding the prime editor protein, and a tpegRNA or a nucleic acid encoding the tpegRNA.

[0018] In certain embodiments, the engineered cell can be obtained by a method comprising introducing a prime editor protein or a nucleic acid encoding the prime editor protein, and a tpegRNA or a nucleic acid encoding the tpegRNA into a cell.

[0019] In certain embodiments, the method may further comprise the step of obtaining DNA from the engineered cells, which step occurs before (b).

[0020] In certain embodiments, the tpegRNA may comprise a spacer; a gRNA core; and an extension region comprising a primer binding site, a tag template, and a reverse transcription template.

[0021] In certain embodiments, the reverse transcription template of the tpegRNA may comprise an editing template and a homology region.

[0022] In certain embodiments, the manipulated genomic DNA may include edits.

[0023] In certain embodiments, the spacer, gRNA core, and extension region may be arranged in the following order in the 5'→3' direction: spacer, gRNA core, and extension region.

[0024] In certain embodiments, the tag template may be located between the primer binding site and the reverse transcription template in the extension region.

[0025] In certain embodiments, the tpegRNA may further comprise a 3' engineered region comprising an RNA protection motif.

[0026] In certain embodiments, the method for predicting off-targets comprises: Information about a given cell, information about a given pegRNA, and information about a given prime editor protein Validating a given prime editing system, including one or more of: It may further comprise:

[0027] In certain embodiments, the cells of interest may be different from the cells used in the method for predicting off-targets.

[0028] In certain embodiments, the sequence of the spacer of the tpegRNA may be the same as the sequence of the spacer of a given pegRNA, and the sequence of the primer binding site of the tpegRNA may be the same as the sequence of the primer binding site of a given pegRNA.

[0029] In certain embodiments, the sequence of the spacer of the tpegRNA may be the same as the sequence of the spacer of a given pegRNA, the sequence of the primer binding site of the tpegRNA may be the same as the sequence of the primer binding site of a given pegRNA, and the sequence of the reverse transcription template of the tpegRNA may be the same as the sequence of the reverse transcription template of a given pegRNA.

[0030] In certain embodiments, the prime editor protein used in the method for predicting off-targets may be the same as or different from a given prime editor protein.

[0031] In certain embodiments, the tag template may be 5 nt to 60 nt in length.

[0032] In certain embodiments, the tag template may be 10 nt to 50 nt in length.

[0033] In certain embodiments, the prime editor protein may be a PE-nuclease, which comprises a Cas protein with double-strand cleavage activity.

[0034] In certain embodiments, the prime editor protein may be PEmax-nuclease.

[0035] In certain embodiments, the Cas protein comprised in the prime editor protein may be a nickase.

[0036] In certain embodiments, the prime editor protein may be a PE2 prime editor protein.

[0037] In certain embodiments, the manipulation of the DNA genome may further involve any one or more of dnMLH1, gRNA, and additional Cas proteins, and additional prime editor proteins.

[0038] In certain embodiments, (b) may comprise specifically analyzing the engineered genomic DNA tags.

[0039] In certain embodiments, (b) may include sequencing the engineered genomic DNA.

[0040] In certain embodiments, (b) is generating a tag-specific library from the engineered genomic DNA; amplifying the tag-specific library to generate an amplified tag-specific library; and sequencing the amplified tag-specific library. Includes:

[0041] Some embodiments of the present application provide a method for predicting off-target events that may occur in the process of genome editing using a prime editing system, the method comprising: (a) preparing a population of cells comprising one or more engineered cells; wherein the engineered cell comprises engineered genomic DNA, wherein the engineered genomic DNA comprises a tag sequence, wherein the engineered genomic DNA engages a prime editor protein and a tpegRNA, as follows: (i) contacting genomic DNA with a prime editor protein and a tpegRNA (tagmentation pegRNA), wherein the prime editor protein comprises a Cas protein and a reverse transcriptase, and wherein the tpegRNA comprises a spacer and an extension region comprising a tag template; (ii) the process of inserting the tag sequence into genomic DNA, where the insertion of the tag sequence is achieved via a reverse transcription process carried out by reverse transcriptase using the tag template of tpegRNA as a reverse transcription template; Produced through a process including (b) obtaining tagmentation information by analyzing results obtained through a process comprising sequencing the engineered genomic DNA of one or more engineered cells; wherein the tagmentation information includes information about one or more sites where each tag sequence is inserted; and (c) obtaining information about off-targets based on the tagmentation information; wherein the information about the off-target includes information about whether or not an off-target candidate exists, and information about the region of one or more off-target candidates; The present invention provides a method comprising:

[0042] Some embodiments of the present application include: The extension region contains the spacer, the gRNA core, and the tag template. The present invention provides a tagmented pegRNA (tpegRNA) comprising:

[0043] In certain embodiments, the extension region comprising the spacer, gRNA core, and tag template may be arranged in the following order in the 5' to 3' direction: spacer, gRNA core, and extension region comprising the tag template.

[0044] In certain embodiments, the extension region may comprise a tag template, a primer binding site, and a reverse transcription template.

[0045] In certain embodiments, the tag template may be located between the primer binding site and the reverse transcription template in the extension region.

[0046] In certain embodiments, the reverse transcription template may be located between the tag template and the primer binding site.

[0047] In certain embodiments, the primer binding site, tag template, and reverse transcription template may be positioned in the extension region in a 5'→3' direction in the following order: reverse transcription template, tag template, and primer binding site.

[0048] In certain embodiments, the reverse transcription template may comprise an editing template and a region of homology.

[0049] In certain embodiments, the tag template may have a length of 5 nt to 60 nt.

[0050] In certain embodiments, the tag template may have a length of 10 nt to 50 nt.

[0051] In certain embodiments, the tpegRNA may further comprise a 3' engineered region comprising an RNA protection motif.

[0052] In certain embodiments, the RNA protection motif may have a length of 10 nt to 60 nt.

[0053] In certain embodiments, the tpegRNA may have a length of 100 nt to 350 nt.

[0054] Some embodiments of the present application provide a composition for predicting potential off-targets in a genome editing process using a prime editing system, the composition comprising: tpegRNA; and Prime editor containing Cas protein and reverse transcriptase A composition comprising:

[0055] beneficial effects The method for predicting off-targets in a prime editing system according to some embodiments of the present application uses the molecular mechanism of the prime editing system, and thus has many advantages in predicting off-targets in a prime editing system compared to other known off-target prediction methods. [Brief explanation of the drawings]

[0056] [Figure 1] Examples of the structures of a conventional guide RNA (gRNA), a prime editing guide RNA (pegRNA), and a tagmentation pegRNA (tpegRNA) are shown. [Figure 2] Regarding an exemplary embodiment of a tpegRNA, the tpegRNA shown in FIG. 2 includes a DNA synthesis template, a tag template, and an extension region including a primer binding site (PBS); [Figure 3]Regarding an exemplary embodiment of a tpegRNA, where the tpegRNA shown in FIG. 3 comprises a primer binding site, a tag template, an editing template, and an extension region comprising a homology region. [Figure 4] Regarding the tag insertion mechanism using tpegRNA in the off-target prediction system of the present application, specifically, examples are shown of a DNA molecule in which a nick has been created at a potential on-target or off-target site, and a prime editor protein / tpegRNA complex that induces the nick. [Figure 5] Regarding the tag insertion mechanism using tpegRNA in the off-target prediction system of the present application, specifically, the primer binding site of tpegRNA functions as a primer and annealing site for genomic DNA, and subsequently indicates the region where reverse transcription is performed by reverse transcriptase (RT) using a tag template or the like as a template. [Figure 6] Regarding the tag insertion mechanism using tpegRNA in the off-target prediction system of the present application, it is shown that tag sequences etc. are added to the endogenous DNA strand (3' DNA flap) by reverse transcription, and then, through a process including removal of the 5' DNA flap and DNA repair, the tag sequence and its complementary sequence are placed at the on-target site or potential off-target site of genomic DNA. [Figure 7] 1 shows an exemplary process of the off-target prediction system of the present application, Tagmentation of Prime Editor sequencing (TAPE-seq). [Figure 8] The results of the insertion rate of the tag sequence depending on the incubation period are shown. [Figure 9] A map of the green fluorescent protein (GFP)-piggyBac vector is shown. [Figure 10] The results of enrichment of GFP-positive cells (HEK293T) are shown. [Figure 11]The results of enrichment of GFP-positive cells (HEK293T) are shown. [Figure 12] The results of enrichment of GFP-positive cells (HeLa) are shown. [Figure 13] The results of enrichment of GFP-positive cells (HeLa) are shown. [Figure 14] The results of enrichment of GFP-positive cells (K562) are shown. [Figure 15] The results of enrichment of GFP-positive cells (K562) are shown. [Figure 16] The number of candidate off-target regions found by TAPE-seq is shown for each incubation period after transfection of HEK294T cells with HEK4(+2G→T)pegRNA. [Figure 17] Figure 17 shows the results of an experiment to find the optimal amount of piggyBac vector for co-transfection with a transposase plasmid, where Figure 17 is a graph showing the copy number of the piggyBac construct found in cells by quantitative polymerase chain reaction (PCR) versus the amount of piggyBac plasmid (PB plasmid). [Figure 18] Figure 18 shows the results of an experiment to find the optimal amount of piggyBac vector for co-transfection with a transposase plasmid, where Figure 18 is a graph showing the tagmentation rate at the on-target site according to the amount (ng) of piggyBac plasmid used for HEK293T transfection. [Figure 19] Figure 19 shows the results of an experiment to find the optimal amount of piggyBac vector for co-transfection with a transposase plasmid, where Figure 19 is a graph showing the tagmentation rate at off-target site 1 according to the amount (ng) of piggyBac plasmid used for HEK293T transfection. [Figure 20] The analysis results of the tagmentation rate by probe sequence length are shown, where the tagmentation rate at the on-target site was analyzed. [Figure 21] The results of an analysis of tagmentation rates by probe sequence length are shown, where the tagmentation rates at off-target sites were analyzed. [Figure 22] The results of analysis of the prime editing rate and on-target tagmentation rate of nine different pegRNAs are shown. [Figure 23] The analysis results of the tagmentation rates at six target sites of HEK4(+2G→T)pegRNA and HBB(+4A→T)pegRNA are shown. [Figure 24] The editing rates of Case 1 and Case 2 are shown as determined by targeted deep sequencing using the PE analyzer, where nine different pegRNAs were analyzed. [Figure 25] 1 shows the results of testing tagmentation with and without prime editing at 10 different on-target and off-target sites. [Figure 26] FIG. 26 shows the comparison results for validated regions with off-target sites of HEK4 pegRNA predicted by TAPE-seq, where FIG. 26 shows the comparison results for validated regions with off-target sites of HEK4 (+2G→T) pegRNA predicted by TAPE-seq. [Figure 27] Figure 27 shows the comparison results for validated regions with off-target sites of HEK4 pegRNA predicted by TAPE-seq, where Figure 27 shows the comparison results for the validated site of HEK4(+3TAA ins), the combination of the off-target site of HEK4(+2G→T) predicted by TAPE-seq using Mi-seq and the off-target site of HEK4(+2G→T) predicted by TAPE-seq using Hi-seq, and the off-target site of HEK4(+3TAA ins) predicted by TAPE-seq (Mi-seq). [Figure 28]Figure 28 shows the comparison results for validated regions with off-target sites of HEK4 pegRNA predicted by TAPE-seq, where Figure 28 shows the comparison results for the validated site of HEK4 (+2G→T), the combination of the off-target site of HEK4 (+2G→T) predicted by TAPE-seq using Mi-seq and the off-target site of HEK4 (+2G→T) predicted by TAPE-seq using Hi-seq, and the off-target site of HEK4 (+3TAA ins) predicted by TAPE-seq (Mi-seq). [Figure 29] Regarding a comparison of the results predicted by TAPE-seq with those predicted by other off-target prediction methods, FIG. 29 shows the results for HEK4(+2G→T)pegRNA. [Figure 30] Regarding a comparison of the results predicted by TAPE-seq with those predicted by other off-target prediction methods, Figure 30 shows the results for HEK4(+3TAA ins)pegRNA. [Figure 31] Regarding a comparison of the results predicted by TAPE-seq with those predicted by other off-target prediction methods, FIG. 31 shows the results for EMX1(+5G→T)pegRNA. [Figure 32] Regarding a comparison of the results predicted by TAPE-seq with those predicted by other off-target prediction methods, FIG. 32 shows the results for FANCF(+6G→C)pegRNA. [Figure 33] Regarding a comparison of the results predicted by TAPE-seq with those predicted by other off-target prediction methods, Figure 33 shows the results for HEK3(+1CTT ins)pegRNA. [Figure 34] Regarding a comparison of the results predicted by TAPE-seq with those predicted by other off-target prediction methods, FIG. 34 shows the results for RNF2(+6G→A)pegRNA. [Figure 35]Regarding a comparison between the results predicted by TAPE-seq and those predicted by other off-target prediction methods, FIG. 35 shows the results for DNMT1(+6G→A)pegRNA. [Figure 36] Regarding a comparison of the results predicted by TAPE-seq with those predicted by other off-target prediction methods, FIG. 36 shows the results for HBB(+4A→T)pegRNA. [Figure 37] Regarding a comparison between the results predicted by TAPE-seq and those predicted by other off-target prediction methods, FIG. 37 shows the results for RUNX1(+6G→C)pegRNA. [Figure 38] Regarding a comparison of the results predicted by TAPE-seq with those predicted by other off-target prediction methods, Figure 38 shows the results for VEGFA(+5G→T)pegRNA. [Figure 39] 29 to 38, the analysis results of validated off-targets overlooked by each prediction method are shown. [Figure 40] The analysis results of tagmentation rates for PE2 TAPE-seq and PE4 TAPE-seq are shown. [Figure 41] Figure 41 shows a comparison of off-targets predicted by PE2 TAPE-seq, off-targets predicted by PE4 TAPE-seq, and bona fide off-targets validated by targeted deep sequencing; Figure 41 shows the results related to HEK293T. [Figure 42] Figure 41 shows a comparison of off-targets predicted by PE2 TAPE-seq, off-targets predicted by PE4 TAPE-seq, and bona fide off-targets validated by targeted deep sequencing; Figure 42 shows the results related to HeLa. [Figure 43]Figure 43 shows the results of a comparison of off-targets predicted by PE2 TAPE-seq, off-targets predicted by PE4 TAPE-seq, and bona fide off-targets validated by targeted deep sequencing; Figure 43 shows the results related to K562. [Figure 44] The analytical results summarizing the number of missed target sites for Figures 41 to 43 are shown, where Figure 44A shows the analytical results by each prediction method, and Figure 44B shows the analytical results for each cell type. [Figure 45] Figure 45 shows a comparison of the TAPE-seq off-target prediction results and the validation results for each cell type. Here, Figure 45 compares the validation results in HEK293T with the TAPE-seq prediction results for each cell type. [Figure 46] Figure 46 shows a comparison of TAPE-seq off-target prediction results and validation results for each cell type. Here, Figure 46 compares the validation results for HeLa cells with the TAPE-seq prediction results for each cell type. [Figure 47] Figure 47 shows a comparison of TAPE-seq off-target prediction results and validation results for each cell type. Here, Figure 47 compares the validation results for K562 cells with the TAPE-seq prediction results for each cell type. [Figure 48] The analysis results of the number of validated off-targets missed by TAPE-seq predictions in each cell type are shown. [Figure 49] TAPE-seq tagmentation rate analysis results using PE2, PE2-nuclease, and PEmax-nuclease used with epegRNA are shown. [Figure 50]Figure 50 shows a comparison of the off-target regions predicted by each TAPE-seq method (PE2 TAPE-seq, PE2-nuclease TAPE-seq, and TAPE-seq using PEmax-nuclease with epegRNA) with the validated off-target regions. Figure 50 shows the results for HEK4(+2G→T)pegRNA (or epegRNA) and HEK4(+3TAA ins)pegRNA. [Figure 51] Figure 51 shows a comparison of the off-target regions predicted by each TAPE-seq method (PE2 TAPE-seq, PE2-nuclease TAPE-seq, and TAPE-seq using PEmax-nuclease with epegRNA) with the validated off-target regions. Figure 51 shows the results for HBB (+4A→T) pegRNA and DNMT1 (+6G→C) pegRNA. [Figure 52] Figure 51 shows a comparison of the off-target regions predicted by each TAPE-seq method (PE2 TAPE-seq, PE2-nuclease TAPE-seq, and TAPE-seq using PEmax-nuclease with epegRNA) with the validated off-target regions. Figure 52 shows the results for VEGFA(+5G→T)pegRNA and EMX1(+5G→T)pegRNA. [Figure 53] Figure 53 shows a comparison of the off-target regions predicted by each TAPE-seq method (PE2 TAPE-seq, PE2-nuclease TAPE-seq, and TAPE-seq using PEmax-nuclease with peG RNA) with the validated off-target regions. Figure 53 shows the results for FANCF (+6G→C)peG RNA and HEK3 (+1CTT ins)peG RNA. [Figure 54] Figure 53 shows a comparison of the off-target regions predicted by each TAPE-seq method (PE2 TAPE-seq, PE2-nuclease TAPE-seq, and TAPE-seq using PEmax-nuclease with peGRNA) with the validated off-target regions. Figure 54 shows the results for RNF2(+6G→A)peGRNA and RUNX1(+6G→C)peGRNA. [Figure 55] Figure 55 shows the comparison of off-target prediction results of nDigenome-seq, GUIDE-seq, and TAPE-seq (TAPE-seq using PEmax-nuclease with pegRNA) with validated off-targets. Figure 55 shows the results for HEK4(+2G→T)pegRNA and HEK4(+3TAA ins)pegRNA. [Figure 56] Figure 56 shows a comparison of the off-target prediction results of nDigenome-seq, GUIDE-seq, and TAPE-seq (TAPE-seq using PEmax-nuclease with pegRNA) with validated off-targets. Figure 56 shows the results for HBB (+4A→T) pegRNA and DNMT1 (+6G→C) pegRNA. [Figure 57] Figure 57 shows a comparison of the off-target prediction results of nDigenome-seq, GUIDE-seq, and TAPE-seq (TAPE-seq using PEmax-nuclease with pegRNA) with validated off-targets. Figure 57 shows the results for VEGFA(+5G→T)pegRNA and EMX1(+5G→T)pegRNA. [Figure 58] Figure 58 shows a comparison of the off-target prediction results of nDigenome-seq, GUIDE-seq, and TAPE-seq (TAPE-seq using PEmax-nuclease with pegRNA) with validated off-targets. Figure 58 shows the results for FANCF(+6G→C)pegRNA and HEK3(+1CTT ins)pegRNA. [Figure 59] Figure 59 shows the comparison of off-target prediction results of nDigenome-seq, GUIDE-seq, and TAPE-seq (TAPE-seq using PEmax-nuclease with pegRNA) with validated off-targets. Figure 59 shows the results for RNF2 (+6G → A) pegRNA and RUNX1 (+6G → C) pegRNA. [Figure 60]The results of an analysis of the oversight rate for GUIDE-seq, nDigenome-seq, TAPE-seq (PE2), TAPE-seq (PE2-nuclease), and TAPE-seq (using PEmax-nuclease with epegRNA) are shown. [Figure 61] Figure 61 shows the results of a comparison of GUIDE-seq, nDigenome-seq, TAPE-seq(PE2), TAPE-seq(PE2-nuclease), and TAPE-seq (using PEmax-nuclease with pegRNA) using receiver operating characteristic (ROC) curves, where Figure 61 shows the results for HEK4(+2G→T)pegRNA and HEK4(+3TAA ins)pegRNA. [Figure 62] Figure 62 shows the results of a comparison of GUIDE-seq, nDigenome-seq, TAPE-seq(PE2), TAPE-seq(PE2-nuclease), and TAPE-seq (using PEmax-nuclease with peGRNA) using receiver operating characteristic (ROC) curves, where Figure 62 shows the results for HBB(+4A→T)peGRNA and DNMT1(+6G→C)peGRNA. [Figure 63] Receiver operating characteristic (ROC) curves are used to compare GUIDE-seq, nDigenome-seq, TAPE-seq(PE2), TAPE-seq(PE2-nuclease), and TAPE-seq (using PEmax-nuclease with peG RNA), where Figure 63 shows the results for HEK3(+1CTT ins)peG RNA. [Figure 64]Figure 64 shows the results of a comparison of GUIDE-seq, nDigenome-seq, TAPE-seq(PE2), TAPE-seq(PE2-nuclease), and TAPE-seq (using PEmax-nuclease with pegRNA) using receiver operating characteristic (ROC) curves, where Figure 64 shows the results for EMX1(+5G→T)pegRNA and FANCF(+6G→C)pegRNA. [Figure 65] Figure 65 shows the results of a comparison of GUIDE-seq, nDigenome-seq, TAPE-seq(PE2), TAPE-seq(PE2-nuclease), and TAPE-seq (using PEmax-nuclease with pegRNA) using receiver operating characteristic (ROC) curves, where Figure 65 shows the results for RNF2(+6G→A)pegRNA and RUNX1(+6G→C)pegRNA. [Figure 66] Figure 66 shows the results of a comparison of GUIDE-seq, nDigenome-seq, TAPE-seq(PE2), TAPE-seq(PE2-nuclease), and TAPE-seq (using PEmax-nuclease with peG RNA) using receiver operating characteristic (ROC) curves, where Figure 66 shows the results for VEGFA(+5G→T)peG RNA. [Figure 67] The analysis results of the area under the ROC curve calculated based on the analysis results of Figures 61 to 66 are shown. [Figure 68] The results of analyzing the editing patterns of off-target sites analyzed by targeted deep sequencing are shown, where Figure 68 shows the results related to the editing pattern induced by HEK4(+3TAA ins)pegRNA. [Figure 69]

[0062] Figure 69 shows the results of analyzing the editing patterns of off-target sites analyzed by targeted deep sequencing, where Figure 69 shows the results related to the editing pattern induced by HEK4 (+2G→T) pegRNA. [Figure 70]

[0073] Figure 70 shows the results of analyzing the editing patterns of off-target sites analyzed by targeted deep sequencing, where Figure 70 shows the results related to the editing pattern induced by HEK4(+2G→T)pegRNA. [Figure 71]

[0073] Figure 71 shows the results of analyzing the editing patterns of off-target sites analyzed by targeted deep sequencing, where Figure 71 shows the results related to the editing pattern induced by HEK4 (+2G→T) pegRNA. [Figure 72]

[0073] Figure 72 shows the results of analyzing the editing patterns of off-target sites analyzed by targeted deep sequencing, where Figure 72 shows the results of editing patterns at validated off-target sites associated with HEK4 (+2G→T) pegRNA. [Figure 73]

[0073] Figure 73 shows the results of analyzing the editing patterns of off-target sites analyzed by targeted deep sequencing, where Figure 73 shows the results of editing patterns at validated off-target sites associated with HEK4 (+2G→T) pegRNA. [Figure 74]

[0073] Figure 74 shows the results of analyzing the editing patterns of off-target sites analyzed by targeted deep sequencing, where Figure 74 shows the results of editing patterns at validated off-target sites associated with HEK4 (+2G→T) pegRNA. [Figure 75] Figure 75 shows the results of analyzing the editing patterns of off-target sites analyzed by targeted deep sequencing. Figure 75 shows the results of editing patterns at validated off-target sites associated with HEK4 (+2G→T) pegRNA. [Figure 76] The results of analyzing the editing patterns of off-target sites analyzed by targeted deep sequencing are shown, where Figure 76 shows the results of editing patterns at validated off-target sites associated with HBB(+4A→T)pegRNA. [Figure 77]The results of analyzing the editing patterns of off-target sites analyzed by targeted deep sequencing are shown, where Figure 77 shows the results of editing patterns at validated off-target sites associated with HEK4(+3TAA ins)pegRNA. [Figure 78] The results of analyzing the editing patterns of off-target sites analyzed by targeted deep sequencing are shown, where Figure 78 shows the results of editing patterns at validated off-target sites associated with HEK4(+3TAA ins)pegRNA. [Figure 79] The results of analyzing the editing patterns of off-target sites analyzed by targeted deep sequencing are shown, where Figure 79 shows the results of editing patterns at validated off-target sites associated with HEK4(+3TAA ins)pegRNA. [Figure 80] The results of analyzing the editing patterns of off-target sites analyzed by targeted deep sequencing are shown, where Figure 80 shows the results of editing patterns at validated off-target sites associated with HEK4(+3TAA ins)pegRNA. [Figure 81]

[0089] Figure 81 shows the results of analyzing the editing patterns of off-target sites analyzed by targeted deep sequencing, where Figure 81 shows the results in HeLa cells, specifically, the results for HEK4(+3TAA ins)pegRNA and HEK4(+2G→T)pegRNA. [Figure 82]

[0089] Figure 82 shows the results of analyzing the editing patterns of off-target sites analyzed by targeted deep sequencing, where Figure 82 shows the results in HeLa cells, specifically, the results for HEK4(+3TAA ins)pegRNA and HEK4(+2G→T)pegRNA. [Figure 83]

[0089] Figure 83 shows the results of analyzing the editing patterns of off-target sites analyzed by targeted deep sequencing, where Figure 83 shows the results in K562 cells, specifically the results for HEK4(+3TAA ins)pegRNA and HEK4(+2G→T)pegRNA. [Figure 84]

[0089] Figure 84 shows the results of analyzing the editing patterns of off-target sites analyzed by targeted deep sequencing, where Figure 84 shows the results in K562 cells, specifically the results for HEK4(+3TAA ins)pegRNA and HEK4(+2G→T)pegRNA. [Figure 85] Figure 85 shows the results of analysis of editing patterns at off-target sites analyzed by targeted deep sequencing, where Figure 85 shows the results of editing patterns at off-target sites validated by TAPE-seq performed using PEmax-nuclease, specifically the results for HEK4 (+2G→T) pegRNA, DNMT1 (+6G→C) pegRNA, HBB (+4A→T) pegRNA, and VEGFA (+5→T) pegRNA. [Figure 86] Figure 86 shows the results of analysis of editing patterns at off-target sites analyzed by targeted deep sequencing, where Figure 86 shows the results of editing patterns at off-target sites validated by TAPE-seq performed using PEmax-nuclease, specifically, the results for HEK4 (+2G→T) pegRNA, DNMT1 (+6G→C) pegRNA, HBB (+4A→T) pegRNA, and VEGFA (+5→T) pegRNA. [Figure 87] Figure 87 shows the results of analysis of editing patterns at off-target sites analyzed by targeted deep sequencing, where Figure 87 shows the results of editing patterns at off-target sites validated by TAPE-seq performed using PEmax-nuclease, specifically, the results for HEK4 (+2G→T) pegRNA, DNMT1 (+6G→C) pegRNA, HBB (+4A→T) pegRNA, and VEGFA (+5→T) pegRNA. [Figure 88] The results of ROC curve analysis constructed using the number of mismatches in each region of tpegRNA (target region, PBS, and RT template) are shown in Figure 88. Figure 88 shows the results for HEK4(+2G→T)pegRNA, HEK4(+3TAA ins)pegRNA, and HBB(+4A→T)pegRNA. [Figure 89] Figure 89 shows the results of ROC curve analysis constructed using the number of mismatches in each region of tpegRNA (target region, PBS, and RT template). Figure 89 shows the results for HEK3 (+1CTT ins)pegRNA, FANCF (+6G→C)pegRNA, and EMX1 (+5G→T)pegRNA. [Figure 90] The analysis results of ROC curves constructed using the number of mismatches in each region of tpegRNA (target region, PBS, and RT template) are shown in Figure 90. Figure 90 shows the results for DNMT1 (+6G→C)pegRNA, RUNX1 (+6G→C)pegRNA, and VEGFA (+5G→T)pegRNA. [Figure 91] The analysis results of the area under the ROC curve calculated based on the analysis results of Figures 88 to 90 are shown. [Figure 92] The results of an analysis of the mismatch rates of false-positive sites predicted by TAPE-seq and validated sites are shown. [Figure 93] The vector map of the piggyBac PE2 all-in-one plasmid (pAllin1-PE2) is shown. DETAILED DESCRIPTION OF THE INVENTION

[0057] Definition of Terms Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains. The following references provide those skilled in the art with general definitions of many of the terms used herein: [Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991)]. As used herein, the following terms have the meanings assigned to them unless otherwise specified.

[0058] "Linked" or "Chained" As used herein, the term "linked" or "linked" means that two or more elements present within a single conceptualizable structure are linked either directly or indirectly (e.g., via other elements such as a linker), and is not intended to imply that there may not be other additional elements between the two or more elements. For example, a statement such as "element B is linked to element A" is intended to include, but is not limited to, both the case where there are one or more other elements between element A and element B (i.e., when element A is linked to element B via one or more other elements) and the case where there are no other elements between element A and element B (i.e., when element A and element B are directly linked).

[0059] Sequence identity As used herein, the term "sequence identity" refers to the degree of similarity between two or more sequences. For example, the term "sequence identity" is used together with a term referring to a reference sequence and a term expressing a ratio (such as a percentage). For example, the term "sequence identity" can be used to describe a sequence that is similar or substantially identical to a reference nucleotide sequence. When a sequence is described as having at least 90% sequence identity with sequence A, the reference sequence herein is sequence A. For example, the percentage of sequence identity can be calculated by aligning the reference sequence and the sequence to be measured for the percentage of sequence identity. Additionally, the percentage of sequence identity can be calculated by including all mismatches, deletions, and insertions of one or more nucleotides. The method for calculating and / or determining the percentage of sequence identity is not particularly limited and can be calculated and / or determined by any reasonable method or algorithm available to those skilled in the art.

[0060] Amino acid sequence notation Unless otherwise specified, amino acid sequences are described herein in an N-terminal to C-terminal direction using either the single-letter or three-letter code for amino acids. For example, RNVP refers to a peptide in which arginine, asparagine, valine, and proline are linked in that order from the N-terminus to the C-terminus. As another example, Thr-Leu-Lys refers to a peptide in which threonine, leucine, and lysine are linked in that order from the N-terminus to the C-terminus. Amino acids that cannot be represented by a single-letter code can be represented using other letters and described in more detail.

[0061] Each amino acid is represented as follows: alanine (Ala, A); arginine (Arg, R); asparagine (Asn, N); aspartic acid (Asp, D); cysteine ​​(Cys, C); glutamic acid (Glu, E); glutamine (Gln, Q); glycine (Gly, G); histidine (His, H); isoleucine (Ile, I); leucine (Leu, L); lysine (Lys, K); methionine (Met, M); phenylalanine (Phe, F); proline (Pro, P); serine (Ser, S); threonine (Thr, T); tryptophan (Trp, W); tyrosine (Tyr, Y); and valine (Val, V).

[0062] Nucleic acid sequence representation As used herein, the symbols A, T, C, G, and U are interpreted as having the meanings understood by those skilled in the art, which may be interpreted as bases, nucleosides, or nucleotides in DNA or RNA, as appropriate, depending on the context and technology. For example, when referring to a base, each symbol may be interpreted as adenine (A), thymine (T), cytosine (C), guanine (G), or uracil (U), and when referring to a nucleoside, each symbol may be interpreted as adenosine (A), thymidine (T), cytidine (C), guanosine (G), or uridine (U). When referring to a nucleotide in a sequence, each symbol may be interpreted as meaning a nucleotide containing the above-mentioned respective nucleosides.

[0063] Orientation of the disclosed sequence Unless otherwise specified or indicated, nucleotide sequences (such as DNA sequences, RNA sequences, or DNA / RNA hybrid sequences) disclosed herein should be understood to be presented in a 5' to 3' orientation. Amino acid sequences disclosed herein should be understood to be presented in an N to C terminal orientation, unless otherwise specified or indicated.

[0064] Target sequence As used herein, the term "target sequence" refers to a specific sequence recognized by a guide RNA or a gene editing tool (e.g., a Cas / conventional gRNA complex, a prime editor enzyme / pegRNA complex, etc.) to cleave a target gene or target nucleic acid. The target sequence may be appropriately selected depending on the purpose. For example, the term "target sequence" may refer to a sequence contained in a target gene or target nucleic acid sequence that is complementary to a spacer sequence contained in a guide RNA (e.g., a pegRNA) (in this case, the target sequence may bind complementarily to the spacer sequence of the guide RNA). In another example, the term "target sequence" contained in a target gene or target nucleic acid sequence may refer to a sequence complementary to a sequence complementary to a spacer sequence contained in a guide RNA (in this case, the target sequence may have substantially the same sequence as the spacer sequence of the guide RNA). As described above, the term "target sequence" may refer to a sequence complementary to a spacer sequence contained in a guide RNA and / or a sequence substantially the same as the spacer sequence of the guide RNA, but should not be construed as being limited thereto. In some embodiments, the target sequence may be disclosed as a sequence including a PAM sequence. In some embodiments, a target sequence may be disclosed as a sequence that does not include a PAM sequence. The term "target sequence" will be interpreted appropriately depending on the context in which it is described. Typically, the spacer sequence is determined taking into account the sequence of the target gene or target nucleic acid and the PAM sequence recognized by the editing protein of the CRISPR / Cas system. The term "target sequence" may refer only to a specific strand sequence that complementarily binds to the guide RNA of a CRISPR / Cas complex, or may refer only to a specific strand sequence that does not complementarily bind to the guide RNA, or may refer to the entire target duplex including a portion of a specific strand, as interpreted appropriately depending on the context. The definition of the term "target sequence" herein is disclosed to describe a strand in which a target sequence may exist, and therefore, the use of the term "target sequence" is not intended to distinguish between on-target and off-target sequences. The term "target sequence" may be used in reference to an on-target sequence.Additionally, the term "target sequence" may be used in reference to an off-target sequence. In other words, in some embodiments, an intended target sequence may be referred to as an on-target sequence, and an unintended target sequence may be referred to as an off-target sequence. For example, in some embodiments, an on-target sequence may be referred to as a target sequence (in this case, the target sequence and the spacer sequence of the guide RNA may, for example, be substantially the same). In another example, in some embodiments, an off-target sequence may be referred to as a target sequence (in this case, there may, for example, be no mismatch or one or more mismatches between the target sequence and the spacer sequence of the guide RNA). The term target sequence may be interpreted appropriately in reference to on-target and off-target depending on the content of the relevant paragraph.

[0065] Spacer linking chain As used herein, the term "spacer-binding strand" refers to a strand comprising a sequence that forms a complementary bond with part or all of the sequence of the spacer region of a guide nucleic acid (such as a guide RNA) in a gene editing system (e.g., a CRISPR / Cas gene editing system, a prime editing system, etc.) involving a guide nucleic acid. Typically, a DNA molecule such as a genome has a double-stranded structure. In the double strand, the strand that has a sequence complementary to part or all of the sequence of the spacer region of the guide nucleic acid and thereby forms a complementary bond may be referred to as the spacer-binding strand.

[0066] Spacer unbound chain As used herein, the term "spacer-unbound strand" refers to a strand other than the "spacer-bound strand," which refers to a strand containing a sequence that forms a complementary bond with part or all of the sequence of the spacer region of a guide nucleic acid in a gene editing system (e.g., a CRISPR / Cas gene editing system, a prime editing system, etc.) involving a guide nucleic acid (e.g., a guide RNA). Typically, DNA molecules such as genomes have a double-stranded structure, and the term "spacer-unbound strand" can be used to refer to a strand other than the spacer-bound strand in the double strand. For example, in editing a DNA molecule using a prime editing system, a strand containing a sequence that forms a complementary bond with part or all of the sequence of the spacer region of a pegRNA may be referred to as the "spacer-bound strand," and a strand containing a sequence that forms a complementary bond with the primer binding site (PBS) of the pegRNA may be referred to as the "spacer-unbound strand." For example, in prime editing version 2, a nick is induced in the spacer-unbound strand by Cas9(H840A), forming a 3' DNA flap on the spacer-unbound strand.

[0067] The first and second strands of a DNA molecule Typically, DNA molecules, such as genomes, have a double helix structure composed of two strands. Such a DNA molecule composed of two strands is sometimes referred to as double-stranded DNA. When describing a CRISPR / Cas-based gene editing system, it may be necessary to distinguish between the two strands of a DNA molecule. A single strand of a DNA molecule may be referred to as the first strand. In this case, the strand other than the first strand of the double-stranded DNA may be referred to as the second strand. In each embodiment, the first and second strands may be randomly determined. For example, in some embodiments, when a single strand of a DNA molecule is referred to as the first strand, the other strand of the DNA molecule may be referred to as the second strand. In some embodiments, for example, a spacer-bound strand may be referred to as the first strand. In some embodiments, a spacer-unbound strand may be referred to as the first strand, as another example. As described above, where necessary, a single strand of a DNA molecule may be referred to as the first strand, and the other strand may be referred to as the second strand.

[0068] Upstream and downstream As used herein, the terms "upstream" and "downstream" are relative terms that define the linear positions of at least two elements located in a nucleic acid molecule (whether single-stranded or double-stranded) oriented in the 5' to 3' direction. For example, when a nucleic acid molecule is described as having a first element upstream of a second element, the first element may be described herein as being located anywhere in the 5' direction relative to the second element. For example, if a single-nucleotide polymorphism (SNP) is located 5' to the nick site, the SNP may be described herein as being located upstream of the Cas9-induced nick site. In another example, when a nucleic acid molecule is described as having a first element downstream of a second element, the first element may be described herein as being located anywhere in the 3' direction relative to the second element. For example, if the SNP is located 3' to the nick site, the SNP may be described herein as being located downstream of the Cas9-induced nick site. The nucleic acid molecule can be DNA (double-stranded or single-stranded), RNA (double-stranded or single-stranded), or a hybrid of DNA and RNA.

[0069] Nuclear localization signal or sequence (NLS) The term "NLS" refers to an amino acid sequence that facilitates the import of a protein into the nucleus of a cell. For example, protein import can be facilitated by nuclear transport. NLSs are well known and will be apparent to those skilled in the art. For example, exemplary sequences of NLSs are described in PCT Application No. PCT / EP2000 / 011690 (Publication No. WO2021 / 038547), the contents of which are incorporated herein by reference for exemplary NLSs. In some embodiments, the NLS is selected from the group consisting of PKKKRKV (SEQ ID NO: 1), KRPAATKKAGQAKKKK (SEQ ID NO: 2), PAAKRVKLD (SEQ ID NO: 3), RQRRNELKRSP (SEQ ID NO: 4), NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 5), RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 6), VSRKRPRP (SEQ ID NO: 7), PPKKARED (SEQ ID NO: 8), P The NLS may include, but is not limited to, the amino acid sequence of QPKKKPL (SEQ ID NO: 9), SALIKKKKKKMAP (SEQ ID NO: 10), DRLRR (SEQ ID NO: 11), PKQKKRK (SEQ ID NO: 12), RKLKKKIKKL (SEQ ID NO: 13), REKKKFLKRR (SEQ ID NO: 14), KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 15), RKCLQAGMNLEARKTKK (SEQ ID NO: 16), or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 17). One or more NLSs can be selectively fused to a gene editing protein, such as a Cas protein or a prime editor protein. An NLS fused to a protein can be used to facilitate the translocation of such a linked protein to the nucleus, which is the desired site.

[0070] Proteins, peptides, and polypeptides As used herein, the terms "protein," "peptide," and "polypeptide" are used interchangeably and refer to a polymer of amino acid residues linked by peptide (amide) bonds. These terms refer to proteins, peptides, or polypeptides of any size, structure, or function. Typically, these proteins, peptides, or polypeptides may be at least three or more amino acids in length. In some embodiments, a protein, peptide, or polypeptide may refer to an individual protein or a combination of proteins. For example, a protein, peptide, or polypeptide may be used to encompass an individual protein, a fusion protein in which two or more elements (where at least one of the two elements is a protein) are fused, and a protein complex in which two or more elements (where at least one of the two elements is a protein) are combined. In some embodiments, one or more amino acids in a protein, peptide, or polypeptide may be modified. In this case, modifications contained in the protein, peptide, or polypeptide may be modifications caused by the addition of chemicals such as carbohydrate groups, hydroxyl groups, phosphate groups, farnesyl groups, isofarnesyl groups, and fatty acid groups, or linkers for conjugation, functionalization, or other modifications. In some embodiments, the protein, peptide, or polypeptide may be a single molecule or a multimolecular complex. In some embodiments, the protein, peptide, or polypeptide may be a naturally occurring protein. In some embodiments, the protein, peptide, or polypeptide may be a protein fragment. In some embodiments, the protein, peptide, or polypeptide may be naturally occurring, recombinant, synthetic, or prepared by any combination thereof. Any protein provided herein may be prepared by any method known in the art.For example, any of the proteins provided herein can be prepared by recombinant protein expression and purification, which is particularly suitable for fusion proteins containing peptide linkers. Techniques for recombinant protein expression and purification are widely known. See Green, Michael R., and Joseph Sambrook. "Molecular cloning." A Laboratory Manual 4th (2012). (The entire contents of which are incorporated herein by reference).

[0071] functional equivalent The term "functional equivalent" or "equivalent" refers to a secondary molecule or conceptualizable element that is functionally equivalent but not necessarily equivalent in structure to the primary molecule or conceptualizable element. For example, a "Cas9 equivalent" refers to a protein that has the same, or substantially the same, or similar function as Cas9, but does not necessarily have the same amino acid sequence. Throughout this application, when a specific protein is referred to, it is intended to include all functional equivalents thereof. For example, when written as "protein X," the term protein X may be interpreted as including its functional equivalent. In this embodiment, a "functional equivalent" or "equivalent" of protein X includes any paralog, ortholog, fragment, homolog, naturally occurring, engineered, mutated, and synthetic version of protein X that has an equivalent function. For example, when the term Cas protein is used, this term may be interpreted to include equivalents of Cas proteins (such as Cas nickases). In another example, when the term reverse transcriptase is used, this term may be interpreted to include equivalents of reverse transcriptase.

[0072] circular permutants As used herein, the term "circular permutation" refers to a polypeptide or protein containing a circular permutation, which is a structural rearrangement of a protein, whereby the order of amino acids found in the amino acid sequence of the protein is altered. A circular permutation is a protein with modified N- and / or C-termini compared to its wild-type counterpart. For example, the wild-type C-terminal half of a protein becomes a new N-terminal half. For example, circular permutation (CP) refers to a topological rearrangement in the primary sequence of a protein, where the sequence is split at a different site to create new adjacent N- and C-termini, which are simultaneously linked by a peptide linker. As a result, proteins with different connectivity but the same or similar overall three-dimensional (3D) shape can be prepared. For example, protein structures with improved or modified characteristics can be prepared, including reduced proteolytic susceptibility, improved catalytic activity, modified matrix or ligand binding, and / or improved thermal stability. Circular permutation proteins can occur in nature (e.g., concanavalin A and lectins). Additionally, circular permutations may occur as a result of post-translational modifications or may be engineered using recombinant techniques. Circular permutations of a particular protein may be included in equivalents of that particular protein.

[0073] As an example of a circular permutant, "circularly permuted Cas9" refers to any Cas9 protein or variant thereof resulting from a circular permutation with locally rearranged N- and C-termini. Such a circularly permuted Cas9 protein ("circularly permuted Cas9: CP-Cas9") or variant thereof has the ability to bind to DNA when complexed with a guide RNA. See Oakes, Benjamin L., Dana C. Nadler, and David F. Savage. "Protein engineering of Cas9 for enhanced function." Methods in enzymology. Vol. 546. Academic Press, 2014. 491-511.; and Oakes, Benjamin L., et al. "CRISPR-Cas9 circular permutants as programmable scaffolds for genome modification." Cell 176.1-2 (2019): 254-267.] (each of which is incorporated herein by reference). The disclosure herein includes new CP-Cas9s, or any previously known CP-Cas9, so long as the resulting circularly permuted protein has the ability to bind to DNA when complexed with a gRNA. Exemplary sequences of CP-Cas9 proteins are disclosed in WO2020191233A1 (Application No. PCT / US2020 / 023712), the entire contents of which are incorporated herein by reference.

[0074] fusion proteins As used herein, the term "fusion protein" refers to a hybrid polypeptide containing domains or proteins derived from at least two different types of elements, where at least one of the elements is a protein. For example, a fusion protein may be a hybrid polypeptide containing proteins derived from two different types of proteins. One type of protein may be located at the amino-terminal (N-terminal) portion of the fusion protein or the carboxy-terminal (C-terminal) portion of the fusion protein, thus forming an "amino-terminal fusion protein" or a "carboxy-terminal fusion protein," respectively. In some embodiments, a fusion protein may be used to refer to an element having a single molecular form in which two or more elements are covalently linked. In other embodiments, a fusion protein may be used to refer to an element having a multimolecular complex form in which two or more elements are non-covalently linked.

[0075] Linker As used herein, the term "linker" may refer to a molecule that links two other molecules or moieties. A linker linking two proteins in a fusion protein may be an amino acid sequence. For example, Cas9 may be linked to a reverse transcriptase by an amino acid linker sequence to form a fusion protein. Additionally, a linker linking two nucleotide sequences may be a nucleotide sequence. For example, in a conventional guide RNA, the crRNA is linked to the tracrRNA via a linker, thus forming a single-stranded guide RNA. In other embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker may have a length of, but is not limited to, 1 to 200 amino acids. In some embodiments, the linker may have a length of, but is not limited to, 1 to 500 nucleotides. Furthermore, longer linkers are also contemplated.

[0076] Dual-specific ligands As used herein, the term "dual-specific ligand" or "dual-specific moiety" refers to a ligand that binds to two different types of ligand-binding domains. In certain embodiments, the ligand is a small molecule, peptide, or polypeptide. In other embodiments, the ligand-binding domain is a dimerization domain, which may be placed as a peptide tag in a protein. In various embodiments, two proteins, each independently containing the same or different dimerization domain, can be induced to dimerize through binding of each dimerization domain to a dual-specific ligand. As used herein, "dual-specific ligand" may equivalently refer to a "chemical inducer of dimerization" or "chemical inducer of dimerization (CID)."

[0077] Dimerization domain The term "dimerization domain" refers to a ligand-binding domain that binds to a binding moiety of a dual-specific ligand. A first dimerization domain binds to a first binding moiety of a dual-specific ligand, and a second dimerization domain binds to a second binding moiety of the same dual-specific ligand. When a first dimerization domain is fused to a first protein and a second dimerization domain is fused to a second protein, the first and second proteins can dimerize in the presence of the dual-specific ligand. In this case, the dual-specific ligand has at least one moiety that binds to the first dimerization domain and at least another moiety that binds to the second dimerization domain. In some embodiments, the dimerization domain (such as the first dimerization domain) can be linked to a Cas protein. In some embodiments, the dimerization domain (such as the second dimerization domain) can be linked to a reverse transcriptase.

[0078] Nickase The term "nickase" refers to a Cas protein in which one of the two nuclease domains is inactivated. Nickases can cleave only one strand of a target DNA molecule.

[0079] Flap endonucleases As used herein, the term "flap endonuclease" refers to an enzyme that catalyzes the removal of 5' single-stranded DNA flaps. Such enzymes process the removal of 5' flaps formed during cellular processes including DNA replication. In some embodiments, prime editing methods can use endogenously or exogenously provided flap endonucleases to remove 5' flaps of endogenous DNA formed at target regions during prime editing. Flap endonucleases are known in the art and are disclosed in detail in [Patel, Nikesh, et al. "Flap endonucleases pass 5'-flaps through a flexible arch using a disorder-thread-order mechanism to confer specificity for free 5'-ends." Nucleic acids research 40.10 (2012): 4507-4519.; and Tsutakawa, Susan E., et al. "Human flap endonuclease structures, DNA double-base flipping, and a unified understanding of the FEN1 superfamily." Cell 145.2 (2011): 198-211.], each of which is incorporated herein by reference. An exemplary flap endonuclease may be flap structure-specific endonuclease 1 (FEN1). The sequence of FEN1 is disclosed in WO2020191233A1 (application number PCT / US2020 / 023712).

[0080] Effective dose As used herein, the term "effective amount" refers to the amount of a biologically active agent sufficient to elicit a desired biological response. For example, in some embodiments, an effective amount of a prime editor protein can refer to the amount of protein sufficient to edit a nucleotide sequence in a target region, such as a genome. In some embodiments, an effective amount of a prime editor protein provided herein, such as a fusion protein comprising a nickase-Cas9 domain and a reverse transcriptase, can refer to the amount of fusion protein sufficient to induce editing of an intended target region specifically bound and edited by the fusion protein. As will be understood by those skilled in the art, the effective amount of an agent, such as a fusion protein, nuclease, hybrid protein, protein dimer, complex of a protein (or protein dimer) and a polynucleotide, or polynucleotide, can vary depending on various factors, such as the desired biological response, the particular gene to be edited, the genome to be edited, the target region to be edited, the cell or tissue being targeted, and the agent used.

[0081] about As used herein, the term "about" means a degree of approximation to any quantity and refers to an amount, level, value, number, frequency, percent, dimension, size, amount, weight, or length that varies by 30%, 25%, 20%, 25%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of a reference amount, level, value, number, frequency, percent, dimension, size, amount, weight, or length.

[0082] CRISPR / Cas system Overview of the CRISPR / Cas system CRISPR This "CRISPR" section is for the convenience of the artisan, and the terms used in this section are not intended to limit the terms disclosed herein.

[0083] CRISPR is a family of DNA sequences (i.e., CRISPR clusters) found in bacteria and archaea that represent snippets of previous viral infection that have invaded prokaryotes. These snippets of DNA are used by prokaryotic cells to detect and destroy DNA from subsequent attacks by similar viruses, and together with CRISPR-associated RNA and CRISPR-associated protein (Cas protein) sequences, they constitute the prokaryotic immune defense system. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). Cas9 / crRNA / tracrRNA then endonucleolytically cleave linear or circular dsDNA targets that are complementary to the RNA. Specifically, target strands that are not complementary to the crRNA are first endonucleolytically cleaved and then exonucleolytically trimmed 3'-5'. DNA binding and cleavage typically require both protein and RNA. However, single-guide RNAs (sgRNAs, or simply gRNAs) have been developed, in which such single-stranded RNAs are engineered to combine aspects of both crRNA and tracrRNA into a single RNA species. See, e.g., Jinek, Martin, et al. "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Science 337.6096 (2012):816-821. (The entire contents of which are incorporated herein by reference.) Cas9 recognizes a short motif (protospacer adjacent motif (PAM)) in the CRISPR repeat sequence, which helps distinguish self from nonself.CRISPR biology, as well as the sequence and structure of the Cas9 nuclease, are well known to those of skill in the art (see, e.g., [Ferretti, Joseph J., et al. "Complete genome sequence of an M1 strain of Streptococcus pyogenes." Proceedings of the National Academy of Sciences 98.8(2001):4658-4663; Deltcheva, Elitza, et al. "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III." Nature 471.7340(2011):602-607; and Jinek, Martin, et al. "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Science 337.6096(2012):816-821], the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to, Streptococcus pyogenes (S. pyogenes) and Streptococcus thermophilus (S. thermophilus). Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on the disclosure herein, including Cas9 sequences from the organisms and loci disclosed in [Chylinski, Krzysztof, Anais Le Rhun, and Emmanuelle Charpentier. "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems." RNA biology 10.5 (2013):726-737.], the entire contents of which are incorporated herein by reference.

[0084] CRISPR / Cas system and its use in editing DNA molecules The CRISPR / Cas system, developed from the CRISPR system described above, is a technology for editing a desired DNA molecule (such as a cell's genome) at a desired site using a Cas protein derived from the cell's CRISPR system and a guide nucleic acid that directs the Cas protein to the target region. For example, the Cas protein forms a Cas / gRNA complex with a guide RNA (gRNA). The Cas / gRNA complex is directed to the desired site by the guide RNA contained therein. The Cas protein contained in the Cas / gRNA complex induces a double-strand break (DSB) or a nick (in the case of a nickase) at the desired site. The CRISPR / Cas system can be used to edit not only the cell's genome, but also DNA molecules not located in the genome. Since the discovery of CRISPR, single-stranded guide RNAs (sgRNAs) linked to tracrRNA and crRNA have been developed for the CRISPR / Cas system (see Jinek, Martin, et al., "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Science 337.6096 (2012):816-821, the entire contents of which are incorporated herein by reference). Additionally, various classes and / or types of Cas proteins have been developed, such as Cas9, Cas12a (cpf1), Cas12b (c2c1), Cas12e (CasX), Cas12k (c2c5), Cas14, Cas14a, Cas13a (c2c2), Cas13b (c2c6), Cas nickases (e.g., Cas9 nickase), and dead Cas. In some embodiments, Cas proteins may be referred to as CRISPR enzymes. For an understanding of CRISPR / Cas systems, see WO2018 / 231018 (International Publication No.), the entire contents of which are incorporated herein by reference. Cas proteins (or CRISPR enzymes) that can be used in CRISPR / Cas systems are further described below for the convenience of skilled artisans.

[0085] Cas proteins Overview of Cas Proteins In the context of the CRISPR / Cas system, the term Cas protein can refer to a protein that creates a nick or DSB in the desired region to complete editing, or a protein that helps induce editing. The term Cas protein can be used to include its equivalents. Typically, Cas proteins have nuclease activity that cleaves nucleic acids. For example, some Cas proteins can induce double-strand breaks (DSBs), which may be referred to as Cas nucleases. In another example, some Cas proteins can induce nicks, which may be referred to as Cas nickases. Some Cas proteins have been modified to lack nuclease activity, which may be referred to as dead Cas. In the CRISPR / Cas system, Cas protein can be used interchangeably with CRISPR enzyme. A representative example of a Cas protein is Cas9.

[0086] As used herein, the term Cas protein is used to collectively refer to editing proteins that can create DSBs or nicks in target regions, or inactive Cas proteins, as used in CRISPR / Cas systems. Examples of Cas proteins include, but are not limited to, Cas9, Cas9 variants, Cas9 nickase (nCas9), dead Cas9, Cpf1 (Cas12a) (type V CRISPR-Cas system), C2c1 (Cas12b) (type V CRISPR-Cas system), C2c2 (Cas13a) (type VI CRISPR-Cas system), and C2c3 (type V CRISPR-Cas system). Additional examples of Cas proteins are described in [Abudayyeh, Omar O., et al. "C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector." Science 353.6299(2016):aaf5573.], the entire contents of which are incorporated herein by reference.

[0087] In one embodiment, the Cas protein is selected from the group consisting of Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Campylobacter jejuni, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp. sp.), Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp.Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicellulosiruptor bescii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldas caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsonii, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp. sp.), Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp.The Cas protein may be derived from various microorganisms, such as Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, or Acaryochloris marina (e.g., Cas9 or Cpf1).

[0088] Below, we will use the Cas9 protein, a representative example of a Cas protein, as an example.

[0089] Cas9 protein In the CRISPR / Cas9 system, a protein with nuclease activity that cleaves nucleic acids, or a protein with inactivated nuclease activity, is called a Cas9 protein. The term Cas9 protein is used to include its equivalents. Additionally, Cas9 protein may be called a Cas9 nuclease, a Casn1 nuclease, or a clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease. The Cas9 protein corresponds to Class 2, Type II of the CRISPR / Cas system classification, examples of which include proteins from Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, and Streptosporangium roseum.The sequence and structure of the Cas9 protein are well known to those of skill in the art (see, e.g., [Ferretti, Joseph J., et al. "Complete genome sequence of an M1 strain of Streptococcus pyogenes." Proceedings of the National Academy of Sciences 98.8(2001):4658-4663; Deltcheva, Elitza, et al. "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III." Nature 471.7340(2011):602-607; and Jinek, Martin, et al. "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Science 337.6096(2012):816-821] (the entire contents of each of which are incorporated herein by reference). Additional Cas9 proteins and sequences are disclosed in [Chylinski, Krzysztof, Anais Le Rhun, and Emmanuelle Charpentier. "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems." RNA biology 10.5 (2013):726-737.], the entire contents of which are incorporated herein by reference. For example, the DNA cleavage domain of Cas9 is known to contain two subdomains: the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, while the RuvC1 subdomain cleaves the non-complementary strand. Inactivating either one of these subdomains can silence the nuclease activity of the inactivated subdomain, and inactivating both subdomains can silence the entire nuclease activity of Cas9. For example, the H840A mutation provides a Cas9 nickase.For example, both the D10A and H840A mutations completely inactivate the nuclease activity of S. pyogenes Cas9 (see Jinek, Martin, et al. "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Science 337.6096 (2012):816-821.). In some embodiments, a protein comprising a Cas9 fragment may be provided. For example, the protein may comprise any one or more selected from the following two Cas9 domains: the Cas9 gRNA binding domain and the Cas9 DNA cleavage domain. In some embodiments, a Cas9 variant may be provided. The Cas9 variant is homologous to Cas9 or a fragment thereof. For example, a Cas9 variant can be at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, at least about 99.6% identical, at least about 99.7% identical, at least about 99.8% identical, or at least about 99.9% identical to a wild-type Cas9 (such as SpCas9). In some embodiments, a Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or more amino acid changes compared to wild-type Cas9 (e.g., SpCas9). In some embodiments, a Cas9 variant may comprise a Cas9 fragment (e.g., a gRNA binding domain and / or a DNA cleavage domain).In some embodiments, a fragment of a Cas9 variant may be at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, at least about 99.6% identical, at least about 99.7% identical, at least about 99.8% identical, or at least about 99.9% identical to a corresponding fragment of wild-type Cas9. In some embodiments, a wild-type Cas9 fragment or a fragment of a Cas9 variant may be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% or more of the amino acid length of the corresponding wild-type Cas9.

[0090] guide RNA Guide RNA Overview In the CRISPR / Cas system, the Cas protein associates with a guide nucleic acid to form a Cas / guide nucleic acid complex. Typically, in the CRISPR / Cas system, a guide RNA (gRNA) is used as the guide nucleic acid, and the Cas protein associates with the guide RNA to form a Cas / gRNA complex. The Cas / gRNA complex is sometimes called a ribonucleoprotein (RNP). The Cas / gRNA complex creates a nick or double-strand break (DSB) in the target region containing a sequence corresponding to (e.g., complementary to) the spacer sequence of the guide RNA (gRNA), and the Cas protein induces the DSB or nick. The site where the DSB or nick is created may be near a PAM sequence in the genome.

[0091] Cas / gRNA targeting involves a protospacer adjacent motif (PAM) in the genome and the spacer sequence of the guide RNA. Once a Cas protein (such as Cas9) is directed to the target region by the PAM and the spacer sequence of the guide RNA, it creates a DSB in the target region.

[0092] In the CRISPR / Cas gene editing system, the RNA that directs the Cas protein to the target region and recognizes a specific sequence in the target DNA molecule is called a guide RNA.

[0093] The functional components of guide RNAs are mainly divided into 1) a scaffold sequence portion and 2) a guide domain containing a guide sequence. The scaffold sequence portion, which interacts with a Cas protein (such as a Cas9 protein), is the portion that binds to the Cas protein to form a resulting complex. Typically, the scaffold sequence portion includes a tracrRNA and a crRNA repeat sequence portion, and the scaffold sequence is determined by the type of Cas protein used. The guide sequence is a portion that can complementarily bind to a certain length of nucleotide sequence portion in a target nucleic acid (such as a cellular genome or a target DNA molecule). The guide sequence may be artificially modified and is determined by the target nucleotide sequence of interest related to the desired gene editing.

[0094] In some embodiments, guide RNAs may be described as including crRNA and tracrRNA. The crRNA may include a spacer and a repeat sequence. A portion of the repeat sequence of the crRNA may interact (e.g., complementarily bind) with the tracrRNA portion. As described above, a single-stranded guide RNA (sgRNA) (see Jinek, Martin, et al., "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Science 337.6096 (2012):816-821.), the entire contents of which are incorporated herein by reference, may be provided. In other words, the guide RNA may be provided as a double-stranded or single-stranded RNA.

[0095] In some embodiments, an sgRNA can be described as comprising a guide domain, a first complementary domain, a linker domain, and a second complementary domain. In this case, the sgRNA can comprise additional domains, including, but not limited to, any one or more of the proximal and tail domains. In this case, the linker domain connects the first and second complementary domains, and part or all of the first complementary domain forms a complementary bond with part or all of the second complementary domain. As a result, the first complementary domain, the linker domain (e.g., including a polynucleotide linker), and the second complementary domain form a secondary structure, such as a loop structure (see PCT Application No. PCT / KR2018 / 006803, Publication No. WO2018 / 231018).

[0096] The term guide RNA also includes equivalent guide nucleic acid molecules that associate with a Cas9 equivalent, homolog, ortholog, or paralog, whether naturally occurring or non-naturally occurring (e.g., engineered, recombinant, etc.), and enable localization of the Cas9 equivalent, etc., to a specific target nucleotide sequence. As noted above, Cas9 equivalents can include other Cas proteins from any type of CRISPR system (such as types II, V, and VI), including Cpf1 (type V CRISPR-Cas system), C2c1 (type V CRISPR-Cas system), C2c2 (type VI CRISPR-Cas system), and C2c3 (type V CRISPR-Cas system). Additional Cas equivalents are described in [Abudayyeh, Omar O., et al. "C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector." Science 353.6299(2016):aaf5573.], the entire contents of which are incorporated herein by reference. Guide RNAs used in conventional CRISPR / Cas systems may be referred to as "conventional" guide RNAs to compare them with modified guide RNAs called prime-editing guide RNAs (pegRNAs) invented for the prime-editing methods and compositions described herein. Prime-editing guide RNAs (pegRNAs) may have a form in which an extension arm is linked to the 3' or 5' end of a conventional guide RNA.

[0097] A guide RNA or pegRNA may include one or more of a spacer, a gRNA core, an extension arm (particularly in pegRNA), and a transcription terminator. Furthermore, various structural elements may be included, without limitation. The spacer includes a spacer sequence, and the spacer sequence refers to a sequence in a guide RNA or pegRNA that binds to a region sequence containing a protospacer sequence of the target region. The gRNA core, sometimes referred to as a gRNA scaffold or backbone sequence, refers to a sequence in a gRNA or pegRNA that is responsible for binding to Cas9 or its equivalent. The gRNA core does not include a spacer or target sequence used to guide Cas9 to the target region (target DNA). The extension arm (particularly in pegRNA) is an element contained in a pegRNA and includes a primer binding site (PBS) and a DNA synthesis template sequence for placement of a single-stranded DNA flap containing the desired genetic modification by a polymerase (e.g., reverse transcriptase). The extension arm may be located at the 3' or 5' end of the pegRNA and is designed to place the desired genetic modification. The extension arm of a pegRNA may be referred to as an extension region. In some embodiments, the guide RNA or pegRNA may further comprise a transcription termination sequence 3' of the molecule.

[0098] Guide sequence of guide RNA The guide RNA may include a guide domain containing a guide sequence. The guide sequence may be used interchangeably with the sequence of a spacer. The guide domain may be used interchangeably with the sequence of a spacer. The guide sequence may be an artificially designed portion and is determined by the intended target nucleotide sequence. In some embodiments, the guide sequence may be designed to target a sequence adjacent to a PAM sequence located in the desired DNA molecule to be edited. As described above, this may guide the localization of the Cas / gRNA complex to the target site (e.g., on-target site). The structure of the guide nucleic acid may vary depending on the type of CRISPR. For example, the guide RNA used in the CRISPR / Cas9 gene editing system may have a 5'-[guide domain]-[scaffold]-3' structure.

[0099] In one embodiment, the guide sequence may have a length of 5 nt to 40 nt. In one embodiment, the guide sequence included in the guide domain of the guide RNA may have a length of 10 nt to 30 nt. In one embodiment, the guide sequence may have a length of 15 nt to 25 nt. In one embodiment, the guide sequence may have a length of 18 nt to 22 nt. In one embodiment, the guide sequence may have a length of 20 nt. In one embodiment, the target sequence (including all target sequences present in the spacer-bound strand and the spacer-unbound strand) that serves as a sequence in the genome that forms a complementary bond with the guide sequence may have a length of 5 nt to 40 nt or 5 bp to 40 bp. In one embodiment, the target sequence that serves as a sequence in the genome that forms a complementary bond with the guide sequence may have a length of 10 nt to 30 nt or 10 bp to 30 bp. In one embodiment, the target sequence may have a length of 15 nt to 25 nt or 15 bp to 25 bp. In one embodiment, the target sequence may have a length of 18 nt to 22 nt or 18 bp to 22 bp. In one embodiment, the target sequence may have a length of 20 nt or 20 bp.

[0100] PAM Conventional CRISPR / Cas systems have two requirements for cleaving a target DNA molecule. First, a base sequence (nucleotide sequence) of a certain length that can be recognized by a Cas protein (such as a Cas9 protein) must be present in the target gene or target nucleic acid. In this case, the base sequence (nucleotide sequence) of a certain length that can be recognized by the Cas9 protein is called a protospacer adjacent motif (PAM) sequence. The PAM sequence is a unique sequence determined by the Cas9 protein. Second, a sequence that can bind complementary to the spacer sequence contained in the guide RNA must be present near the PAM sequence of a certain length. In this case, the PAM sequence can be used to include all sequences present in the spacer-unbound strand and the spacer-bound strand.

[0101] As described above, the Cas / gRNA complex of the CRISPR / Cas system is directed to a target region by the guide sequence of the gRNA and the PAM sequence of the target DNA molecule (e.g., the genome of a cell). In the target DNA molecule, the PAM sequence may be located on the strand to which the guide sequence of the guide RNA does not bind, rather than on the strand to which the guide sequence does not bind. The PAM sequence can be determined independently depending on the type of Cas protein used. In one embodiment, the PAM sequence may be any one selected from the following (disclosed in the 5' to 3' direction): NGG (SEQ ID NO: 19); NNNNRYAC (SEQ ID NO: 20); NNAGAAW (SEQ ID NO: 21); NNNNGATT (SEQ ID NO: 22); NNGRR(T) (SEQ ID NO: 23); TTN (SEQ ID NO: 24); and NNNVRYAC (SEQ ID NO: 25). Each N may independently be A, T, C, or G. Each R may independently be A or G. Each Y may independently be C or T. Each W may independently be A or T. For example, when SpCas9 is used as the Cas protein, the PAM sequence may be NGG (SEQ ID NO: 19). For example, when Streptococcus thermophilus Cas9 (StCas9) is used as the Cas protein, the PAM sequence may be NNAGAAW (SEQ ID NO: 21). For example, when Neisseria meningitidis Cas9 (NmCas9) is used, the PAM sequence may be NNNNGATT (SEQ ID NO: 22). For example, when Campylobacter jejuni Cas9 (CjCas9) is used, the PAM may be NNNVRYAC (SEQ ID NO: 25). In one embodiment, the PAM sequence may be linked to the 3' end of the target sequence present in the non-spacer-bound strand (in this case, the target sequence present in the non-spacer-bound strand refers to a sequence that does not bind to the guide RNA). In one embodiment, the PAM sequence may be located at the 3' end of the target sequence present in the non-spacer-bound strand. The target sequence present in the non-spacer-bound strand refers to a sequence that does not bind to the guide sequence of the guide RNA.The target sequence present in the spacer-unbound strand is complementary to the target sequence present in the spacer-bound strand.

[0102] The site where the DSB or nick is created may be near the PAM sequence in the genome. In one embodiment, the site where the DSB or nick is created may be within a range of -0 to -20 or +0 to +20 relative to the 5' or 3' end of the PAM sequence present in the spacer-unbound strand. In one embodiment, the site where the DSB or nick is created may be within a range of -1 to -5 or +1 to +5 relative to the PAM sequence in the spacer-unbound strand. For example, in a CRISPR / Cas system using SpCas9, it is known that SpCas9 cleaves between the third and fourth nucleotides located upstream of the PAM sequence.

[0103] Genome editing process using conventional CRISPR / Cas systems For the convenience of technical personnel, the genome editing process using the conventional CRISPR / Cas system is briefly disclosed using the following example, in which the conventional CRISPR / Cas system refers to a system that can edit DNA molecules using Cas proteins and conventional gRNAs.

[0104] For example, an environment can be provided in which the desired DNA molecule to be edited can be brought into contact with the Cas / gRNA complex. For intracellular genome editing, a Cas protein or a nucleic acid encoding the protein, and a guide RNA or a nucleic acid encoding the guide RNA are introduced into the cell, thereby creating an environment in which the Cas protein and guide RNA can contact the cellular genomic DNA. In an environment in which the Cas protein and guide RNA can contact the cellular genomic DNA, the Cas protein and guide RNA can form a Cas / gRNA complex. Even in the absence of cellular genomic DNA, a Cas / gRNA complex can be formed if both the Cas protein and gRNA are present in an appropriate environment. The Cas / gRNA complex is directed to a target region containing a predesigned target sequence through the interaction of the genomic PAM sequence and the guide sequence of the gRNA contained in the Cas / gRNA complex. The Cas / gRNA complex directed to the target region creates a DSB (e.g., in the case of Cas9) in the target region. The DSB-created (cleaved) DNA is then repaired via a DNA repair process, thereby completing gene editing of the target region or target site. The two main pathways for repairing DSBs in DNA are homology-directed repair (HDR) and nonhomologous end joining (NHEJ). HDR, a naturally occurring DNA repair system intermediate between the two pathways, can be used to modify the genomes of various organisms, including humans. HDR-mediated repair can be primarily used to insert a desired sequence into a target region or site or to induce specific point mutations, but is not limited to these. HDR-mediated repair can be performed using HDR, a DNA repair system, and an HDR template (e.g., a donor template that can be provided extracellularly). NHEJ refers to the DNA DSB repair process; in contrast to HDR, it joins the cleaved ends without an HDR template; i.e., this repair process does not require an HDR template. NHEJ may be the DNA repair mechanism of choice primarily for indel induction.Indels (insertion / deletion) may refer to variations in which some nucleotides are deleted from the nucleotide sequence of a nucleic acid before gene editing, any nucleotides are inserted, and / or a combination of such insertions and deletions. The occurrence of several indels in a target gene can lead to the inactivation of the corresponding gene. The DNA repair mechanisms HDR and NHEJ are described in detail in Sander, Jeffry D., and J. Keith Joung. "CRISPR-Cas systems for editing, regulating, and targeting genomes." Nature biotechnology 32.4 (2014): 347-355. The entire contents of which are incorporated herein by reference.

[0105] The conventional CRISPR / Cas system, which is the basis of the prime editing system, has been described in detail above for the convenience of technical personnel. The present application relates to a new system for predicting possible off-targets in the DNA editing process using a prime editing system. Prior to describing the off-target prediction system in the prime editing system provided by the present application, the prime editing system, which is the basis of the off-target prediction system, and the process of editing DNA molecules using the prime editing system will be described in detail below.

[0106] Prime Editing System Prime Editing System Overview Prime editing, developed by David R. Liu et al., is a technology that uses Cas proteins, polymerases (e.g., reverse transcriptase), and specialized guide RNAs containing DNA synthesis templates to edit DNA molecules (e.g., genomes) to incorporate or insert desired edits into target regions of the DNA molecule. Descriptions and various embodiments of prime editing are disclosed in [Anzalone, Andrew V., et al. "Search-and-replace genome editing without double-strand breaks or donor DNA." Nature 576.7785(2019):149-157.; Chen, Peter J., et al. "Enhanced prime editing systems by manipulating cellular determinants of editing outcomes." Cell 184.22(2021):5635-5652.; and PCT Application No. PCT / US2020 / 023712, Publication No. WO2020191233A1], the entire contents of each of which are incorporated herein by reference.

[0107] In prime editing, a genome is edited using (1) a prime editor protein, including a Cas protein and a polymerase (such as a reverse transcriptase), and (2) a prime editing guide RNA (pegRNA) to introduce a desired edit into a target region of a target DNA molecule. Various embodiments of prime editing are disclosed in PCT Application No. PCT / US2020 / 023712 (Publication No. WO2020191233A1), the entire contents of which are incorporated herein by reference.

[0108] Prime editing, a versatile and precise genome editing method that uses prime editor proteins, including Cas proteins, to directly write new genetic information into target regions of DNA molecules (e.g., genomes), is a new platform genome editing method developed by David R. Liu et al. Prime editing primarily uses Cas proteins, polymerases, and pegRNAs, where the pegRNA has a form in which an extension arm is linked to a conventional guide RNA. In this case, the extension arm includes an extension region. The extension region contains an editing template that serves as a template for the desired edit, so that the desired edit is inserted into the target region. In this case, insertion of the desired edit into the target region is achieved through multiple processes, including polymerization by a polymerase (e.g., reverse transcriptase) linked to the Cas protein. Polymerization by the polymerase is carried out on the spacer-unbound strand using the DNA synthesis template contained in the extension region of the pegRNA as the polymerization template.

[0109] For example, in prime editing version 2 (PE2), a nick is created in the spacer-unbound strand (induced and / or generated by a Cas protein contained in the PE2 prime editor protein), followed by polymerization (reverse transcription) on the DNA synthesis template strand in the 5' to 3' direction from the site where the nick was created in the spacer-unbound strand using a reverse transcriptase. Reverse transcription is performed using the DNA synthesis template contained in the extension region as a template for reverse transcription. During this polymerization process, a sequence complementary to part or all of the DNA synthesis template is encoded at the site where the nick was created in the spacer-unbound strand. This encoded sequence forms a 3' DNA flap. The 3' DNA flap contains an edit, and the edit has a DNA sequence complementary to the edit template contained in the DNA synthesis template. The 5' DNA flap is then removed via a 5' DNA flap cleavage process (which may involve, for example, the 5' DNA flap endonuclease FEN1). Additionally, the desired edit is incorporated into the desired site through the process of ligation of the 3' DNA flap and repair and / or replication of the cellular DNA. The process of editing DNA molecules using PrimeEditing Version 2 (PE2) is described in detail in [Anzalone, Andrew V., et al. "Search-and-replace genome editing without double-strand breaks or donor DNA." Nature 576.7785(2019):149-157.], the entire contents of which are incorporated herein by reference.

[0110] The term "prime editing" used in reference to prime editing can refer to edits incorporated into DNA molecules as a result of the prime editing system. For example, the term "edit" can refer to edits incorporated into the spacer-unbound strand, the spacer-bound strand, and / or the double-stranded strand. This is because an edit placed in the 3' flap is ultimately placed in the spacer-unbound strand and the spacer-bound strand through a process involving ligation of the 3' DNA flap and repair and / or replication of cellular DNA as described above. Edits can include any one or combination of insertion of one or more nucleotides, deletion of one or more nucleotides, and substitution of one or more nucleotides with other nucleotides.

[0111] For example, editing can involve the insertion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or more nucleotides, where the inserted nucleotides may or may not be located contiguously in the nucleic acid. For example, editing can involve the deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or more nucleotides, where the deleted nucleotides may or may not be located consecutively in the nucleic acid. For example, the editing may include the substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or more nucleotides, where the substituted nucleotides may or may not be consecutively located in the nucleic acid. In another example, the editing may include the insertion and substitution described above. In another example, the editing may include the deletion and substitution described above. In another example, the editing may include the insertion, deletion, and substitution described above.The publication first disclosing prime editing by David R. Liu et al. (Anzalone, Andrew V., et al. "Search-and-replace genome editing without double-strand breaks or donor DNA." Nature 576.7785(2019):149-157.) describes the scope of prime editing as "all four transition point mutations; all eight transition point mutations; insertions (1 bp to ≧44 bp); deletions (1 bp to ≧80 bp); and combinations of the above," indicating that there are a variety of editing embodiments that can be introduced into DNA molecules by prime editing. Furthermore, prime editing technology is still being developed and improved, and therefore the scope of prime editing is not limited to the scope disclosed in the above publication. [Anzalone, Andrew V., et al. "Search-and-replace genome editing without double-strand breaks or donor DNA." Nature 576.7785(2019):149-157.] (the entire contents of which are incorporated herein by reference) describes prime editing as a versatile and precise genome editing method that directly "writes" new genetic information into specific DNA regions. In this regard, any statements herein regarding genetic information that can be inserted or installed into DNA through prime editing should not be construed as limiting.

[0112] In some cases, prime editing can be considered a "search and replace" genome editing technique because the prime editor (or prime editor complex) used to perform prime editing can search for and locate the desired target region to be edited, while replacing the corresponding endogenous DNA strand of the target region with an alternative strand containing the desired edit. PCT Application No. PCT / US2020 / 023712 (Publication No. WO2020191233A1) (the entire contents of which are incorporated herein by reference) discloses that the prime editors described therein are not limited to reverse transcriptases; such reverse transcriptases are merely one type of DNA polymerase that can be used in prime editing. Therefore, whenever reverse transcriptase is mentioned, those skilled in the art will understand that any suitable DNA polymerase can be used in place of reverse transcriptase. Similarly, it will be appreciated that not only Cas9, nCas9, etc., but also proteins or domains functionally equivalent to Cas9 may be used in prime editing.

[0113] A guide RNA (i.e., pegRNA) specialized for prime editing is complexed with a Cas protein (e.g., complexed with a fusion protein containing the Cas protein), ultimately undergoing the prime editing process to place the desired edit at a target site in a target region of a DNA molecule (e.g., a genome). The pegRNA contains an editing template for delivering the desired information to the target DNA. An alternative strand containing a sequence corresponding to the editing template is prepared from the editing template, and this alternative strand is used to replace the corresponding endogenous DNA strand. The prime editing mechanism may involve nicking the target region in a single strand of DNA, exposing a 3'-hydroxyl group to deliver information from the pegRNA to the target DNA. The prime editing mechanism then involves a DNA polymerization process based on the sequence capable of delivering the desired information to the pegRNA, and using the exposed 3'-hydroxyl group to deliver the desired information into the target region. In various embodiments, the extension region providing the polymerization template for the edit-containing alternative strand may be formed from RNA or DNA. In the case of an RNA extension region, the polymerase used for prime editing may be an RNA-dependent DNA polymerase (e.g., reverse transcriptase). In the case of a DNA extension region, the polymerase used for prime editing may be a DNA-dependent DNA polymerase. The newly synthesized strand (i.e., the alternative DNA strand containing the desired edit) by prime editing may be homologous to the genomic target sequence, except for the desired nucleotide modification. The newly synthesized DNA strand may be referred to as a single-stranded DNA flap (e.g., a 3' single-stranded DNA flap), which replaces the corresponding endogenous strand. In various embodiments, prime editing works by contacting a target DNA molecule with a Cas protein complexed with a prime editing guide RNA (pegRNA) (in this case, the Cas protein is contained in the prime editor protein). An example of editing a DNA molecule (e.g., a genome) by prime editing can be described as follows: After contacting the nCas9 (which may, for example, be contained in the prime editor protein) / pegRNA complex with the DNA molecule, the pegRNA guides nCas9 to bind to the target region.A nick is introduced into one strand of the DNA strand in the target region (nCas9 introduces the nick), thus preparing an available 3' end of one strand of the DNA strand. The available 3' end is located in the target region. In certain embodiments, the nick may be created in a strand that does not hybridize with a portion of the pegRNA sequence, i.e., the spacer-unbound strand. In other specific embodiments, the nick may be created in a strand that hybridizes with a portion of the pegRNA sequence, i.e., the spacer-bound strand. The region located at the 3' end of the DNA strand formed by nicking by Cas9 nickase (the region located upstream of the nick site) interacts with a portion of the extension region of the pegRNA for reverse transcription priming. In certain embodiments, the 3'-end DNA strand hybridizes to a primer binding site (PBS) or reverse transcriptase priming sequence contained in the extension region of the pegRNA. A single strand of DNA is synthesized by reverse transcriptase (which may be contained, for example, in a prime editing fusion protein) in the direction from the 3' end of the primed region toward the 5' end of the pegRNA. In other words, a single strand of DNA is synthesized in the 5'→3' direction relative to the spacer-unbound strand (PAM-containing sequence) hybridized to the primer binding site. The single strand of DNA thus synthesized contains the desired nucleotide modification (such as one or more base modifications, one or more insertions, one or more deletions, or a combination thereof). The synthesized single strand of DNA is sometimes called a 3' single-stranded DNA flap. When the 3' single strand invades the endogenous DNA, the formed (unedited) 5' endogenous DNA flap is removed. The 5' endogenous DNA flap can be removed via a 5' flap cleavage process. The 3' single-stranded DNA flap that invaded the endogenous DNA is ligated. DNA repair then takes place. As a result, the desired edit is completely integrated into the target region.

[0114] The objectives of the prime editing system can be achieved by factors including, for example, a prime editor protein, pegRNA, etc.

[0115] The prime editor protein and pegRNA used for prime editing are described below.

[0116] Prime Editor Protein Prime Editor Protein Overview In some embodiments, a prime editor protein (or prime editing construct) refers to a construct in the form of a complex or fusion protein comprising a Cas protein and a polymerase. Prime editor proteins may also be referred to by terms such as prime editing protein, prime editing construct, prime editing enzyme, prime editor enzyme, and prime editing fusion protein. A prime editor protein may comprise a structure represented by [Cas]-[P] or [P]-[Cas], where "P" refers to any polymerase (such as reverse transcriptase) or an element derived therefrom, and "Cas" refers to a Cas protein (such as Cas9 nickase or an SpCas9 variant, including wild-type SpCas9) or an element derived therefrom. The "]-[" or "-" symbol indicating that the Cas protein and polymerase are linked may refer to any element, such as a linker, or bond that functions to covalently or non-covalently link the Cas protein and polymerase.

[0117] As described above, the prime editor protein comprises a Cas protein (such as Cas9 nickase) and a reverse transcriptase (or DNA polymerase). The prime editor protein may be in the form of a fusion protein composed of a single molecule, or in the form of a complex composed of two or more molecules, but is not particularly limited thereto. Prime editing can be performed on a target region by the prime editor protein in the presence of a pegRNA. The prime editor protein forms a complex with the pegRNA; in this case, the complex may be referred to as a prime editor protein / pegRNA complex. In some embodiments, the prime editor protein may be referred to as a prime editing protein.

[0118] In some embodiments, the term "prime editing system" may refer to editing of a DNA molecule performed using a prime editor protein and a pegRNA, or a prime editor protein and a pegRNA.

[0119] As described above, the term "prime editing system" can be used as a comprehensive concept to describe content related to prime editing. In some embodiments, a prime editing system can further include other elements or uses thereof in addition to a prime editor protein and a pegRNA. For example, a prime editing system can further include a conventional guide RNA or use thereof that can direct nicking of a second site in the unedited strand.

[0120] In some embodiments, the prime editor protein (i) a Cas protein; and (ii) polymerase Includes:

[0121] The Cas protein and polymerase contained in the prime editor protein are described below.

[0122] Prime Editor Protein Element 1-Cas Protein Prime editor proteins include Cas proteins and polymerases. Prime editor proteins may include Cas proteins, which are described in detail in the "CRISPR / Cas System" section. The term Cas9 protein is used to include its equivalents. Cas proteins are sometimes referred to as CRISPR enzymes, nucleic acid programmable DNA binding proteins (napDNAbp), CRISPR proteins, etc.

[0123] In some embodiments, the Cas protein is Cas12a, Cas12b1 (C2c1), Cas12c (C2c3), Cas12e (CasX), Cas12d (CasY), Cas12g, Cas12h, Cas12i, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, The Cas protein may be, but is not limited to, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cas13a (C2c2), Cas13b, Cas13c, Cas13d, Cas14, xCas9, circularly permuted Cas9, Argonaute (Ago) domain, or a fragment, homolog, or variant thereof. In some embodiments, the Cas protein may be a Cas protein with nickase activity. The Cas protein having nickase activity may be, but is not limited to, a Cas9 nickase or a Cas12 nickase (e.g., a Cas12a nickase, a Cas12b1 nickase, etc.). In some embodiments, the Cas protein may be a Cas protein having nuclease activity. In some embodiments, the Cas protein may comprise one or more amino acid substitutions or variations in the HNH domain and / or the RuvC domain.

[0124] For example, a variant can comprise an amino acid sequence having about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity compared to the amino acid sequence of a wild-type or parent Cas protein. For example, a variant may contain one or more insertions, one or more deletions, one or more substitutions, or a combination thereof, compared to the amino acid sequence of a wild-type or parent Cas protein. For example, the Cas protein may be Cas9 from Streptococcus pyogenes (SpCas9), Cas9 from Campylobacter jejuni (CjCas9), Cas9 from Staphylococcus aureus (SaCas9), or a variant thereof. For example, the Cas protein may be SpyMac, iSpymac, GeoCas9, xCas9, circularly permuted Cas9, or a variant thereof. For example, SpCas9 variants may contain one or more amino acid residue variations, such as one or more insertions, one or more deletions, one or more substitutions, or a combination thereof, compared to the amino acid sequence of wild-type SpCas9. For example, an SpCas9 variant containing an H840A substitution provides a Cas protein with nickase activity. For example, an SpCas9 variant containing a D10A substitution provides a Cas protein with nickase activity. For example, an SpCas9 variant may contain an R221K and an N394K substitution. For example, an SpCas9 variant may have one or more amino acid residues selected from D10, R221, L244, N394, H840, K1211, and L1245 of wild-type SpCas9 substituted with another amino acid residue.For example, the SpCas9 variant can include any one or more selected from D10A, R221K, L244Q, N394K, H840A, K1211Q, and L1245V. In some embodiments, the Cas protein is an SpCas9 variant with nickase activity, including H840A; an SpCas9 variant with nickase activity, including R221K, N394K, and H840A (see, e.g., Chen, Peter J., et al. "Enhanced prime editing systems by manipulating cellular determinants of editing outcomes." Cell 184.22(2021):5635-5652.); a wild-type SpCas9 variant with nuclease activity (i.e., inducing DSBs) (see, e.g., Adikusuma, Fatwa, et al. "Optimized nickase- and nuclease-based prime editing in human and mouse cells." Nucleic acids research 49.18(2021):10785-10795.] or an SpCas9 variant with nuclease activity, including, but not limited to, R221K and N394K. In some embodiments, the Cas protein may be codon-optimized. In some embodiments, the prime editor protein may comprise a PAM-less Cas protein.

[0125] Various examples of Cas proteins that can be included in a prime editor protein are described in detail in [US Patent Application No. 17 / 219,672].

[0126] In some embodiments, wild-type SpCas9 can comprise the following amino acid sequence of SEQ ID NO: 28:

[0127] In some embodiments, a wild-type SpCas9 variant comprising the H840A variation can comprise the following amino acid sequence of SEQ ID NO: 29:

[0128] In some embodiments, a wild-type SpCas9 variant comprising the R221K and N394K variations can comprise the following amino acid sequence of SEQ ID NO: 30:

[0129] In some embodiments, a wild-type SpCas9 variant comprising the R221K, N394K, and H840A variations can comprise the following amino acid sequence of SEQ ID NO: 31:

[0130] Prime Editor Protein Element 2-Polymerase Overview of polymerases used in prime editing Prime editor proteins include Cas proteins and polymerases. Polymerase refers to an enzyme or protein that synthesizes a nucleotide chain and may be used in connection with the prime editing system or prime editing-based system described herein. The polymerase is a "template-dependent polymerase" (i.e., a polymerase that synthesizes a nucleotide chain based on the order of nucleotide bases in a template strand). The polymerase may also be a "template-independent" polymerase. Polymerases are sometimes categorized as "DNA polymerases" or "RNA polymerases."

[0131] In various embodiments, the prime editing system or prime editor protein can include a DNA polymerase that synthesizes a DNA strand.

[0132] In some embodiments, the DNA polymerase may be a DNA-dependent DNA polymerase, in which case the pegRNA may comprise a DNA template that serves as a template for polymerization by the DNA-dependent DNA polymerase. In this case, the pegRNA may be referred to as a hybrid pegRNA or chimera that comprises an RNA portion (a guide RNA component including a spacer and a gRNA core) and a DNA portion (a DNA template).

[0133] In various embodiments, the DNA polymerase may be an "RNA-dependent DNA polymerase." In this case, the pegRNA may include an RNA template that serves as a template for polymerization by the RNA-dependent DNA polymerase. In other words, the pegRNA may be composed of an RNA component and may include an RNA extension region.

[0134] Polymerase may also refer to an enzyme that catalyzes the polymerization of nucleotides. Typically, polymerization by a polymerase begins at the 3' end of a primer annealed to a polynucleotide template sequence (such as the primer sequence annealed to the primer binding site of a pegRNA in prime editing) and proceeds toward the 5' end of the template strand. DNA polymerases can catalyze the polymerization of deoxynucleotides. As used herein, the term polymerase can refer to enzymes, proteins, variants, and fragments thereof that catalyze and / or carry out the polymerization of nucleotides. In this context, a polymerase fragment refers to any portion of a wild-type or mutant (variant) DNA polymerase that contains an amino acid sequence of less than the full length of the wild-type polymerase and that retains the ability to catalyze and / or carry out the polymerization of deoxynucleotides under at least one condition. Such fragments may exist as separate entities or may constitute a larger polypeptide, such as a fusion protein.

[0135] Example of a polymerase: reverse transcriptase For example, one element used in prime editing, the polymerase, may be reverse transcriptase (RT). Reverse transcriptase refers to a class of polymerases characterized as RNA-dependent DNA polymerases. All known reverse transcriptases require a primer to synthesize DNA transcripts from an RNA template. As used herein, the term reverse transcriptase may be used to include its variants and fragments. For example, a variant can comprise an amino acid sequence having about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity compared to the amino acid sequence of a wild-type or parent reverse transcriptase. For example, a variant can contain one or more insertions, one or more deletions, one or more substitutions, or a combination thereof, compared to the amino acid sequence of the wild-type or parent reverse transcriptase.

[0136] Reverse transcriptase can be derived from many different sources, including, but not limited to, Moloney murine leukemia virus (M-MLV or MLVRT), human T-cell leukemia virus type 1 (HTLV-1), bovine leukemia virus (BLV), Rous sarcoma virus (RSV), human immunodeficiency virus (HIV), yeast such as Saccharomyces, Neurospora, and Drosophila, primates, and rodents.

[0137] Examples of reverse transcriptases include avian myeloblastosis virus (AMV) reverse transcriptase, reverse transcriptase derived from Moloney murine leukemia virus (M-MLV) (see Gerard, Gary F., et al., "Influence on stability in Escherichia coli of the carboxy-terminal structure of cloned Moloney murine leukemia virus reverse transcriptase." Dna 5.4 (1986): 271-279; and Kotewicz, Michael L., et al., "Cloning and overexpression of Moloney murine leukemia virus reverse transcriptase in Escherichia coli." Gene 35.3 (1985): 249-258). RNase Examples of reverse transcriptases that may be used include, but are not limited to, M-MLV reverse transcriptase substantially lacking H activity (see Application No. US07 / 671,156, Publication No. US5244797A), human immunodeficiency virus (HIV) reverse transcriptase, avian sarcoma and leukosis virus (ASLV) reverse transcriptase, Rous sarcoma virus (RSV) reverse transcriptase, avian erythroblastosis virus (AEV) helper virus MCAV reverse transcriptase, avian myelocytomatosis virus MC29 helper virus MCAV reverse transcriptase, avian reticuloendotheliosis virus (REV-T) helper virus REV-A reverse transcriptase, avian sarcoma virus UR2 helper virus UR2AV reverse transcriptase, avian sarcoma virus Y73 helper virus YAV reverse transcriptase, Rous-associated virus (RAV) reverse transcriptase, myeloblastosis-associated virus (MAV) reverse transcriptase, variants thereof, or fragments thereof. In some embodiments, the reverse transcriptase may be a retroviral reverse transcriptase. In some embodiments, the reverse transcriptase may be an error-prone reverse transcriptase. An "error-prone" reverse transcriptase (or any polymerase in the broader sense) refers to a reverse transcriptase derived from another reverse transcriptase that has an error rate that is lower than the error rate of naturally occurring or wild-type M-MLV reverse transcriptase.An error-prone reverse transcriptase may have a higher error rate than the wild-type reverse transcriptase to which it is compared, e.g., 6.7 x 10. -5 , 7.14×10 -5 , 7.7×10 -5 , 9.1×10 -5 , or 1×10 -4 It is possible to obtain an error rate of 100%. [Bebenek, K., et al. "Error-prone polymerization by HIV-1 reverse transcriptase. Contribution of template-primer misalignment, miscoding, and termination probability to mutational hot spots." Journal of Biological Chemistry 268.14(1993):10324-10334.; and Sebastian-Martin, Alba, Veronica Barrioluengo, and Luis Menendez-Arias. "Transcriptional inaccuracy threshold attenuates differences in RNA-dependent DNA synthesis fidelity between retroviral reverse transcriptases." Scientific Reports 8.1(2018):1-13.] can be referenced, the entire contents of each of which are incorporated herein by reference.

[0138] In some embodiments, the reverse transcriptase may be M-MLV reverse transcriptase. The term M-MLV reverse transcriptase may be used to include variants and fragments thereof. Examples of M-MLV reverse transcriptase may include wild-type M-MLV reverse transcriptase, an M-MLV reverse transcriptase variant, a wild-type M-MLV reverse transcriptase fragment, or a fragment of a wild-type M-MLV reverse transcriptase variant. For example, an M-MLV reverse transcriptase variant may have a form in which one or more amino acid residues selected from P51, S67, E69, L139, T197, D200, H204, F209, E302, E302, T306, F309, W313, T330, L345, L435, N454, D524, E562, D583, H594, L603, E607, and D653 of wild-type M-MLV reverse transcriptase or another wild-type reverse transcriptase are substituted with another amino acid residue. The amino acid sequence of wild-type M-MLV reverse transcriptase is disclosed in SEQ ID NO:26. For example, the M-MLV reverse transcriptase variant can include any one or more amino acid variations selected from P51L, S67K, E69K, L139P, T197A, D200N, H204R, F209N, E302K, E302R, T306K, F309N, W313F, T330P, L345G, L435G, N454K, D524G, E562Q, D583N, H594Q, L603W, E607K, and D653N (wherein the sequence on which the amino acid variations are based is the amino acid sequence of the wild-type M-MLV reverse transcriptase of SEQ ID NO: 26). In certain embodiments, the reverse transcriptase may be an M-MLV reverse transcriptase variant containing the amino acid variations D200N, T306K, W313F, T330P, and L603W (e.g., an M-MLV reverse transcriptase quintuple mutant). In certain embodiments, the reverse transcriptase may be a truncated M-MLV reverse transcriptase. In this case, the truncated M-MLV reverse transcriptase may contain four mutations (D200N, T306K, W313F, and T330P). In this case, the L603W mutation present in the M-MLV reverse transcriptase quintuple mutant described above is no longer present due to the truncation. In some embodiments, the polymerase or reverse transcriptase may be codon-optimized.

[0139] The reverse transcriptase (RT) gene (or the genetic information contained therein) can be obtained from many different sources. For example, the gene can be obtained from eukaryotic cells infected with a retrovirus or from various plasmids containing part or all of the retroviral genome. Additionally, messenger RNA-like RNA containing the RT gene can be obtained from a retrovirus. Various examples of reverse transcriptases that can be included in a prime editor protein are described in detail in [U.S. Patent Application No. 17 / 219,672].

[0140] In some embodiments, the wild-type M-MLV reverse transcriptase may comprise the following amino acid sequence of SEQ ID NO:26: (SEQ ID NO: 26).

[0141] In some embodiments, a wild-type M-MLV reverse transcriptase containing the D200N, T306K, W313F, T330P, and L603W variations can comprise the following amino acid sequence of SEQ ID NO:27: (SEQ ID NO: 27).

[0142] Additional elements that may be included in the prime editor protein A prime editor protein comprises a Cas protein and a polymerase (such as a reverse transcriptase). In some embodiments, in addition to these two elements, the prime editor protein may further comprise additional elements such as one or more linkers (such as a linker for connecting elements contained in the prime editor protein) and one or more nuclear localization sequences (NLSs).

[0143] A prime editor protein may comprise one or more linkers. For example, a linker may be used to link a Cas protein to another structure included in the prime editor protein. The linker may be any linker known in the art. For example, a linker may be used to link a polymerase to another structure included in the prime editor protein. For example, a linker may be used to link an NLS to another structure included in the prime editor protein. For example, a linker may be used to link a Cas protein and a polymerase. For example, a linker may be used to link a linker to another independently selected linker. In some embodiments, a linker may be a covalent bond, an organic molecule, a group, a polymer, or a chemical moiety. In some embodiments, each linker may be independently selected. The linker may have a length of 3 to 100 or more amino acids. For example, the linker may be about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acids in length, or within a range set by any two values ​​selected from the above values. In some embodiments, the linker may comprise the following amino acid sequences: one or more G, one or more XP (wherein X is any amino acid), one or more EAAAK (SEQ ID NO: 35), one or more GGS (SEQ ID NO: 36), one or more SGGS (SEQ ID NO: 37), or one or more GGGGS (SEQ ID NO: 38). In some embodiments, the linker may comprise, but is not limited to, the amino acid sequence of SGSETPGTSESATPES (SEQ ID NO: 39) or SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 40).In some embodiments, the linker may be an XTEN linker (e.g., linker XTEN16). As noted above, a prime editor protein may include one or more linkers, each of which may be independently selected or determined. Various examples of linkers are described in detail in [U.S. Patent Application No. 17 / 219,672].

[0144] A prime editor protein may contain one or more NLSs. In some embodiments, a prime editor protein may contain two or more NLSs. When a prime editor protein contains multiple NLSs, each NLS may be independently selected or determined. The NLS may be any NLS known in the art. The NLS may be any later discovered NLS for nuclear localization. The NLS may be any naturally occurring NLS or any non-naturally occurring NLS (e.g., with one or more mutations).In some embodiments, the NLS can include, but is not limited to, the SV40 virus large T antigen NLS having the amino acid sequence of PKKKRKV (SEQ ID NO: 1); a bipartite SV40 NLS having the amino acid sequence of KRTADGSEFESPKKKRKVE (SEQ ID NO: 18) (or a bipartite SV40 NLS containing a single amino acid deletion in the non-PKKKRKV portion); an NLS from nucleoplasmin (such as the nucleoplasmin bipartite NLS having the sequence of KRPAATKKAGQAKKKK (SEQ ID NO: 2)); a c-myc NLS having the amino acid sequence of PAAKRVKLD (SEQ ID NO: 3) or RQRRNELKRSP (SEQ ID NO: 4); or a hRNPA1 M9 NLS having the sequence of NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 5). NLS; an IBB domain sequence from importin alpha of RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 6); fibroid T protein sequences of VSRKRPRP (SEQ ID NO: 7) and PPKKARED (SEQ ID NO: 8); a human p53 sequence of PQPKKKPL (SEQ ID NO: 9); a mouse c-abl IV sequence of SALIKKKKKKMAP (SEQ ID NO: 10); an influenza virus NS sequence of DRLRR (SEQ ID NO: 11) and PKQKKRK (SEQ ID NO: 12); a hepatitis virus delta antigen sequence of RKLKKKIKKL (SEQ ID NO: 13); a mouse Mx1 protein sequence of REKKKFLKRR (SEQ ID NO: 14); a human poly(ADP-ribose) polymerase sequence of KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 15); or an NLS sequence from a steroid hormone receptor (human) glucocorticoid sequence of RKCLQAGMNLEARKTKK (SEQ ID NO: 16). In some embodiments, the NLS may be codon optimized.

[0145] Various examples of NLS are described in detail in [US Patent Application Serial No. 17 / 219,672].

[0146] Prime-edited guide RNA (pegRNA) Overview of pegRNA As used herein, the terms "prime editing guide RNA," "pegRNA," or "extended guide RNA" refer to a specialized form of guide RNA that contains one or more additional sequences so that the prime editing methods and compositions disclosed herein can be implemented. A pegRNA is used in conjunction with a prime editor protein in a prime editing system. As described herein, a pegRNA contains an extension arm or extension region. The extension arm may comprise, but is not limited to, a single-stranded RNA sequence and / or a DNA sequence. As described above, a guide RNA used in a conventional CRISPR / Cas system (i.e., a guide RNA that does not contain the extension arm of a pegRNA) is referred to as a conventional guide RNA and can thus be distinguished from a pegRNA. For example, an extension arm may be formed at the 3' end of a conventional guide RNA. In another example, an extension arm may be formed at the 5' end of a conventional guide RNA. In some embodiments, a pegRNA may contain a spacer region, a gRNA core, and an extension arm formed at the 3' or 5' end of a conventional guide RNA.

[0147] Extension arm The term "extension arm" refers to a portion of a pegRNA nucleotide sequence that serves various functions, including a DNA synthesis template (e.g., including an editing template) and a primer binding site (PBS) for a polymerase (such as a reverse transcriptase). In a pegRNA, an extension arm may be described as an extension region. In some embodiments, an extension arm may be located at the 3' end of a guide RNA. In some embodiments, an extension arm located at the 3' end of a guide RNA may be referred to as a 3' extension arm. In other embodiments, an extension arm may be located at the 5' end of a guide RNA. In some embodiments, an extension arm located at the 5' end of a guide RNA may be referred to as a 5' extension arm. In some embodiments, an extension arm may comprise a homology arm. In some embodiments, an extension arm may comprise an editing template. In some embodiments, an extension arm may comprise a primer binding site. In various embodiments, an extension arm (such as a 3' extension arm) may comprise the following elements in the 5' to 3' direction: a DNA synthesis template and a primer binding site. In other words, when described based on the entire pegRNA, the pegRNA may comprise the following elements in the 5'→3' direction: a spacer, a gRNA core, a DNA synthesis template, and a primer binding site. The DNA synthesis template may comprise a homology region and an editing template. In various embodiments, the extension arm may comprise the following elements in the 5'→3' direction: a homology region, an editing template, and a primer binding site. In other words, when described based on the entire pegRNA, the pegRNA may comprise the following elements in the 5'→3' direction: a spacer, a gRNA core, a homology region, an editing template, and a primer binding site. In some embodiments, the 5' extension arm may comprise the following elements in the 5'→3' direction: a DNA synthesis template and a primer binding site.

[0148] The polymerization activity of reverse transcriptase, an example of a polymerase, is in the 5' to 3' direction relative to the strand that will ultimately bind to the template strand. When a primer is annealed to the primer binding site (PBS), the reverse transcriptase polymerizes a single strand of DNA using the complementary template strand (DNA synthesis template) as a template for reverse transcription. Various embodiments of extension arms used in prime editing are described in detail in [U.S. Patent Application No. 17 / 219,672].

[0149] Typically, the extension arm of a PEG-RNA can be described as containing two regions: a primer binding site (PBS) and a DNA synthesis template (e.g., a reverse transcription template). For example, in PE2, the primer binding site binds to a primer sequence formed from an endogenous DNA strand at a nicking target site caused by a prime editor protein, thereby exposing the 3' end of the nicked strand. As described herein, binding of the primer sequence to the primer binding site in the extension arm of the PEG-RNA generates a double-stranded region with an exposed 3' end (i.e., the 3' end of the primer sequence), providing a matrix that allows reverse transcriptase to subsequently polymerize a single strand of DNA from the exposed 3' end along the length of the DNA synthesis template. The sequence of the resulting single-stranded DNA product is the complement of the DNA synthesis template. Polymerization continues toward the 5' end of the DNA synthesis template (or extension arm) until polymerization terminates. In response, the DNA synthesis template is polymerized by the prime editor protein's polymerase to produce a resulting single-stranded DNA product (i.e., a 3' single-stranded DNA flap containing the desired gene editing information). As a result, a 3' single-stranded DNA flap (e.g., complementary to the DNA synthesis template) is formed, replacing the endogenous DNA strand corresponding to the target region located immediately downstream of the PE-induced nick site. Polymerization of the DNA synthesis template can continue toward the 5' end of the extension arm until polymerization terminates, but is not limited to this. Polymerization can terminate in a variety of ways, including, but not limited to, (a) reaching the 5' end of the pegRNA, (b) reaching an impassable RNA secondary structure (such as a hairpin or stem / loop), or (c) reaching a replication termination signal, such as a specific nucleotide sequence that blocks or inhibits the polymerase, or a nucleic acid phase signal, such as supercoiled DNA or RNA. However, polymerization termination is not limited to this. Several prime editing-related documents have reported that sequences homologous to parts of the gRNA core of pegRNA are found in the 3' DNA flap or editing site.Based on this, it will be understood by those skilled in the art that the above embodiments are merely exemplary, and that polymerization termination is not limited to the above embodiments.

[0150] Primer binding site (PBS) In the prime editing system, information contained in the DNA synthesis template contained in the pegRNA is transferred to the endogenous strand through polymerization by a polymerase. Polymerization by the polymerase requires binding of a primer to the template strand, and primer binding or annealing enables DNA polymerization. In the prime editing system, a portion of the region where a DSB or nick has been created by a Cas protein is used as a primer. For example, in a description based on PE2, a portion of the region upstream of the nick in the spacer-unbound strand induced by the Cas protein of the prime editor protein is used as a primer. In this case, the region designed to bind complementarily to the sequence of the region upstream of the nick is called the primer binding site, and the primer binding site is located in the extension region of the pegRNA. The prime editing process in PE2 is further described below. Once the primer binding site and the region used as a primer in the endogenous DNA (e.g., genome) are bound, reverse transcription is performed by reverse transcriptase using the primer as a template for reverse transcription. Those skilled in the art will appreciate that reverse transcription is performed in the 3'→5' direction relative to the reverse-transcribed template strand (i.e., pegRNA). Upon reverse transcription, a sequence complementary to the sequence of the DNA template is included in the 3' flap of the genomic DNA, and information about the DNA template is transferred to the 3' flap by reverse transcription. Then, through processes including 5' flap removal and processes including repair and / or replication of cellular DNA, the information about the DNA template is ultimately transferred to another desired strand of DNA to be edited. The desired result of prime editing is to transfer or install the information about the DNA template into the first strand (in this case, the first strand is the spacer-unbound strand) and / or the second strand (in this case, the second strand is the spacer-bound strand) of the desired site to be edited. In other words, as a result of exemplary PE2-primed editing, a DNA sequence complementary to the sequence of the DNA template is present at the desired site of the first strand, and a DNA sequence identical to the sequence of the DNA template sequence is present at the desired site of the second strand.

[0151] In some embodiments, the primer binding site of the pegRNA can be designed as a sequence complementary to the sequence of a region located upstream of the site where a nick or DSB has been created in a DNA molecule (e.g., genomic DNA). In some embodiments, the primer binding site can be designed as a sequence complementary to the sequence of a region located upstream of the site where a nick or DSB has been created in the spacer-unbound strand of the DNA molecule. In other words, the sequence of the region located upstream of the site where a nick or DSB has been created in the spacer-unbound strand of the DNA molecule can function as a primer in the prime editing process. As described above, in one example of PE2, the sequence located 5' of the nick functions as a primer, and the nick end of the DNA molecule is exposed to reverse transcriptase through binding of the primer to the primer binding site.

[0152] In some embodiments, a primer may have a length of 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, 35 nt, 36 nt, 37 nt, 38 nt, 39 nt, 40 nt, 41 nt, 42 nt, 43 nt, 44 nt, 45 nt, 46 nt, 47 nt, 48 nt, 49 nt, or 50 nt or more, or may have a length within a range formed by two values ​​selected from the above values. In certain embodiments, the primer may have a length of 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, or 25 nt, or may have a length within a range formed by two values ​​selected from the above values.

[0153] In some embodiments, a primer binding site may have a length of 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, 35 nt, 36 nt, 37 nt, 38 nt, 39 nt, 40 nt, 41 nt, 42 nt, 43 nt, 44 nt, 45 nt, 46 nt, 47 nt, 48 nt, 49 nt, or 50 nt or more, or may have a length within a range formed by two values ​​selected from the above values. In certain embodiments, the primer binding site may have a length of 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, or 25 nt, or may have a length within a range formed by two values ​​selected from the above values. The length of the primer binding site can be selected appropriately depending on the purpose and is not particularly limited.

[0154] DNA synthesis template As used herein, the term "DNA synthesis template" in prime editing refers to a region or portion that is used as a template strand by the polymerase of a prime editor protein to encode a 3' single-stranded DNA flap containing a desired edit. Furthermore, the term DNA synthesis template refers to a region or portion contained in the extension region of a pegRNA, which replaces the corresponding endogenous DNA strand at the target site through the prime editing mechanism. Various embodiments of DNA synthesis templates and extension regions of pegRNAs are described in detail in U.S. Patent Application No. 17 / 219,672, the entire contents of which are incorporated herein by reference.

[0155] The extension region comprising the DNA synthesis template may be composed of DNA, RNA, or a DNA / RNA hybrid. In the case of RNA, the polymerase of the prime editor protein may be an RNA-dependent DNA polymerase (such as a reverse transcriptase). The DNA synthesis template may be referred to as a DNA polymerization template or a reverse transcription template (RT template), where the RT template is intended for use with reverse transcriptase in the prime editing system. In the case of DNA, the polymerase of the prime editor may be a DNA-dependent DNA polymerase. In various embodiments, the DNA synthesis template (such as an RT template) may comprise an "editing template" and a "region of homology."

[0156] In some embodiments, the DNA synthesis template may include, in addition to the editing template and homology region, part or all of the optional 5'-end modifier region e2. Depending on the nature of region e2 (e.g., whether it contains a secondary structure such as a hairpin, T-loop, or stem / loop), the polymerase may encode none, part, or all of the e2 region. In some embodiments, in the case of a 3' extension arm, the DNA synthesis template may include a portion of the extension arm covering from the 5' end of the primer binding site (PBS) to the 3' end of the gRNA core. In other embodiments, in the case of a 5' extension arm, the DNA synthesis template may include a portion of the extension arm covering from the 5' end of the pegRNA molecule to the 3' end of the primer binding site. Preferably, the DNA synthesis template excludes the primer binding site (PBS) of a pegRNA having a 3' extension arm or a 5' extension arm.

[0157] In certain embodiments described herein, the DNA synthesis template may be referred to as a "reverse transcription template (RT template)" that includes the editing template and the homology arm. The RT template may refer to a portion of the sequence of the pegRNA extension arm that is used as a template in DNA synthesis. The term "RT template" may be used interchangeably with the DNA synthesis template.

[0158] In the case of trans-prime editing, the primer binding site (PBS) and DNA synthesis template can be engineered using individual molecules called trans prime editing RNA templates (tPERT) (see U.S. Patent Application No. 17 / 219,672).

[0159] DNA Synthesis Template Element 1 - Edit Template The term "editing template" refers to a portion of an extension arm that encodes a desired edit in a single-stranded 3' DNA flap synthesized by a polymerase, such as a DNA-dependent DNA polymerase and an RNA-dependent DNA polymerase (such as a reverse transcriptase). In other words, the editing template may be complementary to the desired edit. In some embodiments, a DNA synthesis template may comprise an editing template and a homology arm. In some embodiments, an RT template may comprise an editing template and a homology arm. The term "RT template" is equivalent to a DNA synthesis template. However, an RT template herein is based on the use of a prime editor protein with a polymerase, i.e., a reverse transcriptase, while a DNA synthesis template is more broadly based on the use of a prime editor protein with any polymerase.

[0160] The desired edits to be made in the target region of a DNA molecule (such as a genome) to be edited can include any one or combination of the insertion of one or more nucleotides, the deletion of one or more nucleotides, and the substitution of one or more nucleotides for other nucleotides. For example, the edits can include the insertion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or more nucleotides, where the inserted nucleotides may or may not be located contiguously in the nucleic acid. For example, editing can involve the deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or more nucleotides, where the deleted nucleotides may or may not be located consecutively in the nucleic acid. For example, the editing may include the substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or more nucleotides, where the substituted nucleotides may or may not be consecutively located in the nucleic acid. In another example, the editing may include the insertion and substitution described above. In another example, the editing may include the deletion and substitution described above. In another example, the editing may include the insertion and deletion described above. In another example, the editing may include the insertion, deletion, and substitution described above.

[0161] DNA synthesis template element 2 - homology arms (or homology regions) The term "homology arm" refers to a portion of an extended arm that is incorporated into a target DNA region through displacement of the endogenous strand. For example, in PE2 prime editing, the term "homology arm" can refer to a portion of an extended arm that encodes a single-stranded DNA flap portion encoded by a reverse transcriptase. For example, in the PE2 system, the portion of the single-stranded DNA flap encoded by the homology arm is complementary to the unedited strand of the target DNA (e.g., the spacer-bound strand). In other words, in PE2, the sequence of the homology arm has a sequence complementary to the corresponding sequence located in the non-spacer-bound strand of the target DNA and has substantially the same sequence as the corresponding DNA sequence located in the spacer-bound strand. The homology arm displaces the endogenous strand and promotes annealing of the single-stranded DNA flap, thus helping to install an edit into the target DNA molecule. By definition, the homology arm is encoded by the polymerase of the prime editor disclosed herein and is thus part of the DNA synthesis template.

[0162] Additional elements that may be included in pegRNA and engineered pegRNA (epegRNA) Engineered pegRNA (epegRNA) is described with reference to Nelson, James W., et al., "Engineered pegRNAs improve prime editing efficiency." Nature biotechnology 40.3 (2022): 402-410. The entire contents of which are incorporated herein by reference. The term "epegRNA," a type of pegRNA, may be used to refer to an improved pegRNA. Specifically, "epegRNA" refers to a pegRNA having an RNA motif added to the 3' or 5' end of the pegRNA. In some embodiments, the pegRNA may be a pegRNA having an RNA motif (or an engineered RNA motif) added to the 3' end. The epegRNA may include, for example, the following elements in the 5' to 3' direction: a spacer, a gRNA core, a DNA synthesis template, a primer binding site, and an RNA motif.

[0163] David R. Liu et al. developed an engineered pegRNA (epegRNA) by adding an RNA motif to the 3' end of pegRNA to improve its stability and prevent degradation of the 3' extension region. Specifically, in the above-mentioned publication, David R. Liu et al. disclosed an epegRNA in which a stable pseudoknot was additionally incorporated at the 3' end of a conventional pegRNA. Examples of pseudoknots include, but are not limited to, evopreQ1 (a modified prequeosine1-1 riboswitch aptamer) and mpknot (a frameshift pseudoknot from Moloney murine leukemia virus) as described in [Nelson, James W., et al., "Engineered pegRNAs improve prime editing efficiency." Nature Biotechnology 40.3(2022):402-410.].

[0164] The epegRNA can be used regardless of the type of prime editor protein used. For example, the epegRNA can be used with a prime editor protein containing prime editing version 2 (PE2) SpCas9 nickase. In another example, the epegRNA can be used with a PE-nuclease-containing Cas9 with nuclease activity (i.e., DSB activity) to edit DNA molecules (such as genomes). As used herein, the term pegRNA includes embodiments of the epegRNA, and references to the peggRNA can be interpreted as including content related to the epegRNA unless otherwise specified.

[0165] In some embodiments, the pegRNA may further comprise a 3' engineering region at the 3' end. A pegRNA comprising a 3' engineering region may be referred to as an epegRNA. In other words, an epegRNA may further comprise a 3' engineering region in addition to the elements of a pegRNA. In some embodiments, the 3' engineering region may comprise an RNA protection motif. In certain embodiments, the RNA protection motif may comprise an RNA sequence. In certain embodiments, the RNA protection motif may comprise a DNA sequence. In certain embodiments, the RNA protection motif may comprise a DNA / RNA hybrid sequence. In certain embodiments, the RNA protection motif may comprise, but is not limited to, evopreQ1 or mpknot, and may include any other structure for preventing RNA degradation and increasing stability.

[0166] In some embodiments, the 3' engineering region may include an RNA protection motif and a linker for linking the RNA protection motif. The linker functions to link the RNA protection motif and a primer binding site in the epegRNA. In some embodiments, the linker for linking the RNA protection motif may include an RNA sequence. In some embodiments, the linker for linking the RNA protection motif may include a DNA sequence. In some embodiments, the linker for linking the RNA protection motif may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nt in length or longer, or may be within a range defined by any two values ​​selected from the above values. In some embodiments, the linker for linking the RNA protection motif can be designed to avoid base pairing interactions between the linker and the PBS or between the linker and the spacer of the pegRNA. In some embodiments, the sequence of the linker for linking the RNA protection motif can be designed taking into account the sequence of the target region of the target DNA molecule.

[0167] Below, we will explain various prime editing versions developed based on the prime editor protein and pegRNA, which are the basic elements of prime editing. Prime editing is not limited to the versions exemplified below.

[0168] Prime Edited Version Example Prime Edited Version Example Overview Various prime editing versions have been developed based on the core mechanism of prime editing described above. For the convenience of those skilled in the art, examples of prime editing versions will be described below. The method for detecting off-targets in prime editing provided by the present application can use additional elements such as, but not limited to, the prime editor proteins in many prime editing versions exemplified below, various types of pegRNA including epegRNA, and / or dnMLH1. Furthermore, the method for detecting off-targets in prime editing provided by the present application can be applied to the prime editing versions exemplified below and new prime editing versions that will be developed later. Therefore, it should be noted that the prime editing versions exemplified below do not limit the scope of application of the method provided by the present application.

[0169] Prime Editing Version 1 (PE1) Prime Editing Version 1 (PE1) describes a Prime Editing system version that includes the use of the following elements: a prime editor protein containing SpCas9(H840A) and wild-type Moloney murine leukemia virus reverse transcriptase (MMLV RT); and pegRNA.

[0170] In other words, the PE1 prime editor protein contains a Cas protein with nickase activity and wild-type MMLV RT. The PE1 prime editor protein has the form of a fusion protein in which the Cas protein and reverse transcriptase are linked via a linker.

[0171] The PE1 prime editor protein and pegRNA form a complex, thereby directing or carrying out editing of the DNA molecule (e.g., genome editing) in the target region.

[0172] PE1 is described in detail in [Anzalone, Andrew V., et al. "Search-and-replace genome editing without double-strand breaks or donor DNA." Nature 576.7785(2019):149-157.]

[0173] Prime Editing Version 2 (PE2) Prime Editing Version 2 (PE2) describes a version of the Prime Editing system that includes the use of the following elements: Prime editor proteins including SpCas9(H840A) and MMLV RT(D200N+L603W+T330P+T306K+W313F); and pegRNA.

[0174] In other words, the PE2 prime editor protein contains a Cas protein with nickase activity and an MMLV RT quintuple mutant. The PE2 prime editor protein has the form of a fusion protein in which the Cas protein and the reverse transcriptase are linked via a linker. Specifically, the PE2 prime editor protein has the following structure: [bpNLS(SV40)]-[SpCas9 H840A]-[SGGSX2-XTEN16-SGGSX2]-[MMLV RT quintuple mutant]-[bpNLS(SV40)].

[0175] In this context, bpNLS(SV40) refers to the bipartite SV40 NLS. MMLV RT quintuple mutant refers to an MMLV RT variant containing the following amino acid variations compared to wild-type MMLV RT: D200N, L603W, T330P, T306K, and W313F.

[0176] The PE2 prime editing system is described in detail in Anzalone, Andrew V., et al. "Search-and-replace genome editing without double-strand breaks or donor DNA." Nature 576.7785(2019):149-157.; and Chen, Peter J., et al. "Enhanced prime editing systems by manipulating cellular determinants of editing outcomes." Cell 184.22(2021):5635-5652.] In some embodiments, the PE2 prime editor protein may comprise the amino acid sequence of SEQ ID NO:32.

[0177] The amino acid sequence of SEQ ID NO: 32 is as follows:

[0178] Prime Editing Version 3 (PE3) The PE3 prime editing system refers to a version of prime editing developed to enhance prime editing efficiency by using a second-strand nicking guide RNA to nick the non-edited strand (i.e., the strand attached to the spacer of the pegRNA). The second-strand guide RNA can be designed in the form of a conventional gRNA (such as an sgRNA) to nick the non-edited strand at the editing site or at a site adjacent to the target site. In some embodiments, PE3 can include the use of a separate Cas9 nickase in addition to the prime editing protein.

[0179] PE3b refers to PE3, but the second-strand nicking guide RNA is designed for temporal control, preventing the second-strand nick from being introduced until the desired edit is in place. This is achieved by designing a gRNA with a spacer sequence that matches only the edited strand, not the original allele. PE3 and PE3b are described in detail in [[Anzalone, Andrew V., et al. "Search-and-replace genome editing without double-strand breaks or donor DNA." Nature 576.7785(2019):149-157.]]

[0180] Prime Editing Version 4 (PE4) Prime editing version 4 (PE4) involves the use of the same mechanisms as PE2, but further involves the use of a plasmid encoding a dominant negative MLH1 (dnMLH1) or the use of dnMLH1. For example, PE4 can be recognized as including the use of the following elements: PE2 prime editing protein; pegRNA; and Dominant-negative MLH1 (dnMLH1). [Chen, Peter J., et al. "Enhanced prime editing systems by manipulating cellular determinants of editing outcomes." Cell 184.22(2021):5635-5652.] describes how dominant-negative MLH1 knocks out endogenous MLH1 by inhibition, thereby reducing cellular measles-mumps-rubella (MMR) response and potentially increasing prime editing efficiency.

[0181] Prime Editing Version 5 (PE5) Prime editing version 5 (PE5) involves the use of the same mechanism as PE3, but further involves the use of a plasmid encoding dominant-negative MLH1 (dnMLH1) or the use of dnMLH1.

[0182] PE5 is described in detail in [Chen, Peter J., et al. "Enhanced prime editing systems by manipulating cellular determinants of editing outcomes." Cell 184.22(2021):5635-5652.]

[0183] PEmax PEmax is an improved prime editor version developed to enhance editing efficiency. PEmax prime editor proteins include SpCas9 variants and MMLV RT variants. Specifically, PEmax prime editor proteins have the following structure: [bpNLS(SV40)]-[SpCas9 R221K N394K H840A]-[SGGSX2-bpNLS(SV40)-SGGSX2]-[MMLV RT quintuple mutant (codon optimized)]-[bpNLS(SV40)]-[NLS(c-Myc)].

[0184] In this context, bpNLS(SV40) refers to the bisection SV40 NLS. MMLV RT quintuple mutant (codon-optimized) refers to a human codon-optimized MMLV RT variant containing the following amino acid variations compared to wild-type MMLV RT: D200N, L603W, T330P, T306K, and W313F. "SpCas9 R221K N394K H840A" refers to a SpCas9 variant containing the following amino acid variations compared to wild-type SpCas9: R221K, N394K, and H840A. NLS(c-Myc) refers to the c-Myc NLS. PEmax is described in detail in [Chen, Peter J., et al. "Enhanced prime editing systems by manipulating cellular determinants of editing outcomes." Cell 184.22(2021):5635-5652.] Furthermore, the above-mentioned documents disclose various versions of prime editor proteins, such as the PE2* prime editor protein, the CMP-PE-V1 prime editor protein, and the CMP-PEmax prime editor protein, all of which can be used in the off-target prediction system for prime editing provided by the present application.

[0185] Nuclease-based prime editing Nuclease-based prime editing is a version of prime editing that uses a Cas protein (such as wild-type SpCas9 or a non-nickase SpCas9 variant) with nuclease activity (i.e., DSB activity) instead of the Cas9(H840A) nickase. The prime editor protein for nuclease-based prime editing is sometimes called a PE nuclease. Unlike PE3, which is designed to induce nicks in the strand that binds to the spacer of the pegRNA, the use of two types of gRNA is not required. Through the prime editing protein, which contains one type of pegRNA and a Cas nuclease (other than a nickase), a DSB is created at the desired site, thereby inducing editing. Nuclease-based prime editing is described in detail in [Adikusuma, Fatwa, et al. "Optimized nickase- and nuclease-based prime editing in human and mouse cells." Nucleic acids research 49.18(2021):10785-10795.], the entire contents of which are incorporated herein by reference. One example of a PE nuclease is PE2-nuclease. PE2-nuclease has the following structure: [bpNLS(SV40)]-[SpCas9(WT)]-[SGGSx2-XTEN16-SGGSx2]-[MMLV RT]-[bpNLS(SV40)].

[0186] In some embodiments, the PE2-nuclease may comprise the amino acid sequence of SEQ ID NO:33.

[0187] The amino acid sequence of SEQ ID NO: 33 is as follows:

[0188] PEmax-Nuclease PEmax-nuclease is a nuclease-based prime editor protein developed based on the PEmax prime editor protein (i.e., a type of PE-nuclease), and is a prime editor protein containing a Cas protein that has nuclease activity (i.e., DSB activity) other than nickase activity. PEmax-nuclease has the following structure: [bpNLS(SV40)]-[SpCas9 R221K N394K]-[SGGSX2-bpNLS(SV40)-SGGSX2]-[MMLV RT quintuple mutant (codon optimized)]-[bpNLS(SV40)]-[NLS(c-Myc)].

[0189] In some embodiments, the PEmax-nuclease may comprise the amino acid sequence of SEQ ID NO:34.

[0190] The amino acid sequence of SEQ ID NO: 34 is as follows:

[0191] Use of epegRNA As described above, epegRNA is an improved version of pegRNA. The pegRNA used in the prime editing system may be, but is not limited to, epegRNA or a pegRNA other than epegRNA.

[0192] Genome editing process using the Prime editing system For the convenience of those skilled in the art, the process of editing a cell's genome using a prime editing system will be described using the example of PE2. An example of the process of editing a cell's genome using a prime editing system in a cell is as follows: PE2 prime editor protein and pegRNA form a complex, and then the complex is contacted with the cell's genome. The spacer of pegRNA binds to the sequence of the corresponding target region. A nick is created in the strand of genomic DNA that is not bound to the spacer. The nick is created between the third and fourth nucleotides located upstream of the 5' end of the PAM sequence. The sequence located upstream of the nick site functions as a primer that forms a complementary bond with the primer binding site of pegRNA, thereby exposing the 3' end of the cleaved strand to the reverse transcription process. Based on the primer that forms a complementary bond with the primer binding site, reverse transcriptase performs a reverse transcription process to form a 3' DNA flap. The reverse transcription template in this reverse transcription process is the RT template of pegRNA. Through cell-specific mechanisms of 5' flap removal, 3' flap ligation, and DNA mismatch repair, the information for the 3' flap is installed within the genomic DNA. As a result of prime editing, the information on the RT template of the pegRNA is delivered to the desired site on both strands of the genomic DNA. The RT template contains the desired editing template (i.e., the editing template), and the information contained in the editing template is ultimately delivered to the target site in the genomic DNA.

[0193] Below, we will describe in detail the methods for predicting or confirming off-targets in prime editing provided by the present application, which have been developed with prime editing as a target so as to be widely usable or applicable for confirming possible off-targets in the above-mentioned or future prime editing. In the following methods for predicting or confirming off-targets in prime editing, the prime editor proteins used in many of the above-mentioned prime editing versions can be used, and are not particularly limited. Furthermore, in the methods for predicting or confirming off-targets in prime editing of the present application, additional elements used in the above-mentioned prime editing versions can also be used. It will be apparent to those skilled in the art that prime editor proteins, pegRNAs, and / or prime editing systems developed based on the technical features of prime editing, which are characterized by the use of Cas proteins and polymerases, can be used in the methods for predicting off-targets of the present application.

[0194] Off-target prediction method provided by the present application Off-target In the field of DNA editing (such as gene editing or genome editing), off-target refers to gene modification that occurs at an unintended site. Gene modification caused by off-target can be nonspecific. Genome editing tools developed to date include conventional CRISPR / Cas systems, base editing systems, primed editing systems, transcription activator-like effector nucleases (TALENs), meganucleases, zinc finger nucleases, and the like. Such genome editing tools or systems may be designed to edit a target region through a specific mechanism that allows binding to a predetermined sequence (such as the sequence of the target region). For example, in the CRISPR / Cas gene editing system, a guide RNA (gRNA) directs the movement of the Cas / gRNA complex to the intended target site. Movement to the target site may involve a PAM sequence in the genome. However, there is still a high possibility that the Cas / gRNA complex will bind to a sequence at an unintended site rather than the sequence of the target region. As described above, unintended gene modifications occur when the Cas / gRNA complex binds to a sequence at an unintended site and creates a nick or DSB there. Off-target effects induce unintended gene modifications such as unintended point mutations, deletions, insertions, inversions, and translocations. Similarly, the process of editing DNA molecules (e.g., genomic DNA) by prime editing also has off-target problems, even though at least the PAM sequence and the spacer sequence of the pegRNA are involved in targeting.

[0195] Binding of genome editing tools to unwanted regions is known to occur through a partial but sufficient match with the target sequence in the unwanted region. Regarding off-target binding mechanisms, please refer to the published literature [Lin, Yanni, et al. "CRISPR / Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences." Nucleic Acids Research 42.11(2014):7473-7485.].

[0196] Off-target binding mechanisms have been described as being divided into two main types: base mismatch tolerance and bulge mismatch. For example, an off-target region may contain, but is not limited to, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mismatches with the guide RNA sequence. For example, an off-target region may contain, but is not limited to, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mismatches with the target site sequence corresponding to each region of the pegRNA. In other words, in prime editing, mismatches in the off-target region may exist in one or more of the spacer region of the pegRNA, the PBS of the pegRNA, the DNA synthesis template of the pegRNA (such as a homology arm), and the region corresponding to the PAM sequence.

[0197] Off-target problems can cause disruption of important coding regions, potentially leading to serious problems such as cancer. Furthermore, off-target problems can lead to confounding variables in biological research, potentially leading to irreproducible results (see Eid, Ayman, and Magdy M. Mahfouz. "Genome editing: the road of CRISPR / Cas9 from bench to clinic." Experimental & Molecular Medicine 48.10(2016):e265-e265.] (the entire contents of which are incorporated herein by reference).

[0198] As described above, such off-target problems still exist not only in the CRISPR / Cas gene editing system but also in base editing and prime editing developed based on the CRISPR / Cas gene editing system. In this specification, off-target may be used as a concept opposite to on-target, and may be used to refer to gene modification at an unintended site.

[0199] The need for a suitable off-target prediction method for prime editing Overview of the need for prime editing-friendly off-target prediction methods As described above, off-targets can cause severe side effects (such as irreversible and / or undetectable side effects) in various ways. Accordingly, identifying potential off-targets when using systems for editing DNA molecules (such as genome editing systems) is extremely important in the research and development of therapeutic drugs. Additionally, identifying genuine off-targets occurring in designed editing systems (such as CRISPR / Cas systems or prime editing systems) is costly and time-consuming. For this reason, research and development of various methods capable of identifying potential off-targets, i.e., predicting off-targets, is underway. However, existing methods for predicting potential off-targets in gene editing processes (such as genome editing processes using genome editing systems) developed prior to the filing date of this application were developed targeting conventional CRISPR / Cas systems or base editing. An off-target prediction method targeting prime editing, i.e., an off-target prediction method developed targeting genome editing using prime editing, has yet to be developed.Despite the unique editing mechanism of prime editing, which differs from conventional CRISPR / Cas systems, off-target prediction systems developed for conventional CRISPR / Cas systems are still used to predict potential off-target sites in the prime editing DNA editing process (Anzalone, Andrew V., et al., "Search-and-replace genome editing without double-strand breaks or donor DNA." Nature 576.7785(2019):149-157.; Kim, Do Yon, et al., "Unbiased investigation of specificities of prime editing systems in human cells." Nucleic acids research 48.18(2020):10576-10589.; Bae, Sangsu, Jeongbin Park, and Jin-Soo Kim. "Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases." Bioinformatics 30.10(2014):1473-1475.; and Jin, Shuai, et al. "Genome-wide specificity of prime editors in plants." Nature Biotechnology 39.10(2021):1292-1299. (The entire contents of each of which are incorporated herein by reference.) Applying such conventional off-target prediction systems to prime editing has many drawbacks. The need for off-target prediction methods suitable for prime editing will be described in more detail below.

[0200] Off-target prediction system used to predict off-target events in genome editing using conventional CRISPR / Cas systems As described above, various methods for predicting off-target events in genome editing using the CRISPR / Cas system have been developed. Conventional off-target prediction and / or confirmation methods (e.g., systems, platforms, etc.) can be categorized into three types based on the mechanism of action (MOA): cell-based off-target prediction systems, in vitro off-target prediction systems, and in silico off-target prediction systems. Examples of prediction systems in each category are listed below.

[0201] - Cell-based off-target prediction systems: GUIDE-seq, GUIDE-tag, BLISS, BLESS, DISCOVER-seq, DNA break capture via integrase-deficient lentiviral vectors, HTGTS, CReVIS-seq, ITR-seq, TAG-seq, INDUCE-seq, etc.

[0202] - In vitro off-target prediction systems: Digenome-seq, DIG-seq, CHANGE-seq, CIRCLE-seq, SITE-seq, etc.

[0203] - In silico off-target prediction systems: Cas-OFFinder, CRISPOR, CHOPCHOP, etc.

[0204] Each of the above off-target prediction systems has different advantages and disadvantages. Typically, two or three systems are used in combination to predict genome-wide off-target activity during CRISPR-based genome editing.

[0205] Application of a CRISPR / Cas-based off-target prediction system to base editing The above system was expected to be used to predict the off-target activity of base editor systems, such as cytidine and adenine base editors, developed using Cas proteins. However, the above system, developed to predict potential off-target events in genome editing using the CRISPR / Cas system, was not suitable for base editing, which has a different mechanism of action. A more suitable off-target prediction system for base editing was needed, and in response, more appropriate and sophisticated off-target activity prediction systems or methods, such as One-seq (cell-based), CBE Digenome-seq (in vitro), and ABE Digenome-seq (in vitro), have been developed.

[0206] Traditional off-target prediction methods used in prime editing The first step in the genome editing mechanism of prime editing, such as prime editing version 2 (PE2), is Cas9-induced nicking of the spacer-unbound strand. Therefore, the off-target activity of PE2 was predicted to be similar to that of Cas9 or Cas9 nickase (nCas9). Accordingly, we attempted to predict off-target activity in prime editing using systems for predicting and / or confirming off-targets in CRISPR / Cas genome editing, such as GUIDE-seq, nDignome-seq, and CAS-OFFinder (in silico). However, experiments disclosed herein confirmed that using such methods to predict and / or confirm off-targets in conventional CRISPR / Cas genome editing is not suitable for predicting off-targets in prime editing.

[0207] Demand for off-target prediction systems suitable for prime editing Genome editing using prime editor proteins and pegRNAs is based on a different mechanism from that of genome editing using conventional CRISPR / Cas systems. Additionally, unlike conventional CRISPR / Cas systems, prime editing involves numerous factors (e.g., primer binding sites, reverse transcription templates, reverse transcriptases, etc.) in addition to guide sequences, and is carried out through a process using numerous enzymes (e.g., flap endonucleases, exonucleases, ligases, etc.). Although prime editing was developed based on the conventional CRISPR / Cas system, the genome editing mechanism of prime editing differs in various ways from that of conventional CRISPR / Cas systems. Therefore, conventional off-target prediction methods developed for genome editing using conventional CRISPR / Cas systems are not suitable for off-target prediction in prime editing. Furthermore, because of the multiple processes involving numerous factors, it is difficult to develop in vitro-based off-target analysis methods that can faithfully mimic such complex intracellular processes. For this reason, conventional off-target prediction methods are expected to be inapplicable to prime editing or to produce inaccurate results.

[0208] In fact, the inventors of the present application have experimentally confirmed that not only mismatches in the spacer region of pegRNA, but also the primer binding site, homology arms, and / or editing template affect off-targets in prime editing (see the sections "Editing patterns at validated off-target sites" and "Mismatch analysis by region" in the experimental examples of the present application).

[0209] No method has yet been reported for predicting off-target activity developed by targeting prime editing that takes into account the mechanism of prime editing. In other words, there is still no reliable method for predicting off-target candidates in prime editing.

[0210] Overview of the off-target prediction method provided by the present application The present application provides a new off-target prediction method suitable for prime editing. The inventors have confirmed that application of conventional off-target prediction systems targeting CRISPR / Cas systems results in inaccurate predictions (false positives and / or false negatives). Therefore, the inventors have developed a new method or system for predicting off-targets in prime editing. By focusing on the ability or effect of prime editing, which can insert (place or write) a desired sequence at a desired site, the inventors have developed a new system or method for predicting off-targets suitable for prime editing using a new prime-editing guide RNA (pegRNA) containing a tag template for tag insertion. Furthermore, the inventors have confirmed that the prediction reliability and / or accuracy of the newly developed off-target prediction system for prime editing is superior to that of conventional off-target prediction systems developed for conventional CRISPR / Cas genome editing systems.

[0211] The off-target prediction system provided herein, which has been developed to target prime editing (i.e., developed to be suitable for prime editing), may be referred to as "tagmentation of prime editor sequencing (TAPE-seq)." Furthermore, the new pegRNA used in TAPE-seq, which contains a tag template for placing tags in the genome, may be referred to as "tagmentation pegRNA (tpegRNA)."

[0212] The present application provides a method or system for predicting potential off-targets in the process of editing a DNA molecule using a prime editing system. The present application provides a method for predicting potential off-targets in the genome editing process using a prime editing system. A method for predicting off-targets may also be referred to as, for example, a method for confirming off-target candidates, a method for confirming information about off-targets, a method for confirming candidate off-target sites, or the like. Additionally, any description related to a method or system for predicting potential off-targets in the process of editing a DNA molecule (such as a genome) or confirming information about off-targets may be used without limitation. As used herein, the term "off-target" includes the concept of an off-target region. For example, an off-target region or site may be described as an off-target. As used herein, off-target prediction may refer to confirming a candidate off-target. As used herein, "off-target prediction" may refer to confirming a candidate off-target region. The terms "off-target," "off-target prediction," and "candidate off-target" should not be construed as limiting. In other words, the method for predicting off-targets in prime editing may be described as any of the following, but is not limited to these, and may be used interchangeably as long as it relates to predicting or confirming off-targets that may occur in prime editing: predicting off-targets that may occur in prime editing; confirming (or screening) candidate off-targets in prime editing (or that may occur in prime editing); confirming (or screening) off-targets in prime editing (or that may occur in prime editing); confirming information about off-targets in prime editing (or that may occur in prime editing); confirming regions where off-targets may occur; confirming off-target sites; etc.

[0213] The terms false positive and / or false negative may be used in connection with off-target prediction. Identifying a region other than a true off-target as a potential off-target may be expressed as a false positive result. A high false positive rate may be associated with a low validation rate. In this case, a true off-target refers to a validated off-target, which is used to refer to an off-target that actually occurs other than a candidate off-target identified by a prediction system. For example, an off-target that occurs when editing a cell's genome using a prime editing system may be referred to as a true off-target. In contrast, a region related to an off-target identified by an off-target prediction system may be referred to as an "off-target candidate," "predicted off-target," or the like, and thus may be distinguished from a true off-target. A candidate off-target identified by an off-target prediction system may or may not be a true off-target. For example, true off-targets can be identified by validating each candidate off-target. It is important for an off-target prediction system to exhibit a low false positive rate. This is because it is difficult to identify a true off-target when a large number of candidate off-targets are derived from the off-target prediction system.

[0214] In another embodiment, the group of off-target candidates identified by the off-target prediction system may not include all true off-targets. This case is associated with a high oversight rate. For example, if true off-target regions are not identified as off-target candidates, the oversight rate will be higher.

[0215] As described above, the system for predicting off-target events that occur during the prime editing process of DNA molecules of the present application is characterized by tagmentation based on the prime editing mechanism using tpegRNA. Below, the tools for predicting off-target events of the present application (prime editor proteins, tpegRNA, etc.) will be described in detail.

[0216] Tools for predicting off-target sequences in prime editing Overview of tools for predicting off-target sequences in prime editing (elements used in TAPE-seq) The present method for predicting off-targets in prime editing requires two elements: a prime editor protein; and Tagmentation pegRNA (tpegRNA) containing tag template.

[0217] The tool for predicting off-targets in prime editing of the present application may include at least a prime editor protein and a tpegRNA.

[0218] The method for predicting off-targets of the present application may be referred to as TAPE-seq. TAPE-seq, a method designed based on the prime editing mechanism and developed to target prime editing for off-target prediction, may use the prime editing mechanism. Accordingly, the method for predicting off-targets provided by the present application includes the use of a prime editor protein used in prime editing. In other words, the off-target prediction system of the present application may use various prime editor proteins described above. Prime editor proteins used in the off-target prediction system for prime editing of the present application include Cas proteins and polymerases (e.g., reverse transcriptase). However, such a description does not necessarily require the use of a prime editor protein of the same species as the prime editor protein in the specific prime editing system being the target of off-target prediction (e.g., the specific prime system being the target of off-target prediction by TAPE-seq). The off-target prediction system of the present application may use a prime editor protein of the same species or a different species as the prime editor protein in the prime editing system being the target of off-target prediction.

[0219] Similarly, the off-target prediction system of the present application does not necessarily require the use of a pegRNA of the same type as the specific prime editing system being targeted for off-target prediction. The off-target prediction system of the present application can use a tpegRNA based on a pegRNA of the same type as the pegRNA used in the specific prime editing system being targeted for off-target prediction. Alternatively, a tpegRNA based on a different type of pegRNA (e.g., epegRNA) other than typical pegRNAs can be used.

[0220] For example, even if the first prime editing system designated to confirm off-target information through an off-target prediction system is a PE2 prime editing system, a prime editor protein with nuclease activity (such as PE2-nuclease or PEmax-nuclease) can be used in TAPE-seq, which is performed to confirm off-target information using the first prime editing system. In another example, if the first prime editing system designated to confirm off-target information through an off-target prediction system is a PE2 prime editing system, a PE2 prime editor protein can be used in TAPE-seq. Similarly, even if the first prime editing system targeted for off-target prediction is a PE2 prime editing system, an engineered tpegRNA (etpegRNA) can be used in TAPE-seq. In another example, if the first prime editing system targeted for off-target prediction is a PE2 prime editing system, a tagmentation pegRNA (tpegRNA) other than the engineered tpegRNA (etpegRNA) can be used.

[0221] Prime Editor Protein The off-target prediction system for prime editing of the present application involves the use of a prime editor protein. Prime editor proteins include Cas proteins and polymerases (such as reverse transcriptases). Prime editor proteins are described in detail in the "Prime Editing System" section of the present application. Examples of prime editor proteins that can be used in the off-target prediction system of the present application include, but are not limited to, the prime editor proteins described above. It will be understood by those skilled in the art that fusion proteins or complexes for prime editing (or inventions that simply inherit the inventive concept of prime editing) developed for prime editing after the filing date of the present application can also be used in the off-target prediction system of the present application.

[0222] Similarly, examples of tpegRNAs that can be used in the off-target prediction system of the present application include, but are not limited to, various tpegRNA embodiments developed based on the above-mentioned pegRNAs. Those skilled in the art will understand that tpegRNAs based on pegRNAs for prime editing (or inventions that inherit the inventive concept of prime editing) developed for prime editing after the filing date of the present application can also be used in the off-target prediction system of the present application.

[0223] In one embodiment, the prime editor protein used in the present prime editing off-target prediction system may include a Cas protein and a polymerase. In one embodiment, the Cas protein includes Cas12a, Cas12b1 (C2c1), Cas12c (C2c3), Cas12e (CasX), Cas12d (CasY), Cas12g, Cas12h, Cas12i, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, and Cs The Cas protein may be, but is not limited to, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cas13a (C2c2), Cas13b, Cas13c, Cas13d, Cas14, xCas9, circularly permuted Cas9, Argonaute (Ago) domain, or a fragment, homolog, or variant thereof. In certain embodiments, the Cas protein may have nickase activity. In certain embodiments, the Cas protein may be nCas9. In certain embodiments, the Cas protein may be SpCas9 nickase. In certain embodiments, the Cas protein may have nuclease activity. In certain embodiments, the Cas protein may be a Cas protein having nuclease activity. In certain embodiments, the Cas protein may be a Cas9 variant having nuclease activity. In certain embodiments, the Cas protein may be SpCas9 or a variant thereof. For example, the SpCas9 variant may have a form in which any one or more amino acid residues selected from D10, R221, L244, N394, H840, K1211, and L1245 of wild-type SpCas9 are substituted with another amino acid residue.In certain embodiments, the Cas protein may comprise an amino acid sequence comprising the H840A variation of the amino acid sequence of wild-type SpCas9 (SEQ ID NO: 28). In certain embodiments, the Cas protein may comprise an amino acid sequence comprising the R221K and N394K amino acid variations of the amino acid sequence of wild-type SpCas9 (SEQ ID NO: 28). In certain embodiments, the Cas protein may comprise an amino acid sequence comprising the R221K and N394K amino acid variations of the amino acid sequence of wild-type SpCas9 (SEQ ID NO: 28). In certain embodiments, the Cas protein may comprise the amino acid sequence of SEQ ID NO: 29, 30, or 31.

[0224] In certain embodiments, the polymerase may be a reverse transcriptase. In certain embodiments, the reverse transcriptase may be a wild-type M-MLV reverse transcriptase. In certain embodiments, the reverse transcriptase may be a wild-type M-MLV reverse transcriptase variant. In certain embodiments, the wild-type M-MLV reverse transcriptase variant may comprise an amino acid sequence containing any one or more amino acid variations selected from D200N, T306K, W313F, T330P, and L603W in the amino acid sequence of wild-type M-MLV reverse transcriptase (SEQ ID NO: 26). In certain embodiments, the wild-type M-MLV reverse transcriptase variant may comprise the amino acid variations D200N, T306K, W313F, T330P, and L603W based on the amino acid sequence of wild-type M-MLV reverse transcriptase of SEQ ID NO: 26. In certain embodiments, a wild-type M-MLV reverse transcriptase variant can include the amino acid variations D200N, T306K, W313F, and T330P based on the amino acid sequence of wild-type M-MLV reverse transcriptase set forth in SEQ ID NO: 26. In certain embodiments, the reverse transcriptase can include the amino acid sequence of SEQ ID NO: 26 or 27.

[0225] As described above, the prime editor protein may further comprise additional elements, such as one or more linkers and / or one or more NLSs.

[0226] Examples of prime editor proteins that can be used in the off-target prediction system of the present application include prime editor proteins in the above-mentioned prime editing versions (e.g., PE1 to PE5, PEmax, nuclease-based prime editing, PEmax-nuclease, etc.). In some embodiments, the prime editor protein may be a PE2 prime editor protein, PE2-nuclease, a PEmax prime editor protein, or PEmax-nuclease. In certain embodiments, the prime editor protein may be PEmax-nuclease.

[0227] Tagmentation pegRNA (tpegRNA) Overview of tpegRNA Tagmentation pegRNA (tpegRNA) is a guide nucleic acid developed from pegRNA designed to insert a tag sequence into a DNA molecule, and is used in the off-target prediction method provided herein (i.e., the off-target prediction method in prime editing). tpegRNA developed from pegRNA may be referred to as a type of pegRNA. The tpegRNA provided herein contains a tag template and can be used to deliver information contained in the tag template (e.g., a tag sequence) to a DNA molecule (e.g., a genome) based on the prime editing mechanism.

[0228] In some embodiments, the tpegRNA may be a single-stranded nucleic acid molecule (such as a single-stranded RNA). In some embodiments, the tpegRNA may be a nucleic acid complex composed of two or more strands (such as a complex of single-stranded RNA and double-stranded RNA). When the tpegRNA is formed to contain two strands, portions of the sequences of the two strands may form complementary bonds with the gRNA core portion, thus forming a double-stranded tpegRNA. In certain embodiments, the tpegRNA may be a single-stranded RNA molecule.

[0229] Some embodiments of the present application provide a tpegRNA. Elements contained in the tpegRNA will be disclosed below.

[0230] The tpegRNA comprises a spacer, a gRNA core, and an extension region. As described above, the pegRNA used for prime editing has a form in which an extension arm is added to the 3' or 5' end of a conventional gRNA. Typically, the pegRNA has a form in which an extension arm is added to the 3' end of a conventional gRNA. Similarly, the tpegRNA has a form in which an extension arm is added to the 3' or 5' end of a conventional gRNA, and the extension arm may include an extension region.

[0231] In some embodiments, the tpegRNA may have a configuration in which an extension arm is added to the 3' end of a conventional gRNA. In some embodiments, the spacer, gRNA core, and extension region may be located in a 5'->3' direction in the tpegRNA. In some embodiments, the tpegRNA may further include, but is not limited to, one or more independently selected additional functional elements (e.g., linkers, transcription terminators, RNA protection motifs, etc.) in one or more regions selected from the group consisting of between the 5' end and the spacer, between the spacer and the gRNA core, between the gRNA core and the extension region, and between the extension region and the 3' end. In other words, in the tpegRNA, such independently selected additional functional elements may or may not be present between the above elements, and are not particularly limited thereto. In some embodiments, the extension region of the tpegRNA includes a tag template. In some embodiments, the tag template may be described separately from the DNA synthesis template (e.g., the RT template). For example, the extension region of a tpegRNA may be described as comprising a primer binding site (PBS), a tag template, and a DNA synthesis template. In this case, the tag template and the DNA synthesis template are described separately, but this describes the tag template separately from the DNA synthesis template of a conventional pegRNA. In another embodiment, the tag template is encoded in a DNA molecule edited by the reverse transcriptase of a prime editor protein, and thus may be described as a component of the DNA synthesis template. For example, the extension region of a tpegRNA may be described as comprising a primer binding site and a DNA synthesis template (in this case, the DNA synthesis template comprises the tag template). In the following description, the tag template and the DNA synthesis template will be described separately. Unless otherwise specified, a tpegRNA is recognized as comprising a tag template.

[0232] Furthermore, the extension region of the tpegRNA may further comprise one or more independently selected additional functional regions in addition to the PBS, tag template, and DNA synthesis template.

[0233] For example, the extension region of the tpegRNA may further comprise a 3' engineering region containing an RNA protection motif. When the extension region of the tpegRNA further comprises a 3' engineering region containing an RNA protection motif, the tpegRNA may be referred to as an engineered tpegRNA (etpegRNA). For example, the RNA protection motif may comprise the sequence CGCGGUUCUAUCUAGUUACGCGUUAAACCAACUAGAA (SEQ ID NO: 41). In some embodiments, the 3' engineering region may further comprise, in addition to the RNA protection motif, a linker for linking the RNA protection motif. In this case, the linker for linking the RNA protection motif may serve to link the RNA protection motif and the PBS. As used herein, the term tpegRNA is used as a concept that includes embodiments of etpegRNA, and references to tpegRNA may be interpreted as including content related to etpegRNA unless otherwise specified. Specific embodiments limited to the use of etpegRNA will be described in conjunction with the context of etpegRNA.

[0234] In some embodiments, the 3' engineering region is 5nt, 6nt, 7nt, 8nt, 9nt, 10nt, 11 nt, 12nt, 13nt, 14nt, 15nt, 16nt, 17nt, 18nt, 19nt, 20nt, 21 nt, 22nt, 23nt, 24nt, 25nt, 26nt, 27nt, 28nt, 29nt, 30nt, 31 nt, 32nt, 33nt, 34nt, 35nt, 36nt, 37nt, 38nt, 39nt, 40nt, 41 nt, 42nt, 43nt, 44nt, 45nt, 46nt, 47nt, 48nt, 49nt, 50nt, 51 nt, 52nt, 53nt, 54nt, 55nt, 56nt, 57nt, 58nt, 59nt, 60nt, 61nt, 62nt, 63nt, 64nt, 65nt, 66nt, 67nt, 68nt, 69nt, 70nt, 71nt, 72nt, 73nt, 74nt, 75nt, 76nt, 77nt, 78nt, 79nt, 80nt, 81nt, 82nt, 83nt, 84nt, 85nt, 86nt, 87nt, 88nt, 89nt, 90nt, 91nt, 92nt, 93nt, 94nt, 95nt, 96nt, 97nt, 98nt, 99nt, 100nt, 101nt, 102nt, 103nt, The 3' engineering region may have a length of, but is not limited to, 59 nt, 60 nt, 61 nt, 62 nt, 63 nt, 64 nt, 65 nt, 66 nt, 67 nt, 68 nt, 69 nt, 70 nt, 71 nt, 72 nt, 73 nt, 74 nt, 75 nt, 76 nt, 77 nt, 78 nt, 79 nt, 80 nt, 81 nt, 82 nt, 83 nt, 84 nt, 85 nt, 86 nt, 87 nt, 88 nt, 89 nt, 90 nt, 91 nt, 92 nt, 93 nt, 94 nt, 95 nt, 96 nt, 97 nt, 98 nt, 99 nt, or 100 nt or more, or may have a length within a range defined by two values ​​selected from the above values. In certain embodiments, the 3' engineering region may have a length of 10 to 70 nt. In certain embodiments, the 3' engineering region may have a length of 20 to 60 nt.

[0235] In some embodiments, the tpegRNA is about 30nt, 40nt, 50nt, 60nt, 70nt, 80nt, 90nt, 100nt, 110nt, 120nt, 130nt, 140nt, 150nt, 160nt, 170nt, 180nt, 190nt, 200nt, 210nt, 220nt, 230nt, 240nt, 250nt, 260nt, 270nt, 280nt, 290nt, 300nt, 310nt, 320nt, 330nt, 340nt, 350nt, 360nt, 370nt, 380nt, 390nt, 400nt, 410nt, 420nt, 430nt, 440nt, 450nt, 460nt, 470nt, 480nt, 490nt, 500nt, 510nt, 520nt, 530nt, 540nt, 550nt, 560nt, 570nt, 580nt, 590nt, 600nt, 610nt, 620nt, 630nt, 640nt, 650nt, 660nt, 670nt, 680nt, 690nt, 700nt, 710nt, 720nt, 730nt, 740nt, 750nt, 760nt, 770nt, 780nt, 790nt, 800nt, 810nt, 820nt, 830nt, 840nt, 850nt, 860nt, 8 In certain embodiments, the tpegRNA may have a length of 0 nt, 350 nt, 360 nt, 370 nt, 380 nt, 390 nt, 400 nt, 410 nt, 420 nt, 430 nt, 440 nt, 450 nt, 460 nt, 470 nt, 480 nt, 490 nt, 500 nt, 520 nt, 540 nt, 560 nt, 580 nt, or 600 nt or more, or may have a length within a range defined by any two values ​​selected from the above values, but is not limited thereto. In certain embodiments, the tpegRNA may have a length of 100 to 300 nt or 100 to 400 nt.

[0236] It should be noted that unlike typical pegRNAs (pegRNAs that do not contain a tag template), the tpegRNA of the present application contains a tag template for inserting a tag sequence into a DNA molecule. For the convenience of those skilled in the art, examples of conventional gRNAs, pegRNAs, and tpegRNAs are disclosed in FIG. 1. The examples of gRNAs, pegRNAs, and tpegRNAs disclosed in FIG. 1 are exemplified based on the essential elements contained in each guide RNA, and it will be apparent to those skilled in the art that additional elements may be contained between or at the ends of each element.

[0237] Below, we will explain each element of tpegRNA in detail.

[0238] Conventional gRNA portion - spacer As described above, the tpegRNA may comprise a spacer, a gRNA core, and an extension region. In this case, the spacer and gRNA core are elements derived from conventional gRNAs. The spacer and gRNA core are fully described in the "CRISPR / Cas System" and "Prime Editing System" sections herein. The spacer comprises a spacer sequence. The spacer sequence can be designed according to the target sequence, without limitation. In this case, the PAM sequence region may be considered. The spacer sequence can be designed as a sequence complementary to the target sequence in the spacer-bound strand of genomic DNA. The spacer sequence can be designed as the same sequence (or a substantially identical sequence or a sequence corresponding to) the target sequence in the spacer-unbound strand of genomic DNA. The spacer sequence may be an RNA sequence, a DNA sequence, or an RNA / DNA hybrid sequence. Typically, the spacer sequence is an RNA sequence. As with conventional gRNAs, the spacer sequence is involved in directing the Cas protein (the Cas protein contained in the prime editor) to the target region. In other words, the spacer sequence and the target sequence form a complementary bond, thus positioning the prime editor protein / tpegRNA complex at the target region, and the prime editor protein creates a nick or DSB in the target region.

[0239] In some embodiments, the spacer sequence may have a length of about 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, 35 nt, 36 nt, 37 nt, 38 nt, 39 nt, 40 nt, 41 nt, 42 nt, 43 nt, 44 nt, 45 nt, 46 nt, 47 nt, 48 nt, 49 nt, or 50 nt or more, or may have a length within a range of two values ​​selected from the above values, but is not limited thereto. In certain embodiments, the spacer sequence may have a length of 10 to 35 nt. In certain embodiments, the spacer sequence may have a length of 13 to 30 nt, In certain embodiments, the spacer sequence may have a length of 15 to 25 nt.

[0240] Conventional gRNA portion - gRNA core As described above, the tpegRNA may include a spacer, a gRNA core, and an extension region. In this case, the spacer and the gRNA core are elements derived from a conventional gRNA. The gRNA core is the portion that interacts with the Cas protein and binds to the Cas protein to form a complex. The gRNA core is sometimes referred to as the scaffold region. The gRNA core or scaffold may be designed differently depending on the type of Cas protein used, for example, depending on the type of microorganism from which the Cas protein is derived and the type of CRISPR system.

[0241] In one embodiment, the gRNA core may comprise a scaffold sequence. The scaffold sequence may be an RNA sequence, a DNA sequence, or an RNA / DNA hybrid sequence. A portion of the sequence of the gRNA core may interact with a portion of another sequence of the gRNA core, thus forming a structure such as a stem / loop or a hairpin.

[0242] In some embodiments, the scaffold sequence is about 5nt, 6nt, 7nt, 8nt, 9nt, 10nt, 11nt, 12nt, 13nt, 14nt, 15nt, 16nt, 17nt, 18nt, 19nt, 20nt, 21nt, 22nt, 23nt, 24nt, 25nt, 26nt, 27nt, 28nt, 29nt, 30nt, 31nt, 32nt, 33nt, 34nt, 35nt, 36nt, 37nt, 38nt, 39nt, 40nt, 41nt, 42nt, 43nt, 44nt, 45nt, 46nt, 47nt, 48nt, 49nt, 50nt, 51nt, 52nt, 53nt, 54nt, 55nt, 56nt, 57nt, 58nt, 59nt, 60nt, 61nt, 62nt, 63nt, 64nt, 65nt, 66nt, 67nt, 68nt, 69nt, 70nt, 71nt, 72nt, 73nt, 74nt, 75nt, 76nt, 77nt, 78nt, 79nt, 80nt, 81nt, 82nt, 83nt, 84nt, 85nt, 86nt, 87nt, 88nt, 89nt, 90nt, 91nt, 92nt, 93nt, 94nt, 95nt, 96nt, 97nt, 98nt, 99nt, 100nt, 101nt, 102nt, 103nt, 104nt, The scaffold sequence may have a length of 9 nt, 50 nt, 55 nt, 60 nt, 65 nt, 70 nt, 75 nt, 80 nt, 85 nt, 90 nt, 95 nt, 100 nt, 110 nt, 120 nt, 130 nt, 140 nt, 150 nt, 160 nt, 170 nt, 180 nt, 190 nt, 200 nt, 210 nt, 220 nt, 230 nt, 240 nt, 250 nt, 260 nt, 270 nt, 280 nt, 290 nt, or 300 nt or more, or may have a length within a range defined by two values ​​selected from the above values. In certain embodiments, the length of the scaffold sequence may be 30 to 200 nt, but is not particularly limited thereto. In certain embodiments, the length of the scaffold sequence may be 50 to 150 nt. In certain embodiments, the length of the scaffold sequence may be 60 to 100 nt.

[0243] Overview of the tpegRNA expansion region As described above, the tpegRNA may include an extension region (or extension arm). The extension region of the tpegRNA is characterized by including a tag template. The extension region of the pegRNA may be located at the 3' or 5' end of the conventional gRNA. For example, the tpegRNA may have the following structure in the 5' to 3' direction: "[conventional gRNA portion]-[extension region]" or "[extension region]-[conventional gRNA portion]." The [conventional gRNA portion] may include the spacer and scaffold (gRNA core) described above. Preferably, the extension region is located at the 3' end of the conventional gRNA portion. For example, the tpegRNA may include a spacer, a gRNA core, and an extension region. In some embodiments, the spacer, gRNA core, and extension region may be located in the 5' to 3' direction in the tpegRNA.

[0244] In some embodiments, the extension region, spacer, and gRNA core may be located in a 5' to 3' direction in the tpegRNA. In some embodiments, the extension region of the tpegRNA may comprise an RNA sequence, a DNA sequence, or a DNA / RNA hybrid sequence. Preferably, the extension region comprises an RNA sequence, but is not limited thereto.

[0245] The extension region of the tpegRNA is characterized by including a tag template. In other words, the extension region includes a primer binding site (PBS), a tag template, and a DNA synthesis template (such as an RT template). The extension region may further include one or more independently selected additional elements (e.g., linkers, RNA protection motifs, etc.) between the above elements or at the terminal elements.

[0246] Additional elements that may be included In some embodiments, the tpegRNA may include one or more independently selected additional elements in addition to the extension region, gRNA core, and spacer. The additional elements may be, for example, but are not limited to, any one of a linker, a poly-U tail, a poly-A tail, and an RNA protection motif. For example, the tpegRNA may include a U-rich, A-rich, or AU-rich sequence at its 3' end. In certain embodiments, the tpegRNA may include a (U)n sequence at its 3' end, where n is an integer between 3 and 20. In certain embodiments, the tpegRNA may include a (U)7 sequence at its 3' end.

[0247] tpegRNA extension region (1) Overview of the tpegRNA expansion region (1) As described above, the tpegRNA comprises an extension region, which may include a tag template and a primer binding site as described in detail for the pegRNA.

[0248] In some embodiments, the extension region of the tpegRNA can be described as comprising a first region comprising a DNA synthesis template, a second region comprising a tag template, and a third region comprising a primer binding site. In this case, part or all of the first region may be a DNA synthesis template. In this case, part or all of the second region may be a tag template. In this case, part or all of the third region may be a primer binding site. The elements contained in the extension region will be described in detail below.

[0249] Tag Template The extension region of the tpegRNA may contain a tag template. The tag template refers to a portion of the extension region that is complementary to the tag sequence to be placed on the spacer-unbound strand of a DNA molecule or a single-stranded DNA flap (e.g., a 3' DNA flap) synthesized by a polymerase such as reverse transcriptase. The tag template may be complementary to the tag sequence to be placed on the spacer-unbound strand of a DNA molecule or a DNA flap (e.g., a 3' DNA flap). The off-target prediction method of the present application can achieve the purpose of off-target prediction in prime editing by confirming information about the tag sequence to be placed on a DNA molecule and the tag containing a sequence complementary to the tag sequence (e.g., information about the site where the tag sequence is inserted, whether the tag sequence or a sequence complementary to the tag sequence exists, and the chromosome into which the tag sequence is inserted, etc.). Examples of tag sequences corresponding to the tag template of tpegRNA can be described in [Tsai, Shengdar Q., et al. "GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases." Nature biotechnology 33.2 (2015): 187-197.], the entire contents of which are incorporated herein by reference.

[0250] The tag template of the tpegRNA and the tag sequence to be inserted into DNA are not particularly limited and can be selected appropriately depending on the intended use of the tpegRNA. For example, the sequence of the tag template can include the sequence AUACCGUUAUUAACAUAUGACAACUCAAUUAAAC (SEQ ID NO: 42), GUUAUUAACAUAUGACAACUCAAUUAAAC (SEQ ID NO: 43), UAUGACAACUCAAUUAAAC (SEQ ID NO: 44), AUUAACAUAUGAC (SEQ ID NO: 45), GACAACUCA (SEQ ID NO: 46), or CUCAAUUA (SEQ ID NO: 47). For example, the tag sequence can include the sequence GTTTAATTGAGTTGTCATATGTTAATAACGGTAT (SEQ ID NO: 48), GTTTAATTGAGTTGTCATATGTTAATAAC (SEQ ID NO: 49), or GTTTAATTGAGTTGTCATA (SEQ ID NO: 50).

[0251] In some embodiments, the tag template may be an RNA sequence, a DNA sequence, or an RNA / DNA hybrid sequence. Preferably, the tag template is an RNA sequence.

[0252] In some embodiments, the tag template may have a length of 1 nt to 500 nt. In some embodiments, the tag template may have a length of 1 nt, 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, 35 nt, 36 nt, 37 nt, 38 nt, 39 nt, 40 nt, 41 nt, 42 nt, 43 nt, 44 nt, 45 nt, 46 nt, 47 nt, 48 nt, 49 nt, 50 nt, 51 nt, 52 nt, 53 nt, 54 nt, 55 nt, It may have a length of 56nt, 57nt, 58nt, 59nt, 60nt, 61nt, 62nt, 63nt, 64nt, 65nt, 66nt, 67nt, 68nt, 69nt, 70nt, 71nt, 72nt, 73nt, 74nt, 75nt, 76nt, 77nt, 78nt, 79nt, 80nt, 81nt, 82nt, 83nt, 84nt, 85nt, 86nt, 87nt, 88nt, 89nt, 90nt, 91nt, 92nt, 93nt, 94nt, 95nt, 96nt, 97nt, 98nt, 99nt, or 100nt or more, or may have a length within a range set by two values ​​selected from the above values. In certain embodiments, the tag template may have a length of 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, 35 nt, 36 nt, 37 nt, 38 nt, 39 nt, 40 nt, 41 nt, 42 nt, 43 nt, 44 nt, 45 nt, 46 nt, 47 nt, 48 nt, 49 nt, or 50 nt or more, or may have a length within a range set by two values ​​selected from the above values. In certain embodiments, the tag template may have a length of 10 to 70 nt.In certain embodiments, the tag template may have a length of 10 to 50 nt. In certain embodiments, the tag template may have a length of 15 to 40 nt. In certain embodiments, the tag template may have a length of 25 to 40 nt. In certain embodiments, the tag template may have a length of 30 to 40 nt. In certain embodiments, the tag template may have a length of 19, 24, 29, or 34 nt.

[0253] The length of the tag template can be appropriately designed according to the objective of the present disclosure, which is to analyze off-targets in prime editing through the tag sequence to be installed. If the tag template length is too short, it may be difficult to obtain information about the tag sequence inserted into the DNA molecule. If the tag template length is too long, it may be difficult to install the tag sequence into the DNA molecule, and the off-target prediction objective may not be achieved.

[0254] Furthermore, the tag template can be designed according to the intended use of the tpegRNA, without limitation. In the off-target prediction method of the present application, the tag template serves as the basis for the tag sequence to be inserted into the genomic DNA. In other words, the tag template is used as a template for reverse transcription, thereby installing the tag sequence in the genomic DNA. The tag sequence thus installed in the genomic DNA, or a sequence complementary to the tag sequence, can be used to identify tagging sites in the genomic DNA. The tagging sites can be used to identify regions where off-targets may occur (such as candidate off-target regions or candidate off-targets). When designing the tag sequence or tag template of the tpegRNA used for off-target prediction, consideration may be given to whether the same sequence exists in the genomic DNA. This is because, for example, if a sequence identical to the sequence of the tag sequence or tag template exists in the genomic DNA, it may affect the off-target prediction results. In another example, even if the same sequence exists, if the site where the same sequence exists is known in advance, the results of the corresponding site can be excluded from the off-target prediction results. As described above, the sequence of the tag template or tag sequence can be designed according to the intended purpose or plan of using the tpegRNA.

[0255] Primer binding site (PBS) The extension region of the tpegRNA may contain a primer binding site (PBS). The PBS of the tpegRNA may play the same or similar role as the primer binding site of the pegRNA in prime editing. The polymerization activity of the polymerase (e.g., reverse transcriptase) of the prime editing protein may be in the 5' to 3' direction relative to the strand that binds to the template strand. When a primer (e.g., a region in the spacer-unbound strand) is annealed to the primer binding site, the polymerase (e.g., reverse transcriptase) can polymerize a single strand of DNA using the template strand as a template. For example, when using a prime editing version 2 prime editing protein, the primer binding site (PBS) of the tpegRNA binds to the primer sequence formed from the endogenous DNA strand at the nicking target site caused by the prime editing protein, thereby exposing the 3' end of the nicked strand. The binding of the primer binding site and primer sequence in the extension region of the tpegRNA provides a matrix that allows reverse transcriptase to polymerize the single strand of DNA. The primer binding site may have a sequence complementary to a primer sequence located upstream (toward the 5' direction) of the cleavage site (caused by a nick or DSB) in the spacer-unbound strand. In some embodiments, the primer sequence may be a portion of a sequence located in a region ranging from -0 to -200 relative to the cleavage site. In certain embodiments, the primer sequence may be a portion of a sequence located in a region ranging from -0 to -50 relative to the cleavage site. In certain embodiments, the primer sequence may be a portion of a sequence located in a region ranging from -0 to -30 relative to the cleavage site. In certain embodiments, the primer sequence may be a portion of a sequence located in a region ranging from -0 to -20 relative to the cleavage site. In this case, - refers to the 5' direction, and numbers such as 30 refer to the number of nucleotides. For example, -30 refers to the 30th nucleotide upstream from the cleavage site. However, 0 refers to the cleavage site.

[0256] In some embodiments, the primer binding site may be an RNA sequence, a DNA sequence, or an RNA / DNA hybrid sequence. Preferably, the primer binding site is an RNA sequence.

[0257] In some embodiments, the primer binding site or primer may have a length of 1 nt to 500 nt. In some embodiments, the primer binding site or primer may have a length of 1 nt, 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, 35 nt, 36 nt, 37 nt, 38 nt, 39 nt, 40 nt, 41 nt, 42 nt, 43 nt, 44 nt, 45 nt, 46 nt, 47 nt, 48 nt, 49 nt, 50 nt, 51 nt, 52 nt, 53 nt, 54 nt, 55 nt, 56 nt, 57 nt, 58 nt, 59 nt, 60 nt, 61 nt, 62 nt, 63 nt, 64 nt, 65 nt, 66 nt, 67 nt, 68 nt, 69 nt, 70 nt, 71 nt, 72 nt, 73 nt, 74 nt, 75 nt, 76 nt, 77 nt, 78 nt, 79 nt, 80 nt, 57nt, 58nt, 59nt, 60nt, 61nt, 62nt, 63nt, 64nt, 65nt, 66nt, 67nt, 68nt, 69nt, 70nt, 71nt, 72nt, 73nt, 74nt, 75nt, 76nt, 77nt, 78nt, 79nt, 80nt, 81nt, 82nt, 83nt, 84nt, 85nt, 86nt, 87nt, 88nt, 89nt, 90nt, 91nt, 92nt, 93nt, 94nt, 95nt, 96nt, 97nt, 98nt, 99nt, or 100nt or more in length, or may have a length within a range set by two values ​​selected from the above values, but is not limited thereto. In certain embodiments, the primer binding site or primer may have a length of 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, may have a length of 29 nt, 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, 35 nt, 36 nt, 37 nt, 38 nt, 39 nt, 40 nt, 41 nt, 42 nt, 43 nt, 44 nt, 45 nt, 46 nt, 47 nt, 48 nt, 49 nt, or 50 nt or more, or may have a length within a range set by two values ​​selected from the above values.In certain embodiments, the primer binding site or primer may have a length of 3 to 30 nt. In certain embodiments, the primer binding site or primer may have a length of 5 to 20 nt. In certain embodiments, the primer binding site or primer may have a length of 5 to 15 nt.

[0258] DNA synthesis template The extension region of the tpegRNA may contain a DNA synthesis template. The DNA synthesis template may be a reverse transcription template (RT template). The DNA synthesis template of the tpegRNA may play the same or similar role as the DNA synthesis template of the pegRNA. The DNA synthesis template of the tpegRNA may optionally contain an editing template. A typical pegRNA used for prime editing necessarily contains an editing template because the prime editing is intended to perform editing. On the other hand, the tpegRNA used in the off-target prediction system of the present application preferentially aims to place tags rather than edit, and thus selectively contains an editing template. In other words, in some embodiments, the DNA synthesis template may or may not contain an editing template. Preferably, the DNA synthesis template contains an editing template, but is not limited to this.

[0259] In some embodiments, the DNA synthesis template may be an RNA sequence, a DNA sequence, or a DNA / RNA hybrid sequence. Preferably, the DNA synthesis template (such as an RT template) is an RNA sequence.

[0260] In some embodiments, the sequence of the DNA synthesis template may correspond to a portion of the sequence present in the region ranging from +0 to +500 of the cleavage site (resulting from a nick or DSB) of the spacer-unbound strand. In this case, + refers to the 3' direction, and a number such as 500 refers to the order of the nucleotide from the cleavage site. For example, 1 refers to the first nucleotide located downstream from the cleavage site. For example, 500 refers to the 500th nucleotide located downstream from the cleavage site. However, 0 refers to the cleavage site. In some embodiments, the sequence of the DNA synthesis template may correspond to a portion of the sequence in the region <+100, <+90, <+80, <+70, <+60, <+50, <+40, <+30, <+20, or <+10 of the cleavage site (resulting from a nick or DSB) of the spacer-unbound strand. For example, the sequence of the DNA synthesis template other than the editing template may be a sequence complementary to a portion of the sequence of the region of the spacer-unbound strand that is <+100, <+90, <+80, <+70, <+60, <+50, <+40, <+30, <+20, or <+10 relative to the cleavage site, and / or may be a sequence substantially identical to the above portion of the sequence of the spacer-unbound strand.

[0261] In some embodiments, the DNA synthesis template may have a length of 1 nt to 500 nt. In some embodiments, the DNA synthesis template may have a length of 1 nt, 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, 30nt, 31nt, 32nt, 33nt, 34nt, 35nt, 36nt, 37nt, 38nt, 39nt, 40nt, 41nt, 42nt, 43nt, 44nt, 45nt, 46 nt, 47nt, 48nt, 49nt, 50nt, 51nt, 52nt, 53nt, 54nt, 55nt, 56nt, 57nt, 58nt, 59nt, 60nt, 61nt, 62nt , 63nt, 64nt, 65nt, 66nt, 67nt, 68nt, 69nt, 70nt, 71nt, 72nt, 73nt, 74nt, 75nt, 76nt, 77nt, 78nt, 79nt, 80nt, 81nt, 82nt, 83nt, 84nt, 85nt, 86nt, 87nt, 88nt, 89nt, 90nt, 91nt, 92nt, 93nt, 94nt, 95nt, 96nt, 97nt, 98nt, 99nt, 100nt, 110nt, 120nt, 130nt, 140nt, 150nt, 160nt, 170nt, 180nt, 190nt, or 200nt or more in length, or may have a length within a range set by two values ​​selected from the above values, but is not limited thereto. In certain embodiments, the DNA synthesis template may have a length of 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, 35 nt, 36 nt, 37 nt, 38 nt, 39 nt, or 40 nt. In certain embodiments, the DNA synthesis template may have a length of 3 to 40 nt.In certain embodiments, the DNA synthesis template may have a length of 5 to 30 nt, In certain embodiments, the DNA synthesis template may have a length of 7 to 30 nt.

[0262] In some embodiments, the DNA synthesis template may include an editing template and a homology region (or homology arm). In some embodiments, the DNA synthesis template may include a homology region. The homology region contained in the DNA synthesis template will be described below.

[0263] The homologous region is a region corresponding to the above-mentioned homologous arm or homologous region of the pegRNA used for prime editing.

[0264] In some embodiments, the homologous region is complementary to a portion of the sequence of the non-spacer-binding strand of the target DNA. In some embodiments, the homologous region has a sequence homologous to a portion of the sequence of the spacer-binding strand of the target DNA.

[0265] The sequence of the homologous region is complementary to a portion of the sequence of a region located downstream (toward the 3' direction) of the resulting cleavage site (caused by a DSB or a nick) in the spacer-unbound strand of the DNA molecule. For example, in prime editing version 2, the homologous region may have a sequence complementary to a sequence located downstream of the site where the nick was created in the spacer-unbound strand. From another perspective, in prime editing version 2, the homologous region may have a sequence homologous to a portion of the sequence located upstream of the region corresponding to the site where the nick was created in the spacer-bound strand.

[0266] On the other hand, homology regions replace sequences in the endogenous strand of a DNA molecule and facilitate the annealing of single-stranded DNA flaps (such as the 3' DNA flap), thus facilitating the placement of editing and / or tag sequences into the DNA molecule. Homology regions are encoded by the polymerase (e.g., reverse transcriptase) of the prime editing protein, and thus can be described as part of the DNA synthesis template.

[0267] In some embodiments, the homology region may comprise an RNA sequence, a DNA sequence, or a DNA / RNA hybrid sequence. Preferably, the homology region comprises an RNA sequence.

[0268] In some embodiments, the homology region may have a length of 1 nt to 500 nt. In some embodiments, the homology region may have a length of 1 nt, 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, 35 nt, 36 nt, 37 nt, 38 nt, 39 nt, 40 nt, 41 nt, 42 nt, 43 nt, 44 nt, 45 nt, 46 nt, 47 nt, 48 nt, 49 nt, 50 nt, 51 nt, 52 nt, 53 nt, 54 nt, 55 nt, 56 nt, 57 nt, 58 nt, 59 nt, 60 nt, 61 nt, 62 nt, 63 nt, 64 nt, 65 nt, 66 nt, 67 nt, 68 nt, 69 nt, 70 nt, 71 nt, 72 nt, 73 nt, 74 nt, 75 nt, 76 nt, 77 nt, 78 nt, 79 nt, 80 nt, 8 It may have a length of 6nt, 57nt, 58nt, 59nt, 60nt, 61nt, 62nt, 63nt, 64nt, 65nt, 66nt, 67nt, 68nt, 69nt, 70nt, 71nt, 72nt, 73nt, 74nt, 75nt, 76nt, 77nt, 78nt, 79nt, 80nt, 81nt, 82nt, 83nt, 84nt, 85nt, 86nt, 87nt, 88nt, 89nt, 90nt, 91nt, 92nt, 93nt, 94nt, 95nt, 96nt, 97nt, 98nt, 99nt, or 100nt or more, or may have a length within a range set by two values ​​selected from the above values. In certain embodiments, the homology region may have a length of 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, 35 nt, 36 nt, 37 nt, 38 nt, 39 nt, or 40 nt. In certain embodiments, the homology region may have a length of 3 to 40 nt. In certain embodiments, the homology region may have a length of 5 to 30 nt. In certain embodiments, the homology region may have a length of 7 to 30 nt.

[0269] In some embodiments, the DNA synthesis template can include an editing template, which refers to a portion of an extension region that encodes an edit to be placed in a spacer-unbound strand or a single-stranded DNA flap (e.g., a 3' DNA flap) synthesized by a polymerase (e.g., reverse transcriptase).

[0270] The editing template can be complementary to the target edit in the non-spacer strand of the DNA molecule or DNA flap (such as a 3' DNA flap). For example, as a result of prime editing, the target edit in the non-spacer strand is located downstream of the resulting cleavage site.

[0271] In some embodiments, an RT template can include an editing template, a region of homology, etc. In this case, the RT template is equivalent to a DNA synthesis template. However, an RT template herein is based on the use of a prime editing protein with a polymerase, i.e., reverse transcriptase, while a DNA synthesis template is more broadly based on the use of a prime editing protein with any polymerase.

[0272] For example, the editing template of the tpegRNA may have the same sequence as the editing template corresponding to the desired edit to encode the desired edit in the DNA molecule (in this case, the desired edit may be a desired edit previously designed for prime editing, which is the subject of off-target analysis via the off-target prediction system of the present application). For example, the editing template of the tpegRNA may have a sequence complementary to the sequence of the desired edit to be placed in the DNA molecule (e.g., genome) or DNA flap (e.g., 3' DNA flap). In another example, the editing template of the tpegRNA may have a sequence different from the editing template corresponding to the desired edit to be encoded in the DNA molecule. In another example, the editing template of the tpegRNA may have a sequence different from part or all of the sequence complementary to the sequence of the desired edit to be placed in the DNA molecule (e.g., genome) or DNA flap (e.g., 3' DNA flap). In some embodiments, two types of tpegRNA may be used for off-target prediction in prime editing, in which case the sequence of the editing template included in each tpegRNA may differ from part or all of the sequence of the editing template for the desired edit.

[0273] In some embodiments, a single tpegRNA may be used for off-target prediction in prime editing, in which case the sequence of the editing template contained in the tpegRNA may have the same sequence as the editing template corresponding to the desired editing. In some embodiments, a single tpegRNA may be used for TAPE-seq, in which case the sequence of the editing template contained in the tpegRNA may have a sequence that is different from part or all of the editing template corresponding to the desired editing.

[0274] As described above, the prime editing technology is a system designed to insert a desired sequence into a desired site (i.e., a system designed to "write" a desired sequence), and the editing is not particularly limited. For example, the length of the editing may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 54, 56, 58, or 60 nt (or bp) or more.

[0275] In some embodiments, the edited DNA molecule may include an insertion of one or more nucleotides, a deletion of one or more nucleotides, a substitution of one or more nucleotides with other nucleotides, or a combination thereof, compared to the original sequence (i.e., the sequence before editing) located in the region of the edited DNA molecule corresponding to the edit. Additionally, the edited DNA molecule may have a region designed to insert a sequence identical to a portion of the sequence of the endogenous DNA strand to be replaced. For example, editing can involve the insertion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or more nucleotides, where the inserted nucleotides may or may not be located contiguously in the nucleic acid. For example, editing can involve the deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or more nucleotides, where the deleted nucleotides may or may not be located consecutively in the nucleic acid. For example, the editing may include the substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or more nucleotides, where the substituted nucleotides may or may not be consecutively located in the nucleic acid. In another example, the editing may include the insertion and substitution described above. In another example, the editing may include the deletion and substitution described above. In another example, the editing may include the insertion and deletion described above. In another example, the editing may include the insertion, deletion, and substitution described above.Any one or more of the above insertions, deletions, and substitutions may occur in the region of the "DNA molecule to be edited" that corresponds to the site where the edit is to be placed.

[0276] Furthermore, the edit to be made in the DNA molecule to be edited may have a region designed to insert a sequence identical to a portion of the sequence of the endogenous DNA strand to be replaced, and the region in the editing template that encodes this may be referred to as a "homology region of the editing template." One or more homology regions may be present in the editing template. In other words, the editing template may contain one or more homology regions within the editing template.

[0277] For the convenience of those skilled in the art, possible structures of the editing template will be exemplified below. The editing template can be designed according to the purpose without limitation, and therefore, possible embodiments should not be construed as being limited to the following examples. For example, the editing template can have the following structure: [first homology region of the editing template]-[nucleotide for G to T substitution]-[second homology region of the editing template]-[nucleotide for A to T substitution]-[third homology region of the editing template]. In another example, the editing template can have the following structure: [first homology region of the editing template]-[nucleotide for A to C substitution]-[second homology region of the editing template]. In a further example, the editing template can have the following structure: [first homology region of the editing template]-[nucleotide for TGG insertion]-[second homology region of the editing template]-[nucleotide for A to G substitution]. In a further example, the editing template can have the following structure: [nucleotides for AGG insertion]-[first homology region of editing template].

[0278] In some embodiments, the site where editing occurs may be within a range of +0 to +100 relative to the cleavage site of the spacer-unbound strand. In certain embodiments, the site where editing occurs may be within a range of +0 to +60. In certain embodiments, the site where editing occurs may be within a range of +1 to +30. In certain embodiments, the site where editing occurs may be within a range of +0 to +20. In certain embodiments, the site where editing occurs may be within a range of +0 to +10. In some embodiments, when a tag is inserted, the site where editing occurs may be located downstream of the installed tag sequence. For example, editing may occur within a range of +10 to +50 relative to the cleavage site.

[0279] In some embodiments, the editing template may be comprised of RNA. In certain embodiments, the editing template may be comprised of DNA. In certain embodiments, the editing template may be comprised of an RNA / DNA hybrid. In certain embodiments, the editing template may be comprised of RNA.

[0280] In some embodiments, the editing template may have a length of 1 nt to 200 nt, In some embodiments, the editing template may have a length of 1 nt, 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, 35 nt, 36 nt, 37 nt, 38 nt, 39 nt, 40 nt, 41 nt, 42 nt, 43 nt, 44 nt, 45 nt, 46 nt, 47 nt, 48 nt, 49 nt, or 50 nt or more. In certain embodiments, the editing template may have a length of 1 nt, 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, or 20 nt or more.

[0281] Relative position of elongation regions The relative positions of the above elements in the extension region will be described below. Each of the tag template, PBS, and DNA synthesis element may be directly linked to the other elements (e.g., via a covalent bond) or may be linked via an additional element such as a linker.

[0282] In the case of a 3' extension region (i.e., a tpegRNA in which the extension region is added to the 3' end of a conventional gRNA), the positional relationship may be as follows:

[0283] In some embodiments, these elements may be located in the 5'→3' direction of the tpegRNA in the extension region in the following order: DNA synthesis template, tag template, and primer binding site. In this case, in the DNA molecule (such as genomic DNA), in the 5'→3' direction relative to the resulting cleavage site of the spacer-unbound strand, the tag sequence delivered by the tag template may be located at the first nucleotide, and the sequence delivered by the DNA synthesis template may be located at the second nucleotide. In other words, the positional relationship between the tag sequence installed in the DNA molecule and the sequence complementary to the DNA synthesis template in the spacer-unbound strand may have the following structure: v-[tag sequence]-[sequence complementary to the DNA synthesis template]. In this case, v refers to the resulting cleavage site.

[0284] In other embodiments, these elements may be located in the 5'→3' extension region of the tpegRNA in the following order: tag template, DNA synthesis template, and primer binding site. In this case, in a DNA molecule (such as genomic DNA), the sequence delivered by the DNA synthesis template may be located at the first nucleotide, and the tag sequence delivered by the tag template may be located at the second nucleotide in the 5'→3' direction relative to the resulting cleavage site in the spacer-unbound strand. In other words, the positional relationship between the tag sequence installed in the DNA molecule and the sequence complementary to the DNA synthesis template in the spacer-unbound strand may have the following structure: v-[sequence complementary to the DNA synthesis template]-[tag sequence]. In this case, v refers to the resulting cleavage site.

[0285] Preferably, the DNA synthesis template, tag template, and primer binding site may be located in such order in the 5'→3' direction of the tpegRNA, but this is not limiting.

[0286] In the case of a 5' extension region (i.e., a tpegRNA in which the extension region is added to the 5' end of a conventional gRNA), the following positional relationship may be present: In some embodiments, the tag template, DNA synthesis template, and primer binding site may be located in the 5'->3' direction of the tpegRNA in the order shown. In some embodiments, the DNA synthesis template, tag template, and primer binding site may be located in the 5'->3' direction of the tpegRNA in the order shown.

[0287] In some embodiments, the tag template can be located between the DNA synthesis template and the primer binding site. In some embodiments, the tag template can be located between the gRNA core and the DNA synthesis template. In some embodiments, the tag template can be located between the spacer and the DNA synthesis template. In some embodiments, the DNA synthesis template can be located between the tag template and the primer binding site. In some embodiments, the DNA synthesis template can be located between the tag template and the gRNA core. In some embodiments, the DNA synthesis template can be located between the tag template and the spacer. An exemplary embodiment of a tpegRNA described as comprising a DNA synthesis template, a tag template, and a primer binding site is disclosed in FIG. 2.

[0288] tpegRNA extension region (2) In some embodiments, the tpegRNA may be described as comprising a homology region, an editing template, a tag template, and a primer binding site, thereby enabling the description of cases where the tag template is located between the editing template and the homology region. In some embodiments, the tpegRNA may comprise a first region comprising the homology region, a second region comprising the editing template, a third region comprising the tag template, and a fourth region comprising the primer binding site. In this case, part or all of the first region may be a homology region. In this case, part or all of the second region may be the editing template. In this case, part or all of the third region may be the tag template. In this case, part or all of the fourth region may be the primer binding site.

[0289] The positional relationship between the primer binding site, tag template, and each element based on the DNA synthesis template has been described in detail in the previous section. Therefore, the positional relationship between the homology region, editing template, and tag template will be described below. As described above, the tag template is installed in genomic DNA by a polymerase and can therefore be described as part of the DNA synthesis template. In some embodiments, including the following embodiments in the "tpegRNA Extension Region (2)" section of this specification, the tag template may be described as being included in the DNA synthesis template, which will not mislead those skilled in the art. A tpegRNA containing a 3' extension region will be exemplified. In some embodiments, the tag template may be located downstream of the editing template, i.e., between the primer binding site and the editing template. In some embodiments, the tag template may be located downstream of the homology region, i.e., between the homology region and the primer binding site. In some embodiments, the tag template may be located between the editing template and the homology region. In some embodiments, the tag template may be located upstream of the homology region, i.e., between the homology region and the gRNA core. In some embodiments, the tag template may be located upstream of the editing template, i.e., between the editing template and the gRNA core. An exemplary embodiment of a tpe gRNA described as comprising a homology region, an editing template, a tag template, and a primer binding site is disclosed in Figure 3.

[0290] Engineered tpegRNA Some embodiments of the present application provide an engineered tpegRNA (etpegRNA). An etpegRNA developed from pegRNA, epegRNA, and tpegRNA may be referred to as a tpegRNA. In other words, the term "tpegRNA" in this application will be recognized as including embodiments of etpegRNA. An etpegRNA refers to a pegRNA in which the extension region of the tpegRNA further comprises a 3' engineering region, which is an element of the epegRNA. In other words, an etpegRNA comprises an extension region including a tag template, a DNA synthesis template, a primer binding site, and a 3' engineering region. In some embodiments, the 3' engineering region may include an RNA protection motif. In some embodiments, the 3' engineering region may further comprise a linker for linking the RNA protection motif in addition to the RNA protection motif. For example, the above elements of an etpegRNA may be arranged in the extension region in the 5'→3' direction in the following order: DNA synthesis template, tag template, primer binding site, and 3' engineering region.

[0291] Note that unlike typical pegRNAs (pegRNAs that do not contain a tag template), tpegRNAs contain a tag template for inserting a tag sequence into a DNA molecule.

[0292] Examples of tools for predicting off-target sites in prime editing As described above, the present tool for predicting off-targets in prime editing includes at least the following two elements: prime editor protein; and tpegRNA.

[0293] In some embodiments, a tool for predicting off-targets in prime editing may further include additional elements. For example, but not limited to, any one or more of a dominant-negative MLH1 (dnMLH1), a Cas protein, a guide RNA (such as a conventional sgRNA), an additional prime editing protein, a pegRNA, and an additional tpegRNA (such as a tpegRNA containing an editing template with a sequence different from that of the tpegRNA used). Those skilled in the art will be able to improve or optimize the present system for predicting off-targets in prime editing using appropriate additional elements.

[0294] The present off-target prediction method, designed based on the prime editing mechanism, is a method for confirming or analyzing information about off-targets in prime editing. The prime editing mechanism is characterized by including a step of installing a desired edit in a target DNA molecule using a pegRNA containing a DNA synthesis template (e.g., an RT template) used as a template in a polymerization process (e.g., reverse transcription). The present off-target prediction method, based on the characteristic mechanism of prime editing, confirms or analyzes off-targets in prime editing by inserting a tag sequence into the target DNA molecule and confirming information about the inserted tag sequence. Therefore, the present off-target prediction method uses the characteristic mechanism of prime editing in the process of inserting the tag sequence.

[0295] Hereinafter, an example of a mechanism for inserting a tag into a DNA molecule to be edited in the off-target prediction method of the present application will be disclosed. This disclosure has been made for the convenience of those skilled in the art who read this specification, and the scope of this specification should not be limited by the following description.

[0296] Below, we will disclose an example of a mechanism for inserting a tag into a DNA molecule using prime editing version 2 tpegRNA and a prime editor protein.

[0297] The prime editing protein (including nCas9 and the reverse transcriptase MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)) and tpegRNA form a complex. The gRNA core of the tpegRNA, sometimes referred to as the gRNA scaffold or backbone sequence, refers to the sequence in the gRNA, pegRNA, or tpegRNA that is responsible for binding to Cas9 or its equivalent. The tpegRNA can bind to the Cas protein contained in the prime editing protein via the gRNA core.

[0298] The prime editor protein / tpegRNA complex localizes to a site where off-targeting is possible based on the spacer sequence and PAM sequence. The spacer sequence of the tpegRNA can form a complementary bond with the target (on-target or off-target) sequence of a complementary DNA molecule. In this case, the complementary bond may contain no mismatches or may contain one or more mismatches. The mismatches may be any one or more selected from, but are not limited to, bulge mismatches and base mismatches, which are known to cause off-targeting. Furthermore, off-targeting may occur due to a mismatch between a portion of the sequence contained in the extension region and the sequence of genomic DNA. Furthermore, the site where the prime editing protein / tpegRNA complex localizes may not be limited by the PAM sequence. When compared with the on-target sequence, the sequence of a predicted off-target (such as a candidate off-target) may contain any one or more mismatches selected from one or more PAM mismatches, one or more spacer mismatches (i.e., mismatches present in a protospacer, which is a sequence corresponding to the spacer sequence), one or more PBS mismatches (i.e., mismatches present in a primer sequence, which is a sequence corresponding to a PBS sequence), and one or more DNA synthesis template mismatches (i.e., mismatches present in a sequence corresponding to a DNA synthesis template).

[0299] The Cas protein of the prime editor protein (nCas9 in PE2) creates a nick between nucleotides -3 and -4 5' of the PAM sequence (5'-NGG-3') located upstream of the spacer-unbound strand. As a result, a tag sequence can be inserted in a window of 1 to 100 nucleotides downstream of the nick site. The tag sequence can be inserted in a region ranging from approximately -4 to +100 of the PAM sequence. Figure 4 shows an example of the resulting DNA molecule with a nick at an off-target site and the prime editor protein / tpegRNA complex that induces the nick.

[0300] Upstream of the nick, the PBS anneals to a region that acts as a primer (a region present on the unbound strand of the DNA molecule, sometimes called a primer). Annealing of the PBS to a primer is shown in Figure 5.

[0301] After annealing, reverse transcription is performed using a reverse transcriptase enzyme, using the tag template and the DNA synthesis template as templates. Reverse transcription is performed in the 5' to 3' direction relative to the nucleotide-polymerized strand, or in other words, in the 5' to 3' direction relative to the spacer-unbound strand. A sequence (tag sequence) having a sequence complementary to the tag template is added to the endogenous DNA strand by reverse transcription, followed by addition of a sequence complementary to the DNA synthesis template to the endogenous DNA strand.

[0302] Figure 6 shows the tag sequence and edits added to the endogenous DNA strand by reverse transcription. The tag sequence added to the endogenous DNA strand and the sequence corresponding to the DNA synthesis template (edits, sequences complementary to homologous regions, etc.) form a 3' DNA flap. The 5' flap is then removed, and the tag sequence and edit are finally incorporated into the DNA molecule via the repair system.

[0303] Through the above process, the tag sequence is inserted into a region where editing may occur by prime editing. This means that the tag sequence can be inserted into sites where not only on-target but also off-target events may occur. Accordingly, by confirming the presence and / or location of the tag sequence, the off-target location and / or possibility can be verified. The tag sequence is then analyzed using a method that can specifically analyze the tag sequence, such as tag-specific amplification or sequencing. Through tag sequence analysis, information about the tag sequence can be obtained, such as the type of DNA molecule into which the tag sequence is inserted (e.g., the type of chromosome), the site into which the tag sequence is inserted (e.g., the site in the DNA molecule into which the tag sequence is inserted), and the insertion rate of the tag sequence at each site. Based on the information about the tag sequence, information about off-target events that may occur during prime editing can be obtained.

[0304] The scenario for inserting a tag sequence into a target DNA molecule (such as genomic DNA) is not particularly limited. In some embodiments, tag insertion may not disrupt the remaining pattern of prime editing. In this case, if the tag sequence is removed from the product resulting from prime editing, the product resulting from prime editing with the tag sequence removed may be identical to the prime editing pattern induced by a pegRNA that does not include a tag template. For example, the tag sequence may be installed along with editing at one or more potential off-target sites and / or on-target sites. In some embodiments, tag insertion may disrupt the remaining pattern of prime editing. For example, the tag sequence may be installed without editing at one or more potential off-target sites and / or on-target sites. In another example, editing may be installed without the tag sequence at one or more potential off-target sites and / or on-target sites. In certain embodiments, the tag sequence may be installed with editing at one or more potential off-target sites and / or on-target sites. The off-target prediction system of the present application includes a process of contacting cellular genomic DNA with a prime editor protein and a tpegRNA, and then analyzing the genomic DNA. The process of the off-target prediction system of the present application will be described in detail below.

[0305] Contact between the cell's genomic DNA and the prime editor protein and tpegRNA Overview of contact with genomic DNA The present off-target prediction method for prime editing relates to identifying off-targets that may occur during the DNA editing process via prime editing. In other words, as a result of the present off-target prediction method for prime editing, information regarding potential off-targets that may occur during the DNA editing process via prime editing can be derived. For example, the present off-target prediction method can determine whether or not an off-target candidate exists, the potential off-target site, and the off-target candidate score associated with the true off-target. To obtain information regarding off-targets that occur during the DNA editing process, the prime editor protein and tpegRNA must be in contact with the target DNA. Once contact with the target DNA is achieved, a tag insertion mechanism, including a DNA cleavage process, can be performed. The target DNA can be, for example, the genomic DNA of a cell. As described above, the present off-target prediction method can be classified as a cell-based off-target prediction method, in which contact between the genomic DNA of a cell and the prime editor protein and tpegRNA can be performed intracellularly.

[0306] The cells used in the off-target prediction method for prime editing are not particularly limited. In some embodiments, the cells may be animal cells or plant cells. In some embodiments, the cells may be human cells or cells of non-human animals (e.g., mouse, rat, monkey, chimpanzee, dog, cat, cow, pig, horse, sheep, etc.), but are not particularly limited thereto. In some embodiments, the cells used in the off-target prediction method of the present application may be cells derived from a patient. In some embodiments, the cells used in the off-target prediction method of the present application may be cells of a cell line (e.g., a human, mouse, monkey, or rat cell line). In certain embodiments, the cells may be human cells or a human cell line. Examples of cells derived from a cell line include, but are not limited to, 3T3 cells, A549 cells, HeLa cells, HEK 293 cells, K562 cells, Huh7 cells, Jurkat cells, OK cells, Ptk2 cells, or Vero cells.

[0307] One embodiment of the off-target prediction system of the present application may include a step of contacting cellular genomic DNA with a prime editor protein and a tpegRNA (or a prime editor protein / tpegRNA complex). The step of contacting genomic DNA with a prime editor protein and a tpegRNA can be carried out intracellularly or in the nucleus of a cell, but is not particularly limited thereto. In order to contact genomic DNA with a prime editor protein and a tpegRNA, cells containing a prime editor protein and a tpegRNA must be prepared. Cells containing a prime editor protein and a tpegRNA and methods for preparing the same will be described in detail below.

[0308] Cells containing tools for predicting off-targets in prime editing In some embodiments, the off-target prediction method of the present application may comprise preparing cells containing a tool for predicting off-targets in prime editing.

[0309] Some embodiments of the present application provide cells containing tools for predicting off-targets in prime editing.

[0310] Tools for predicting off-targets in prime editing include a prime editor protein and a tpegRNA. In some embodiments, tools for predicting off-targets in prime editing may further include additional elements. For example, but not limited to, a tool for predicting off-targets in prime editing may further include one or more of a dominant-negative MLH1 (dnMLH1), a Cas protein, a guide RNA (such as a conventional sgRNA), an additional prime editing protein, a pegRNA, and an additional tpegRNA (such as a tpegRNA containing an editing template with a sequence different from that of the tpegRNA used).

[0311] Methods for preparing cells containing off-target prediction tools in prime editing Preparation of cells containing a tool for predicting off-target events in prime editing can be achieved by introducing each element of the prime editing tool into cells (e.g., by electroporation) or by introducing nucleic acids encoding each element of the prime editing tool into cells. The process for preparing cells containing a tool for predicting off-target events in prime editing will be described in detail below.

[0312] In some embodiments, preparing a cell containing a tool for predicting off-targets in prime editing may comprise contacting a cell with a prime editor protein or a nucleic acid encoding the prime editor protein, and a tpegRNA or a nucleic acid encoding the tpegRNA.

[0313] In some embodiments, the step of preparing a cell containing a tool for predicting off-targets in prime editing may include the step of introducing a prime editor protein or a nucleic acid encoding the prime editor protein, and a tpegRNA or a nucleic acid encoding the tpegRNA into a cell. A cell in this manner contacted with a prime editor protein or a nucleic acid encoding the prime editor protein and a tpegRNA or a nucleic acid encoding the tpegRNA, or a cell into which a prime editor protein or a nucleic acid encoding the prime editor protein and a tpegRNA or a nucleic acid encoding the tpegRNA have been introduced may be referred to as a cell to be analyzed.

[0314] Contact of cells with each component of the tool for predicting off-target events in prime editing may be performed simultaneously (e.g., using a single composition or an all-in-one vector) or at different times. For example, these tools can be introduced into cells by contacting cells with a composition comprising a prime editor protein or a nucleic acid encoding the prime editor protein and a tpegRNA or a nucleic acid encoding the tpegRNA. In another example, these tools can be introduced into cells by contacting cells with a first composition comprising a prime editor protein or a nucleic acid encoding the prime editor protein, and subsequently (or subsequently) with a second composition comprising a tpegRNA or a nucleic acid encoding the tpegRNA. As described above, the process for introducing the tool for predicting off-target events in prime editing into cells is not particularly limited.

[0315] In some embodiments, a prime editor protein or a nucleic acid encoding the prime editor protein, and / or a tpegRNA or a nucleic acid encoding the tpegRNA may be introduced into a cell in the form of a vector or a non-vector.

[0316] In some embodiments, the prime editor protein may be a fusion protein composed of a single molecule, or may be in the form of a complex containing two or more molecules. For example, when the prime editor protein is in the form of a fusion protein in the form of a single molecule, the prime editor protein or a nucleic acid encoding the prime editor protein can be introduced into a cell. In another example, when the prime editor protein is in the form of a complex containing two or more molecules, each element constituting the prime editor protein or each nucleic acid encoding each element can be introduced or delivered into a cell simultaneously (e.g., in the form of an assembled complex or encoded in a single vector) or separately (e.g., in the form of separate elements, encoded in separate vectors, or at appropriate time intervals).

[0317] In some embodiments, a prime editor protein or a nucleic acid encoding the prime editor protein and a tpegRNA or a nucleic acid encoding the tpegRNA can be introduced into a cell simultaneously (e.g., in the form of an assembled complex or encoded in a single vector) or separately (e.g., in the form of separate elements, encoded in separate vectors, or at an appropriate time interval). In some embodiments, the prime editor protein can be delivered or introduced into a cell in the form of a protein. In some embodiments, the prime editor protein can be delivered or introduced into a cell in the form of a nucleic acid encoding the prime editor protein. In some embodiments, the tpegRNA can be delivered or introduced into a cell in the form of an RNA. In some embodiments, the tpegRNA can be delivered or introduced into a cell in the form of a nucleic acid encoding the tpegRNA.

[0318] In some embodiments, a prime editor protein or a nucleic acid encoding the prime editor protein (e.g., DNA encoding the prime editor protein) and / or a tpegRNA or a nucleic acid encoding the tpegRNA (e.g., DNA encoding the tpegRNA) may be introduced into a cell in the form of a liposome, a plasmid, a viral vector, a nanoparticle, or a protein translation domain (PTD).

[0319] In some embodiments, a prime editor protein or a nucleic acid encoding the prime editor protein, and / or a tpegRNA or a nucleic acid encoding the tpegRNA can be delivered or introduced into a cell by any one selected from electroporation, lipofection, microinjection, a gene gun, virosomes, liposomes, immunoliposomes, and lipid-mediated transfection.

[0320] In some embodiments, nucleic acids encoding a prime editor protein (such as DNA, RNA, or a hybrid of DNA or RNA encoding a prime editor protein) and / or nucleic acids encoding a tpegRNA (such as DNA, RNA, or a hybrid of DNA or RNA encoding a tpegRNA) can be delivered or introduced into cells by methods known in the art. Alternatively, nucleic acids encoding a prime editor protein and / or nucleic acids encoding a tpegRNA can be delivered into a target cell by a vector, a non-vector, or a combination thereof.

[0321] The vector may be a viral vector or a non-viral vector (e.g., a plasmid). The non-vector may be naked DNA, a DNA complex, or mRNA.

[0322] Vector-based implementation In some embodiments, a prime editor protein or a nucleic acid encoding the prime editor protein and / or a tpegRNA and a nucleic acid encoding the tpegRNA can be introduced into a cell in the form of a vector, in other words, can be delivered or introduced into a target cell by a vector.

[0323] In some embodiments, a vector may contain a nucleic acid encoding a prime editor protein and / or a nucleic acid encoding a tpegRNA. In some embodiments, the nucleic acid encoding the prime editor protein may be contained in a single vector or may be divided and contained in multiple vectors. For example, the nucleic acid encoding the prime editor protein may be introduced or delivered into a cell by one, two, three, four, five, or more vectors. In some embodiments, the nucleic acid encoding the tpegRNA may be contained in a single vector or may be divided and contained in multiple vectors. For example, the nucleic acid encoding the tpegRNA may be introduced or delivered into a cell by one, two, three, four, five, or more vectors. In some embodiments, the nucleic acid encoding the prime editor protein and the nucleic acid encoding the tpegRNA may be contained in a single vector or may be divided and contained in multiple vectors. For example, the nucleic acid encoding the prime editor protein and the nucleic acid encoding the tpegRNA can be introduced or delivered into a cell by one, two, three, four, or five or more vectors.

[0324] In some embodiments, the vector may include one or more regulatory / control elements.

[0325] In this case, the regulatory / control element may be any one or more selected from a promoter, an enhancer, an intron, a polyadenylation signal, a Kozak consensus sequence, an internal ribosome entry site (IRES), a nuclear localization signal (NLS) or a nucleic acid encoding the same, polyA, a splice acceptor, and a 2A sequence. The promoter may be a promoter recognized by RNA polymerase II. The promoter may be a promoter recognized by RNA polymerase III. The promoter may be an inducible promoter. The promoter may be a target-specific promoter. The promoter may be a viral or non-viral promoter. An appropriate promoter may be selected depending on the control region.

[0326] In some embodiments, the vector may be a viral vector or a recombinant viral vector. The virus may be a DNA virus or an RNA virus.

[0327] In this case, the DNA virus may be a double-stranded DNA (dsDNA) virus or a single-stranded DNA (ssDNA) virus.

[0328] In this case, the RNA virus may be a single-stranded RNA (ssRNA) virus. The virus may be, but is not limited to, a retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV), vaccinia virus, poxvirus, or herpes simplex virus. The AAV vector may be, for example, any one selected from AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, AAVrh.10, AAVrh.74, and AAVhu.37, but is not limited to these. Examples of AAV vectors used in research or clinical practice are disclosed in detail in [Wang, Dan, Phillip WL Tai, and Guangping Gao. "Adeno-associated virus vector as a platform for gene therapy delivery." Nature reviews Drug discovery 18.5 (2019): 358-378.], the entire contents of which are incorporated herein by reference. Typically, a virus infects a host (such as a cell) and either introduces a nucleic acid encoding genetic information on the virus into the host or inserts a nucleic acid encoding genetic information into the host genome. Viruses with such characteristics can be used to introduce a nucleic acid encoding a target protein or target sequence into a target (such as a cell). Furthermore, the target protein and target sequence can be expressed in the host.

[0329] Non-vector-based transfer In one embodiment, a prime editor protein or a nucleic acid encoding the prime editor protein and / or a tpegRNA and a nucleic acid encoding the tpegRNA can be introduced into a cell by non-vector-based introduction.

[0330] In some embodiments, one or more of a prime editor protein or a nucleic acid encoding the prime editor protein and / or a tpegRNA and a nucleic acid encoding the tpegRNA can be introduced into a cell by non-vector-based introduction.

[0331] In some embodiments, one or more of a prime editor protein or a nucleic acid encoding the prime editor protein and / or a tpegRNA and a nucleic acid encoding the tpegRNA may be introduced into a cell by one or more non-vectors. For example, a protein editor protein or a nucleic acid encoding the protein editor protein, and a tpegRNA or a nucleic acid encoding the tpegRNA may be introduced or delivered into a cell by one, two, three, four, or five or more non-vectors.

[0332] The non-vector may include a protein editor protein or a nucleic acid encoding the protein editor protein and a tpegRNA or a nucleic acid encoding the tpegRNA. The non-vector may be naked DNA, a DNA complex, mRNA, or a mixture thereof. The non-vector may be delivered or introduced into a target by electroporation, a gene gun, sonoporation, magnetofection, temporary cell compaction or squeezing (as disclosed in Lee, et al., (2012) Nano Lett., 12, 6322-6327), lipid-mediated transfection, dendrimers, nanoparticles, calcium phosphate, silica, ormosil, or a combination thereof. For example, delivery by electroporation can be performed by mixing cells with a nucleic acid encoding the desired element in a cartridge, chamber, or cuvette, and then applying an electrical stimulus of defined duration and amplitude. In another example, the non-vector may be delivered using nanoparticles. The nanoparticles may be inorganic nanoparticles (e.g., magnetic nanoparticles, silica, etc.) or organic nanoparticles (e.g., polyethylene glycol (PEG)-coated lipids, etc.). The outer surface of the nanoparticles may be conjugated with a positively charged polymer (eg, polyethyleneimine, polylysine, polyserine, etc.) to allow for attachment.

[0333] Delivery or introduction in the form of peptides, polypeptides, proteins, or RNA In one embodiment, the prime editor protein and / or tpegRNA can be delivered or introduced into a target cell by methods known in the art. Peptide, polypeptide, protein, or RNA forms can be delivered or introduced into cells by electroporation, microinjection, transient cell compaction or squeezing (as disclosed in Lee, et al., (2012) Nano Lett., 12, 6322-6327), lipid-mediated transfection, nanoparticles, liposomes, peptide-mediated delivery, or a combination thereof.

[0334] As described above, cells containing a prime editor protein and a tpegRNA are obtained. The prime editor protein and the tpegRNA (or the prime editor protein / tpegRNA complex) in the cells can contact the genomic DNA of the cells. Below, the results that can be achieved by contacting the genomic DNA of a cell with the prime editor protein and the tpegRNA are described in detail.

[0335] Genomic DNA was contacted with the prime editor protein and tpegRNA (tagmentation). As a result of contacting genomic DNA with a prime editor protein and tpegRNA, a tag sequence and a sequence complementary to the tag sequence may be inserted into the genomic DNA. In other words, a tag may be installed in the genomic DNA. Such a process of installing a tag in genomic DNA may be referred to as tagmentation. As a result of the contact, a tag may be installed in a candidate off-target region and / or an on-target region. After contacting genomic DNA with a prime editor protein and tpegRNA, the resulting genomic DNA may be referred to as analyzed genomic DNA. In some embodiments, the analyzed genomic DNA may not contain a tag. This applies when there are no candidate off-targets, when installation of a tag sequence in the genomic DNA has failed, or the like. In some embodiments, the analyzed genomic DNA may contain a tag. The analyzed genomic DNA containing a tag may be referred to as tagged DNA (or tagmented DNA). The tag is present in a candidate off-target site (i.e., a candidate off-target region) and / or an on-target region. By analyzing the tags inserted in genomic DNA, it may be possible to identify potential candidate off-target regions that may be genuine off-targets. For example, the genomic DNA to be analyzed may contain one or more tags. By analyzing the presence or absence of each tag, each tagging site, etc., one or more off-target candidates can be identified. For example, the off-target prediction method of the present application can be performed on a population of cells. The genomic DNA to be analyzed of some cells in the cell population may contain one or more tags. The genomic DNA to be analyzed of some cells in the cell population may not contain tags. Analyzing the genomic DNA of each cell in the cell population may make it possible to identify one or more off-target candidates. When tags are inserted into the off-target candidate regions, the tagmentation rate for each candidate off-target region can be obtained. Furthermore, by inserting tags into the on-target region as well, the tagmentation rate for the on-target region can be obtained.The tagmentation rate may be, for example, about 0.001%, 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%, or may fall within a range of two values ​​selected from the above values.

[0336] Engineered cells containing engineered genomic DNA In some embodiments, engineered cells containing engineered genomes can be provided. For example, if the analyzed genomic DNA contains a tag, i.e., if the tag has been successfully installed in the analyzed genomic DNA, the analyzed genomic DNA can be referred to as an engineered genome. For example, if the analyzed genomic DNA contains an edit, i.e., if the edit has been successfully installed in the analyzed genomic DNA, the analyzed genomic DNA can be referred to as engineered genomic DNA. In some embodiments, the engineered genomic DNA can contain any one or more of a tag and an edit. In some embodiments, a cell population containing engineered cells can be provided.

[0337] Analysis of target DNA Overview of the analysis of the target DNA The off-target prediction system for prime editing of the present application includes a step of analyzing target DNA. When the off-target prediction system for prime editing of the present application is performed on cells, the target DNA may be target genomic DNA. Analysis of target DNA will be described using analysis of target genomic DNA as an example. The target genomic DNA may be DNA of one or more genomes. Analysis of target genomic DNA may include, but is not limited to, analyzing DNA of one or more genomes. Analysis of target genomic DNA makes it possible to obtain tagmentation information about the genomic DNA. For example, the tagmentation information may include, but is not limited to, whether or not tag sequences are contained in the target genomic DNA, the site (e.g., tagging site) of each tag sequence relative to one or more tag sequences in the genomic DNA, the tagmentation rate at one or more tagging sites, etc. Information about off-target candidates can be obtained based on the tagmentation information. For example, the information about off-target candidates may include, but is not limited to, information about one or more off-target candidates, the off-target score of one or more off-target candidates, etc.

[0338] Analysis method The genomic DNA to be analyzed can be analyzed to obtain tagmentation information. The genomic DNA to be analyzed may be engineered genomic DNA. The off-target prediction system of the present application is characterized by confirming information about potential off-target sites based on tag sequences incorporated into the engineered genome. Information about one or more tag sequences contained in the engineered genome can be confirmed using methods known in the art or methods currently under development, but is not limited to these. The information about the tag sequences may include, but is not limited to, one or more of the following: whether or not each tag sequence has been inserted; the chromosome into which each tag sequence has been inserted; the site into which each tag sequence has been inserted (e.g., the site within the chromosome); the insertion rate of the tag sequence; and the insertion rate at each site into which the tag sequence has been inserted. For example, the information about the tag sequences can be confirmed using tag sequence analysis methods, including, but not limited to, tag-specific amplification, sequencing, etc. For methods of analyzing information about tag sequences, see, for example, [Tsai, Shengdar Q., et al. "GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases." Nature biotechnology 33.2 (2015): 187-197.; Kim, Daesik, et al. "Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells." Nature methods 12.3 (2015): 237-243.; and Kim, Do Yon, et al. "Unbiased investigation of specificities of prime editing systems in human cells." Nucleic acids research 48.18 (2020): 10576-10589.] The entire contents of each of these publications are incorporated herein by reference.

[0339] In some embodiments, the analysis of the genomic DNA to be analyzed may be tag-specific analysis (e.g., analysis to identify sites where a tag sequence is present). In some embodiments, the method of analyzing the genomic DNA to be analyzed may include tag-specific amplification. In some embodiments, the method of analyzing the genomic DNA to be analyzed may include sequencing. In some embodiments, the analysis of the genomic DNA to be analyzed may include tag-specific amplification and sequencing.

[0340] In some embodiments, the genomic DNA to be analyzed can be analyzed by DNA analysis methods well known to those skilled in the art. In some embodiments, analysis of the genomic DNA to be analyzed can be performed by a process including any one or more selected from PCR-based analysis (see, for example, Cameron, Peter, et al., "Mapping the genomic landscape of CRISPR-Cas9 cleavage." Nature methods 14.6 (2017): 600-606) and sequencing (see, for example, Metzker, Michael L., "Sequencing technologies—the next generation." Nature reviews genetics 11.1 (2010): 31-46; and Kumar, Kishore R., Mark J. Cowley, and Ryan L. Davis. "Next-generation sequencing and emerging technologies." Seminars in thrombosis and hemostasis. Vol. 45. No. 07. Thieme Medical Publishers, 2019).

[0341] For example, any one or more of the sequencing methods referred to as whole genome sequencing (WGS), deep sequencing, high-throughput sequencing (HTS), de novo sequencing, second-generation sequencing, next-generation sequencing, third-generation sequencing, large-scale sequencing, shotgun sequencing, long-read sequencing, and short-read sequencing can be used, but the sequencing method is not particularly limited.For example, Hi-seq sequencing method can be used.For example, Mi-seq sequencing method can be used.For example, two or more sequencing methods can be used to analyze the target DNA.In a specific example, a process including Hi-seq and Mi-seq may be included in the analysis of the target DNA. In one embodiment, the sequencing depth of the sequencing method used to analyze the genomic DNA to be analyzed can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000×. In one embodiment, the sequencing depth can be within a range of two values ​​selected from the above values. In one embodiment, the sequencing depth can be less than, equal to, or greater than the above values. In a specific embodiment, the sequencing depth used for analysis may be about 10 to 40. The sequencing depth is not particularly limited, and any sequencing depth that allows confirmation of the presence or absence and / or location of a tag sequence in the genomic DNA to be analyzed is acceptable.

[0342] In some embodiments, the analysis of the genomic DNA to be analyzed can include a tag-specific amplification process.Through tag-specific amplification, an amplified tag-specific library can be generated.In some embodiments, the analysis of the genomic DNA to be analyzed can include sequencing the amplified tag-specific library.

[0343] Tagmentation information can be obtained through analysis of the genomic DNA to be analyzed. In some embodiments, analysis of the genomic DNA to be analyzed can include generating a tag-specific library from the genomic DNA to be analyzed; and sequencing the tag-specific library. In some embodiments, analysis of the genomic DNA to be analyzed can include generating an amplified tag-specific library from the genomic DNA to be analyzed; and sequencing the amplified tag-specific library. In some embodiments, analysis of the genomic DNA to be analyzed can include generating a tag-specific library from the genomic DNA to be analyzed; amplifying the tag-specific library; and sequencing the amplified tag-specific library. For example, tag-specific amplification can use tag-specific primers and / or adapter-specific primers. For example, tag-specific amplification can be performed by PCR.

[0344] In some embodiments, generating a tag-specific library from the genomic DNA to be analyzed may include one or more processes selected from the group consisting of shearing the genomic DNA to be analyzed and ligating the sheared genomic DNA with adapters to generate a tag-specific library. For the amplification process of the tag-specific library, see Tsai, Shengdar Q., et al., "GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases," Nature Biotechnology 33.2 (2015): 187-197; and Liang, Shun-Qing, et al., "Genome-wide detection of CRISPR editing in vivo using GUIDE-tag," Nature Communications 13.1 (2022): 1-14.

[0345] In some embodiments, for analysis of the genomic DNA to be analyzed, any one or more of the following processes may be further included: cell disruption, incubation, RNA removal, and DNA purification. The above processes may be performed, for example, after contacting the genomic DNA with the prime editor protein and the tpegRNA.

[0346] Obtaining tagmentation information Through the above analysis of the target DNA, tagmentation information can be obtained. The tagmentation information is information obtained based on the tag sequence and / or information related to the tag sequence present in the target genomic DNA.

[0347] For example, the tagmentation information may be information obtained based on information about tag sequences present in the DNA of one genome to be analyzed. In another example, the tagmentation information may be information obtained based on information about tag sequences present in the DNA of multiple genomes to be analyzed. It will be understood that the analysis of the genome DNA to be analyzed includes all embodiments of the analysis of the DNA of one or more genomes to be analyzed. For example, the tagmentation information may include, but is not limited to, one or more of the following: whether each tag sequence has been inserted, the chromosome into which each tag sequence has been inserted, the site into which each tag sequence has been inserted (e.g., the site within the chromosome), the insertion rate of the tag sequence, and the insertion rate for each site into which the tag sequence has been inserted.

[0348] In some embodiments, tagmentation information may include any one or more of the following: Whether the tag sequence is contained in the genomic DNA being analyzed; For one or more tag sequences, the site in the genomic DNA of each tag sequence; and The tagmentation rate of one or more tag sequences.

[0349] For example, if the genomic DNA to be analyzed contains a tag sequence, the presence of the tag sequence may be related to the presence of an on-target or candidate off-target region. As described above, one or more tag sequences may be contained in the DNA of one genome, or one or more tag sequences may be contained in the DNA of multiple genomes to be analyzed for analysis. As a result, whether or not a tag sequence is contained in the DNA to be analyzed provides information regarding whether or not one or more tag sequences are present in the DNA of one or more genomes to be analyzed. For example, in the case of DNA of multiple genomes to be analyzed, if the first genomic DNA to be analyzed does not contain a tag sequence, but the second genomic DNA to be analyzed contains a tag sequence, it can be determined that the tag sequence is present in the genomic DNA to be analyzed.

[0350] For example, for one or more tag sequences, the location of each tag sequence in genomic DNA can be derived through analysis of the site where the tag sequence is present (this may be referred to as a "tagging site"). For example, when the DNA of one analyzed genome (first analyzed genomic DNA) contains a first tag sequence and another analyzed genomic DNA (second analyzed genomic DNA) contains a second tag sequence among the DNAs of multiple analyzed genomes, the site of the first tag sequence may be referred to as the first site, and the site of the second tag sequence may be referred to as the second site. In another example, multiple tag sequences may be present in the DNA of one analyzed genome, in which case one tag sequence may be referred to as the first tag sequence, and another tag sequence may be referred to as the second tag sequence. In this case, the site of each tag sequence in genomic DNA for one or more tag sequences may include the first site, the second site, or both the first and second sites. Here, the first and second sites are relative to the target site (on-target site and / or candidate off-target site). The first and second sites other than the on-target site may both be candidate off-target sites. The first and second sites may refer to the same or different sites. In this case, information about sites such as the first and second sites includes information about the chromosome number and information about the sites on a specific chromosome.

[0351] For example, the tagmentation rate of one or more tag sequences can be derived from the frequency of discovery at each tagging site.For example, in the analysis of the genomic DNA to be analyzed, if a tag sequence is discovered 10 times at the first site and 5 times at the second site, the tagmentation rate of the first site is twice that of the second site.The tagmentation rate can be related to, but is not limited to, the probability that the corresponding off-target candidate is a genuine off-target.

[0352] In some embodiments, the process of obtaining tagmentation information by analyzing the genomic DNA to be analyzed may further include an additional process for obtaining tagmentation information. For example, this process may further include a process for processing information (or data) and / or a process for normalizing the obtained information (or data). For example, a process may further include a process for comparing the obtained cleavage information with information about a predetermined on-target. The process of obtaining cleavage information may further include additional processes as described above, and is not particularly limited thereto.

[0353] In some embodiments, the tagmentation information may further include, but is not limited to, other information obtainable through analysis of the genomic DNA being analyzed (such as DNA sequencing).

[0354] Obtaining information about off-target events Based on tagmentation information, information about off-targets can be obtained. A person skilled in the art to which the present application relates can easily obtain information about off-targets based on cleavage information. Accordingly, the disclosure of this specification does not limit the process of the off-target prediction system of the present application. A person skilled in the art to which the present application relates can obtain information about off-targets using tagmentation information obtained by analyzing the genomic DNA to be analyzed, with or without a separate process.

[0355] In some embodiments, the off-target prediction methods of the present application may include a process for confirming information about off-target candidates based on tagmentation information.

[0356] In some embodiments, the information about the off-target candidates may include information about the sites in genomic DNA of one or more off-target candidates (such as information about candidate off-target regions). For example, the information about the off-target candidate sites may include information about each site (site in genomic DNA) of all off-target candidates. For example, the information about the off-target candidate sites may include information about each site of one or more off-target candidate sites. In other words, information about all candidate off-target regions can be obtained. Alternatively, information about one or more candidate off-target regions can be obtained, rather than all candidate off-target regions. Among all the off-target candidates, there may be genuine off-targets (such as actual off-targets resulting from the use of a prime editing system). Information about the off-target candidate sites can be obtained based on the above-mentioned tagmentation information.

[0357] In one embodiment, the information about the off-target candidates may include off-target scores (e.g., off-target prediction scores) of one or more off-target candidates. For example, the information about the off-target candidates may include the off-target scores of each off-target candidate for all off-target candidates. For example, the information about the off-target candidates may include the off-target scores of each off-target candidate for one or more off-target candidates. In other words, the off-target scores of all candidate off-target regions may be obtained. Alternatively, the off-target scores of one or more candidate off-target regions may be obtained, rather than all candidate off-target regions. The information about the off-target scores of the off-target candidates may be obtained based on the above-mentioned tagmentation information (e.g., information about the tagmentation rate). In one embodiment, the rank of each off-target candidate may be calculated based on the obtained off-target scores. For example, an off-target candidate (e.g., a candidate off-target region) with a high off-target score may be ranked higher. For example, the off-target candidate with the highest off-target score may be ranked first. For example, a high off-target score of an off-target candidate may be associated with a true off-target, but is not particularly limited thereto.

[0358] In one embodiment, the information about off-target candidates may include information about the number of off-target candidates. For example, the total number of off-target candidates can be calculated. For example, in calculating the number of off-target candidates, overlapping sites may be counted as one. In another example, in calculating the number of off-target candidates, overlapping sites may be counted as multiple. For example, if the number of candidate off-target regions X identified is 5, the total number can be counted as 1 or 5. Through information about the number of such off-target candidates, the total number of off-target candidates that may occur in the genome editing process using prime editing can be confirmed. In other words, the total number of predicted off-targets can be confirmed.

[0359] In one embodiment, information regarding the off-target or potential off-target may include, but is not limited to, any one or more of the following: one or more potential off-target sites in the genomic DNA of the off-target candidates; an off-target score for each off-target candidate for one or more off-target candidates; and Number of predicted off-target candidates.

[0360] In some embodiments, the process of obtaining information about off-target candidates may further include additional processes for obtaining information about the off-target candidates. For example, this process may further include a process for processing the information (or data) and / or a process for normalizing the obtained information (or data). For example, a process may further include a process for comparing the obtained information about the off-target candidates with information about the predetermined on-target. The process of obtaining information about the off-target candidates may further include additional processes, as described above, and is not limited to any other processes.

[0361] In some embodiments, the information regarding potential off-targets may further include, but is not limited to, additional information that aids in predicting potential off-targets from use of the prime editing system.

[0362] Comparison of off-target candidates with tpegRNA As described above, the tag can be inserted into a potential off-target site (i.e., a potential off-target region). In conventional CRISPR / Cas systems, it is known that off-targeting can occur due to a partial but sufficient match between the guide sequence and the target sequence. Similarly, in prime editing systems, off-targeting is expected to result from a partial but sufficient match between the sequence of each element of the tpegRNA and the target sequence, although the reasons for off-targeting are not limited herein. In some embodiments, off-targeting can result from one or more mismatches between the sequence of the tpegRNA and the sequence of the off-target. In this case, mismatches include base mismatches (e.g., a difference of one or more nucleotides) and bulge mismatches (e.g., an addition of one or more nucleotides or a deletion of one or more nucleotides). In some embodiments, the sequence of the off-target (or potential off-target) may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more mismatches compared to the corresponding sequence of the tpegRNA. In some embodiments, the sequence of the off-target (or potential off-target) may have 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity compared to the corresponding sequence of the tpegRNA, or may have sequence identity within a range defined by two values ​​selected from the above values. For example, the sequence of the spacer of the tpegRNA and the sequence corresponding to the spacer of the off-target (or potential off-target) may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more mismatches. In another example, the sequence corresponding to the PAM sequence and the PAM sequence of the off-target (or potential off-target) may contain 1, 2, 3, 4, or 5 or more mismatches. For example, the sequence corresponding to the DNA synthesis template of the tpegRNA and the DNA synthesis template of the off-target (or potential off-target) may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more mismatches.For example, the sequences corresponding to the homologous region of the tpegRNA and the homologous region of the off-target (or off-target candidate) may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mismatches. For example, the sequences corresponding to the primer binding site of the tpegRNA and the primer binding site of the off-target (or off-target candidate) (such as a sequence that functions as a primer) may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mismatches. For example, one or more mismatches may be present in one or more of the sequence corresponding to the spacer of the off-target (or off-target candidate), the sequence corresponding to the PAM sequence of the off-target (or off-target candidate), the sequence corresponding to the DNA synthesis template of the off-target (or off-target candidate), and the sequence corresponding to the primer binding site of the off-target (or off-target candidate).

[0363] Comparison of off-target candidates with on-target As described above, the tag can be inserted into a potential off-target site (i.e., a potential off-target region). A potential off-target refers to an off-target predicted through a prediction system, which may or may not be a genuine off-target. In some embodiments, a potential off-target region can refer to a designated site. In some embodiments, an on-target site or region, or a potential off-target site or region, may be understood as a specific region, where the specific region may refer to a region consisting of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 450, or 500 consecutive nucleotides, or a region consisting of more than the above-mentioned number of consecutive nucleotides. In some aspects, the greater the number of consecutive nucleotides, the more precisely the off-target region or on-target region can be specified. This is because the greater the number of nucleotides, the lower the possibility of identical sequences (duplicate sequences) existing in genomic DNA.

[0364] The off-target or candidate off-target can be compared with the on-target sequence. In some embodiments, the candidate off-target or genuine off-target can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more mismatches (on-target mismatches) compared with the on-target sequence. In some embodiments, the off-target (candidate off-target) sequence can have 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with the corresponding on-target sequence, or can have a sequence identity within a range defined by two values ​​selected from the above values. The mismatches used in the comparison between the on-target and off-target are used to account for the sequence differences between the off-target and on-target. Furthermore, the term "mismatch" is used to include both nucleotide mismatches (e.g., a nucleotide difference) and bulge mismatches (e.g., an addition of one or more nucleotides or a deletion of one or more nucleotides). For example, if the sequence corresponding to the off-target candidate spacer is GGCACTGaGGgTGGAGGTGG (SEQ ID NO: 51) and the sequence corresponding to the on-target spacer is GGCACTGCGGCTGGAGGTGG (SEQ ID NO: 52), the sequence corresponding to the off-target candidate spacer can be described as having two nucleotide mismatches (written in lowercase) compared to the on-target sequence. In another example, if the sequence corresponding to the off-target candidate spacer is GGCACTGC-CTGGAGGTGG (SEQ ID NO: 53) and the sequence corresponding to the on-target spacer is GGCACTGCGGCTGGAGGTGG (SEQ ID NO: 54), the sequence corresponding to the off-target candidate spacer can be described as having two bulge mismatches (e.g., two bulge-on-target mismatches) compared to the on-target sequence.In a further example, if the sequence corresponding to the spacer of the off-target candidate is GGCACTGCGGCTGGAGgTGG (SEQ ID NO: 55) and the sequence corresponding to the spacer of the on-target is GGCACT-GGCTGGAGGTGG (SEQ ID NO: 56), the sequence corresponding to the spacer of the off-target candidate can be described as having one nucleotide mismatch and two bulge mismatches (a total of three mismatches) compared to the on-target sequence. Below, the sequence of the off-target (or off-target candidate) will be explained in comparison with the on-target sequence.

[0365] In some embodiments, a sequence corresponding to an off-target (or potential off-target) spacer may contain 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more mismatches (such as on-target mismatches). In some embodiments, a sequence corresponding to an off-target (or potential off-target) PAM sequence may contain 0, 1, 2, 3, 4, or 5 or more mismatches. In some embodiments, a sequence corresponding to an off-target (or potential off-target) DNA synthesis template may contain 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more mismatches. In some embodiments, a sequence corresponding to an off-target (or potential off-target) homology region may contain 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more mismatches. In some embodiments, the sequence corresponding to the primer binding site of the off-target (or potential off-target) may contain 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more mismatches. In some embodiments, the one or more mismatches may be present in any one or more of the sequence corresponding to the spacer of the off-target (or potential off-target), the sequence corresponding to the PAM sequence of the off-target (or potential off-target), the sequence corresponding to the DNA synthesis template of the off-target (or potential off-target), and the primer binding site of the off-target (or potential off-target).

[0366] In some embodiments, in any one or more of the regions corresponding to the spacer, PAM, PBS, and DNA synthesis template, the potential off-target (or off-target) region may contain 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more on-target mismatches, or may contain on-target mismatches within a range set by two values ​​selected from the above values. In some embodiments, a potential off-target (or off-target) region may contain 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more on-target mismatches in the region corresponding to the spacer and the DNA synthesis template, or may contain on-target mismatches within a range defined by two values ​​selected from the above values. In certain embodiments, a potential off-target (or off-target) region may contain 0 to 20 on-target mismatches in the region corresponding to the spacer and the DNA synthesis template. In certain embodiments, a potential off-target (or off-target) region may contain 1 to 15 on-target mismatches in the region corresponding to the spacer and the DNA synthesis template. In certain embodiments, a potential off-target (or off-target) region may contain 1 to 10 on-target mismatches in the region corresponding to the spacer and the DNA synthesis template. In this case, on-target mismatches refer to mismatches determined through comparison of the sequence of the on-target region with the sequence of the corresponding region. On-target mismatches may be counted in one strand or in both strands. For example, a potential off-target (or off-target) region may contain 0 to 10 on-target mismatches in the following regions of the spacer-unbound strand: (i) a region corresponding to the protospacer, and (ii) a region consisting of 5 to 20 nucleotides located downstream of the region corresponding to the protospacer.For example, a potential off-target (or off-target) region may contain 0 to 10 on-target mismatches in a region ranging from -30 to +10 or -20 to +10 relative to the cleavage site (nick or DSB).

[0367] Relationship with the prime editing system being predicted The off-target prediction system of the present application can be associated with a prime editing system to be predicted. In this case, the prime editing system to be predicted can refer to, but is not limited to, a prime editing system used in research or a prime editing system determined to be used in therapy. In other words, the prime editing system to be predicted refers to a prime editing system (or a genome editing process using a prime editing system) for which off-targets need to be predicted.

[0368] For example, when specific cells are used in the prime editing system to be predicted, these specific cells can also be used in the method for predicting off-targets of the present application. In another example, when specific cells are used in the prime editing system to be predicted, cells other than the specific cells can be used in the method for predicting off-targets of the present application. For example, patient-derived cells can be used in the prime editing system to be predicted, and the cells used in the off-target prediction system of the present application can be a human cell line.

[0369] In the methods for predicting off-targets of the present application, for example, when a tpegRNA having a specific sequence is used in the prime editing system to be predicted, a tpegRNA having a sequence that is the same as or partially different from the above sequence can be used. Similarly, when a specific prime editor protein is used in the prime editing system to be predicted, a prime editor protein of the same or different type as the above protein can be used in the methods for predicting off-targets of the present application. In another example, in addition to the elements of the prime editing system to be predicted, additional elements (e.g., dnMLH1, sgRNA, additional tpegRNA, etc.) may be used in the methods for predicting off-targets of the present application, without particular limitation.

[0370] In this aspect, the method for predicting off-targets according to one embodiment of the present application may further include a process for confirming the predicted prime editing system. The predicted prime editing system may be referred to as a predetermined prime editing system. The predetermined prime editing system may include using any one or more of a predetermined cell (e.g., a cell to be subjected to genome editing by the prime editing system), a predetermined prime editor protein, and a predetermined pegRNA, or a combination thereof.

[0371] In one embodiment, the method for predicting off-target genes of the present application may further include a step of confirming or designing a predetermined prime editing system. Confirming the predetermined prime editing system makes it possible to design appropriate elements to be used in the off-target prediction system. In this case, the process of confirming the predetermined gene editing system can be carried out before contacting the genomic DNA of a cell with the prime editor protein and tpegRNA. An example of confirming a predetermined prime editing system (i.e., the target of prediction) will be described below.

[0372] In one embodiment, the method for predicting off-targets of the present application may include identifying a predetermined prime editing system. In this case, identifying the predetermined prime editing system may include identifying any one or more of information about a predetermined cell, a predetermined prime editor protein, and a predetermined pegRNA. The predetermined prime editing system, the predetermined cell, the predetermined prime editor protein, the predetermined pegRNA, etc. may be used together with an ordinal determinant, for example, a first prime editing system, a first cell, a first prime editor protein, a first pegRNA, etc.

[0373] In certain embodiments, the step of identifying the predetermined prime editing system may include the step of identifying a predetermined cell. In certain embodiments, the same cell as the predetermined cell may be used in the off-target prediction system of the present application. In certain embodiments, a cell different from the predetermined cell may be used in the off-target prediction system of the present application. For example, the predetermined cell may be a human cell rather than a cell line, and a human cell line may be used in the off-target prediction system of the present application. In some embodiments, the predetermined cell may be an animal cell or a plant cell. In some embodiments, the predetermined cell may be a human cell or a cell of a non-human animal (e.g., mouse, rat, monkey, chimpanzee, dog, cat, cow, pig, horse, sheep, etc.), but is not limited thereto. In some embodiments, the predetermined cell may be a cell derived from a patient. In some embodiments, the predetermined cell may be a cell from a cell line (e.g., a human, mouse, monkey, or rat cell line). Examples of cells from a cell line may include, but are not limited to, 3T3 cells, A549 cells, HeLa cells, HEK 293 cells, K562 cells, Huh7 cells, Jurkat cells, OK cells, Ptk2 cells, or Vero cells.

[0374] In certain embodiments, the step of confirming the predetermined prime editing system may include the step of confirming a predetermined prime editor protein. In certain embodiments, the same prime editor protein as the predetermined prime editor protein may be used in the off-target prediction system of the present application. In certain embodiments, a prime editor protein different from the predetermined prime editor protein may be used in the off-target prediction system of the present application. For example, the predetermined prime editor protein may be a PE2 prime editor protein, but the prime editor protein used in the off-target prediction system of the present application may be a PE2-nuclease prime editor protein or a PEmax-nuclease prime editor protein. Other types of prime editor proteins may be used to increase the tagmentation rate.

[0375] In certain embodiments, the step of confirming the predetermined prime editing system may include the step of confirming a predetermined pegRNA. In certain embodiments, a tpegRNA identical to the predetermined pegRNA may be used in the off-target prediction system of the present application (in this case, a tpegRNA identical to the predetermined pegRNA indicates that all sequences except for the tag template are the same). In certain embodiments, a tpegRNA different from the predetermined pegRNA may be used in the off-target prediction system of the present application. The relationship between the predetermined pegRNA and the tpegRNA used in the off-target prediction system of the present application will be described below.

[0376] A given pegRNA can be referred to as a first pegRNA, and the first pegRNA includes a first spacer, a first DNA synthesis template, and a first primer binding site. For convenience, the tpegRNA used in the off-target prediction system of the present application is referred to as a second tpegRNA. The second tpegRNA includes a second spacer...

Claims

1. A method for predicting off-targets occurring in a process of editing a genome using a prime editing system, comprising: (a) obtaining an engineered cell; wherein the engineered cell comprises engineered genomic DNA, the engineered genomic DNA comprises a tag sequence, and the engineered genomic DNA associates with a prime editor protein and a tpegRNA, as follows: (i) contacting the genomic DNA with a prime editor protein and a tpegRNA (tagmentation pegRNA), wherein the prime editor protein comprises a Cas protein and a reverse transcriptase, and the tpegRNA comprises a spacer and an extension region comprising a tag template; (ii) inserting the tag sequence into the genomic DNA by a reverse transcription process carried out by the reverse transcriptase using the tag template of the tpegRNA as a reverse transcription template; It is generated through a process that includes (b) analyzing the engineered genomic DNA to obtain information regarding tagmentation; wherein the information regarding the tagmentation includes information regarding a region of genomic DNA into which the tag sequence is inserted; A method comprising:

2. The method for predicting off-targets comprises: Obtaining information about the off-target based on the information about the tagmentation, wherein the information about the off-target includes information about whether an off-target candidate exists, and information about the region of the off-target candidate if the off-target candidate exists. The method of claim 1 further comprising:

3. The method for predicting off-targets comprises: Verifying information about the on-target and comparing the information about the on-target with the information about the tagmentation. The method of claim 1 further comprising:

4. The method for predicting off-targets comprises: Ascertaining information regarding whether off-target candidates exist by identifying on-target information and comparing the on-target information with the information regarding the tagmentation. The method of claim 1 further comprising:

5. The method of claim 1 , wherein the tag sequence is inserted into the region of the genomic DNA specified by the spacer of tpegRNA.

6. The method of claim 1, wherein the region into which the tag sequence is inserted is associated with (corresponds to) a potential off-target region or an on-target region.

7. 2. The method of claim 1, wherein information about the region into which the tag sequence is inserted comprises information about the chromosome in which the tag sequence is located and information about the region within the chromosome in which the tag sequence is present.

8. 3. The method of claim 2, wherein the information regarding the region of the off-target candidate includes information regarding the chromosome on which the off-target candidate is located and the region within the chromosome in which the off-target candidate is located.

9. The information regarding the tagmentation, The insertion rate of the tag sequence for each region into which the tag sequence is inserted The method of claim 1 further comprising:

10. The information regarding the off-target is an off-target prediction score for said off-target candidates; The method of claim 2 further comprising:

11. The information regarding the off-target is Number of predicted off-target candidates The method of claim 2 further comprising:

12. The engineered cell comprises: contacting the prime editor protein or a nucleic acid encoding the prime editor protein, and the tpegRNA or a nucleic acid encoding the tpegRNA with a cell. The method of claim 1 , wherein the nucleic acid is obtained by a method comprising:

13. The engineered cell comprises: Introducing the prime editor protein or a nucleic acid encoding the prime editor protein, and the tpegRNA or a nucleic acid encoding the tpegRNA into a cell. The method of claim 1 , wherein the nucleic acid is obtained by a method comprising:

14. The method further comprising: obtaining DNA from the engineered cells, said step occurring prior to (b); The method of claim 1 further comprising:

15. The tpegRNA is a spacer; a gRNA core; and an extension region that includes a primer binding site, a tag template, and a reverse transcription template. The method of claim 1 , comprising:

16. The method of claim 15, wherein the reverse transcription template of the tpegRNA comprises an editing template and a homology region.

17. 16. The method of claim 15, wherein the manipulated genomic DNA comprises an edit.

18. The method of claim 15, wherein the spacer, the gRNA core, and the extension region are arranged in the following order in the 5'→3' direction: the spacer, the gRNA core, and the extension region.

19. 2. The method of claim 1, wherein the tag template is located in the extension region between the primer binding site and the reverse transcription template.

20. The method of claim 1 , wherein the tpegRNA further comprises a 3′ engineered region comprising an RNA protection motif.

21. The method further comprising: Information about a specific cell, information about a specific pegylated RNA, and information about a specific prime editor protein Validating a given prime editing system, comprising one or more of: The method of claim 1 further comprising:

22. 22. The method of claim 21, wherein the predetermined cell is different from the cell used in the method for predicting off-targets.

23. the sequence of the spacer of the tpegRNA is the same as the sequence of the spacer of the given pegRNA; 22. The method of claim 21 , wherein the sequence of the primer binding site of the tpegRNA is the same as the sequence of the primer binding site of the given pegRNA.

24. the sequence of the spacer of the tpegRNA is the same as the sequence of the spacer of the given pegRNA; the sequence of the primer binding site of the tpegRNA is the same as the sequence of the primer binding site of the given pegRNA; 22. The method of claim 21 , wherein the sequence of the reverse transcription template of the tpegRNA is the same as the sequence of the reverse transcription template of the given pegRNA.

25. 22. The method of Claim 21, wherein the prime editor protein used in the method for predicting off-targets is the same as or different from the predetermined prime editor protein.

26. The method of claim 1, wherein the tag template is between 5 nt and 60 nt in length.

27. The method of claim 1, wherein the tag template is between 10 nt and 50 nt in length.

28. 2. The method of claim 1, wherein the prime editor protein is a PE-nuclease comprising a Cas protein having double-strand cleavage activity.

29. 2. The method of claim 1, wherein the prime editor protein is PEmax-nuclease.

30. The method of claim 1, wherein the Cas protein comprised in the prime editor protein is a nickase.

31. 2. The method of claim 1, wherein the prime editor protein is a PE2 prime editor protein.

32. 2. The method of claim 1, wherein said manipulation of the DNA genome further involves any one or more of dnMLH1, gRNA, and an additional Cas protein, and an additional prime editor protein.

33. The (b) is specifically analyzing the engineered genomic DNA tags. The method of claim 1 , comprising:

34. The (b) is Sequencing the engineered genomic DNA. The method of claim 1 , comprising:

35. The (b) is generating a tag-specific library from the engineered genomic DNA; amplifying the tag-specific library to generate an amplified tag-specific library; and sequencing the amplified tag-specific library. The method of claim 1 , comprising:

36. A method for predicting off-targets occurring in a genome editing process using a prime editing system, comprising: (a) preparing a cell population comprising one or more engineered cells; wherein the engineered cell comprises engineered genomic DNA, the engineered genomic DNA comprises a tag sequence, and the engineered genomic DNA associates with a prime editor protein and a tpegRNA, as follows: (i) contacting the genomic DNA with a prime editor protein and a tpegRNA (tagmentation pegRNA), wherein the prime editor protein comprises a Cas protein and a reverse transcriptase, and wherein the tpegRNA comprises a spacer and an extension region comprising a tag template; (ii) a process of inserting said tag sequence into said genomic DNA, wherein said insertion of said tag sequence is achieved via a reverse transcription process carried out by said reverse transcriptase using the tag template of said tpegRNA as a reverse transcription template; Produced through a process including (b) obtaining tagmentation information by analyzing the results obtained through a process comprising sequencing the engineered genomic DNA of one or more engineered cells; wherein the tagmentation information includes information regarding one or more sites into which each tag sequence is inserted; and (c) obtaining information about the off-target based on the tagmentation information; wherein the information regarding the off-target includes information regarding whether off-target candidates are present, and information regarding the region of one or more off-target candidates. A method comprising:

37. A spacer; a gRNA core; and an extension region comprising a tag template. A tagmentation pegRNA (tpegRNA).

38. 38. The tpegRNA of claim 37, wherein the extension region comprising the spacer, the gRNA core, and the tag template is arranged in the following order in the 5'→3' direction: the spacer, the gRNA core, and the extension region comprising the tag template.

39. 38. The tpegRNA of claim 37, wherein the extension region comprises the tag template, a primer binding site, and a reverse transcription template.

40. 40. The tpegRNA of claim 39, wherein the tag template is located between the primer binding site and the reverse transcription template in the extension region.

41. 40. The tpegRNA of claim 39, wherein the reverse transcription template is located between the tag template and the primer binding site.

42. The tpegRNA of claim 39, wherein the primer binding site, the tag template and the reverse transcription template are positioned in the extension region in a 5' to 3' direction in the following order: the reverse transcription template, the tag template, and the primer binding site.

43. 40. The tpegRNA of claim 39, wherein the reverse transcription template comprises an editing template and a homology region.

44. 38. The tpegRNA of claim 37, wherein the tag template has a length of 5 nt to 60 nt.

45. The tpegRNA of claim 37, wherein the tag template has a length of 10 nt to 50 nt.

46. 38. The tpegRNA of claim 37, wherein the tpegRNA further comprises a 3' engineered region comprising an RNA protection motif.

47. The tpegRNA of claim 46, wherein the RNA protection motif has a length of 10 nt to 60 nt.

48. The tpegRNA of claim 47, wherein the tpegRNA has a length of 100 nt to 350 nt.

49. A composition for predicting possible off-targets in the process of genome editing by a prime editing system, A tpegRNA according to any one of claims 37 to 48; and Prime editor containing Cas protein and reverse transcriptase A composition comprising:

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