Increasing cas9 genome editing fidelity through attenuation of guide RNA watson-crick base pairing potential

Abstract

Systems including a nucleic acid programmable DNA binding protein and a guide RNA, having reduced off-target activity, are described. Off-target activity is reduced by attenuating the base pairing potential of a spacer sequence of the guide RNA with its target sequence by selective base substitutions in the spacer sequence.

Description

Attorney Docket: 4634 / 1036WO Increasing Cas9 Genome Editing Fidelity Through Attenuation of Guide RNA Watson- Crick Base Pairing Potential Cross-Reference to Related Applications

[0001] The present application claims priority to U.S. Provisional Patent Application Serial No.63 / 673,293, filed July 19, 2024, the content of which is hereby incorporated herein by reference in its entirety. Technical Field

[0002] The present invention relates to improved genome editing systems, and more particularly to gene editing systems having increased fidelity through attenuation of base pairing potential of a guide RNA. Background Art

[0003] The efficacy and precision of gene editing reagents are critical components in the development of therapeutics based on somatic cell genome editing[1]. Derived from a prokaryotic adaptive immune system, CRISPR (clustered regularly interspaced short palindromic repeats) guide RNAs and CRISPR-associated (Cas) proteins have been repurposed for genome engineering[2-5]. Class II systems utilize single multidomain Cas effector proteins with unique characteristics, such as recognition of specific nucleic acid species (DNA or RNA) and different sequence requirements for target engagement, and are further divided in types II, V and VI[6-8]. The signature type II endonuclease derived from the Streptococcus pyogenes Cas9 (SpyCas9) utilizes bound crRNA:tracrRNA components to achieve target site recognition upon PAM sequence recognition within the DNA[9, 10]. Sufficient complementarity between the spacer sequence within the guide RNA and the DNA leads to A-form-like helical RNA:DNA hybridization, termed an R-loop, triggering structural rearrangements in the nuclease effector protein to enable DNA cleavage[10-12]. Tight allosteric control through PAM recognition, RNA:DNA base pairing and concerted firing of both Cas9 nuclease domains provides multiplecheckpoints that ensure sequence-specific DNA cleavage. It is the intrinsic programmability of Cas9 to generate a double-strand break at any genomic site of interest with a compatible PAM that defines the utility of the CRISPR-Cas technology. However, nuclease activity at near- cognate (off-target) sites remains a concern for therapeutic applications employing Cas9 [13, 14]. Notably, multiple studies have established the tolerance of mismatches in the PAM distal region of the spacer sequence for DNA cleavage by Cas9 nuclease[15-17]. The reorganization of a loop located within the RuvC domain allows for a highly stabilized duplex conformation between guide RNA and PAM in the presence of distally mismatched DNA that facilitates cleavage

[0017] . Moreover, kinetic studies describe enzyme dissociation rates at near-cognate target sites that are only modestly faster than a cognate sequence, which may lead to spurious edits[15, 18]. Summary of the Embodiments

[0004] In accordance with one aspect, the disclosure provides a programmable genome editing system for modification of a double-stranded target DNA sequence comprising a target strand having a target sequence, and a non-target strand having a sequence complementary to the target sequence, the system comprising: a nucleic acid programmable nuclease, and a guide RNA comprising, in a 5′ to 3′ direction, a spacer sequence having a region of complementarity to the target sequence; wherein the spacer sequence comprises a base substitution in the region of complementarity to the target sequence, the base substitution being selected to reduce hydrogen bonding potential of the spacer sequence with the target sequence, thereby reducing off-target activity of the programmable genome editing system.

[0005] In some aspects, the spacer sequence may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 base substitutions selected to reduce hydrogen bonding potential of the spacer sequence with the target sequence. The spacer sequence may have 2–10, 2–8, 2–6, 4–8, or 4–6 base substitutions selected to reduce hydrogen bonding potential of the spacer sequence with the target sequence.

[0006] In some aspects, the base substitution is a non-canonical base. The base substitution may be a base selected from the group consisting of N4-ethyl-cytosine, N4-methyl- cytosine, N6-ethyl-adenine, N6-methyl-adenine, hypoxanthine, and combinations thereof. The base substitution may be a base selected from the group consisting of N4-ethyl-cytosine, N4-methyl-cytosine, N6-ethyl-adenine, N6-methyl-adenine, and combinations thereof and may further include a hypoxanthine base substitution.

[0007] In some aspects the base substitution is a substitution selected from the group consisting of cytosine to N4-ethyl-cytosine, cytosine to N4-methyl-cytosine, adenine to N6-ethyl- adenine, adenine to N6-methyl-adenine, guanine to hypoxanthine (wherein no base of the spacer sequence other than guanine is substituted with hypoxanthine), and combinations thereof. The base substitution may be a substitution selected from the group consisting of cytosine to N4- ethyl-cytosine, cytosine to N4-methyl-cytosine, adenine to N6-ethyl-adenine, adenine to N6- methyl-adenine, and combinations thereof and may further include a guanine to hypoxanthine base substitution (wherein no base of the spacer sequence other than guanine is substituted with hypoxanthine).

[0008] In some aspects, the nucleic acid programmable nuclease is Cas9, HiFiCas9, eSpCas9, SpCas9-HF1, Hypa-SpCas9, SuperFi-Cas9, Sniper-Cas9, or evoCas9. In other aspects, the nucleic acid programmable nuclease is Cas12.

[0009] In accordance with another aspect, the disclosure provides a method for site- specific modification of the double-stranded target DNA sequence comprising the target strand and the non-target strand, the method comprising: contacting the double-stranded target DNA sequence with the programmable genome editing system, wherein the contacting results in cleavage of (i) the target sequence of the target strand and (ii) the sequence complementary to the target sequence of the non-target strand. The double-stranded target DNA sequence may comprise a mutation at a locus selected from the group consisting of HPS1 and HBB.

[0010] In accordance with another aspect, the disclosure provides a method of treating a subject having or suspected of having a disease or disorder, the method comprising administering the programmable genome editing system, ex vivo, to a cell from the subject. The disease or disorder may be associated with a mutation at a locus selected from the group consisting of HPS1 and HBB.

[0011] In accordance with yet another aspect, the disclosure provides a programmable prime editing system for modification of a double-stranded target DNA sequence comprising a target strand having a target sequence, and a complementary non-target strand, the system comprising: a prime editor protein, the prime editor protein being a fusion protein comprising a nucleic acid programmable DNA binding domain fused to a reverse transcriptase domain, theDNA binding domain having nickase activity, and a pegRNA comprising, in a 5′ to 3′ direction: (i) a spacer sequence comprising a region of complementarity to the target sequence; (ii) a gRNA core that interacts with the DNA binding domain; and (iii) an extension arm, the extension arm comprising, in a 5′ to 3′ orientation: (a) a DNA synthesis template encoding one or more nucleotide changes compared to a region downstream of a nick site in the non-target strand of the double-stranded target DNA sequence, and (b) a primer binding sequence comprising a region of complementarity to a primer sequence upstream of the nick site in the non-target strand of the double-stranded target DNA sequence; wherein the spacer sequence comprises a base substitution in the region of complementarity to the target sequence, the base substitution being selected to reduce hydrogen bonding potential of the spacer sequence with the target sequence, thereby reducing off-target activity of the programmable prime editing system.

[0012] In some aspects, the spacer sequence may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 base substitutions selected to reduce hydrogen bonding potential of the spacer sequence with the target sequence. The spacer sequence may have 2–10, 2–8, 2–6, 4–8, or 4–6 base substitutions selected to reduce hydrogen bonding potential of the spacer sequence with the target sequence.

[0013] In some aspects, the base substitution is a non-canonical base. The base substitution may be a base selected from the group consisting of N4-ethyl-cytosine, N4-methyl- cytosine, N6-ethyl-adenine, N6-methyl-adenine, hypoxanthine, and combinations thereof. The base substitution may be a base selected from the group consisting of N4-ethyl-cytosine, N4- methyl-cytosine, N6-ethyl-adenine, N6-methyl-adenine, and combinations thereof and may further include a hypoxanthine base substitution.

[0014] In some aspects the base substitution is a substitution selected from the group consisting of cytosine to N4-ethyl-cytosine, cytosine to N4-methyl-cytosine, adenine to N6-ethyl- adenine, adenine to N6-methyl-adenine, guanine to hypoxanthine (wherein no base of the spacer sequence other than guanine is substituted with hypoxanthine), and combinations thereof. The base substitution may be a substitution selected from the group consisting of cytosine to N4- ethyl-cytosine, cytosine to N4-methyl-cytosine, adenine to N6-ethyl-adenine, adenine to N6- methyl-adenine, and combinations thereof and may further include a guanine to hypoxanthine base substitution (wherein no base of the spacer sequence other than guanine is substituted with hypoxanthine).

[0015] In accordance with another aspect, the disclosure provides a method for site- specific modification of the double-stranded target DNA sequence comprising the target strand and the non-target strand, the method comprising: contacting the double-stranded target DNA sequence with the programmable prime editing system, wherein the contacting results in: nicking the non-target strand of the double-stranded target DNA sequence to form a free 3′ end at the nick site; annealing the primer binding sequence with the primer sequence upstream of the nick site in the non-target strand of the double-stranded target DNA; synthesizing a single strand of DNA encoded by the DNA synthesis template from the free 3′ end of the non-target strand of the double-stranded target DNA sequence; and replacing the region downstream of the nick site in the non-target strand of the double-stranded target DNA sequence with the single strand of DNA encoded by the DNA synthesis template, thereby modifying the sequence of the double-stranded target DNA sequence. The double-stranded target DNA sequence may comprise a mutation at a locus selected from the group consisting of HPS1 and HBB.

[0016] In accordance with another aspect, the disclosure provides a method of treating a subject having or suspected of having a disease or disorder, the method comprising administering the programmable prime editing system, ex vivo, to a cell from the subject. The disease or disorder may be associated with a mutation at a locus selected from the group consisting of HPS1 and HBB.

[0017] In accordance with an alternative aspect, the disclosure provides a programmable base editing system for modification of a double-stranded target DNA sequence comprising a target strand having a target sequence, and a non-target strand having a sequence complementary to the target sequence, the system comprising: a base editor protein, the base editor protein being a fusion protein comprising a nucleic acid programmable DNA binding domain fused to a nucleoside deaminase domain, the DNA binding domain having nickase activity, and a guide RNA comprising, in a 5′ to 3′ direction, a spacer sequence having a region of complementarity to the target sequence; wherein the spacer sequence comprises a base substitution in the region of complementarity to the target sequence, the base substitution being selected to reduce hydrogen bonding potential of the spacer sequence with the target sequence, thereby reducing off-target activity of the programmable base editing system.

[0018] In some aspects, the spacer sequence may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 base substitutions selected to reduce hydrogen bonding potential of the spacer sequence withthe target sequence. The spacer sequence may have 2–10, 2–8, 2–6, 4–8, or 4–6 base substitutions selected to reduce hydrogen bonding potential of the spacer sequence with the target sequence.

[0019] In some aspects, the base substitution is a non-canonical base. The base substitution may be a base selected from the group consisting of N4-ethyl-cytosine, N4-methyl- cytosine, N6-ethyl-adenine, N6-methyl-adenine, hypoxanthine, and combinations thereof. The base substitution may be a base selected from the group consisting of N4-ethyl-cytosine, N4- methyl-cytosine, N6-ethyl-adenine, N6-methyl-adenine, and combinations thereof and may further include a hypoxanthine base substitution.

[0020] In some aspects the base substitution is a substitution selected from the group consisting of cytosine to N4-ethyl-cytosine, cytosine to N4-methyl-cytosine, adenine to N6-ethyl- adenine, adenine to N6-methyl-adenine, guanine to hypoxanthine (wherein no base of the spacer sequence other than guanine is substituted with hypoxanthine), and combinations thereof. The base substitution may be a substitution selected from the group consisting of cytosine to N4- ethyl-cytosine, cytosine to N4-methyl-cytosine, adenine to N6-ethyl-adenine, adenine to N6- methyl-adenine, and combinations thereof and may further include a guanine to hypoxanthine base substitution (wherein no base of the spacer sequence other than guanine is substituted with hypoxanthine).

[0021] The base editor protein may comprise a uracil glycosylase inhibitor (UGI) domain and wherein the nucleoside deaminase domain is a cytosine deaminase. In an alternative aspect, the nucleoside deaminase domain may be an adenine deaminase.

[0022] In accordance with another aspect, the disclosure provides a method for site- specific modification of the double-stranded target DNA sequence comprising the target strand and the non-target strand, the method comprising: contacting the double-stranded target DNA sequence with the programmable base editing system, wherein the contacting results in nicking the target sequence of the target strand; and deaminating a nucleobase of the sequence complementary to the target sequence, the nucleobase being selected from the group consisting of cytosine and adenine. The double-stranded target DNA sequence may comprise a mutation at a locus selected from the group consisting of HPS1 and HBB.

[0023] In accordance with another aspect, the disclosure provides a method of treating a subject having or suspected of having a disease or disorder, the method comprising administeringthe programmable base editing system, ex vivo, to a cell from the subject. The disease or disorder may be associated with a mutation at a locus selected from the group consisting of HPS1 and HBB.

[0024] In accordance with yet another aspect, the disclosure provides system for modification of a double-stranded target DNA sequence of a double-stranded target DNA sequence comprising a target strand having a target sequence, and a non-target strand having a sequence complementary to the target sequence, the system comprising: a nucleic acid programmable DNA binding protein; and a guide RNA comprising, in a 5′ to 3′ direction, a spacer sequence having a region of complementarity to the target sequence; wherein the spacer sequence comprises a base substitution in the region of complementarity to the target sequence, the base substitution being selected to reduce hydrogen bonding potential of the spacer sequence with the target sequence, thereby reducing off-target activity of the system.

[0025] In some aspects, the spacer sequence may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 base substitutions selected to reduce hydrogen bonding potential of the spacer sequence with the target sequence. The spacer sequence may have 2–10, 2–8, 2–6, 4–8, or 4–6 base substitutions selected to reduce hydrogen bonding potential of the spacer sequence with the target sequence.

[0026] In some aspects, the base substitution is a non-canonical base. The base substitution may be a base selected from the group consisting of N4-ethyl-cytosine, N4-methyl- cytosine, N6-ethyl-adenine, N6-methyl-adenine, hypoxanthine, and combinations thereof. The base substitution may be a base selected from the group consisting of N4-ethyl-cytosine, N4- methyl-cytosine, N6-ethyl-adenine, N6-methyl-adenine, and combinations thereof and may further include a hypoxanthine base substitution.

[0027] In some aspects the base substitution is a substitution selected from the group consisting of cytosine to N4-ethyl-cytosine, cytosine to N4-methyl-cytosine, adenine to N6-ethyl- adenine, adenine to N6-methyl-adenine, guanine to hypoxanthine (wherein no base of the spacer sequence other than guanine is substituted with hypoxanthine), and combinations thereof. The base substitution may be a substitution selected from the group consisting of cytosine to N4- ethyl-cytosine, cytosine to N4-methyl-cytosine, adenine to N6-ethyl-adenine, adenine to N6- methyl-adenine, and combinations thereof and may further include a guanine to hypoxanthinebase substitution (wherein no base of the spacer sequence other than guanine is substituted with hypoxanthine).

[0028] In accordance with another aspect, the disclosure provides a method for site- specific modification of the double-stranded target DNA sequence comprising the target strand and the non-target strand, the method comprising: contacting the double-stranded target DNA sequence with the system, wherein the contacting results in specific modification of the double- stranded target DNA sequence. The double-stranded target DNA sequence may comprise a mutation at a locus selected from the group consisting of HPS1 and HBB.

[0029] In accordance with yet another aspect, the disclosure provides a method, the method comprising administering the system, ex vivo, to a cell from the subject. The disease or disorder may be associated with a mutation at a locus selected from the group consisting of HPS1 and HBB. Brief Description of the Drawings

[0030] The foregoing features of embodiments will be more readily understood by reference to the following detailed description, taken with reference to the accompanying drawings, in which:

[0031] Figs.1A–E: On-Target Activity of Inosine Substituted Guide RNAs. Fig.1A shows chemical structures of Guanosine-Cytosine (G-C) and Inosine-Cytosine (I-C) base pairs. Dotted lines represent hydrogen bonds (H-bond). G-C base pairs make three H-bonds while I-C base pairs only make two H-bonds with the loss of the 2-amino group. An estimate in the loss of free energy accrued from a G-C bond to an I-C bond for RNA duplexes as described by Siegfried et al.2007. Fig.1B shows a previously described HPS1 guide RNA design for SpyCas9, denoted by the underlined segment, targeting the 16bp pathogenic microduplication (top). The PAM sequence is denoted by the boxed sequence and cut site is denoted by the scissor symbol. The percentage G-C content of the guide RNA is indicated above the spacer sequence (top). The secondary structure of HPS1 spacer sequence along with free energy estimated by RNAfold is shown on the bottom left and 2 Inosine substituted HPS1 guide RNAs (4 inosine (4i) and 6 inosine (6i)) are shown on the bottom right, according to an aspect of this disclosure. Fig.1B discloses SEQ ID NOS 160, 161 and 161-163, respectively, in order of appearance. Fig.1C is a bar graph showing HPS1 on-target editing for unmodified and substituted guide RNA withWTCas9 and HiFiCas9 in HPS1 B-LCLs measured by deep-sequencing, according to an embodiment of this disclosure. Error bars represent standard deviation (s.d.) for n=3. Indels quantified via CRISPResso2. Fig.1D shows a previously described HBB guide RNA design for SpyCas9 denoted by the underlined segment. The PAM is boxed and cutting site is denoted by the scissor symbol. The percent G-C content of the guide RNA is indicated above the spacer sequence (top). The secondary structure of HPS1 spacer sequence along with free energy estimated by RNAfold (bottom left). Depiction of 5 Inosine substituted HBB guide RNAs (3 inosine (3a-c) and 4 inosine (4a-b)) are shown on the bottom right, according to an embodiment of this disclosure. Fig.1D discloses SEQ ID NOS 164-171, respectively, in order of appearance. Fig.1E is a bar chart showing HBB On-target editing for unmodified and inosine substituted guide RNA with WTCas9 and HiFiCas9 in HEK293Ts measured by deep sequencing, according to an embodiment of this disclosure.

[0032] Figs.2A–2D: Editing at active off-target sites are mitigated with WTCas9 and inosine guide RNAs at G-C rich therapeutic targets. Fig.2A is a chart showing quantification of editing efficiencies at 16 HPS1 off-target sites (identified via Guide-Tag analysis and amplified with rhAmpSeq technology) by WTCas9-standard unmodified RNPs (left most bar for each grouping) or WTCas9-inosine modified RNPs (4-inosine (4i) (second bar for each grouping) or 6-inosine (6i) (third bar for each grouping) in patient-derived HPS1 B-LCLs, according to an aspect of this disclosure. Untreated sample (background) is denoted by the right most bar for each grouping. Sequences of four off-target sites (OT2, OT5, OT6, OT16) are depicted at the top. Fig.2A discloses SEQ ID NOS 172-175, respectively, in order of appearance. Fig.2B is a chart showing relative specificity ratios (on-target WTCas9 editing / off-target WTCas9 editing) of inosine modified guide RNAs at each of the 16 off-target site normalized to unmodified guide RNA based on data from Fig.1C and Fig.2A, according to an embodiment of this disclosure. Fig.2C is a chart showing quantification of editing efficiencies at three previously identified active off-target sites (OT1, OT2, OT3 sequences depicted above plot) by WTCas9-standard unmodified RNPs or WTCas9-inosine modified guides in HEK293Ts. For each grouping, bars are shown in the order presented in the legend. Fig.2C discloses SEQ ID NOS 176-177 and 106, respectively, in order of appearance. Fig.2D is a bar graph showing relative specificity ratios (on-target editing / off-target editing) of inosine modified guide RNAs at each of the 3 off-target site normalized to unmodified guide RNA based on data from Fig.1E and Fig.2C, according toan embodiment of this disclosure. For each grouping, bars are shown in the order presented in the legend. Editing levels are quantified from deep-sequencing via CRISPResoo2 pipeline. n=3 independent replicates data presented as the mean (SD). P-values calculated via Levene’s test and adjusted using Hochberg method where “*” p-value <0.05, “**” p-value<0.01, “***” p- value<0.001.

[0033] Fig.3 is a bar graph showing HPS1 on-target editing efficiencies as quantified in Fig.1C, according to an aspect of this disclosure. Percentage of 16 bp precise deletions (lower darker segment of each bar) out of total editing alleles at HPS1 associated 16bp duplication by WTCas9 / HiFiCas9-standard unmodified RNPs or WTCas9 / HiFiCas9-inosine RNPs.

[0034] Figs.4A–B: WTCas9 RNP titrations with HBB unmodified and inosine modified guide RNAs. Fig.4A is a bar graph showing quantification of editing efficiencies at the HBB ON-target site by WTCas9-standard unmodified RNPs or WTCas9-inosine RNPs at five increasing RNP concentrations (5uM-80uM) with constant 3x excess guide RNA molar ratio in HEK293Ts, according to an aspect of this disclosure. Arrow at 40uM concentration indicates RNP concentration used in experiments throughout this study (unless otherwise stated). For each grouping, bars are shown in the order presented in the legend. Fig.4B is a bar chart showing quantification of editing efficiencies at HBB off-target site 1 (OT1) by WTCas9-standard unmodified RNPs or WTCas9-inosine RNPs at five increasing RNP concentrations (5uM-80uM) with constant 3x excess guide RNA molar ratio in HEK293Ts, according to an aspect of this disclosure. For each grouping, bars are shown in the order presented in the legend. Editing levels are quantified from deep-sequencing via CRISPResoo2 pipeline. n=3 independent replicates data presented as the mean (SD). P-values calculated via Levene’s test and adjusted using Hochberg method where “*” p-value <0.05, “**” p-value<0.01, “***” p-value<0.001.

[0035] Figs.5A–D: Inosine substitutions combined with HiFiCas9 result in additive improvements at active off-target sites at G-C rich therapeutic targets. Fig.5A is a bar chart showing quantification of editing efficiencies at 16 HPS1 off-target sites (identified via Guide- Tag analysis and amplified with rhAmpSeq technology) by HiFiCas9-standard unmodified RNPs (left most bar for each grouping) or HiFiCas9-inosine modified RNPs (4-inosine (4i) (second bar for each grouping) or 6-inosine (6i) (third bar for each grouping) in patient-derived HPS1 B- LCLs, according to an aspect of this disclosure. Untreated sample (background) is denoted by the by the right most bar for each grouping. Sequences of four off-target sites (OT2, OT5, OT6,OT16) are depicted at the top. Fig.5B is a bar chart showing relative specificity ratios (on-target HiFiCas9 editing / off-target HiFiCas9 editing) of inosine modified guide RNAs at each of the 16 off-target site normalized to unmodified guide RNA based on data from Fig.1C and Fig.2A, according to an aspect of this disclosure. For each grouping, bars are shown in the order presented in the legend. Fig.5C is a bar chart showing quantification of editing efficiencies at three previously identified active off-target sites (OT1, OT2, OT3 sequences depicted above plot) by HiFiCas9-standard unmodified RNPs or HiFiCas9-inosine modified guides in HEK293Ts, according to an aspect of this disclosure. For each grouping, bars are shown in the order presented in the legend. Fig.5D is a bar chart showing relative specificity ratios (on-target editing / off-target editing) of inosine modified guide RNAs at each of the 3 off-target site normalized to unmodified guide RNA based on data from Fig.1C and Fig.2C, according to an aspect of the disclosure. For each grouping, bars are shown in the order presented in the legend. Editing levels are quantified from deep-sequencing via CRISPResoo2 pipeline. n=3 independent replicates data presented as the mean (SD). P-values calculated via Levene’s test and adjusted using Hochberg method where “*” p-value <0.05, “**” p-value<0.01, “***” p-value<0.001.

[0036] Fig.6A–D: HBB inosine modified RNPs display improved in vitro cleavage rate over unmodified RNPs. Fig.6A is a plot depicting percent substrate cleaved over time (seconds) with WTCas9 RNPs with HBB ON-target substrate, according to an aspect of this disclosure. Fig.6B is a plot depicting percent substrate cleaved over time (seconds) with WTCas9 RNPs with HBB off-target site 1 (OT1) substrate, according to an aspect of this disclosure. Fig.6C is a plot depicting percent substrate cleaved over time (seconds) with HiFiCas9 RNPs with ON-target substrate, according to an aspect of this disclosure. Fig.6D is a plot depicting percent substrate cleaved over time (seconds) with HiFiCas9 RNPs with HBB off-target site 1 (OT1) substrate, according to an aspect of this disclosure. Bands were quantified via Fiji ImageJ. Data presented as s.e.m, n=2

[0037] Figs.7A–C: Inosine substituted crRNA:tracrRNA guide RNAs reproduce reduced off-target edits like single-guide RNA format. Fig.7A is a bar graph showing HPS1 on-target editing efficiencies as quantified in Fig.1C, according to an aspect of this disclosure. Percentage of 16 bp precise deletions (lower darker segment of each bar) out of total editing alleles at HPS1 associated 16bp duplication by WTCas9 / HiFiCas9 programme by standard unmodified crRNA:tracRNAs or inosine modified crRNA:tracRNAs. Fig.7B is a bar chart showingquantification of editing efficiencies at 16 HPS1 off-target sites (identified via Guide-Tag analysis and amplified with rhAmpSeq technology) by WTCas9 programmed with either standard unmodified crRNA:tracrRNA (left most bar for each grouping) or inosine modified crRNA:tracrRNA (4-inosine (4i) (second bar for each grouping) or 6-inosine (6i) (third bar for each grouping) in patient-derived HPS1 B-LCLs, according to an aspect of this disclosure. Untreated sample (background) is denoted by the by the right most bar for each grouping. Fig. 7C is a bar chart showing quantification of editing efficiencies at 16 HPS1 off-target sites (identified via Guide-Tag analysis and amplified with rhAmpSeq technology) by HiFiCas9 programmed with either standard unmodified crRNA:tracrRNA (second bar for each grouping) or inosine modified crRNA:tracrRNA (4-inosine (4i) (third bar for each grouping) or 6-inosine (6i) (right most bar for each grouping) in patient-derived HPS1 B-LCLs, according to an aspect of this disclosure. Untreated sample (background) is denoted by the left most bar of each grouping. Editing levels are quantified from deep-sequencing via CRISPResoo2 pipeline. Independent replicates data presented as the mean (SD).

[0038] Figs.8A–H: WTCas9 and HiFiCas9 RNP titrations with HBB unmodified and inosine modified guide RNAs in CD34+ HSCPs. Fig.8A is a bar graph showing quantification of editing efficiencies by WTCas9-standard unmodified RNPs or WTCas9-inosine RNPs at five increasing RNP concentrations (5µM-50µM, bars for each grouping shown in increasing order of concentration) with constant 3x excess guide RNA molar ratio in healthy human CD34+ stem and progenitor cells (HSPCs) at HBB ON-target site, according to an aspect of this disclosure. Fig.8B is a bar graph showing quantification of editing efficiencies by WTCas9-standard unmodified RNPs or WTCas9-inosine RNPs at five increasing RNP concentrations (5µM-50µM, bars for each grouping shown in increasing order of concentration) with constant 3x excess guide RNA molar ratio in healthy human CD34+ stem and progenitor cells (HSPCs) at HBB off-target site 1 (OT1), according to an aspect of this disclosure. Fig.8C is a bar graph showing quantification of editing efficiencies by WTCas9-standard unmodified RNPs or WTCas9-inosine RNPs at five increasing RNP concentrations (5µM-50µM, bars for each grouping shown in increasing order of concentration) with constant 3x excess guide RNA molar ratio in healthy human CD34+ stem and progenitor cells (HSPCs) at HBB off-target site 2 (OT2), according to an aspect of this disclosure. Fig 8D is a bar graph showing quantification of editing efficiencies by WTCas9-standard unmodified RNPs or WTCas9-inosine RNPs at five increasing RNPconcentrations (5µM-50µM, bars for each grouping shown in increasing order of concentration) with constant 3x excess guide RNA molar ratio in healthy human CD34+ stem and progenitor cells (HSPCs) at HBB off-target site 3 (OT3), according to an aspect of this disclosure. Fig.8E is a bar graph showing quantification of editing efficiencies by HiFiCas9-standard unmodified RNPs or HiFiCas9-inosine RNPs at five increasing RNP concentrations (5µM-50µM, bars for each grouping shown in increasing order of concentration) with constant 3x excess guide RNA molar ratio in healthy human CD34+ stem and progenitor cells (HSPCs) at HBB ON-target site, according to an aspect of this disclosure. Fig.8F is a bar graph showing quantification of editing efficiencies by HiFiCas9-standard unmodified RNPs or HiFiCas9-inosine RNPs at five increasing RNP concentrations (5µM-50µM, bars for each grouping shown in increasing order of concentration) with constant 3x excess guide RNA molar ratio in healthy human CD34+ stem and progenitor cells (HSPCs) at HBB off-target site 1 (OT1), according to an aspect of this disclosure. Fig.8G is a bar graph showing quantification of editing efficiencies by HiFiCas9- standard unmodified RNPs or HiFiCas9-inosine RNPs at five increasing RNP concentrations (5µM-50µM, bars for each grouping shown in increasing order of concentration) with constant 3x excess guide RNA molar ratio in healthy human CD34+ stem and progenitor cells (HSPCs) at HBB off-target site 2 (OT2), according to an aspect of this disclosure. Fig.8H is a bar graph showing quantification of editing efficiencies by HiFiCas9-standard unmodified RNPs or HiFiCas9-inosine RNPs at five increasing RNP concentrations (5µM-50µM, bars for each grouping shown in increasing order of concentration) with constant 3x excess guide RNA molar ratio in healthy human CD34+ stem and progenitor cells (HSPCs) at HBB off-target site 3 (OT3), according to an aspect of this disclosure. Editing levels are quantified from deep- sequencing via CRISPResoo2 pipeline. n=3 independent replicates data presented as the mean (SD).

[0039] Figs.9A–E: HBB inosine RNPs efficiently mitigate off-target editing in CD34+ HSPCs. Fig.9A is a bar graph showing quantification of editing efficiencies at HBB ON-target site by either WTCas9 or HiFiCas9 programmed with the standard unmodified (n=2), 3b-inosine guide RNA (n=4) or 4a-inosine guide RNAs (n=3) in healthy human CD34+ stem and progenitor cells (HSPCs), according to an aspect of this disclosure. For each grouping, bars are shown in the order presented in the legend. Fig.9B is a bar graph showing quantification of editing efficiencies at three active HBB off-target sites (OT1, OT2, OT3) by WTCas9-standardunmodified RNPs (second bar for each grouping) or WTCas9-3b inosine RNPs (third bar for each grouping) or WTCas9-4a inosine RNPs (right most bar for each grouping) in healthy human CD34+ HSPCs, according to an aspect of this disclosure. Untreated sample (background) is denoted by the left most bar for each grouping. Fig.9C is a bar graph showing specificity ratios (on-target WTCas9 editing / off-target WTCas9 editing) for WTCas9 RNPs plotted at each of the 3 off-target sites, according to an aspect of this disclosure. Bars for each grouping represent, in the following order, unmodified, 3b, and 4a, respectively. Fig.9D is a bar graph showing quantification of editing efficiencies at three active HBB off-target sites (OT1, OT2, OT3) by HiFiCas9-standard unmodified RNPs (second bar for each grouping) or HiFiCas9-3b inosine RNPs (third bar for each grouping) or HiFiCas9-4a inosine RNPs (right most bar for each grouping) in healthy human CD34+ HSPCs, according to an aspect of this disclosure. Untreated sample (background) is denoted by the left most bar for each grouping. Fig.9E is a bar graph showing specificity ratios (on-target HiFiCas9 editing / off-target HiFiCas9 editing) for HiFiCas9 RNPs plotted at each of the 3 off-target sites, according to an aspect of this disclosure. Bars for each grouping represent, in the following order, unmodified, 3b, and 4a, respectively. Editing levels are quantified from deep-sequencing via CRISPResoo2 pipeline. Independent replicates data presented as the mean (SD).

[0040] Figs.10A–E: HBB Inosine substituted guide RNAs do not cause additional off- target editing. Fig.10A displays off-target sites identified by GUIDE-Tag performed in HEK293T cells treated with standard HBB unmodified guide RNA and WTCas9, according to an aspect of this disclosure. Table at top depicts top sites identified ranked by UMI reads (>2reads). Manhattan plot (bottom) depicts sites predicted to have high CFD scores (cutoff>0.2 CFD score=dotted line). ON-target site is denoted by the “ON” row in the table (the first non- heading row) and the arrow labeled “ON” in the Manhattan plot. Fig.10A discloses SEQ ID NOS 139-140, 142-155 and 158, respectively, in order of appearance. Fig.10B displays off- target sites identified by GUIDE-Tag performed in HEK293T cells treated with 4a-inosine guide RNA and WTCas9, according to an aspect of this disclosure. Table at top depicts top sites identified ranked by UMI reads (>2reads). Manhattan plot (bottom) depicts sites predicted to have high CFD scores (cutoff>0.2 CFD score=dotted line). ON-target site is denoted by the “ON” row in the table (the first non-heading row) and the arrow labeled “ON” in the Manhattan plot. Fig.10B discloses SEQ ID NOS 139-143 and 157, respectively, in order of appearance.Fig.10C shows Manhattan plots depicting sites predicted to have high CFD scores (cutoff>0.2 CFD score=dotted line) from off-target sites identified by GUIDE-Tag performed in HEK293T cells treated with (3a, 3b, 3c)-inosine guide RNA and WTCas9, according to an aspect of this disclosure. Fig.10D is a bar chart showing quantification of editing efficiencies to validate select five off-target sites (OT4, OT6, OTss13, OTss15, OTss17) in HEK293T cells treated with WTCas9 RNPs, according to an aspect of this disclosure. For each grouping, bars are shown in the order presented in the legend. Fig.10E is a bar chart showing quantification of editing efficiencies to validate select five off-target sites (OT4, OT6, OTss13, OTss15, OTss17) in HEK293T cells treated with HiFiCas9 RNPs, according to an aspect of this disclosure. For each grouping, bars are shown in the order presented in the legend. Editing levels are quantified from deep-sequencing via CRISPResoo2 pipeline. Independent replicates data presented as the mean (SD).

[0041] Figs.11A–B: Analysis of large deletions and translocations by UDiTaS. Fig.11A shows fraction of edits that are the precise HPS116 bp collapse / deletion (second from right most segment of each bar), small indels (<50bp) (third from right most segment of each bar), large indels (>50bp) (fourth from right most segment of each bar), translocations (fifth from right most segment of each bar (percentage indicated on right)) and unedited (right most segment of each bar) observed in HEK293T-HPS1 cells treated with WTCas9-standard unmodified RNPs (below dotted line), and HiFiCas9-standard unmodified RNPs (above dotted line), according to an aspect of this disclosure. Fig.11B shows fraction of edits that are the precise HPS116 bp collapse / deletion (second from right most segment of each bar), small indels (<50bp) (third from right most segment of each bar), large indels (>50bp) (fourth from right most segment of each bar), translocations (fifth from right most segment of each bar (percentage indicated on right)) and unedited (right most segment of each bar) observed in HPS1 B-LCLs WTCas9-standard unmodified RNPs (bottom) and WTCas9-inosine modified RNPs (4i (middle) and 6i (top)), according to an aspect of this disclosure.5p and 3p labels represent locus forward or reverse reads. Detailed Description of Specific Embodiments

[0042] Definitions. As used in this description and the accompanying claims, the following terms shall have the meanings indicated, unless the context otherwise requires:

[0043] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: 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 ascribed to them unless specified otherwise.

[0044] The terms “a” and “an” and “the” and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0045] “Nucleobase” or “base,” as used herein, means a group of atoms that can be linked to a sugar moiety to create a nucleoside that is capable of incorporation into a nucleic acid molecule as a nucleotide, and wherein the group of atoms is capable of hydrogen bonding with a complementary nucleobase. Nucleobases may be naturally occurring or may be modified. “Nucleobase” and “base” are used interchangeably herein.

[0046] As used herein, a “canonical base” means a base selected from the group consisting of adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U).

[0047] As used herein, a “non-canonical base” means a base that is not a canonical base.

[0048] As used herein, “base substitution” means replacing (i.e., substituting) a canonical (A, G, C, T, U) base of a nucleotide of a guide RNA spacer sequence with a different, non- canonical, base. For example, guanine may be substituted with hypoxanthine. As used herein, such a substitution may also be referred to using the corresponding names of nucleosides. For example, a guanine to hypoxanthine substitution means the same as a guanosine to inosine substitution because the base of the nucleoside guanosine is guanine and the base of thenucleoside inosine is hypoxanthine. “Guanine to inosine,” and the like, is sometimes also used herein to refer to a guanine to hypoxanthine / guanosine to inosine substitution.

[0049] As disclosed herein, a spacer sequence base substitution that reduces hydrogen bonding potential of the spacer sequence with a target sequence means substituting a canonical base of the spacer sequence with a substitute base, wherein the substitute base is able to pair (form at least one hydrogen bond) with the unsubstituted base’s complementary base, but with reduced free energy (a weaker pairing) compared to the unsubstituted base when paired with its complementary base. With respect to hypoxanthine base substitutions, although hypoxanthine can form wobble base pairs with uracil, adenine, and cytosine, the present disclosure contemplates substituting only guanine with hypoxanthine so as to pair with cytosine.

[0050] A spacer sequence may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 base substitutions selected to reduce hydrogen bonding potential of the spacer sequence with a target sequence. A spacer sequence may have 2–10, 2–8, 2–6, 4–8, or 4–6 base substitutions selected to reduce hydrogen bonding potential of the spacer sequence with a target sequence

[0051] In accordance with aspects of the present disclosure, the following base substitutions may be used to reduce hydrogen bonding potential of a spacer sequence with a target sequence.

[0052] Table 1: Base substitutions that reduce hydrogen bonding potential of a spacer sequence with a target sequence Canonical Base of Substituted Base Complementary Weaker Pairing S r S n B f T r t w / C m l m nt rbonding efficiency with guanine wn, , y g g p spacer sequence with a target sequence.

[0054] The term “Cas9” or “Cas9 nuclease” refers to a nucleic acid programmable nuclease (an RNA-guided nuclease) comprising a Cas9 protein, or a fragment thereof (e.g., a protein comprising an active or inactive DNA cleavage domain of Cas9, and / or the gRNA binding domain of Cas9). A “Cas9 domain” as used herein, is a protein fragment comprising an active or inactive cleavage domain of Cas9 and / or the gRNA binding domain of Cas9. A “Cas9 protein” is a full length Cas9 protein. A Cas9 nuclease is also referred to sometimes as a casn1 nuclease or a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-associated nuclease. CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and a Cas9 domain. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently, Cas9 / crRNA / tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer. The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3′-5′ exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNAs. However, single guide RNAs (“sgRNA”, or simply “gRNA”) can be engineered to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science 337:816-821(2012), the entire contents of which are hereby incorporated by reference. Cas9 recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus non-self. Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g., “Complete genome sequence of an M1 strain of Streptococcus pyogenes.” Ferretti et al., J. J., McShan W. M., Ajdic D. J., Savic D. J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A. N., Kenton S., Lai H. S., Lin S. P., Qian Y., Jia H. G., Najar F. Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S. W., Roe B. A., McLaughlin R. E., Proc. Natl. Acad. Sci. U.S.A.98:4658-4663(2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor Rnase III.” Deltcheva E., Chylinski K., Sharma C. M., Gonzales K., Chao Y., Pirzada Z. A., Eckert M. R., Vogel J., Charpentier E., Nature 471:602-607(2011); and “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science 337:816- 821(2012), 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, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on U.S. Patent No.11,447,770, which is hereby incorporated by reference for its disclosure of Cas9 nucleases, and such Cas9 nucleases and Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737, the entire contents of which are incorporated herein by reference. In some embodiments, a Cas9 nuclease comprises one or more mutations that partially impair or inactivate the DNA cleavage domain. U.S. Patent No.11,447,770 is hereby incorporated by reference for its disclosure of Cas9.

[0055] In some embodiments, Cas9 refers to Cas9 protein from: Streptococcus pyogenes (NCBI Ref: NC_002737.2) (SpCas9); Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquisl (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1); Listeria innocua (NCBI Ref:NP_472073.1); Campylobacter jejuni (NCBI Ref: YP_002344900.1); or Neisseria. meningitidis (NCBI Ref: YP_002342100.1), Staphylococcus aureus (NCBI Ref: AYD60528.1). In some embodiments, Cas9 refers to a SpCas9 variant with a PAM requirement that is not NGG (where N is A, C, G, or T), including, but not limited to: (1) SpCas9-VQR(NGA) (Nature, 2015, 523, 481-5, PMID: 26098369), (2) SpCas9-NG(NG) (Science, 2018, 361,1259-1262, PMID: 30166441), (3) SpCas9-NRNH(NRNH) (Nat Biotechnol, 2020, 38, 471-481, PMID: 32042170), (4) SpG(NG) or (5) SpRY(NR>NY) (Science, 2020, 368, 290-296, PMID: 32217751). In some embodiments, Cas9 refers to a high fidelity SpCas9 variant, including, but not limited to: (1) eSpCas9 (Science.2016, 351, 84–88, PMID: 26628643), (2) SpCas9-HF1 (Nature, 2016, 529, 490-5, PMID: 26735016), (3) HypaSpCas9 (Nature.2017, 550, 407-410, PMID: 28931002), (4) HeFSpCas9 (Genome Biol.2017, 18, 190, PMID: 28985763), (5) HiFi Cas9 (Nat Med.2018, 24, 1216–1224, PMID: 30082871), (6) Sniper (Nat Commun.2018, 9, 3048, PMID: 30082838), (7) evoSpCas9 (Nat Biotechnol.2018, 36, 265-271. PMID: 29431739), (8) Blackjack, eSpCas9- plus, and SpCas9-HF1-plus (Nat Commun.2020, 11, 1223, PMID: 32144253), or (9) SuperFi- Cas9 (Nature.2022, 603, 343-347. PMID: 35236982).

[0056] In some embodiments, a nucleic acid programmable nuclease is selected from the group consisting of Cas9, HiFiCas9, eSpCas9, SpCas9-HF1, Hypa-SpCas9, SuperFi-Cas9, Sniper-Cas9, evoCas9, and Cas12.

[0057] The term “nickase,” “Cas9 nickase,” and “nCas9” refers to a Cas9 with one of its two nuclease domains inactivated. This enzyme is capable of cleaving only one strand of a target DNA. U.S. Patent No.11,447,770 is hereby incorporated by reference for its disclosure of Cas9 nickases.

[0058] As used herein, the terms “DNA synthesis template,” “reverse transcriptase template,” and “RTT” are used interchangeably to refer to the region or portion of the extension arm of a pegRNA that is utilized as a template strand by a polymerase, such as reverse transcriptase, of a prime editor to encode a 3′ replacement DNA flap that contains a desired edit and which then, through the mechanism of prime editing, replaces the corresponding endogenous strand of DNA at the target site. US Patent No.11,447,770 is hereby incorporated by reference for its disclosure of DNA synthesis templates, the mechanisms of prime editing, components of prime editing systems, and methods of using prime editors and prime editing systems.

[0059] As used herein, the terms “upstream” and “downstream” are terms of relativity that define the linear position of at least two elements located in a nucleic acid molecule (whether single or double-stranded) that is orientated in a 5′-to-3′ direction. In particular, a first element is upstream of a second element in a nucleic acid molecule where the first element is positioned somewhere that is 5′ to the second element. For example, a SNP is upstream of a Cas9-induced nick site if the SNP is on the 5′ side of the nick site. Conversely, a first element is downstream of a second element in a nucleic acid molecule where the first element is positioned somewhere that is 3′ to the second element. For example, a SNP is downstream of a Cas9- induced nick site if the SNP is on the 3′ side of the nick site. The nucleic acid molecule can be a DNA (double or single stranded). RNA (double or single stranded), or a hybrid of DNA and RNA. The analysis is the same for single strand nucleic acid molecule and a double strand molecule since the terms upstream and downstream are in reference to only a single strand of a nucleic acid molecule, except that one needs to select which strand of the double stranded molecule is being considered. Often, the strand of a double stranded DNA which can be used to determine the positional relativity of at least two elements is the “sense” or “coding” strand. In genetics, a “sense” strand is the segment within double-stranded DNA that runs from 5′ to 3′, and which is complementary to the antisense strand of DNA, or template strand, which runs from 3′ to 5′. Thus, as an example, a SNP nucleobase is “downstream” of a promoter sequence in a genomic DNA (which is double-stranded) if the SNP nucleobase is on the 3′ side of the promoter on the sense or coding strand.

[0060] The term “extension arm” refers to a nucleotide sequence component of a pegRNA which provides several functions, including a primer binding site (PBS) and a DNA synthesis template (also referred to as an “reverse transcriptase template” or “RTT”) for reverse transcriptase. In some embodiments the extension arm is located at the 3′ end of the pegRNA. In various embodiments, the extension arm comprises the following components in a 5′ to 3′ direction: the DNA synthesis template and the primer binding site. Since polymerization activity of the reverse transcriptase is in the 5′ to 3′ direction, the preferred arrangement of the DNA synthesis template and primer binding site is in the 5′ to 3′ direction such that the reverse transcriptase, once primed by an annealed primer sequence, polymerases a single strand of DNA using the DNA synthesis template as a complementary template strand.

[0061] The extension arm may also be described as comprising generally two regions: a primer binding site (PBS) and a DNA synthesis template, as known in the art. The primer binding site binds to a primer sequence that is formed from the endogenous DNA strand of the target site when it becomes nicked by the prime editor complex, thereby exposing a 3′ end on the endogenous nicked strand. The binding of the primer sequence to the primer binding site on the extension arm of the pegRNA creates a duplex region with an exposed 3′ end (i.e., the 3′ of the primer sequence), which then provides a substrate for reverse transcriptase to begin polymerizing a single strand of DNA from the exposed 3′ end along the length of the DNA synthesis template. The sequence of the single strand DNA product is the complement of the DNA synthesis template. Polymerization continues towards the 5′ of the DNA synthesis template (or extension arm) until polymerization terminates. Thus, the DNA synthesis template represents the portion of the extension arm that is encoded into a single strand DNA product (i.e., the 3′ single strand DNA flap containing the desired genetic edit information) by the polymerase of the prime editor complex and which ultimately replaces the corresponding endogenous DNA strand of the target site that sits immediate downstream of the PE-induced nick site. Without being bound by theory, polymerization of the DNA synthesis template continues towards the 5′ end of the extension arm until a termination event. Polymerization may terminate in a variety of ways, including, but not limited to (a) reaching a 5′ terminus of the pegRNA (e.g., in the case of the 5′ extension arm wherein the DNA polymerase simply runs out of template), (b) reaching an impassable RNA secondary structure (e.g., hairpin or stem / loop), or (c) reaching a replication termination signal, e.g., a specific nucleotide sequence that blocks or inhibits the polymerase, or a nucleic acid topological signal, such as, supercoiled DNA or RNA. US Patent No.11,447,770 is hereby incorporated by reference for its disclosure of extension arms.

[0062] The term “fusion protein” as used herein refers to a hybrid polypeptide which comprises protein domains from at least two different proteins. One protein may be located at the amino-terminal (N-terminal) portion of the fusion protein or at the carboxy-terminal (C-terminal) protein thus forming an “amino-terminal fusion protein” or a “carboxy-terminal fusion protein,” respectively. A protein may comprise different domains, for example, a nucleic acid binding domain (e.g., the gRNA binding domain of Cas9 that directs the binding of the protein to a target site) and a nucleic acid cleavage domain or a catalytic domain of a nucleic-acid editing protein.For example, a fusion protein may comprise a Cas9 nickase fused to a reverse transcriptase. Such a fusion protein is referred to herein as a “prime editor protein.”.

[0063] As used herein, the term “guide RNA” (gRNA) is a particular type of guide nucleic acid which is mostly commonly associated with a Cas protein of a CRISPR-Cas9 and which associates with Cas9, directing the Cas9 protein to a specific sequence in a DNA molecule that includes complementarity a spacer sequence of the guide RNA. A gRNA spacer sequence is approximately 20 nts in length and binds to the protospacer (target sequence) of the target DNA Guide RNA also embraces the equivalent guide nucleic acid molecules that associate with Cas9 equivalents, homologs, orthologs, or paralogs, whether naturally occurring or non-naturally occurring (e.g., engineered or recombinant), and which otherwise program the Cas9 equivalent to localize to a specific target nucleotide sequence. U.S. Patent No.8,697,359 for its disclosure of Cas9 genome editing systems and guide RNAs for use in such systems.

[0064] As used herein, a spacer sequence having “a region of complementarity” to a target sequence means a spacer sequence having a region of at least 15 contiguous nucleotides that are at least 90% complementarity to a contiguous sequence of the target sequence. In some aspects, the region of complementarity is 17–24 contiguous nucleotides in length. In other aspects, the region of complementarity is 20 contiguous nucleotides in length. In some aspects, the region of complementarity is 95% complementary to a contiguous sequence of the target sequence. In other aspects, the region of complementarity is 100% complementary to a contiguous sequence of the target sequence.

[0065] Aspects of the present disclosure also contemplate that a spacer sequence of a guide RNA suitable for use with various nucleic acid programmable systems (comprising programmable nucleic acid binding proteins) known in the art now or in the future, including, but not limited to, Cas12 systems (Cas 12 is a programmable nucleic acid binding protein), base editor systems, Cas9 systems, and other prime editor systems, may comprise a base substitution to reduce hydrogen bonding potential of the spacer sequence of the guide RNA with a corresponding target sequence.

[0066] “Prime editing guide RNA” (or “pegRNA”) is a guide RNA that has been modified and designed for the prime editing methods and systems disclosed herein. A pegRNA associates with a prime editor protein. U.S. Patent No.11,447,770 is hereby incorporated by reference for its disclosure of guide RNA and pegRNA.

[0067] A pegRNA may comprise various structural elements that include, but are not limited to:

[0068] Spacer sequence—the sequence in the pegRNA (in some embodiments, having about 20 nts in length) which binds to the protospacer (target sequence) in the target DNA.

[0069] gRNA core (or gRNA scaffold or backbone sequence)—refers to the sequence within the gRNA that is responsible for Cas9 binding, it does not include a spacer sequence that is used to guide Cas9 to target DNA.

[0070] Extension arm—a single strand extension at the 3′ end of the pegRNA which comprises a primer binding sequence and a DNA synthesis template sequence that encodes via a polymerase (e.g., a reverse transcriptase) a single stranded DNA flap containing the genetic change of interest, which then integrates into the endogenous DNA by replacing the corresponding endogenous strand, thereby installing the desired genetic change.

[0071] Transcription terminator—the pegRNA may comprise a transcriptional termination sequence at the 3′ of the molecule.

[0072] The term “homology arm” refers to a portion of the extension arm that encodes a portion of the resulting reverse transcriptase-encoded single strand DNA flap that is to be integrated into the target DNA site by replacing the endogenous strand. The portion of the single strand DNA flap encoded by the homology arm is complementary to the non-edited strand of the target DNA sequence, which facilitates the displacement of the endogenous strand and annealing of the single strand DNA flap in its place, thereby installing the edit. The homology arm is part of the DNA synthesis template since it is by definition introduced into the target DNA by the polymerase of the prime editors described herein. U.S. Patent No.11,447,770 is hereby incorporated by reference for its disclosure of homology arms.

[0073] As used herein, the term “nucleic acid programmable DNA binding protein,” “programmable nucleic acid binding protein,” or “napDNAbp,” of which Cas9 is an example, refers to a protein which use RNA:DNA hybridization to target and bind to specific sequences in a DNA molecule. Each napDNAbp is associated with at least one guide nucleic acid (e.g., guide RNA), which localizes the napDNAbp to a DNA sequence that comprises a DNA strand (i.e., a target strand) that is complementary to the guide nucleic acid, or a portion thereof (e.g., a spacer sequence of a guide RNA). In other words, the guide nucleic-acid “programs” the napDNAbp(e.g., Cas9 or equivalent) to localize and bind to a complementary sequence. U.S. Patent No. 11,447,770 is hereby incorporated by reference for its disclosure of napDNAbps.

[0074] “Off-target activity,” and the like, means activity (e.g., nuclease activity, nickase activity, base editing activity, prime editing activity, etc.) of a programmable nucleic acid binding protein system (for example, programmable genomic editing systems, programmable prime editing systems, and programmable base editing systems) at a genomic site other than the intended target DNA site having an intended target sequence. Off-target activity typically occurs at near-cognate sequences, i.e., sequences that are similar, but not identical, to the target sequence.

[0075] A “nucleic acid programmable nuclease” means a napDNAbp having nuclease activity.

[0076] The term “nuclear localization sequence,” “nuclear localization signal,” “nuclear localization signal sequence,” or “NLS” refers to an amino acid sequence that promotes import of a protein into the cell nucleus, for example, by nuclear transport. Nuclear localization sequences are known in the art and would be apparent to the skilled artisan. For example, NLS sequences are described in Plank et al., international PCT application, PCT / EP2000 / 011690, filed Nov.23, 2000, published as WO / 2001 / 038547 on May 31, 2001, the contents of which are incorporated herein by reference for its disclosure of exemplary nuclear localization sequences.

[0077] As used herein, the terms “prime editing guide RNA” or “pegRNA” refers to a specialized form of a guide RNA that has been modified to include one or more additional sequences for implementing the prime editing methods and systems described herein. The additional sequences comprise (i) a “DNA synthesis template” which encodes (copied by the polymerase, e.g., reverse transcriptase, of the prime editor) a single-stranded DNA which, in turn, has been designed to be (a) homologous with the endogenous target DNA to be edited, and (b) which comprises at least one desired nucleotide change (e.g., a transition, a transversion, a deletion, or an insertion) to be introduced or integrated into the endogenous target DNA; and (ii) a “primer binding site.” As used herein the “primer binding site” comprises a sequence that hybridizes to a single-strand DNA sequence having a 3′ end generated from the nicked DNA of the R-loop. U.S. Patent No.11,447,770 is hereby incorporated by reference for its disclosure of pegRNA.

[0078] As used herein, the term “polymerase” refers to an enzyme that synthesizes a nucleotide strand and which may be used in connection with the prime editor systems described herein. Reverse transcriptase is a polymerase. U.S. Patent No.11,447,770 is hereby incorporated by reference for its disclosure of polymerases.

[0079] As used herein, the term “prime editing” refers to an approach for gene editing using napDNAbps (e.g., a Cas9 nickase), a polymerase (e.g., a reverse transcriptase), and specialized guide RNAs that include a DNA synthesis template for encoding desired new genetic information (or deleting genetic information) that is then incorporated into a target DNA sequence. U.S. Patent No.11,447,770 is hereby incorporated by reference for its disclosure of prime editing.

[0080] The term “prime editor protein” refers to fusion constructs comprising a napDNAbp (e.g., Cas9 nickase) and a polymerase (e.g., reverse transcriptase) and is capable of carrying out prime editing on a target nucleotide sequence in the presence of a pegRNA. The term “prime editor” may refer to the fusion protein or to the fusion protein complexed with a pegRNA, and / or further complexed with a second-strand nicking sgRNA. U.S. Patent No. 11,447,770 is hereby incorporated by reference for its disclosure of prime editors and prime editor proteins.

[0081] The terms “primer binding site,” “primer binding sequence,” and “PBS” are used interchangeably to refer to the nucleotide sequence located on a pegRNA as component of the extension arm (typically at the 3′ end of the extension arm) and serves to bind to the primer sequence that is formed after Cas9 nicking of the target site sequence by the prime editor. U.S. Patent No.11,447,770 is hereby incorporated by reference for its disclosure of primer binding sites.

[0082] The term “reverse transcriptase” describes a class of polymerases characterized as RNA-dependent DNA polymerases. All known reverse transcriptases require a primer to synthesize a DNA transcript from an RNA template. Avian myoblastosis virus (AMV) reverse transcriptase was the first widely used RNA-dependent DNA polymerase (Verma, Biochim. Biophys. Acta 473:1 (1977)). The enzyme has 5′-3′ RNA-directed DNA polymerase activity, 5′- 3′ DNA-directed DNA polymerase activity, and RNase H activity. RNase H is a processive 5′ and 3′ ribonuclease specific for the RNA strand for RNA-DNA hybrids (Perbal, A Practical Guide to Molecular Cloning, New York: Wiley & Sons (1984)). Another reverse transcriptasewhich is used extensively in molecular biology is reverse transcriptase originating from Moloney murine leukemia virus (M-MLV). See, e.g., Gerard, G. R., DNA 5:271-279 (1986) and Kotewicz, M. L., et al., Gene 35:249-258 (1985). M-MLV reverse transcriptase substantially lacking in RNase H activity has also been described. See, e.g., U.S. Pat. No.5,244,797. U.S. Patent No.11,447,770 is hereby incorporated by reference for its disclosure of reverse transcriptases.

[0083] The term “target site” refers to a sequence within a nucleic acid molecule that is edited by a prime editor (PE) disclosed herein. The target site further refers to the target sequence within a nucleic acid molecule to which a complex of the prime editor (PE) and gRNA binds.

[0084] As used herein the term “variant” should be taken to mean the exhibition of qualities that have a pattern that deviates from what occurs in nature, e.g., a variant Cas9 is a Cas9 comprising one or more changes in amino acid residues as compared to a wild type Cas9 amino acid sequence. The term “variant” encompasses homologous proteins having at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% percent identity with a reference sequence and having the same or substantially the same functional activity or activities as the reference sequence. The term also encompasses mutants, truncations, or domains of a reference sequence, and which display the same or substantially the same functional activity or activities as the reference sequence.

[0085] As used herein, the term “3′ replacement DNA flap” or simply, “replacement DNA flap,” refers to the strand of DNA that is synthesized by the prime editor and which is encoded by the extension arm of the prime editor pegRNA. More in particular, the 3′ replacement DNA flap is encoded by the DNA synthesis template of the pegRNA. The 3′ replacement DNA flap comprises the same sequence as the 5′ endogenous DNA flap except that it also contains the edited sequence (e.g., single nucleotide change). The 3′ replacement DNA flap anneals to the target DNA, displacing or replacing a 5′ endogenous DNA flap (which can be excised, for example, by a 5′ flap endonuclease, such as FEN1 or EXO1) and then is ligated to join the 3′ end of the 3′ replacement DNA flap to the exposed 5′ hydoxyl end of endogenous DNA (exposed after excision of the 5′ endogenous DNA flap, thereby reforming a phosophodiester bond and installing the 3′ replacement DNA flap to form a heteroduplex DNA containing one edited strand and one unedited strand. DNA repair processes resolve theheteroduplex by copying the information in the edited strand to the complementary strand permanently installs the edit into the DNA. This resolution process can be driven further to completion by nicking the unedited strand, i.e., by way of “second-strand nicking,” U.S. Patent No.11,447,770 is hereby incorporated by reference for its disclosure of 3′ replacement DNA flaps, 5′ endogenous DNA flaps, and 5′ endogenous DNA flap removal, and second-strand nicking.

[0086] The term “base editor (BE)” refers to an agent comprising a polypeptide that is capable of making a modification to a base (e.g., A, T, C, G, or U) within a nucleic acid sequence (e.g., DNA or RNA). In some embodiments, the base editor is capable of deaminating a base within a nucleic acid. In some embodiments, the base editor is capable of deaminating a base within a DNA molecule. In some embodiments, the base editor is a “cytosine base editor” (CBE). In some embodiments, a “cytosine base editor” is capable of deaminating a cytosine (C) in DNA. In some embodiments, the base editor is a protein (e.g., a fusion protein) comprising a nucleic acid (guide RNA) programmable DNA binding protein (napDNAbp) such as a Cas9 domain, fused to a cytidine deaminase. In some embodiments, the base editor is a protein (e.g., a fusion protein) comprising a nucleic acid (guide RNA) programmable DNA binding protein (napDNAbp) such as a Cas9 domain, fused to an adenine deaminase. In some embodiments, the base editor comprises a napDNAbp, a cytidine deaminase, and a uracil glycosylase inhibitor (UGI). U.S. Patent No.11,542,496 is hereby incorporated by referenced for its disclosure of base editors and base editing, including cytosine base editors and cytosine base editing. U.S. Publication No. US2023 / 0235309 is hereby incorporated by referenced for its disclosure of base editors and base editing, including adenine base editors and adenine base editing.

[0087] The term “subject,” as used herein, refers to an individual organism, for example, an individual mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, a goat, a cattle, a cat, or a dog. In some embodiments, the subject is a vertebrate, an amphibian, a reptile, a fish, an insect, a fly, or a nematode. In some embodiments, the subject is a research animal. In some embodiments, the subject is genetically engineered, e.g., a genetically engineered non- human subject. The subject may be of either sex and at any stage of development.

[0088] “Melting temperature” (“Tm”) is the temperature at which one half of the strands of a population of duplexed nucleic acid will dissociate to become single-stranded. In some embodiments, the duplexed nucleic acid is a RNA:DNA duplex. Melting temperature is a function of both the sequence and the length of the duplex. Methods of calculating Tm are well known in the art. See, e.g., Dumousseau et al. (2012) BMC Bioinformatics, 13, 101, hereby incorporated by reference for its disclosure of melting temperature calculation.

[0089] By “hybridizable” (and derivatives thereof) or “complementary” it is meant that a nucleic acid (e.g. RNA) comprises a sequence of nucleotides that enables it to non-covalently bind, i.e. form Watson-Crick base pairs and / or G / U base pairs, “anneal”, or “hybridize,” to another nucleic acid in a sequence-specific, antiparallel, manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength. As is known in the art, standard Watson-Crick base- pairing includes; adenine (A) pairing with thymidine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C) [DNA, RNA]. In addition, it is also known in the art that for hybridization between two RNA molecules (e.g., dsRNA), guanine (G) base pairs with uracil (U). For example, G / U base-pairing is partially responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anti-codon base-pairing with codons in mRNA. In the context of this disclosure, a guanine (G) of a protein-binding segment (dsRNA duplex) of a subject DNA-targeting RNA molecule is considered complementary to a uracil (U), and vice versa. As such, when a G / U base-pair can be made at a given nucleotide position a protein-binding segment (dsRNA duplex) of a subject DNA-targeting RNA molecule, the position is not considered to be non-complementary, but is instead considered to be complementary. U.S. Publication No. US 2019 / 0010520 is hereby incorporated by reference for its disclosure of complementarity (including “complementary”) and hybridization (including “hybridizable”).

[0090] CRISPR-Cas9 Genome Editing

[0091] For therapeutic applications of CRISPR-Cas9, off-target cleavage poses a risk. Unintended breaks in the genome can lead to aberrant genomic modifications such as loss-of- function mutations, large deletions, translocations and loss of chromosomal arms[19-23]. Consequently, substantial efforts have been made to improve and optimize the specificity of the CRISPR-Cas9 system over the past decade. A variety of high-fidelity Cas9 variants withimproved nuclease specificities have been described[4, 24-26]. Many structurally-guided engineered Cas9 variants introduce substitutions that attenuate residues that make non-specific contacts with the phosphodiester backbone[27-30]. Cell-based selection methods employing directed evolution have isolated improved Cas9 variants from randomized libraries carrying mutations in the REC domains in yeast

[0031] and bacteria

[0032] . Despite improvement in specificity, many of these variants come at the cost of reduced on-target editing efficiency[33-35]. Although recent efforts have described new variants that overcome the on-target activity and off-target specificity trade-off, such as HiFi Cas9, off-target editing at some near cognate sequences remains an issue for some target sequences[30, 32, 35]. Thus, there is a continued need for developing strategies that will improve on-target accuracy.

[0092] Careful target site selection and modifications to the guide RNA have also proven to be impactful in decreasing off-target editing events. Several groups have developed general design criteria for selecting guide RNAs that display high activity in complex with Cas9 based on large guide RNA screens in mammalian cells[36-38] and zebrafish embryos

[0039] . Various prediction algorithms trained on these datasets rank guide RNAs based on their predicted activity at the target site and at potential off-target sites[38, 40]. However, the predicted activity of guide RNA at off-target sites typically is a poor predictor of cellular activity. In some cases, the guide RNA targeting window may be restricted at therapeutic target sites, which can limit the choices for minimizing off-target sites with small numbers of mismatches. In addition to optimal target site selection, guide-centric approaches to improving Cas9 specificity have been developed, such as: shortening the region of complementarity between the guide RNA and target DNA

[0041] , chemical modifications at select positions within the sugar and phosphate backbone of the guide RNA[42-46], incorporation of an extra G residue or hairpin structure 5’ to spacer region[47, 48] and substitution of 2’-deoxynucleosides within the spacer sequence of the guide RNA[49, 50]. These improvements typically come at a cost of reduced on-target activity, especially when used in combination with high-fidelity Cas9 variants

[0050] .

[0093] The specificity and versatility of the CRISPR-Cas9 systems are best harnessed when both engineered nuclease and sgRNA can function synergistically to achieve precise control of DNA cleavage. R-loop formation is an important conformational checkpoint for nucleolytic cleavage[51, 52]. The strength of guide RNA and target DNA hybridization impacts the stability of the R-loop structure. Due to the presence of the additional hydrogen bond, G-Cbase pairs are stronger and have a higher melting temperature compared to an A-T base pairs. Spacers capable of forming a large fraction of G-C base pairs likely form stronger RNA:DNA heteroduplexes at near-cognate targets. We hypothesized that replacing guanosines within the spacer region for a base with weaker base pairing ability, such as inosine, would increase the thermodynamic barrier for cleavage and thereby achieve enhanced discrimination of near- cognate sites. Inosines are naturally-occurring nucleosides that differ from guanosines by the absence of the 2-amino group (Fig.1A). First discovered as a component of tRNAs

[0053] , inosines play important roles in purine metabolism and RNA biology

[0054] . Inosine:cytosine (I-C) base pairs are well tolerated and have little impact on the canonical structural features of a duplex, but they reduce the stability of the duplex[55, 56]. Thus, substituting inosines for guanosines within G-C rich guide RNAs could reduce nuclease activity at near-cognate sites.

[0094] Here, we disclose that inosine substituted guide RNAs targeting two therapeutically relevant genes HPS1 and HBB can mitigate editing at highly active off-target sites while maintaining high on-target activity. These inosines substituted guide RNAs can also act synergistically when employed in combination with a high-fidelity variant, HiFi Cas9, while maintaining high on-target activity. We demonstrate inosine guide RNAs designed to the sickle- cell disease-associated locus, HBB, improve nuclease fidelity in healthy human CD34+ HSPCs. We observe robust on-target editing with complete reduction in off-target editing to background levels at the most highly active off-target site (OT1). Importantly, our inosine substituted guide RNAs do not lead to unwanted editing at additional off-target sites. These findings represent a novel method for mitigating off-target editing by attenuating Watson-Crick base pairing potential within the spacer sequence through base substitution, which provides an additional tool that can be combined with high-fidelity Cas9 variants to achieve precision at G-C rich target sites.

[0095] Because many nucleic acid programmable DNA binding proteins comprise Cas9 or a Cas9 domain (or a comparable programmable nuclease, such as Cas12 or a Cas12 domain), the results disclosed herein are broadly applicable to all systems for DNA modification that comprise a nucleic acid programmable DNA binding protein, wherein the nucleic acid is a guide RNA having a spacer sequence configured to facilitate sequence-specific targeting of a genomic DNA sequence.

[0096] Example 1: Increased fidelity of WTCas9 activity when complexed with inosine substituted guide RNAs at HPS1 target site

[0097] Studies describing optimal guide RNA designs correlate well-balanced G-C content (~50%) with ideal on-target cleavage efficiency[36, 57, 58]. Replacing guanosines for a weaker base could likely offset the strong binding energetics of a G-C rich guide RNA which is unfavorable at off-target sites. SpyCas9 programmed with guide RNAs targeting G-C rich target sites (VEGFA site 2 and HEK293 site 4) display editing at a large number of off-target sites[59- 64]. To test this, we selected two therapeutically relevant target genes with high G-C content within their Cas9 target sites to determine the impact of inosine modifications on nuclease activity genome-wide.

[0098] We initially investigated the effects of substituting inosines for guanosines within a G-C rich guide RNA (80%) targeting the HPS1 gene. Hermansky-Pudlak syndrome (HPS) is a heterogeneous group of autosomal recessive disorders typified by hypopigmentation, impaired blood clotting and in many cases pulmonary fibrosis[65-67]. A common loss-of-function mutation in the HPS1 gene caused by a 16bp microduplication has a high carrier frequency (1:21) in the northwestern Puerto Rico region[68, 69]. Previously, we demonstrated this disease allele can be efficiently reverted to the wild-type (WT) sequence by generating a double-strand break near the center of the microduplication, which utilizes cellular DNA repair machinery to collapse the duplication

[0070] . Given the potential therapeutic utility of these genome editing reagents, we performed GUIDE-Tag

[0071] to identify the genome-wide off-target profile of Cas9 nuclease in complex with the HPS1 guide RNA in 2 cell lines: a patient-derived HPS1 B- lymphoblastoid cells and an HEK293T cell line engineered to harbor the pathogenic 16bp duplication. Our analysis identified over 100 potential off-target sites for the HPS1 guide RNA (Table 2). To validate these potential sites for unintended editing, we employed rhAmpSeq[72, 73], a high-throughput amplicon sequencing methodology, to characterize the genome at the top 85 identified sites, as well as an additional 38 computationally predicted off-target sites from CRISPOR

[0074] (Table 3). Fifteen of the 123 sites analyzed displayed statistically significant editing above background levels (Fig.2A). The large number of off-target sites that have been edited with Cas9-unmodified (standard guide RNA with end modifications) HPS1 RNP is not surprising considering the guide RNA is G-C rich. In fact, CRISPOR specificity analysis predicts this guide RNA to share sequence similarity with over 500 putative off-target sites, most of which are likely non-functional (data not shown).

[0099] Table 2: Off-Target Sites Identified from GUIDE-Tag Analysis in HEK293T- HPS1 Cells HEK293T-HPS1 Cell Line - GUIDE-Tag Results Label Target_sequence location UMI S Cas9 HiFiCas9OT46 CAGCAGtGccGGCCCCCAGCTGG (SEQ ID NO: 47) chr4:27025006 50 0 OT47 CtGCAGGGaAGaCCCaCAGCAGG (SEQ ID NO: 48) chr2:109428063 50 0 OT48 atGaAGGGGtGaCCCCCAGCGGG (SEQ ID NO: 49) chr15:25177791 48 0aAaCAGaGGAacCCCCCAGCTGG (SEQ ID NO: 91) chr2:58569267 6 0 agGCtGGGGAGGCCCCCAGgAGG (SEQ ID NO: 92) chr12:108284508 5 0 CAGCAGGGcAGGCaCCCAGCAGG (SEQ ID NO: 60) chr19:49375937 5 0HEK293T-HPS1 cells treated with WTCas9-standard unmodified RNPs or HiFiCas9-standard unmodified RNPs. Top 85 sites (OT1-OT85) chosen to be validated by rhAmpSeq technology.

[0101] Table 3: Compiled Top Off-Target Sites for rhAmp-Seq Panel HEK-HPS1 GUIDE-Tag rhAmpseq Primer Panel Input Target Sequence location name BED Chr start endCctCAGGaGAGaCCCCCAGC chr19:550986 OT1 5509860 chr1 5509860 5509862 CGG (SEQ ID NO: 11) 05 0 5 9 5 4 gtGCAGaGGAGGCtCCCAGC chr1:2502804 OT1 2502804 chr1 2502804 2502806aAGCAGcaGAGcCCCCCAGC chr12:130701 OT3 1307012 chr1 1307012 1.31E+0 AGG (SEQ ID NO: 38) 200 7 00 2 00 8 CAGCAcaGGAGGCCCtCAGt chr15:780686 OT3 7806860 chr1 7806860 7806862 O O OCAGCAGGGcAGGCaCCCAG chr22:189117 OT6 1891175 chr2 1891175 1891177 Merged CAGG (SEQ ID NO: 60) 55 2 5 2 5 4 OT61__O T62AAGCAGAAGAGGCCCCCA Guide- OT8 COT-3 chr1 3860626 3860648 GATGG (SEQ ID NO: 85) tag_Site89 8 7 COT-2AGGCTGGGGAGGCCCCCA Guide- OT1 MOT-12 chr 1536148 1536149 GGAGG (SEQ ID NO: 92) tag_Site96 14 X 85 07 MOT10(UMI)) from GUIDE-Tag from HEK293T-HPS1 knock-in cell line. Includes additional 38 computationally predicted off-target sites from CRISPOR (top sites ranked by CFD score (termed COT1-17) and by MIT score (termed MOT1-21). Compiled list for rhAmp-Seq panel (right).

[0103] The high G-C content of the HPS1 guide RNA provides additional hybridization energy that may allow for stable mismatched RNA:DNA heteroduplex formation at many off- target sites[17, 18]. This encouraged us to consider destabilizing base substitutions (guanine -> inosine) to potentially lower the high hybridization potential energy and thereby reduce off-target editing. Moreover, HPS1 guide RNA is predicted to have a strong propensity to form a hairpin secondary structure which could limit its functionality (Fig.1B). We took advantage of the 6 G- C hydrogen bonds involved in this intramolecular structure to define the position of the inosine substitutions. We designed 2 modified guide RNAs to include 4 inosine (4i) or 6 inosine (6i) at the indicated positions (Fig.1B). Wild-type (WT) SpyCas9 protein complexed with either the unmodified or substituted guide RNA targeting the HPS1 locus was introduced into patient- derived HPS1 B-lymphoblastoid cells and subjected to deep-sequencing following genome editing to quantify indels at the 15 active off-target sites via Illumina sequencing

[0075] . Weobserved that Cas9-inosine RNPs (4i or 6i) had comparable on-target activity to the unmodified guide RNA (Fig.1C). In addition, HPS1 inosine substituted sgRNAs do not change the on-target indel profiles and preserve the 16bp deletion allele, which restores the wild-type sequence, as the most frequent editing outcome (Fig.3). Encouragingly, when tested for activity at the 15 off- target sites, 4i RNP was able to mitigate editing at 11 out of 15 off-target sites (Fig.2A). However, 4i RNP retained activity at off-target (OT) sites OT2, OT5, OT6, and OT16. OT sites contain mismatches at the inosine substituted positions likely leading to reduced impact on R- loop formation for the inosine substituted guide relative to the unmodified guide RNA (Fig.2A). The incorporation of 2 additional inosine substitutions, the last guanosine within the G-quartet and one in the PAM-proximal position (Fig.1B), reduced editing at three of the remaining off- target sites to near background levels.

[0104] The relative specificity ratios, where the specificity ratio is defined as the percent of edited alleles at the on-target over the percent of edited alleles at an off-target site

[0076] , were calculated for Cas9 editing with each modified guide RNA and then was normalized against the specificity ratio for Cas9 editing with the unmodified guide RNA to ascertain improvements in specificity. The 6i guide RNA showed the largest improvement in specificity ratio at all but 1 off-target site (Fig.2A and 2B). Editing levels at OT16 with the heavier modified guide RNA remained comparable to the unmodified guide RNA. Nevertheless, we observe increased fidelity in nuclease activity of WT Cas9 when complexed with inosine guide RNAs at fourteen of the fifteen off-target sites surveyed. Substituting inosines in the spacer regions of a G-C rich guide RNA demonstrates an efficient strategy in enhancing specificity.

[0105] Example 2: Increased fidelity of WTCas9 activity when complexed with inosine substituted guide RNAs at the HBB target site

[0106] We chose an additional target site with an elevated G-C-content with a challenging off-target site to evaluate the efficacy of our strategy. Sickle cell disease (SCD) is an autosomal recessive disorder that affects millions of people worldwide

[0077] . A single point mutation altering adenine to thymidine in codon 6 of the HBB locus leads to the formation of sickle beta-globin protein in red blood cells. Several groups have investigated a homology- directed recombination (HDR) based correction of the sickled HBB gene via CRISPR-Cas9 editing, as a pathway towards an autologous stem cell therapy[78-81]. One of the most favorable SpCas9 target sites for gene correction of the SCD mutation has a single off-target site 1 (OT1)with high editing rates observed[78, 79]. Others have utilized chemically modified guide RNAs incorporated with 2′-O-methyl-3′-phosphonoacetate

[0044] and even high-fidelity variants of Cas9 (e.g. HiFi-Cas9[30, 80, 81] and rCas9HF

[0082] ) to reduce indels at OT1. Nonetheless, indels at this off-target site are not completely abrogated which could contribute to unwanted consequences such as translocation events at low levels

[0080] .

[0107] We sought to investigate whether the replacement of bases within the spacer region with inosines might help to limit off-target activity of this G-C rich (60%) HBB guide RNA. To optimize the positional effects of inosine substitutions within the spacer region, five configurations of the sgRNA targeting the HBB locus were designed containing inosine substitutions at various positions. As positions for substitution, we selected four guanines predicted to form G-C base pairs involved in intramolecular secondary structure formation (Fig. 1D). Guanosines at the PAM proximal, PAM distal and / or the G-triplet in the seed regions of the guide RNA were chosen for combined substitution. These guide RNAs were tested for their editing efficiencies at the target site and at three previously described off-target sites for this guide RNA[78-81, 83]. WT Cas9 RNPs were electroporated into HEK293T cells targeting the HBB locus and subjected to deep-sequencing to quantify indel percentages at the target and three off-target loci. All 3-inosine substituted guide RNAs (HBB guides 3a, 3b, 3c) regardless of inosine positions retained comparable on-target editing to the unmodified guide RNA in complex with WT Cas9 (Fig.1E). The destabilizing effects of base substitution were more evident with the addition of a 4thinosine. Complete substitution within the G-triplet in combination with the PAM-proximal substitution (HBB guide 4b) led to a reduction in on-target activity. However, replacing 1 guanosine in the PAM-distal position in combination with the full G-triplet substitution (HBB guide 4a) retained on-target activity at this Cas9 RNP dose, consistent with PAM-proximal substitutions being more destabilizing for proper R-loop formation[15, 18]. At off-target site 1 (OT1), WT Cas9 and standard unmodified (standard guide RNA with end modifications) guide RNA retained comparable editing to the target site, as previously described (Fig.2C). The 3-inosine substituted guides varied in their ability to reduce editing by WT Cas9 at OT1. The HBB 3a and 3b sgRNAs had on average ~20% and ~40% lower editing rates at OT1 over unmodified guide RNA respectively, while the HBB 3c sgRNA retained similar editing levels to the unmodified RNP. Both 4-inosine substituted sgRNAs in complex with WT Cas9 had on average ~60% and ~40% lower editing rates at OT1. The improvement in specificity wasmost pronounced for the 4-inosine substituted sgRNAs, as measured by their specificity ratios

[0076] , relative to the unmodified sgRNA at OT1 (Fig.2D). Encouragingly, all WT Cas9- inosine RNPs showed improved specificity over the unmodified RNP at OT2 and OT3 (Fig.2D).

[0108] Differences in off-target editing rates for WT Cas9-inosine RNPs could result from differences in the level of saturation of the nuclease complex within the cell. We performed a titration experiment in HEK293T cells to test editing rates for the HBB inosine guide RNAs with WT Cas9 at increasing RNP concentrations. We observed concentration dependentincreases in the editing rate for all of the treatment groups except for higher RNP concentrations≥60uM suggesting that all are at saturating levels (Fig. 4A). A similar concentration-dependenttrend was also observed at the OT1 site for all of the except for the 4-inosine substituted sgRNAs where the increase in RNP concentrations did not result in substantial increases in editing rates (Fig.4B).

[0109] Example 3: Inosine guide RNAs complexed with HiFiCas9 display enhanced fidelity without affecting on-target editing

[0110] Engineered variants of SpyCas9 with high-fidelity are often used to reduce off- target editing[27-31, 82, 84-86]. We evaluated the ability of HiFi-Cas9 to mitigate off-target editing at the HPS1 target site with both unmodified and inosine-modified guides. All HPS1 sgRNAs, including the unmodified sgRNA, had reduced on-target activity when complexed with HiFi Cas9 (Fig.1C and Fig.3A). HiFi Cas9-standard unmodified HPS1 RNPs was able to efficiently decrease indel rates at all of the OT sites with indel rates reduced to background levels at OT7, OT11, OT18, OT29, and OT37 (while 8 out of 15 active OT sites retain significant editing levels) (Fig.5A). Programming HiFi-Cas9 with the HPS14i guide RNA improved specificity at 6 of the 8 remaining HPS1 off-target sites. Editing rates at OT2 and OT5 were mitigated to background levels with the HPS16i guide RNA while OT16 retained editing levels slightly above background, potentially due to the mismatches present within the target site at inosine substituted positions. HiFi Cas9-inosine RNPs improved specificity at 11 OT sites, thereby demonstrating increased fidelity over the editing with the unmodified guide RNA (Fig. 5B).

[0111] To ensure these trends were not specific to the HPS1 locus, we evaluated whether HiFi-Cas9 programmed with HBB inosine guide RNAs could mitigate off-target editing at the previously identified off-target sites 1, 2 and 3. Others have shown improvement in specificitywhen editing the HBB locus with HiFi-Cas9 in CD34+ hematopoietic stem cells

[0030] . HiFi Cas9- unmodified RNP retained on-target activity comparable to WT Cas9 at the HBB target site in HEK293T cells, consistent with previously reported editing outcomes (Fig.1E). The HBB 3c sgRNA complexed with HiFi Cas9 maintained an on-target editing rate comparable to unmodified guide RNA while HBB 3a and 3b guide RNAs complexed with HiFi Cas9 displayed a 10% and 20% reduction in activity, respectively. The HBB 4a and 4b substituted guide RNAs complexed with HiFi Cas9 also displayed reduced editing efficiently at the target site when complexed with HiFi Cas9; with a 20% and 45% reduction in activity respectively when compared to unmodified guide RNA (Fig.1E). At OT1, HiFi Cas9 programmed with the standard unmodified guide RNA displayed a modest edited rate (5%). HiFi Cas9 programmed with the 3-inosine substituted guide RNAs further reduced editing at OT1 (2.5-1%; Fig.5C). HiFi Cas9 programmed with the 4-inosine guide RNAs reduced editing to near background levels at OT1. Specificity was improved by >5 fold at OT1 when HiFi Cas9 was programmed with the 4-inosine guide RNAs (Fig.5D). HiFi Cas9 was effective in diminishing editing to background levels at OT2 and OT3 in HEK293Ts irrespective of the sgRNA (standard / unmodified or inosine-modified) that was loaded.

[0112] In vitro cleavage analysis demonstrated that the majority of the WT Cas9-inosine RNPs displayed improved cleavage rates relative to the WT Cas9- standard unmodified RNPs at the target site (Fig.6A). WT Cas9-3b inosine RNPs were an outlier, displaying a slower rate of target site cleavage, similar to the unmodified RNPs. Similar cleavage trends were observed at the OT1 site across all WT Cas9 RNPs (Fig.6B). These data suggest that the HBB inosine- containing sgRNAs are not defective for DNA cleavage relative to the unmodified sgRNA when bound to WT Cas9. In vitro DNA cleavage efficiencies for HiFi Cas9-inosine RNPs were also evaluated. Similar to results observed with WT Cas9 RNPs, all HiFi Cas9- inosine RNPs with the exception of the 3b sgRNA displayed similar rates of cleavage at the target site (Fig.6C). As expected, HiFi Cas9 RNPs overall displayed slower rates of cleavage of the OT1 sequence compared to WT Cas9 RNPs likely due to the nuclease’s R691A mutation inhibiting its transition to active cleavage competent state in the presence of near-cognate targets (Fig.6D).

[0113] Interestingly, when evaluating editing outcomes for WT Cas9-inosine RNPs targeting in the HPS1 locus in the crRNA:tracrRNA format, we observed an improvement in editing levels for inosine substituted crRNAs relative to the unmodified crRNA (Fig.9A). Asimilar trend was observed for the 6i crRNA with HiFi Cas9. The inosine substitutions within the spacer of the crRNA may increase editing rates by reducing unwanted intra- and intermolecular interactions that might hinder spacer function (Fig.1B). However, Cas9 programmed with the two-part guide RNA system displays lower on-target editing efficiency compared with Cas9 complexes containing the corresponding sgRNAs in most instances, which is likely due to the additional RNA species that may be present in the annealed crRNA:tracrRNA product. Both WT Cas9 and HiFi Cas9 nucleases complexed with inosine substituted crRNAs display lower indel rates at HPS1 off-target sites consistent with the observations made when complexed with the inosine containing sgRNAs (Fig.9B, 9C). Working in combination HiFi Cas9 and the 6-inosine guide RNA effectively suppress editing at all the 15 identified off-target sites.

[0114] Example 4: Improvements in nuclease fidelity when complexed with inosine guide RNAs reduce off-target editing at HBB target site in CD34+ HSPCs

[0115] To ensure the effects of inosine substituted guide RNAs can be recapitulated in a primary cell type of interest, we tested the efficacy of our HBB inosine guide RNA designs in human CD34+ stem and progenitor cells (HSPCs). Healthy donor CD34+ HSPCs were electroporated with both WT Cas9 and HiFi Cas9 programmed with our best performing 4a guide RNA, 3b guide RNA which harbors a similar substitution configuration with a lower specificity profile compared to 4a, and the unmodified guide RNA. We performed a titration experiment to ensure the inosine RNPs were not being delivered at sub-saturating levels which may mask true comparisons in mitigating off-target editing. The 3b and 4a inosine guide RNAs complexed with WT Cas9 or HiFi Cas9 were electroporated into CD34+ HSPCs at increasing RNP concentrations (5 to 50uM) with a constant 3x excess guide RNA molar ratio. As expected, we observed progressive increases in editing levels with higher RNP concentrations until saturation was achieved (Fig.8A). Editing by WT Cas9-3b inosine RNPs approached saturation at concentrations of 10uM and above, while WT Cas9-4a inosine RNPs approached saturation at 20uM and above. HiFi Cas9-3b inosine RNPs approached saturation at concentrations of 20uM and above, while HiFi Cas9-4a inosine RNPs approached saturation at 30uM and above (Fig. 8E). These saturated dose concentrations were treated as individual replicates to demonstrate that on average all guide RNAs retained >90% edited alleles when complexed with either WT Cas9 or HiFi Cas9 (Fig.8A).

[0116] WT Cas9 complexed with either the unmodified or 3b inosine guide RNAs in CD34+ HSPCs displayed substantial levels of editing at ~75% and ~50% respectively at OT1, while WT Cas9 complexed with 4a inosine guide RNA was effective in reducing indels down to ~20% (Fig.8B). These trends were sustained at even the highest RNP concentrations tested for the HBB inosine sgRNAs. We note that WT Cas9 was inactive at OT2 when in complex with the unmodified guide RNA in CD34+ HSPCs at all concentrations tested (Fig.8C) while retaining editing levels above background in HEK293Ts (Fig.2C). WT Cas9 sufficiently reduced editing levels at OT3 to background when complexed with either the 3b or 4a inosine-modified guide RNA at all concentrations tested (Fig.8D, Fig.9B). Although HiFi Cas9 provides improved specificity with an unmodified guide RNA, there is still considerable activity (~4%) at OT1 (Fig. 9D). HiFi Cas9-3a inosine RNPs reduced the editing rate at OT1 to ~1.4%. The potent combination of HiFi Cas9 and HBB 4a-inosine modified guide RNA reduced the editing rate to background levels at OT1. The specificity ratio at OT1 increased over 10-fold for HiFi Cas9 complexed with the HBB 4a-inosine modified guide RNA in comparison to HiFi Cas9 complexed with the unmodified guide RNA (Fig.4E). Overall, Cas9-inosine RNPs sustain editing levels at the HBB target site while providing a substantial reduction of off-target editing rates in CD34+ HSPCs.

[0117] Example 5: Inosine modified guide RNAs programmed with WTCas9 do not lead to additional off-target activity

[0118] To test whether Cas9 programmed with the inosine modified HBB guide RNA produced editing at additional off-target sites within the genome beyond those previously identified for editing with the unmodified guide RNA[78, 81, 87], we employed GUIDE-tag

[0071] to identify potential off-target sites genome-wide. GUIDE-tag is a variant of GUIDE-seq

[0059] that employs Tn5 tagmentation to generate NGS libraries following incorporation of a double-strand DNA tag at sites of double-strand break formation. We performed GUIDE-tag analysis using WT SpyCas9- programmed with the unmodified HBB sgRNA or each of the five different inosine modified HBB guide RNAs (3a, 3b, 3c, 4a). Our analysis curated 19 putative off-targets among which were the previously defined OT1, OT2, OT3, OT4, and OT6 off-target sites[30, 42, 81, 87] sites (Table 4). Ranking the curated list of off-target sites by their CFD scores

[0038] , we retrieved 6 off-target sites predicted to have high cleavage activity (arbitrary score of >0.2 denoted by dotted line) by WTCas9-standard unmodified RNPs (Fig.10A). Two off-target sites(OTss18 and OTss19) have not been described before for the standard HBB guide RNA. WTCas9-3 inosine RNPs do not reveal any new potential off-target sites (Fig.10C). WTCas9-4a inosine RNPs revealed an off-target site (OTss17) with very low UMI reads (2 UMIs) that is not identified in the standard unmodified guide RNA treated sample. To validated these potential off-target sites for their activity in cells, we amplified 5 of the identified sites (OT4, OT6, OTss13, OTsss15, and OTss17) with high predicted cleavage scores (> 0.2 CFD cutoff ) to detect any measurable levels of editing resulting from either WT Cas9 or HiFi Cas9 RNP combinations in HEK293Ts. Despite WT Cas9 and unmodified RNP retaining slight above background editing at OT4 and OT6, inosine RNP combinations maintained indels at background levels across all sites tested (Fig.10D, 10E). These results demonstrate inosine-modified guide RNAs do not increase Cas9’s nuclease activity at additional off-target sites.

[0119] Table 4: Guide-Tag Data for Inosine modified guide RNAs with WTCas9 at HBB locus in HEK293T cells OT OT total chr offT offT offTa offTarget_ S1_UMI: S2_UMI:r S3_UMI S4_UMI S5_UMI Site # Site .mis om arge arge rgetS sequence reads+po eads+posi :reads+ :reads+ :reads+ Descri mat oso t St t E trand sition tion position position position ng 2 1(SEQ ID NO: 144)(OTss2 ID NO: 6) 155) s9-c- nos ne ; as - -nos ne ; as - c-nos ne ; as - 4a-inosine RNP.

[0121] Double-strand breaks at the target site within the genome can produce large segmental deletions and in conjunction with double-stranded breaks at an off-target site can produce translocations within the genome[19-22, 88, 89]. To examine whether inosine substitutions when complexed with either WT Cas9 or HiFi Cas9 introduce unintended complex genomic modifications, we performed UDiTaS

[0090] to characterize the HPS1 inosine guide RNAs in both patient-derived HPS1 B-LCLs and the HEK293T cell line harboring the HPS1 associated 16bp microduplication. Overall, we show that Cas9 in complex with HPS1 inosine modified guide RNAs maintained similar frequencies of mutagenesis when compared to with unmodified guide RNA, consistent with our amplicon sequencing data. Low levels (<0.3%) of translocation were detected in the HEK293Ts treated with Cas9 and unmodified guide RNA and in B-LCLs treated with Cas9 and 6i guide RNA (Figs.11A, 11B). However, due to the low read counts (<3) associated with the observed translocations in each treatment group, further studies repeating this result with higher read counts including performing validation experiments via junction qPCRs will be necessary to more rigorously define the changes in translocations within the population of treated cells for each editing condition.

[0122] Materials and Methods

[0123] Oligo synthesis

[0124] All DNA oligonucleotides were purchased from Integrated DNA Technologies (IDT). Chemical synthesis of HPS1 unmodified and HPS1 modified inosine guide RNAs and crRNAs along with SpyCas9 tracrRNAs were performed by IDT.

[0125] Production of HBB inosine guide RNA

[0126] The inosine modified guide RNAs were synthesized at 1 μmole scale on and Expedite DNA synthesizer using 1000 Å standard controlled pore glass (CPG) solid support. Inosine phosphoramidites were (purchased from ChemGenes Corporation, MA) and incorporated at desired positions. BTT (0.25M) in acetonitrile was used as activator.0.05M iodine in pyridine:water (9:1), DDTT (0.1 M in Pyridine) and 3% DCA in dichloromethane was used as oxidizer, sulfurizing reagent and deblock solution, respectively. All phosphoramidites were prepared as 0.15 M solution in acetonitrile and coupled for 10 mins for each base. After completion of the syntheses, cleavage and deprotection was done using ammonium hydroxide- methylamine (AMA) for 15 mins for 65 °C. Oligonucleotides were analyzed on an Agilent 6530 Q-TOF LC-MS system equipped with electrospray ionization and time-of-flight ion separation in negative ionization mode, using 100 mM hexafluoroisopropanol with 9 mM triethylamine in water as buffer A and 100 mM hexafluoroisopropanol with 9 mM trimethylamine in methanol as buffer B. Finally, samples were desalted using 10K cutoff amicon desalting columns and sent for biological experiments.

[0127] Expression and Purification of SpyCas9

[0128] The generation and characterization of the 3xNLS-SpCas9 (Addgene #114365)) constructs have been recently described

[0105] . The pET21a plasmid backbone (Novagen) is used to drive the expression of a hexa-His (SEQ ID NO: 159) tagged version of each protein. The plasmid expressing 3xNLS-SpCas9 was transformed into E. coli Rosetta (DE3) pLysS cells (EMD Millipore) for protein production. Cells were grown at 37°C to an OD600 of ~0.2, then shifted to 18°C and induced at an OD600 of ~0.4 for 16 hours with IPTG (1 mM final concentration). Following induction, cells were pelleted by centrifugation and then resuspended with Nickel-NTA buffer (20 mM TRIS pH 7.5 + 1 M NaCl + 20 mM imidazole + 1 mM TCEP) supplemented with HALT Protease Inhibitor Cocktail, EDTA-Free (100X) [ThermoFisher] and lysed with M-110s Microfluidizer (Microfluidics) following the manufacturer’s instructions. The protein was purified from the cell lysate using Ni-NTA resin, washed with five volumes of Nickel-NTA buffer and then eluted with elution buffer (20 mM TRIS, 500 mM NaCl, 500 mMImidazole, 10% glycerol, pH 7.5). The 3xNLS-SpCas9 was dialyzed overnight at 4°C in 20 mM HEPES, 500 mM NaCl, 1 mM EDTA, 10% glycerol, pH 7.5. Subsequently, the protein was step dialyzed from 500 mM NaCl to 200 mM NaCl (Final dialysis buffer: 20 mM HEPES, 200 mM NaCl, 1 mM EDTA, 10% glycerol, pH 7.5). Next, the protein was purified by cation exchange chromatography (Column = 5ml HiTrap-S, Buffer A = 20 mM HEPES pH 7.5 + 1 mM TCEP, Buffer B = 20 mM HEPES pH 7.5 + 1 M NaCl + 1 mM TCEP, Flow rate = 5 ml / min, CV = column volume = 5ml) followed by size-exclusion chromatography (SEC) on Superdex-200 (16 / 60) column (Isocratic size-exclusion running buffer = 20 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP for 3xNLS-SpCas9). The primary protein peak from the SEC was concentrated in an Ultra-15 Centrifugal Filters Ultracel-30K (Amicon) to a concentration around 100 μM based on absorbance at 280nm. The purified protein quality was assessed by SDS- PAGE / Coomassie staining to be >95% pure and protein concentration was quantified with Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific). Protein was stored at -80°C until further use.

[0129] Cell culture:

[0130] A lymphoblastoid cell line from B-lymphoblastoid cells (B-LCL) derived from a HPS1 patient-homozygous for the 16 bp microduplication was purchased from Coriell (Catalog GM14606). The cell line was cultured following the recommended procedure using RPM11640 with 2mM L-Glutamine, 15% FBS and 1% Pen / Strep.

[0131] To construct a HEK293T based cell line that is homozygous for the HPS116bp duplication, we designed a donor template to knock-in the HPS1 associated 16bp duplication into the sec61b locus in HEK293T cells.20pmols of SpyCas9 and 25pmols of sgRNA targeting the sec61b locus were complexed to form RNPs at room temperature in a final volume of 12ul. 200,000 cells per reaction were resuspended in 10ul of RNP-buffer R mix and electroporated into HEK293Ts along with 30pmols of donor template using the ThermoFisher Neon transfection system (1150V, 20ms, 2pulses). Cells were plated into 24 well plates with pre- equilibrated 500ul of antibiotic free culture media and grown in a humidified incubator at 37C and 5% CO2 for 3 days.100ul of electroporated cells were harvested for indel analysis and rest were further propagated to establish clones. The remaining cells were counted and appropriate amounts were plated into 96 wells such that each well contained at least one cell via serial dilutions. After 20 days of culturing, wells containing individual clones were further propagatedinto 24 well plates.100ul of the clonal population was harvested for indel analysis and rest were subjected to further propagation. Cells homozygous for the HPS116bp duplication were frozen down and used in subsequent experiments. HEK293T-HPS1 cells were cultured following the recommended procedure using DMEM, 10% FBS and 1% Pen / Strep.

[0132] Healthy human CD34 HSPCs were obtained from the Fred Hutchinson Cancer Research Center (Seattle, WA). Human CD34 HSPCs were thawed and cultured into serum-free medium Stem Cell Growth Medium (CellGenix, 20806-0500) supplemented with human Stem Cell Factor (SCF,100 ng / ml) (CellGenix, 1418-050), FMS-like Tyrosine Kinase 3 Ligand (Flt3L, 100 ng / ml) (CellGenix, 1415-050) and Thrombopoietin (TPO, 100 ng / ml) (CellGenix, 1417- 050).

[0133] All cultures were maintained in a humidified incubator with 5% CO2at 37°C.

[0134] Electroporation of patient-derived HPS1 B-lymphoblastoid (B-LCL) cells:

[0135] 40 pmol of 3xNLS-SpyCas9 or HiFiCas9 protein was precomplexed with 50 pmol of chemically synthesized sgRNA (or crRNA:tracrRNA) in buffer R for 20 minutes at room temperature.300,000 cells per reaction were resuspended in 10μl of RNP-buffer R mix and electroporated with 2 pulses of 1700V for 20ms using the 10μl tip. Cells were then plated in 24 well plates with pre-equilibrated 500μl of antibiotic free culture media and grown for 72 hours before harvesting for subsequent indel analysis.

[0136] Electroporation of HEK293T-HPS1 cells:

[0137] 40pmol of 3xNLS-SpyCas9 protein or HiFiCas9 and 120pmol of chemically synthesized sgRNA were pre-complexed in buffer R for 20 minutes at room temperature. 200,000 cells per reaction were resuspended in 10μl of RNP-buffer R mix and nucleofected with SpyCas9 guide RNA complex using 2 pulses of 1150V for 20ms using the 10μl tip. Cells were then plated in 24 well plates with pre-equilibrated 500μl of antibiotic free culture media and grown for 72 hours before harvesting for subsequent indel analysis.

[0138] Electroporation of HSPCs:

[0139] After 48 hours of pre-stimulation, HSPCs were harvested for electroporation. Electroporation was performed using Lonza 4D Nucleofector. The RNP complex was prepared by mixing 100 pmol-1000 pmol of 3xNLS wt spCas9 or 3xNLS HiFi spCas9 with 300 pmol- 3000 pmol of sgRNA, adding P3 solution up to 10 µl. The RNP was incubated at room temperature for 10-15 min.200,000 cells were suspended in 10 µl of P3 solution. The cellsuspension was mixed with RNP and transferred to cuvette (Lonza 4D, V4XP-3032) for electroporation with program EO-100. The P3 solution was removed after 15 min of incubation at room temperature. The electroporated cells were cultured in SCGM medium supplemented with cytokines. Cells were harvested 5 days after electroporation. Genomic DNA was isolated using the Blood and Tissue Kit (Qiagen, 69506) according to the vendor’s recommendations. gDNA was eluted in 50 µl of nuclease free water.

[0140] Concentration of SpyCas9 and HiFiCas9 for Titration Experiments in HSPCs

[0141] For CD34+ dose titration experiments, SpyCas9 [45uM] and HiFiCas9 [60uM] were diluted in 400ul of Lonza P3 electroporation buffer for concentration using Amicon Ultra 30 kDa MWCO Concentrator. Diluted protein was spun at 12,000g for 10 mins at 4C to bring volume down to 50ul.1x volume of P3 was mixed again and spun as described above. This was repeated 3x times until volume ~100ul and concentration was measured via Nanodrop Protein A280. Protein was stored at -80.

[0142] In vitro cleavage assay

[0143] 5’-Cy3 labeled target strand (TS) single-stranded oligo and unlabeled non-target strand (NTS) single-stranded oligo for HBB on-target and HBB OT1 target sites were ordered for in-vitro cleavage assays.40uM Labeled TS and 33uM unlabeled NTS were resuspended in IDT duplex buffer and annealed by heating to 94C for 2-5mins and slow cooled down to room temperature.5uM RNP complexes were made by incubating SpyCas9 / HiFiCas9 protein with 3x times synthetic HBB standard / unmodified or inosine sgRNAs in ThermoFisher Neon Nucleofector R buffer in separate 15ul reactions for 15-20 minutes at room temperature.6pmols of RNP with 0.3pmols of annealed substrate was added to NEB Buffer 3.1. The in vitro cleavage reactions were incubated at 37C and quenched at various time-points with 0.5M EDTA and treated with proteinase K at 50C for 15 mins. Lastly reactions were incubated at 95C for 5 mins and 2x Novex TBE-Urea Sample buffer was added. Reactions were gel on Novex 15% TBE- Urea gels Gels were visualized on the BioRad Gel Doc Imager under Cy3 filter. Bands were quantified using Fiji ImageJ densitometry: (cleaved band / (sum of un-cleaved and cleaved bands))*100 = % subtrate cleaved.

[0144] Illumina Amplicon sequencing library preparation and analysis

[0145] Library construction for deep sequencing was performed using our previously described protocol[70, 106]. Briefly, cells were harvested following nuclease treatment and genomic DNA was extracted with the GenElute Mammalian Genomic DNA Miniprep Kit (Sigma G1N350) or the Blood and Tissue Kit (Qiagen, 69506). Genomic loci spanning the target sites were PCR amplified with locus-specific primers carrying tails complementary to the TruSeq adapters.50 ng input genomic DNA was PCR amplified with Q5 High-Fidelity DNA Polymerase (New England Biolabs): (98°C, 15s; 67°C 25s; 72°C 20s) x30 cycles. Next, 0.1 µl of each PCR reaction was amplified with barcoded primers to reconstitute the TruSeq adaptors using the Q5 High-Fidelity DNA Polymerase (New England Biolabs): (98°C, 15s; 67°C, 25s; 72°C, 20s) x10 cycles. Products were qualitatively analyzed by gel electrophoresis. Equal amounts of the products were pooled and gel purified using QIAquick Gel Extraction Kit (Qiagen Cat. #28704). The purified library was deep sequenced using a paired-end 150bp Illumina MiniSeq run. MiniSeq data analysis for indel frequencies at on-target and off-target sites was performed using CRISPResso2 software

[0075]

[0146] RhAmp-Seq library preparation

[0147] The off-target panel design and library preparation for high-throughput amplicon sequencing was performed following IDT rhAmpSeq Technology protocol

[0107] . The purified library was deep sequenced using a paired-end 150bp Illumina MiniSeq run. MiniSeq data analysis for indel frequencies at on-target and off-target sites was performed using CRISPResso2 software

[0075]

[0148] GUIDE-Tag and UDiTaS Library Preparation

[0149] Followed as previously described[71, 108]

[0150] For tagmentation, transposome was assembled as previously described

[0090] using purified Tn5 protein and oligonucleotides purchased from IDT. Two hundred nanogram of genomic DNA was incubated with 2ul of assembled transposome at 55 degrees for 7 mins, and the product was cleaned up (20 μl) with a Zymo column (Zymo Research, #D4013). Tagmented DNA was used for the 1st PCR using PlatinumTM SuperFi DNA polymerase (Thermo) with i5 primer and gene-specific primers (Supplementary Table 3). Two different libraries were prepared for gDNA from each mouse with different combinations of primers. The i7 index was added in the 2nd PCR and the PCR product was cleaned up with Ampure XP SPRI beads (Agencourt,0.9X reaction volume). Completed libraries were quantified by Tapestation and Qubit (Agilent), pooled with equal amounts, and sequenced with 150 bp paired-end reads on an Illumina MiniSeq instrument.

[0151] GUIDE-Tag Analysis

[0152] Cells treated with RNPs (wild-type SpCas9 or SpCas9-HiFi (R691A) protein with different modified sgRNA) and dsODN donors were harvested 72 hours after nucleofection, then genomic DNA was isolated using DNeasy blood & tissue kits (Qiagen). Tn5 Adaptor oligonucleotides were synthesized by IDT. Transposon assembly was done by incubating 158ug Tn5 with 1.4nmol annealed oligo (contains the full-length Illumina forward (i5) adapter, a sample barcode, and unique molecule identifier (UMI) at room temperature for 60mins.

[0153] Genomic DNA tagmentation and library was performed as previous descrbibed

[0071] Briefly, 200ng of genomic DNA was incubated with 2ul of assembled transposome at 55 degree for 7 mins, and the product was cleaned up (20ul) with a Zymo column (Zymo Research, #D4013). Tagmented DNA was used for the 1st PCR using PlatinumTM SuperFi DNA polymerase (Thermo) with i5 primer and GUIDE-Tag specific primers. The i7 index was added in the 2nd PCR and the PCR product was cleaned up with Ampure XP SPRI beads (Agencourt, 0.9X reaction volume). Completed libraries were quantified by Tapestation and Qubit (Agilent), pooled with equal mole and sequenced with 150 bp paired-end reads on an Illumina MiniSeq instrument.

[0154] The GUIDE-tag raw sequencing data pre-processing and analysis pipeline is available at:github.com / umasstr / GS-Preprocess and rdrr.io / github / LihuaJulieZhu / GUIDEseq / .

[0155] Briefly, it consists of the following steps: i. Demultiplexing and UMI extraction. Raw BCL files were converted and demultiplexed using the appropriate i5 and i7 sequencing barcodes, allowing up to one mismatch in each barcode. Unique molecular identifiers (UMIs) for each read were extracted for further downstream analysis. ii. Raw reads were processed with fastqc (Version 0.11.9) and trim_galore (Version 0.6.5) (https: / / www.bioinformatics.babraham.ac.uk / projects / ) to remove reads with low quality and trim adapters. iii. Create a reference sequence based on the UDiTaS locus-specific primer position and donor map separately. Build index files for the reference using bowtie2-index, version 2.4.0.iv. Alignment analysis. Paired reads were then globally aligned (end-to-end mode) to mouse genome (mm10) and all the reference amplicons using bowtie2’s very sensitive parameter. Finally, Samtools (version 0.1.19) was used to create an index-sorted bam file.

[0156] Data analysis:

[0157] GUIDE-Tag sequencing data were run through the Bioconductor GUIDE-seq analysis pipeline https: / / github.com / umasstr / GS-Preprocess). Briefly, for GUIDE-seq analysis processed paired reads were merged (if they overlap) and then globally aligned to the human genome (hg38) using bowtie2. Then BAM files and UMI files were used to aggregate unique reads. Mapped BAM files, sgRNA / crRNA fasta files and presorted UMI fastq file as inputs to run Bioconductor GUIDE-Seq package to get potential Off-targets across the whole genome. For off target site identification within potential peaks required the presence of a near-cognate recognition sequence for Cas9 with these parameters: the maximum number of mismatches is 6 positions with one DNA / RNA bulge allowed and the presence of an NNG / NGN for spCas9 PAM. For more stringent UMI or reads number filtering, we use parameter” ignoreTagmSite “as True to ollapse reads with the same integration site and UMI but with different tagmentation site. To collapse reads with the same integration and tagmentation site but with different UMIs, set parameter “ignoreUMI” to TRUE and retain the UMI that appears most frequently for each combination of integration and tagmentation site. In case of ties, randomly select one UMI.

[0158] UDiTas analysis

[0159] The analysis pipeline is similar to GUIDE-Tag and is adapted from our previously described paper

[0108] . Briefly, the analysis steps are as follows: i. Demultiplexing and UMI extraction. Raw BCL files were converted and demultiplexed using the appropriate i5 and i7 sequencing barcodes, allowing up to one mismatch in each barcode. UMIs for each read were extracted into UMI.fastq files after filtering out the UMIs containing ‘N’ for further downstream analysis. ii. Raw reads were processed with fastqc (Version 0.11.9) and trim_galore (Version 0.6.5) (www.bioinformatics.babraham.ac.uk / projects / ) to remove reads with low quality and trim adapters (regular Illumina adapter sequences), inserted tag (GUIDE-seq, iGUIDE) sequences, locus-specific sequences in UDiTaS (gene-specific primers) or IRES-GFP and FAH repair cassette for GUIDE-tag.iii. For UDiTaS create a reference sequence based on the UDiTaS locus-specific primer position and donor map separately. Build index files for the reference using bowtie2- index, version 2.4.0. iv. Alignment analysis. Paired reads were then globally aligned (end-to-end mode) to mouse genome (mm10) and all the reference amplicons using bowtie2’s very sensitive parameter. Finally, Samtools (version 0.1.19) was used to create an index-sorted bam file.

[0160] Data analysis:

[0161] For UDiTaS analysis at each target site, locus-specific primers were used to construct UDiTaS libraries, precise editing or small indels were analyzed as previously described

[0109] . Pindel (version 0.2.5b8) was used to detect breakpoints of large deletions and donor integration. Raw sequencing reads that align to the reference sequence were collapsed to a single read by common UMI and categorized as an exemplar for each UMI to a specific category—for example, Wild Type, precise editing, small indel / substitution (<50 bp), and Large Deletions / Insertions (>50 bp). Then the number of UMIs assigned per category was determined to define the ratio of each event.

[0162] Statistical Analysis

[0163] R, a system for statistical computation and graphics, was used for the analysis

[0110] . Percentage of indel rate was first arcsin transformed to homogenize the variance. When Levene's test indicates that the assumption of homogeneity of variances was met, one-way analysis of variance (ANOVA) with Randomized Complete Block Design was performed for each off-target site and on-target. Treatments were compared to the corresponding negative control under the ANOVA framework using lsmeans package

[0111] . P values were adjusted using hochberg method to correct for multiple inferences

[0112] . When the assumption of homogeneity of variances was not met, unequal variance t-test was performed.

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[0165] The publications (including patent publications), web sites, company names, books, manuals, treatise, and scientific literature referred to herein establish the knowledge that is available to those with skill in the art and are hereby incorporated by reference in their entirety to the same extent as if each was specifically and individually indicated to be incorporated byreference. Any conflict between any reference cited herein and the specific teachings of this specification shall be resolved in favor of the latter.

[0166] Various embodiments of the present invention may be characterized by the potential claims listed in the paragraphs following this paragraph (and before the actual claims provided at the end of this application). These potential claims form a part of the written description of this application. Accordingly, subject matter of the following potential claims may be presented as actual claims in later proceedings involving this application or any application claiming priority based on this application. Inclusion of such potential claims should not be construed to mean that the actual claims do not cover the subject matter of the potential claims. Thus, a decision to not present these potential claims in later proceedings should not be construed as a donation of the subject matter to the public.

[0167] Without limitation, potential subject matter that may be claimed (prefaced with the letter “P” to avoid confusion with the actual claims presented below) includes: P1. A programmable genome editing system for modification of a double-stranded target DNA sequence comprising a target strand having a target sequence, and a non-target strand having a sequence complementary to the target sequence, the system comprising: a nucleic acid programmable nuclease, and a guide RNA comprising, in a 5′ to 3′ direction, a spacer sequence having a region of complementarity to the target sequence; wherein the spacer sequence comprises a base substitution in the region of complementarity to the target sequence, the base substitution being selected to reduce hydrogen bonding potential of the spacer sequence with the target sequence, thereby reducing off-target activity of the programmable genome editing system. P2. The programmable genome editing system of potential claim P1, wherein the base substitution is a non-canonical base (and, optionally, if the base substitution includes hypoxanthine, no base of the spacer sequence other than guanine is substituted with hypoxanthine).P3. The programmable genome editing system according to any one of potential claims P1 and P2, wherein the base substitution is a base selected from the group consisting of N4-ethyl- cytosine, N4-methyl-cytosine, N6-ethyl-adenine, N6-methyl-adenine, hypoxanthine, and combinations thereof. P4. The programmable genome editing system according to any one of potential claims P1 and P2, wherein the base substitution is a base selected from the group consisting of N4-ethyl- cytosine, N4-methyl-cytosine, N6-ethyl-adenine, N6-methyl-adenine, and combinations thereof. P5. The programmable genome editing system of potential claim P4, wherein the base substitution further includes hypoxanthine. P6. The programmable genome editing system according to any one of potential claims P1–P2, wherein the base substitution is a substitution selected from the group consisting of cytosine to N4-ethyl-cytosine, cytosine to N4-methyl-cytosine, adenine to N6-ethyl-adenine, adenine to N6- methyl-adenine, guanine to hypoxanthine (wherein no base of the spacer sequence other than guanine is substituted with hypoxanthine), and combinations thereof. P7. The programmable genome editing system according to any one of potential claims P1–P2, wherein the base substitution is a substitution selected from the group consisting of cytosine to N4-ethyl-cytosine, cytosine to N4-methyl-cytosine, adenine to N6-ethyl-adenine, adenine to N6- methyl-adenine, and combinations thereof. P8. The programmable genome editing system of potential claim P7, wherein the base substitution further includes a guanine to hypoxanthine base substitution, wherein no base of the spacer sequence other than guanine is substituted with hypoxanthine. P9. The programmable genome editing system according to any one of the preceding potential claims, wherein the nucleic acid programmable nuclease is selected from the group consisting of Cas9, HiFiCas9, eSpCas9, SpCas9-HF1, Hypa-SpCas9, SuperFi-Cas9, Sniper-Cas9, evoCas9, and Cas12.P10. A method for site-specific modification of the double-stranded target DNA sequence comprising the target strand and the non-target strand, the method comprising: contacting the double-stranded target DNA sequence with the programmable genome editing system according to any one of the preceding potential claims, wherein the contacting results in cleavage of (i) the target sequence of the target strand and (ii) the sequence complementary to the target sequence of the non-target strand. P11. The method of potential claim P10, wherein the double-stranded target DNA sequence comprises a mutation at a locus selected from the group consisting of HPS1 and HBB. P12. A method of treating a subject having or suspected of having a disease or disorder, the method comprising administering the programmable genome editing system according to any one of potential claims P1–P9, ex vivo, to a cell from the subject. P13. The method of potential claim P12, wherein the disease or disorder is associated with a mutation at a locus selected from the group consisting of HPS1 and HBB. P14. A programmable prime editing system for modification of a double-stranded target DNA sequence comprising a target strand having a target sequence, and a complementary non-target strand, the system comprising: a prime editor protein, the prime editor protein being a fusion protein comprising a nucleic acid programmable DNA binding domain fused to a reverse transcriptase domain, the DNA binding domain having nickase activity, and a pegRNA comprising, in a 5′ to 3′ direction: (i) a spacer sequence comprising a region of complementarity to the target sequence; (ii) a gRNA core that interacts with the DNA binding domain; and (iii) an extension arm, the extension arm comprising, in a 5′ to 3′ orientation:(a) a DNA synthesis template encoding one or more nucleotide changes compared to a region downstream of a nick site in the non-target strand of the double-stranded target DNA sequence, and (b) a primer binding sequence comprising a region of complementarity to a primer sequence upstream of the nick site in the non-target strand of the double- stranded target DNA sequence; wherein the spacer sequence comprises a base substitution in the region of complementarity to the target sequence, the base substitution being selected to reduce hydrogen bonding potential of the spacer sequence with the target sequence, thereby reducing off-target activity of the programmable prime editing system. P15. The programmable prime editing system of potential claim P14, wherein the base substitution is a non-canonical base (and, optionally, if the base substitution includes hypoxanthine, no base of the spacer sequence other than guanine is substituted with hypoxanthine). P16. The programmable prime editing system according to any one of potential claims P14 and P15, wherein the base substitution is a base selected from the group consisting of N4-ethyl- cytosine, N4-methyl-cytosine, N6-ethyl-adenine, N6-methyl-adenine, hypoxanthine, and combinations thereof. P17. The programmable prime editing system according to any one of potential claims P14 and P15, wherein the base substitution is a base selected from the group consisting of N4-ethyl- cytosine, N4-methyl-cytosine, N6-ethyl-adenine, N6-methyl-adenine, and combinations thereof. P18. The programmable prime editing system of potential claim P17, wherein the base substitution further includes hypoxanthine. P19. The programmable prime editing system according to any one of potential claims P14–P15, wherein the base substitution is a substitution selected from the group consisting of cytosine to N4-ethyl-cytosine, cytosine to N4-methyl-cytosine, adenine to N6-ethyl-adenine, adenine to N6-methyl-adenine, guanine to hypoxanthine (wherein no base of the spacer sequence other than guanine is substituted with hypoxanthine), and combinations thereof. P20. The programmable prime editing system according to any one of potential claims P14–P15, wherein the base substitution is a substitution selected from the group consisting of cytosine to N4-ethyl-cytosine, cytosine to N4-methyl-cytosine, adenine to N6-ethyl-adenine, adenine to N6- methyl-adenine, and combinations thereof. P21. The programmable prime editing system of potential claim P20, wherein the base substitution further includes a guanine to hypoxanthine base substitution, wherein no base of the spacer sequence other than guanine is substituted with hypoxanthine. P22. A method for site-specific modification of the double-stranded target DNA sequence comprising the target strand and the non-target strand, the method comprising: contacting the double-stranded target DNA sequence with the programmable prime editing system according to any one of potential claims P14–P21, wherein the contacting results in: nicking the non-target strand of the double-stranded target DNA sequence to form a free 3′ end at the nick site; annealing the primer binding sequence with the primer sequence upstream of the nick site in the non-target strand of the double-stranded target DNA; synthesizing a single strand of DNA encoded by the DNA synthesis template from the free 3′ end of the non-target strand of the double-stranded target DNA sequence; and replacing the region downstream of the nick site in the non-target strand of the double- stranded target DNA sequence with the single strand of DNA encoded by the DNA synthesis template, thereby modifying the sequence of the double-stranded target DNA sequence. P23. The method of potential claim P22, wherein the double-stranded target DNA sequence comprises a mutation at a locus selected from the group consisting of HPS1 and HBB.P24. A method of treating a subject having or suspected of having a disease or disorder, the method comprising administering the programmable prime editing system according to any one of potential claims P14–P21, ex vivo, to a cell from the subject. P25. The method of potential claim P24, wherein the disease or disorder is associated with a mutation at a locus selected from the group consisting of HPS1 and HBB. P26. A programmable base editing system for modification of a double-stranded target DNA sequence comprising a target strand having a target sequence, and a non-target strand having a sequence complementary to the target sequence, the system comprising: a base editor protein, the base editor protein being a fusion protein comprising a nucleic acid programmable DNA binding domain fused to a nucleoside deaminase domain, the DNA binding domain having nickase activity, and a guide RNA comprising, in a 5′ to 3′ direction, a spacer sequence having a region of complementarity to the target sequence; wherein the spacer sequence comprises a base substitution in the region of complementarity to the target sequence, the base substitution being selected to reduce hydrogen bonding potential of the spacer sequence with the target sequence, thereby reducing off-target activity of the programmable base editing system. P27. The programmable base editing system of potential claim P26, wherein the base substitution is a non-canonical base (and, optionally, if the base substitution includes hypoxanthine, no base of the spacer sequence other than guanine is substituted with hypoxanthine). P28. The programmable base editing system according to any one of potential claims P26 and P27, wherein the base substitution is a base selected from the group consisting of N4-ethyl- cytosine, N4-methyl-cytosine, N6-ethyl-adenine, N6-methyl-adenine, hypoxanthine, and combinations thereof.P29. The programmable base editing system according to any one of potential claims P26 and P27, wherein the base substitution is a base selected from the group consisting of N4-ethyl- cytosine, N4-methyl-cytosine, N6-ethyl-adenine, N6-methyl-adenine, and combinations thereof. P30. The programmable base editing system of potential claim P29, wherein the base substitution further includes hypoxanthine. P31. The programmable base editing system according to any one of potential claims P26–P27, wherein the base substitution is a substitution selected from the group consisting of cytosine to N4-ethyl-cytosine, cytosine to N4-methyl-cytosine, adenine to N6-ethyl-adenine, adenine to N6- methyl-adenine, guanine to hypoxanthine (wherein no base of the spacer sequence other than guanine is substituted with hypoxanthine), and combinations thereof. P32. The programmable base editing system according to any one of potential claims P26–P27, wherein the base substitution is a substitution selected from the group consisting of cytosine to N4-ethyl-cytosine, cytosine to N4-methyl-cytosine, adenine to N6-ethyl-adenine, adenine to N6- methyl-adenine, and combinations thereof. P33. The programmable base editing system of potential claim P32, wherein the base substitution further includes a guanine to hypoxanthine base substitution, wherein no base of the spacer sequence other than guanine is substituted with hypoxanthine. P34. The programmable base editing system according to any one of potential claims P26–P33, wherein the base editor protein comprises a uracil glycosylase inhibitor (UGI) domain and the nucleoside deaminase domain is a cytosine deaminase. P35. The programmable base editing system according to any one of potential claims P26–P33, wherein the nucleoside deaminase domain is an adenine deaminase. P36. A method for site-specific modification of the double-stranded target DNA sequence comprising the target strand and the non-target strand, the method comprising:contacting the double-stranded target DNA sequence with the programmable base editing system according to any one of potential claims P26–P35, wherein the contacting results in: nicking the target sequence of the target strand; and deaminating a nucleobase of the sequence complementary to the target sequence, the nucleobase being selected from the group consisting of cytosine and adenine. P37. The method of potential claim P36, wherein the double-stranded target DNA sequence comprises a mutation at a locus selected from the group consisting of HPS1 and HBB. P38. A method of treating a subject having or suspected of having a disease or disorder, the method comprising administering the programmable base editing system according to any one of potential claims P26–P35, ex vivo, to a cell from the subject. P39. The method of potential claim P38, wherein the disease or disorder is associated with a mutation at a locus selected from the group consisting of HPS1 and HBB. P40. A system for modification of a double-stranded target DNA sequence of a double-stranded target DNA sequence comprising a target strand having a target sequence, and a non-target strand having a sequence complementary to the target sequence, the system comprising: a nucleic acid programmable DNA binding protein; and a guide RNA comprising, in a 5′ to 3′ direction, a spacer sequence having a region of complementarity to the target sequence; wherein the spacer sequence comprises a base substitution in the region of complementarity to the target sequence, the base substitution being selected to reduce hydrogen bonding potential of the spacer sequence with the target sequence, thereby reducing off-target activity of the system. P41. The system of potential claim P40, wherein the base substitution is a non-canonical base(and, optionally, if the base substitution includes hypoxanthine, no base of the spacer sequence other than guanine is substituted with hypoxanthine).P42. The system according to any one of potential claims P40 and P41, wherein the base substitution is a base selected from the group consisting of N4-ethyl-cytosine, N4-methyl- cytosine, N6-ethyl-adenine, N6-methyl-adenine, hypoxanthine, and combinations thereof. P43. The system according to any one of potential claims P40 and P41, wherein the base substitution is a base selected from the group consisting of N4-ethyl-cytosine, N4-methyl- cytosine, N6-ethyl-adenine, N6-methyl-adenine, and combinations thereof. P44. The system of potential claim P43, wherein the base substitution further includes hypoxanthine. P45. The system according to any one of potential claims P40–P41, wherein the base substitution is a substitution selected from the group consisting of cytosine to N4-ethyl-cytosine, cytosine to N4-methyl-cytosine, adenine to N6-ethyl-adenine, adenine to N6-methyl-adenine, guanine to hypoxanthine (wherein no base of the spacer sequence other than guanine is substituted with hypoxanthine), and combinations thereof. P46. The system according to any one of potential claims P40–P41, wherein the base substitution is a substitution selected from the group consisting of cytosine to N4-ethyl-cytosine, cytosine to N4-methyl-cytosine, adenine to N6-ethyl-adenine, adenine to N6-methyl-adenine, and combinations thereof. P47. The system of potential claim P46, wherein the base substitution further includes a guanine to hypoxanthine base substitution, wherein no base of the spacer sequence other than guanine is substituted with hypoxanthine. P48. A method for site-specific modification of the double-stranded target DNA sequence comprising the target strand and the non-target strand, the method comprising: contacting the double-stranded target DNA sequence with the system according to any one of potential claims P40–P47,wherein the contacting results in specific modification of the double-stranded target DNA sequence. P49. The method of potential claim P48, wherein the double-stranded target DNA sequence comprises a mutation at a locus selected from the group consisting of HPS1 and HBB. P50. A method of treating a subject having or suspected of having a disease or disorder, the method comprising administering the system according to any one of potential claims P40–P47, ex vivo, to a cell from the subject. P51. The method of potential claim P50, wherein the disease or disorder is associated with a mutation at a locus selected from the group consisting of HPS1 and HBB.

[0168] The embodiments of the invention described above are intended to be merely exemplary; numerous variations and modifications will be apparent to those skilled in the art. All such variations and modifications are intended to be within the scope of the present invention as defined in any appended claims.

Claims

What is claimed is:

1. A programmable genome editing system for modification of a double-stranded target DNA sequence comprising a target strand having a target sequence, and a non-target strand having a sequence complementary to the target sequence, the system comprising: a nucleic acid programmable nuclease, and a guide RNA comprising, in a 5′ to 3′ direction, a spacer sequence having a region of complementarity to the target sequence; wherein the spacer sequence comprises a base substitution in the region of complementarity to the target sequence, the base substitution being selected to reduce hydrogen bonding potential of the spacer sequence with the target sequence, thereby reducing off-target activity of the programmable genome editing system.

2. The programmable genome editing system of claim 1, wherein the base substitution is a non- canonical base.

3. The programmable genome editing system of claim 1, wherein the base substitution is a base selected from the group consisting of N4-ethyl-cytosine, N4-methyl-cytosine, N6-ethyl-adenine, N6-methyl-adenine, hypoxanthine, and combinations thereof.

4. The programmable genome editing system of claim 1, wherein the base substitution is a base selected from the group consisting of N4-ethyl-cytosine, N4-methyl-cytosine, N6-ethyl-adenine, N6-methyl-adenine, and combinations thereof.

5. The programmable genome editing system of claim 4, wherein the base substitution further includes hypoxanthine.

6. The programmable genome editing system of claim 1, wherein the base substitution is a substitution selected from the group consisting of cytosine to N4-ethyl-cytosine, cytosine to N4- methyl-cytosine, adenine to N6-ethyl-adenine, adenine to N6-methyl-adenine, guanine tohypoxanthine (wherein no base of the spacer sequence other than guanine is substituted with hypoxanthine), and combinations thereof.

7. The programmable genome editing system of claim 1, wherein the base substitution is a substitution selected from the group consisting of cytosine to N4-ethyl-cytosine, cytosine to N4- methyl-cytosine, adenine to N6-ethyl-adenine, adenine to N6-methyl-adenine, and combinations thereof.

8. The programmable genome editing system of claim 7, wherein the base substitution further includes a guanine to hypoxanthine base substitution, wherein no base of the spacer sequence other than guanine is substituted with hypoxanthine.

9. The programmable genome editing system of claim 6, wherein the nucleic acid programmable nuclease is selected from the group consisting of Cas9, HiFiCas9, eSpCas9, SpCas9-HF1, Hypa- SpCas9, SuperFi-Cas9, Sniper-Cas9, evoCas9, and Cas12.

10. A method for site-specific modification of the double-stranded target DNA sequence comprising the target strand and the non-target strand, the method comprising: contacting the double-stranded target DNA sequence with the programmable genome editing system of claim 6, wherein the contacting results in cleavage of (i) the target sequence of the target strand and (ii) the sequence complementary to the target sequence of the non-target strand.

11. The method of claim 10, wherein the double-stranded target DNA sequence comprises a mutation at a locus selected from the group consisting of HPS1 and HBB.

12. A method of treating a subject having or suspected of having a disease or disorder, the method comprising administering the programmable genome editing system of claim 6, ex vivo, to a cell from the subject.

13. The method of claim 12, wherein the disease or disorder is associated with a mutation at a locus selected from the group consisting of HPS1 and HBB.

14. A programmable prime editing system for modification of a double-stranded target DNA sequence comprising a target strand having a target sequence, and a complementary non-target strand, the system comprising: a prime editor protein, the prime editor protein being a fusion protein comprising a nucleic acid programmable DNA binding domain fused to a reverse transcriptase domain, the DNA binding domain having nickase activity, and a pegRNA comprising, in a 5′ to 3′ direction: (i) a spacer sequence comprising a region of complementarity to the target sequence; (ii) a gRNA core that interacts with the DNA binding domain; and (iii) an extension arm, the extension arm comprising, in a 5′ to 3′ orientation: (a) a DNA synthesis template encoding one or more nucleotide changes compared to a region downstream of a nick site in the non-target strand of the double-stranded target DNA sequence, and (b) a primer binding sequence comprising a region of complementarity to a primer sequence upstream of the nick site in the non-target strand of the double- stranded target DNA sequence; wherein the spacer sequence comprises a base substitution in the region of complementarity to the target sequence, the base substitution being selected to reduce hydrogen bonding potential of the spacer sequence with the target sequence, thereby reducing off-target activity of the programmable prime editing system.

15. The programmable prime editing system of claim 14, wherein the base substitution is a non- canonical base.

16. The programmable prime editing system of claim 14, wherein the base substitution is a base selected from the group consisting of N4-ethyl-cytosine, N4-methyl-cytosine, N6-ethyl-adenine, N6-methyl-adenine, hypoxanthine, and combinations thereof.

17. The programmable prime editing system of claim 14, wherein the base substitution is a base selected from the group consisting of N4-ethyl-cytosine, N4-methyl-cytosine, N6-ethyl-adenine, N6-methyl-adenine, and combinations thereof.

18. The programmable prime editing system of claim 17, wherein the base substitution further includes hypoxanthine.

19. The programmable prime editing system of claim 14, wherein the base substitution is a substitution selected from the group consisting of cytosine to N4-ethyl-cytosine, cytosine to N4- methyl-cytosine, adenine to N6-ethyl-adenine, adenine to N6-methyl-adenine, guanine to hypoxanthine (wherein no base of the spacer sequence other than guanine is substituted with hypoxanthine), and combinations thereof.

20. The programmable prime editing system of claim 14, wherein the base substitution is a substitution selected from the group consisting of cytosine to N4-ethyl-cytosine, cytosine to N4- methyl-cytosine, adenine to N6-ethyl-adenine, adenine to N6-methyl-adenine, and combinations thereof.

21. The programmable prime editing system of claim 20, wherein the base substitution further includes a guanine to hypoxanthine base substitution, wherein no base of the spacer sequence other than guanine is substituted with hypoxanthine.

22. A method for site-specific modification of the double-stranded target DNA sequence comprising the target strand and the non-target strand, the method comprising: contacting the double-stranded target DNA sequence with the programmable prime editing system of claim 19, wherein the contacting results in: nicking the non-target strand of the double-stranded target DNA sequence to form a free 3′ end at the nick site;annealing the primer binding sequence with the primer sequence upstream of the nick site in the non-target strand of the double-stranded target DNA; synthesizing a single strand of DNA encoded by the DNA synthesis template from the free 3′ end of the non-target strand of the double-stranded target DNA sequence; and replacing the region downstream of the nick site in the non-target strand of the double- stranded target DNA sequence with the single strand of DNA encoded by the DNA synthesis template, thereby modifying the sequence of the double-stranded target DNA sequence.

23. The method of claim 22, wherein the double-stranded target DNA sequence comprises a mutation at a locus selected from the group consisting of HPS1 and HBB.

24. A method of treating a subject having or suspected of having a disease or disorder, the method comprising administering the programmable prime editing system of claim 19, ex vivo, to a cell from the subject.

25. The method of claim 24, wherein the disease or disorder is associated with a mutation at a locus selected from the group consisting of HPS1 and HBB.

26. A programmable base editing system for modification of a double-stranded target DNA sequence comprising a target strand having a target sequence, and a non-target strand having a sequence complementary to the target sequence, the system comprising: a base editor protein, the base editor protein being a fusion protein comprising a nucleic acid programmable DNA binding domain fused to a nucleoside deaminase domain, the DNA binding domain having nickase activity, and a guide RNA comprising, in a 5′ to 3′ direction, a spacer sequence having a region of complementarity to the target sequence; wherein the spacer sequence comprises a base substitution in the region of complementarity to the target sequence, the base substitution being selected to reduce hydrogen bonding potential of the spacer sequence with the target sequence, thereby reducing off-target activity of the programmable base editing system.

27. The programmable base editing system of claim 26, wherein the base substitution is a non- canonical base.

28. The programmable base editing system of claim 26, wherein the base substitution is a base selected from the group consisting of N4-ethyl-cytosine, N4-methyl-cytosine, N6-ethyl-adenine, N6-methyl-adenine, hypoxanthine, and combinations thereof.

29. The programmable base editing system of claim 26, wherein the base substitution is a base selected from the group consisting of N4-ethyl-cytosine, N4-methyl-cytosine, N6-ethyl-adenine, N6-methyl-adenine, and combinations thereof.

30. The programmable base editing system of claim 29, wherein the base substitution further includes hypoxanthine.

31. The programmable base editing system of claim 26, wherein the base substitution is a substitution selected from the group consisting of cytosine to N4-ethyl-cytosine, cytosine to N4- methyl-cytosine, adenine to N6-ethyl-adenine, adenine to N6-methyl-adenine, guanine to hypoxanthine (wherein no base of the spacer sequence other than guanine is substituted with hypoxanthine), and combinations thereof.

32. The programmable base editing system of claim 26, wherein the base substitution is a substitution selected from the group consisting of cytosine to N4-ethyl-cytosine, cytosine to N4- methyl-cytosine, adenine to N6-ethyl-adenine, adenine to N6-methyl-adenine, and combinations thereof.

33. The programmable base editing system of claim 32, wherein the base substitution further includes a guanine to hypoxanthine base substitution, wherein no base of the spacer sequence other than guanine is substituted with hypoxanthine.

34. The programmable base editing system of claim 26, wherein the base editor protein comprises a uracil glycosylase inhibitor (UGI) domain and the nucleoside deaminase domain is a cytosine deaminase.

35. The programmable base editing system of claim 26, wherein the nucleoside deaminase domain is an adenine deaminase.

36. A method for site-specific modification of the double-stranded target DNA sequence comprising the target strand and the non-target strand, the method comprising: contacting the double-stranded target DNA sequence with the programmable base editing system of claim 31, wherein the contacting results in: nicking the target sequence of the target strand; and deaminating a nucleobase of the sequence complementary to the target sequence, the nucleobase being selected from the group consisting of cytosine and adenine.

37. The method of claim 36, wherein the double-stranded target DNA sequence comprises a mutation at a locus selected from the group consisting of HPS1 and HBB.

38. A method of treating a subject having or suspected of having a disease or disorder, the method comprising administering the programmable base editing system of claim 31, ex vivo, to a cell from the subject.

39. The method of claim 38, wherein the disease or disorder is associated with a mutation at a locus selected from the group consisting of HPS1 and HBB.

40. A system for modification of a double-stranded target DNA sequence of a double-stranded target DNA sequence comprising a target strand having a target sequence, and a non-target strand having a sequence complementary to the target sequence, the system comprising: a nucleic acid programmable DNA binding protein; anda guide RNA comprising, in a 5′ to 3′ direction, a spacer sequence having a region of complementarity to the target sequence; wherein the spacer sequence comprises a base substitution in the region of complementarity to the target sequence, the base substitution being selected to reduce hydrogen bonding potential of the spacer sequence with the target sequence, thereby reducing off-target activity of the system.

41. The system of claim 40, wherein the base substitution is a non-canonical base.

42. The system of claim 40, wherein the base substitution is a base selected from the group consisting of N4-ethyl-cytosine, N4-methyl-cytosine, N6-ethyl-adenine, N6-methyl-adenine, hypoxanthine, and combinations thereof.

43. The system of claim 40, wherein the base substitution is a base selected from the group consisting of N4-ethyl-cytosine, N4-methyl-cytosine, N6-ethyl-adenine, N6-methyl-adenine, and combinations thereof.

44. The system of claim 43, wherein the base substitution further includes hypoxanthine.

45. The system of claim 40, wherein the base substitution is a substitution selected from the group consisting of cytosine to N4-ethyl-cytosine, cytosine to N4-methyl-cytosine, adenine to N6-ethyl-adenine, adenine to N6-methyl-adenine, guanine to hypoxanthine (wherein no base of the spacer sequence other than guanine is substituted with hypoxanthine), and combinations thereof.

46. The system of claim 40, wherein the base substitution is a substitution selected from the group consisting of cytosine to N4-ethyl-cytosine, cytosine to N4-methyl-cytosine, adenine to N6-ethyl-adenine, adenine to N6-methyl-adenine, and combinations thereof.

47. The system of claim 46, wherein the base substitution further includes a guanine to hypoxanthine base substitution, wherein no base of the spacer sequence other than guanine is substituted with hypoxanthine.

48. A method for site-specific modification of the double-stranded target DNA sequence comprising the target strand and the non-target strand, the method comprising: contacting the double-stranded target DNA sequence with the system of claim 45, wherein the contacting results in specific modification of the double-stranded target DNA sequence.

49. The method of claim 48, wherein the double-stranded target DNA sequence comprises a mutation at a locus selected from the group consisting of HPS1 and HBB.

50. A method of treating a subject having or suspected of having a disease or disorder, the method comprising administering the system of claim 45, ex vivo, to a cell from the subject.

51. The method of claim 50, wherein the disease or disorder is associated with a mutation at a locus selected from the group consisting of HPS1 and HBB.

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