Optimized mrnas encoding casx proteins

Abstract

Provided herein are optimized messenger ribonucleic acids (mRNA) sequences encoding CasX proteins for modifying a cell, in vitro, in vivo, or ex vivo. The mRNA sequences may be optimized for improved protein expression, reduced immunogenicity, increased stability, and enhanced manufacturability. Also provided are methods for delivery of the optimized mRNA sequences.

Description

Attorney Docket No. SCRB-061 / 01WO 333322-2448 EXAMPLES Example 1: Design of optimized coding sequences encoding CasX

[0193] This example describes methods used to design optimized mRNA sequences encoding CasX. Briefly, an algorithm was developed to generate optimized sequences encoding CasX based on codon usage, GC content, secondary structure, and other sequence considerations. The designs of the 5’UTR, 3’UTR, and poly(A) tail sequences are discussed in Examples 2-4, below. Design of optimized coding sequences encoding CasX:

[0194] Protein coding sequences can be optimized based on features including codon usage, GC content, secondary structure, CpG frequency, and uridine frequency. As described in detail below, a computational strategy was developed to generate candidate CasX coding sequences that are predicted to have high translation efficiency and / or high stability to mRNA degradation. Specifically, a strategy was developed to optimize the CasX variant protein 515 coding sequence based on codon usage, GC content, and free energy of folding at specific positions within the coding sequence, as described in detail below, and summarized in FIG.1.

[0195] Three CasX coding sequences were used as benchmarks in the coding sequence design. First, the original sequence encoding CasX variant protein 515 sequence was used (SEQ ID NO: 78). Second, a codon-optimized version of the 515 coding sequence was created using the most frequently occurring human codon at each position (SEQ ID NO: 79). Finally, a 515 coding sequence was generated by sampling alternate sequences within regions of the coding sequence and choosing codons based on the top Codon Adaptation Index (CAI) values while excluding rare codons, and was termed “maxCAI” (SEQ ID NO: 80). The CAI was calculated relative to the codon usage of all human genes, as shown in Table 1A. Table 1A: Reference human codon usage DNA codon EncodedAmino AcidCodon frequency in all human genesAGA Arg 0.2 AGG Arg 0.2 CGA Arg 0.11 CGC Arg 0.19 CGG Arg 0.21 73Attorney Docket No. SCRB-061 / 01WO 333322-2448 DNA codon EncodedAmino AcidCodon frequency in all human genesCGT Arg 0.08 CAC His 0.59 CAT His 0.41 AAA Lys 0.42 AAG Lys 0.58 GAC Asp 0.54 GAT Asp 0.46 GAA Glu 0.42 GAG Glu 0.58 AGC Ser 0.24 AGT Ser 0.15 TCA Ser 0.15 TCC Ser 0.22 TCG Ser 0.06 TCT Ser 0.18 ACA Thr 0.28 ACC Thr 0.36 ACG Thr 0.12 ACT Thr 0.24 AAC Asn 0.54 AAT Asn 0.46 CAA Gln 0.25 CAG Gln 0.75 TGC Cys 0.55 TGT Cys 0.45 GGA Gly 0.25 GGC Gly 0.34 GGG Gly 0.25 74Attorney Docket No. SCRB-061 / 01WO 333322-2448 DNA codon EncodedAmino AcidCodon frequency in all human genesGGT Gly 0.16 CCA Pro 0.27 CCC Pro 0.33 CCG Pro 0.11 CCT Pro 0.28 GCA Ala 0.23 GCC Ala 0.4 GCG Ala 0.11 GCT Ala 0.26 ATA Ile 0.16 ATC Ile 0.48 ATT Ile 0.36 CTA Leu 0.07 CTC Leu 0.2 CTG Leu 0.41 CTT Leu 0.13 TTA Leu 0.07 TTG Leu 0.13 ATG Met 1 TTC Phe 0.55 TTT Phe 0.45 TGG Trp 1 TAC Tyr 0.57 TAT Tyr 0.43 GTA Val 0.11 GTC Val 0.24 GTG Val 0.47 GTT Val 0.18 75Attorney Docket No. SCRB-061 / 01WO 333322-2448 DNA codon EncodedAmino AcidCodon frequency in all human genesTAA stop codon 0.28 TAG stop codon 0.2 TGA stop codon 0.52

[0196] mRNA translation efficiency is a function of translation initiation efficiency at the 5’ end of the mRNA and conversion into translation elongation complexes. To model this spatial dependence into the CasX coding sequence optimizations, a first set of optimized CasX coding sequences was designed by engineering just the 5’ end of the coding sequence (see left side of FIG.1). It is anticipated that the 5’ end of the coding sequence has a larger effect on translation speed and efficiency than does the 3’ end. Further, it is believed that certain properties that might be beneficial for mRNA stability and translation efficiency at certain positions might have negative effects if applied to the entire coding sequence. For example, reducing GC content or secondary structure stability in an mRNA overall may be detrimental to translation initiation, while a localized reduction in GC content or secondary structure stability in just the 5’ end of a coding sequence may be helpful for initiating translation efficiently. Similarly, ribosome pausing in the 5’ end of a transcript is thought to be important for translation. Therefore, including low- frequency codons in the 5’ end of a coding sequence might be beneficial, but could be detrimental if included throughout the transcript.

[0197] First, a sequence encoding an N-terminal c-MYC nuclear localization signal (NLS), with the amino acid sequence of APAAKRVKLDSR (SEQ ID NO: 695), was optimized for minimum free energy, GC content, and CAI parameters. Specifically, two sequences were chosen that represented the maximum, median, or minimum value for each parameter. One duplicate sequence was removed, for a total of 17 optimized NLS sequences. CAIs for the sequences were calculated relative to the codon usage of all human genes (see Table 1A).

[0198] The DNA and RNA sequences of the optimized N-terminal c-MYC NLS are provided in Table 1B, below. Table 1C, below, provides a brief description of each of the optimized N- terminal c-MYC NLS coding sequences and the CAI score (relative to all human genes, see Table 1A), rare codon frequency, CpG frequency, GC frequency, uridine frequency, change in minimum free energy upon in silico folding (kcal / mol), and DegScore (see Leppek, K., et al., Nat Commun.2022 Mar 22;13(1):1536) for each NLS coding sequence. 76Attorney Docket No. SCRB-061 / 01WO 333322-2448 Table 1B: DNA and RNA sequences of optimized N-terminal c-MYC NLS sequences N-terminal SEQ ID DescrNO:DNA sequenceSEQ ID NO:NLS IDiption RNA sequence(RNA)(DNA)NT_NLS_1 Low MFE GCUCCGGCUGCCAA 8 GCTCCGGCTGCCA 25 GCGGGUGAAGCUG AGCGGGTGAAGC GAUAGCAGG TGGATAGCAGG NT_NLS_2 Low MFE GCUCCUGCUGCUAA 9 GCTCCTGCTGCTA 26 AAGGGUGAAGCUG AAAGGGTGAAGC GACAGCAGG TGGACAGCAGG NT_NLS_3 Mid MFE GCCCCAGCAGCUAA 10 GCCCCAGCAGCT 27 GCGGGUGAAACUG AAGCGGGTGAAA GACUCUCGG CTGGACTCTCGG NT_NLS_4 Mid MFE GCACCCGCUGCCAA 11 GCACCCGCTGCCA 28 GAGAGUGAAACUC AGAGAGTGAAAC GAUAGCCGG TCGATAGCCGG NT_NLS_5 High MFE GCCCCCGCAGCAAA 12 GCCCCCGCAGCA 29 ACGUGUAAAGUUA AAACGTGTAAAG GACAGUAGG TTAGACAGTAGG NT_NLS_6 High MFE GCCCCUGCAGCCAA 13 GCCCCTGCAGCCA 30 GCGAGUUAAACUG AGCGAGTTAAAC GACAGUCGA TGGACAGTCGA NT_NLS_7 Low GC GCACCAGCGGCAAA 14 GCACCAGCGGCA 31 AAGGGUUAAAUUA AAAAGGGTTAAA GACUCUCGA TTAGACTCTCGA NT_NLS_8 Low GC GCACCAGCCGCUAA 15 GCACCAGCCGCT 32 AAGGGUAAAACUC AAAAGGGTAAAA GACAGUAGA CTCGACAGTAGA NT_NLS_9 Mid GC GCUCCCGCGGCAAA 16 GCTCCCGCGGCA 33 AAGAGUCAAACUG AAAAGAGTCAAA GACAGCCGC CTGGACAGCCGC NT_NLS_10 High GC GCCCCGGCCGCCAA 17 GCCCCGGCCGCC 34 GCGCGUGAAGUUG AAGCGCGTGAAG GACUCGCGC TTGGACTCGCGC NT_NLS_11 High GC GCCCCCGCUGCGAA 18 GCCCCCGCTGCGA 35 GAGGGUGAAGCUU AGAGGGTGAAGC GACUCGCGG TTGACTCGCGG NT_NLS_12 Low CAI, no GCUCCGGCAGCUAA 19 GCTCCGGCAGCTA 36 rare codons GCGUGUCAAGCUC AGCGTGTCAAGCT GAUAGCCGG CGATAGCCGG NT_NLS_13 Low CAI, no GCUCCUGCCGCAAA 20 GCTCCTGCCGCAA 37 rare codons GCGAGUUAAACUU AGCGAGTTAAAC 77Attorney Docket No. SCRB-061 / 01WO 333322-2448 N-terminal SEQ ID Description RNANO:DNA sequenceSEQ ID NO:NLS IDsequence(RNA)(DNA)GACAGUCGC TTGACAGTCGC NT_NLS_14 Mid CAI, no GCACCUGCCGCCAA 21 GCACCTGCCGCCA 38 rare codons GCGAGUGAAAUUG AGCGAGTGAAAT GAUUCCAGG TGGATTCCAGG NT_NLS_15 Mid CAI, no GCACCUGCCGCUAA 22 GCACCTGCCGCTA 39 rare codons GAGGGUGAAAUUG AGAGGGTGAAAT GAUUCUCGC TGGATTCTCGC NT_NLS_16 High CAI, no GCUCCAGCAGCCAA 23 GCTCCAGCAGCC 40 rare codons GCGCGUGAAGCUG AAGCGCGTGAAG GAUUCCCGG CTGGATTCCCGG NT_NLS_17 High CAI, no GCCCCCGCCGCCAA 24 GCCCCCGCCGCCA 41 rare codons GAGAGUCAAACUG AGAGAGTCAAAC GACUCAAGG TGGACTCAAGG Table 1C: Properties of optimized N-terminal c-MYC NLS sequences Δ Minimu Rare m free N- codon CpG GC Uridine energy terminal Descripti CAI frequenc frequenc frequenc frequenc (kcal / mo DegSco NLS ID on score y y y y l) re NT_NLS_ Low MFE 0.834622 0 0.057142 0.666666 0.138888 -14.2 12.972 1 4 86 67 89 NT_NLS_ Low MFE 0.855202 0 0 0.583333 0.166666 -11.7 15.47 2 4 33 67 NT_NLS_ Mid MFE 0.861045 0 0.057142 0.638888 0.138888 -6.4 13.846 3 6 86 89 89 NT_NLS_ Mid MFE 0.830141 0 0.085714 0.611111 0.111111 -6.5 14.294 4 2 29 11 11 NT_NLS_ High MFE 0.599828 0.166666 0.057142 0.527777 0.138888 -1.2 15.997 5 8 67 86 78 89 NT_NLS_ High MFE 0.730793 0 0.057142 0.583333 0.138888 -2.1 17.062 6 3 86 33 89 NT_NLS_ Low GC 0.560154 0.166666 0.057142 0.472222 0.166666 -5.5 15.959 7 6 67 86 22 67 NT_NLS_ Low GC 0.683402 0.083333 0.057142 0.5 0.111111 -3.2 16.614 8 1 33 86 11 NT_NLS_ Mid GC 0.732123 0.083333 0.085714 0.611111 0.083333 -8.3 13.697 9 9 33 29 11 33 NT_NLS_ High GC 0.726530 0.083333 0.171428 0.75 0.111111 -8 13.987 10 6 33 57 11 NT_NLS_ High GC 0.698521 0.166666 0.114285 0.694444 0.138888 -8.7 12.618 11 4 67 71 44 89 78Attorney Docket No. SCRB-061 / 01WO 333322-2448 Δ Minimu Rare m free N- codon CpG GC Uridine energy terminal Descripti CAI frequenc frequenc frequenc frequenc (kcal / mo DegSco NLS ID on score y y y y l) re NT_NLS_ Low CAI, 0.657653 0 0.114285 0.638888 0.166666 -11.1 15.768 12 no 8 71 89 67 rare codons NT_NLS_ Low CAI, 0.670523 0 0.085714 0.555555 0.194444 -5.2 15.642 13 no 6 29 56 44 rare codons NT_NLS_ Mid CAI, 0.770290 0 0.057142 0.583333 0.166666 -7.1 16.352 14 no 2 86 33 67 rare codons NT_NLS_ Mid CAI, 0.764859 0 0.057142 0.555555 0.222222 -5.7 16.296 15 no 86 56 22 rare codons NT_NLS_ High CAI, 0.880126 0 0.085714 0.666666 0.138888 -8.8 14.531 16 no 4 29 67 89 rare codons NT_NLS_ High CAI, 0.877916 0 0.057142 0.638888 0.083333 -6.6 14.479 17 no 1 86 89 33 rare codons

[0199] Separately, sequences encoding the N-terminal region of the CasX protein were optimized. For these optimizations, the 5’ end of the coding sequence was defined as the first 150 nucleotides, encoding the first 50 amino acids of the CasX protein. This 5’ end of the coding sequence was optimized for folding stability (minimum free energy), GC content, and CAI parameters. Specifically, the first 150 nucleotides were split into two 75-nucleotide stretches, 100 alternate sequences were generated for each 75-nucleotide region by sampling from codons at the naturally occurring frequency in the human transcriptome, and two sequences were chosen that represented the maximum, median, or minimum value for each parameter. CAIs were calculated relative to the codon usage of all human genes. Each of these six N-terminal regions was combined with sequences encoding the C-terminal 928 amino acids from the three CasX variant 515 benchmark sequences described above, as diagrammed in FIG.1.

[0200] The DNA and RNA sequences of the optimized 5’ region of the coding sequences, encoding the N-terminal portion of CasX (but not the NLS), are provided in Table 1D. Table 1E, below, provides a brief description of each of the optimized 5’ region coding sequences and the CAI score (relative to all human genes, see Table 1A), rare codon frequency, CpGAttorney Docket No. SCRB-061 / 01WO 333322-2448 frequency, GC frequency, uridine frequency, change in minimum free energy upon in silico folding (kcal / mol), and DegScore for each coding sequence. Table 1D: DNA and RNA sequences of optimized coding sequences encoding the N- terminal portion (first 150 nucleotides) of CasX variant 515 NT_CasX CDS ID Parameter(s) optimized SEQ ID NO: (RNA) SEQ ID NO: (DNA) NT_CasX_CDS_1 Low MFE 42 60 NT_CasX_CDS_2 Low MFE 43 61 NT_CasX_CDS_3 Mid MFE 44 62 NT_CasX_CDS_4 Mid MFE 45 63 NT_CasX_CDS_5 High MFE 46 64 NT_CasX_CDS_6 High MFE 47 65 NT_CasX_CDS_7 Low GC 48 66 NT_CasX_CDS_8 Low GC 49 67 NT_CasX_CDS_9 Mid GC 50 68 NT_CasX_CDS_10 Mid GC 51 69 NT_CasX_CDS_11 High GC 52 70 NT_CasX_CDS_12 High GC 53 71 NT_CasX_CDS_13 Low CAI, no rare codons 54 72 NT_CasX_CDS_14 Low CAI, no rare codons 55 73 NT_CasX_CDS_15 Mid CAI, no rare codons 56 74 NT_CasX_CDS_16 Mid CAI, no rare codons 57 75 NT_CasX_CDS_17 High CAI, no rare codons 58 76 NT_CasX_CDS_18 High CAI, no rare codons 59 77Attorney Docket No. SCRB-061 / 01WO 333322-2448 Table 1E: Properties of optimized coding sequences encoding the N-terminal portion of CasX variant 515 Δ Rare scriptio CAI codon CpG GC Ur Minimu NT_CasX De idine m free DegSco CDS ID n score frequenc frequenc frequenc frequenc y y energy re y y (kcal / mol) NT_CasXLow0.827528 0.067114 0.526666 0.166666 _CDS_1MFE65 0 09 67 67 -46.9 61.485 NT_CasXLow MFE0.762382 0.087248 0.526666 0.146666 _CDS_293 0 32 67 67 -48 59.883 NT_CasXMid MFE0.720660 0.060402 0.166666 _CDS_332 0.04 68 0.48 67 -38.9 63.81 NT_CasX 0.671422 0.060402 4Mid M0.486666 0.173333 _CDS_FE48 0.06 68 67 33 -30.1 64.45 NT_CasXHigh M0.767949 0.033557 0.466666 0.153333 _CDS_5FE16 0.02 05 67 33 -22.6 65.271 NT_CasX 0.792082 0.040268 0.446666 0.1666 _6High66 _CDSMFE5 0 46 67 67 -20.7 65.244 NT_CasX 0.678835 0.02 _7Lo0134 _CDSw GC82 0.06 23 0.38 0.16 -20.3 63.184 NT_CasX 0.714679 0.046979 0.413333 0.146666 _CDS_8Low GC77 0.08 87 33 67 -23.3 64.438 NT_CasXMi0.722785 0.053691 0.173333 _CDS_9d GC39 0.02 28 0.48 33 -37.8 63.525 NT_CasXMi0.758977 0.053691 0.153333 _CDS_10d GC77 0.06 28 0.48 33 -34.7 62.952 NT_CasX 0.813730 0.087248 0.573333 0.126666 _CDS_11High GC64 0.02 32 33 67 -40.3 56.657 NT_CasX 0.808268 0.087248 0.133333 _CDS_12High GC66 0 32 0.56 33 -49.5 61.221 NT_CasX Low CAI, _CDS_13 no rare 0.697120 0.067114 0.166666 codons 2 0 09 0.5 67 -42.9 62.061 NT_CasX Low CAI, _CDS_14 no rare 0.700113 0.067114 0.486666 0.173333 codons 22 0 09 67 33 -34.2 65.416 NT_CasX Mid CAI, _CDS_15 no rare 0.797022 0.046979 0.133333 codons 36 0 87 0.5 33 -30.1 64.884 NT_CasX Mid CAI, _CDS_16 no rare 0.795122 0.033557 0.146666 codons 26 0 05 0.5 67 -37.1 60.841 NT_CasX High CAI, _CDS_17 no rare 0.893834 0.040268 0.126666 codons 09 0 46 0.52 67 -33.5 61.485 NT_CasX High CAI, _CDS_18 no rare 0.881172 0.067114 0.126666 codons 41 0 09 0.56 67 -50 58.21

[0201] In another separate approach, nine full-length coding sequences of CasX variant 515 were generated with a range of properties. In contrast to the 5’ end optimization approach asAttorney Docket No. SCRB-061 / 01WO 333322-2448 described above, these sequences were generated by optimizing the full-length of the coding sequence. Specifically, the full-length coding sequences were split into 75-nucleotide stretches, and 100 alternate sequences were generated for each 75-nucleotide region using the sampling method described above. Three sequences were chosen for each 75-nucleotide region that represented the maximum CAI, maximum free energy, or minimum free energy, which were combined to generate full-length sequences. CAIs were calculated relative to the codon usage of all human genes. Any duplicate sequences generated by the approaches described above were discarded. Taken together, 64 full-length sequences encoding CasX variant 515 were generated.

[0202] The DNA and RNA sequences of the 515 coding full-length sequences (but not the NLS) are provided in Table 1F. Table 1G, below, provides a brief description of each of the CasX variant 515 coding sequences and the CAI score (relative to all human genes, see Table 1A), rare codon frequency, CpG frequency, GC frequency, uridine frequency, change in minimum free energy upon in silico folding (kcal / mol), and DegScore for each coding sequence. In the Description column of Table 1F, a “+” indicates sequences in which an optimized 5’ region coding sequence was combined with the 3’ end of a benchmark sequence. Table 1F: DNA and RNA sequences of optimized full-length CasX 515 coding sequences CasX CDS ID Description SEQ ID NO: (RNA) SEQ ID NO: (DNA) CasX515_CDS_1 Original sequence (“original”) 78 142 CasX515_CDS_2 Most frequently used human codons 79 143 (“highcodonfreq”) CasX515_CDS_3 Maximum CAI (“maxCAI”) 80 144 CasX515_CDS_4 Low MFE + original 81 145 CasX515_CDS_5 Low MFE + original 82 146 CasX515_CDS_6 Low MFE + highcodonfreq 83 147 CasX515_CDS_7 Low MFE + highcodonfreq 84 148 CasX515_CDS_8 Low MFE + maxCAI 85 149 CasX515_CDS_9 Low MFE + maxCAI 86 150 CasX515_CDS_10 Mid MFE + original 87 151 CasX515_CDS_11 Mid MFE + original 88 152 CasX515_CDS_12 Mid MFE + highcodonfreq 89 153 82Attorney Docket No. SCRB-061 / 01WO 333322-2448 CasX CDS ID Description SEQ ID NO: (RNA) SEQ ID NO: (DNA) CasX515_CDS_13 Mid MFE + highcodonfreq 90 154 CasX515_CDS_14 Mid MFE + maxCAI 91 155 CasX515_CDS_15 Mid MFE + maxCAI 92 156 CasX515_CDS_16 High MFE + original 93 157 CasX515_CDS_17 High MFE + original 94 158 CasX515_CDS_18 High MFE + highcodonfreq 95 159 CasX515_CDS_19 High MFE + highcodonfreq 96 160 CasX515_CDS_20 High MFE + maxCAI 97 161 CasX515_CDS_21 High MFE + maxCAI 98 162 CasX515_CDS_22 Low GC + original 99 163 CasX515_CDS_23 Low GC + original 100 164 CasX515_CDS_24 Low GC + highcodonfreq 101 165 CasX515_CDS_25 Low GC + highcodonfreq 102 166 CasX515_CDS_26 Low GC + maxCAI 103 167 CasX515_CDS_27 Low GC + maxCAI 104 168 CasX515_CDS_28 Mid GC + original 105 169 CasX515_CDS_29 Mid GC + original 106 170 CasX515_CDS_30 Mid GC + highcodonfreq 107 171 CasX515_CDS_31 Mid GC + highcodonfreq 108 172 CasX515_CDS_32 Mid GC + maxCAI 109 173 CasX515_CDS_33 Mid GC + maxCAI 110 174 CasX515_CDS_34 High GC + original 111 175 CasX515_CDS_35 High GC + original 112 176 CasX515_CDS_36 High GC + highcodonfreq 113 177 CasX515_CDS_37 High GC + highcodonfreq 114 178 CasX515_CDS_38 High GC + maxCAI 115 179 CasX515_CDS_39 High GC + maxCAI 116 180 CasX515_CDS_40 Low CAI, no rare codons + original 117 181 83Attorney Docket No. SCRB-061 / 01WO 333322-2448 CasX CDS ID Description SEQ ID NO: (RNA) SEQ ID NO: (DNA) CasX515_CDS_41 Low CAI, no rare codons + original 118 182 CasX515_CDS_42 Low CAI, no rare codons + 119 183 highcodonfreq CasX515_CDS_43 Low CAI, no rare codons + 120 184 highcodonfreq CasX515_CDS_44 Low CAI, no rare codons + maxCAI 121 185 CasX515_CDS_45 Low CAI, no rare codons + maxCAI 122 186 CasX515_CDS_46 Mid CAI, no rare codons + original 123 187 CasX515_CDS_47 Mid CAI, no rare codons + original 124 188 CasX515_CDS_48 Mid CAI, no rare codons + 125 189 highcodonfreq CasX515_CDS_49 Mid CAI, no rare codons + 126 190 highcodonfreq CasX515_CDS_50 Mid CAI, no rare codons + maxCAI 127 191 CasX515_CDS_51 Mid CAI, no rare codons + maxCAI 128 192 CasX515_CDS_52 High CAI, no rare codons + original 129 193 CasX515_CDS_53 High CAI, no rare codons + original 130 194 CasX515_CDS_54 High CAI, no rare codons + 131 195 highcodonfreq CasX515_CDS_55 High CAI, no rare codons + 132 196 highcodonfreq CasX515_CDS_56 High CAI, no rare codons + 133 197 maxCAI CasX515_CDS_57 MaxCAI, no rare codons 134 198 CasX515_CDS_58 MaxCAI, no rare codons 135 199 CasX515_CDS_59 Max MFE, no rare codons 136 200 CasX515_CDS_60 Max MFE, no rare codons 137 201 CasX515_CDS_61 Max MFE, no rare codons 138 202 CasX515_CDS_62 Min MFE, no rare codons 139 203 CasX515_CDS_63 Min MFE, no rare codons 140 204 CasX515_CDS_64 Min MFE, no rare codons 141 205 84Attorney Docket No. SCRB-061 / 01WO 333322-2448 Table 1G: Properties of optimized CasX 515 coding sequences Δ CasX CDS CAI Rare codon CpG GC Uridine Minimum frequenc frequenc frequenc fre DegSco IDDescriptionscore frequency e y y y energy re (kcal / mol) Original CasX515_CD sequence 0.937326 0.042277 0.553169 0.150647 1125.02 S_1 (“original”) 49 0 53 73 58 -945.6 2 Most frequently used human codons CasX515_CD (“highcodonfre 0.992142 0.049437 0.599863 0.123721 1046.50 S_2 q”) 1 0 44 67 88 -1038.4 4 Maximum CasX515_CD CAI 0.893131 0.048414 0.543967 0.174164 1143.46 S_3 (“maxCAI”) 63 0 59 28 96 -985.3 8 CasX515_CD Low MFE + 0.933098 0.044664 0.553851 0.154055 1127.52 S_4 original 45 0 17 4 9 -972.3 7 CasX515_CD Low MFE + 0.929220 0.045687 0.553851 0.153033 1123.39 S_5 original 35 0 01 4 4 -953.6 2 CasX515_CD Low MFE + 0.983053 0.051824 0.598841 0.127471 1065.17 S_6 highcodonfreq 51 0 07 17 03 -1057.4 2 CasX515_CD Low MFE + 0.978967 0.052846 0.598841 0.126448 1062.34 S_7 highcodonfreq 79 0 91 17 53 -1044.8 1 CasX515_CD Low MFE + 0.889641 0.049778 0.544308 0.176209 1133.64 S_8 maxCAI 86 0 38 11 95 -981 9 CasX515_CD Low MFE + 0.885944 0.050801 0.544308 0.175187 1145.10 S_9 maxCAI 37 0 23 11 46 -976 5 CasX515_CD Mid MFE + 0.926567 0.002044 0.044323 0.551465 0.154055 1129.83 S_10 original 76 99 22 58 9 -942.7 9 CasX515_CD Mid MFE + 0.923243 0.003067 0.044323 0.551806 0.154396 1130.42 S_11 original 07 48 22 41 73 -951.7 1 CasX515_CD Mid MFE + 0.976173 0.002044 0.051483 0.596455 0.127471 1073.66 S_12 highcodonfreq 19 99 12 35 03 -1039 5 CasX515_CD Mid MFE + 0.972670 0.003067 0.051483 0.596796 0.127811 1068.74 S_13 highcodonfreq 5 48 12 18 86 -1020.8 6 CasX515_CD Mid MFE + 0.883415 0.002044 0.049437 0.541922 0.176209 1156.76 S_14 maxCAI 32 99 44 29 95 -969.1 6 CasX515_CD Mid MFE + 0.880245 0.003067 0.049437 0.542263 0.176550 1156.63 S_15 maxCAI 47 48 44 12 78 -964 8 CasX515_CD High MFE + 0.929563 0.001022 0.042959 0.550783 0.153374 1140.07 S_16 original 76 49 43 91 23 -932.2 5 CasX515_CD High MFE + 0.931025 0.043300 0.549761 0.154055 S_17 original 86 0 38 42 9 -940 1140.6 CasX515_CD High MFE + 0.979329 0.001022 0.050119 0.595773 0.126789 S_18 highcodonfreq 59 49 33 69 37 -1013.2 1062.95 CasX515_CD High MFE + 0.980869 0.050460 0.594751 0.127471 1066.75 S_19 highcodonfreq 97 0 28 19 03 -1013.3 9 CasX515_CD High MFE + 0.886271 0.001022 0.048073 0.541240 0.175528 1148.42 S_20 maxCAI 79 49 64 63 29 -957.4 1 CasX515_CD High MFE + 0.887665 0.048414 0.540218 0.176209 S_21 maxCAI 8 0 59 13 95 -949.1 1149.61 85Attorney Docket No. SCRB-061 / 01WO 333322-2448 Δ CasX CDS CAI Rare codon CpG GC Uridine Minimum ncy frequenc frequenc f DegSco IDDescriptionscore freque requenc free y y y energy re (kcal / mol) CasX515_CD Low GC + 0.923758 0.003067 0.042277 0.546353 0.153715 1138.70 S_22 original 15 48 53 1 06 -922.3 1 CasX515_CD Low GC + 0.926175 0.004089 0.043641 0.548057 0.153033 1134.76 S_23 original 64 98 32 26 4 -937 6 CasX515_CD Low GC + 0.973213 0.003067 0.049437 0.591342 0.127130 1080.67 S_24 highcodonfreq 16 48 44 88 2 -1010.9 3 CasX515_CD Low GC + 0.975760 0.004089 0.050801 0.593047 0.126448 1075.85 S_25 highcodonfreq 08 98 23 03 53 -1018.8 6 CasX515_CD Low GC + 0.880736 0.003067 0.047391 0.536809 0.175869 1157.23 S_26 maxCAI 56 48 75 82 12 -947.3 9 CasX515_CD Low GC + 0.883041 0.004089 0.048755 0.538513 0.175187 1142.61 S_27 maxCAI 47 98 54 97 46 -949.2 9 CasX515_CD Mid GC + 0.926706 0.001022 0.043982 0.551465 0.154396 1133.77 S_28 original 35 49 27 58 73 -950.7 9 CasX515_CD Mid GC + 0.929009 0.003067 0.043982 0.551465 0.153374 1130.64 S_29 original 09 48 27 58 23 -948.6 3 CasX515_CD Mid GC + 0.976319 0.001022 0.051142 0.596455 0.127811 1070.39 S_30 highcodonfreq 2 49 18 35 86 -1029 1 CasX515_CD Mid GC + 0.978745 0.003067 0.051142 0.596455 0.126789 1063.17 S_31 highcodonfreq 22 48 18 35 37 -1037.6 9 CasX515_CD Mid GC + 0.883547 0.001022 0.049096 0.541922 0.176550 1141.91 S_32 maxCAI 45 49 49 29 78 -971.7 5 CasX515_CD Mid GC + 0.885742 0.003067 0.049096 0.541922 0.175528 1133.49 S_33 maxCAI 95 48 49 29 29 -974.6 2 CasX515_CD High GC + 0.932301 0.001022 0.045687 0.556237 0.152010 1122.83 S_34 original 86 49 01 22 91 -962.4 3 CasX515_CD High GC + 0.931982 0.045687 0.555555 0.152351 1124.41 S_35 original 99 0 01 56 74 -977.6 4 CasX515_CD High GC + 0.982214 0.001022 0.052846 0.601226 0.125426 1047.03 S_36 highcodonfreq 28 49 91 99 04 -1049.2 5 CasX515_CD High GC + 0.981878 0.052846 0.600545 0.125766 S_37 highcodonfreq 33 0 91 33 87 -1059.8 1040.98 CasX515_CD High GC + 0.888882 0.001022 0.050801 0.546693 0.174164 1151.15 S_38 maxCAI 37 49 23 93 96 -959.9 7 CasX515_CD High GC + 0.888578 0.050801 0.546012 0.174505 1150.43 S_39 maxCAI 35 0 23 27 79 -975.9 9 Low CAI, no rare CasX515_CD codons + 0.925006 0.044664 0.552488 0.154055 1138.08 S_40 original 09 0 17 07 9 -951.7 2 Low CAI, no rare CasX515_CD codons + 0.925207 0.044664 0.551806 0.154396 S_41 original 4 0 17 41 73 -945.9 1148.95 Low CAI, no rare CasX515_CD codons + 0.974527 0.051824 0.597477 0.127471 1057.50 S_42 highcodonfreq 91 0 07 85 03 -1047.3 8 Low CAI, no rare CasX515_CD codons + 0.051824 0.596796 0.127811 1029.67 S_43 highcodonfreq 0.97474 0 07 18 86 -1043.5 7 86Attorney Docket No. SCRB-061 / 01WO 333322-2448 Δ CasX CDS CAI Rare codon CpG GC Uridine Minimum fre DegSco IDDescriptionscore frequency quenc frequenc frequenc free y y y energy re (kcal / mol) Low CAI, no rare CasX515_CD codons + 0.881926 0.049778 0.542944 0.176209 1145.40 S_44 maxCAI 38 0 38 79 95 -973.8 6 Low CAI, no rare CasX515_CD codons + 0.882118 0.049778 0.542263 0.176550 1150.10 S_45 maxCAI 32 0 38 12 78 -977.8 1 Mid CAI, no rare CasX515_CD codons + 0.931319 0.043641 0.552488 0.152351 1126.92 S_46 original 92 0 32 07 74 -945 8 Mid CAI, no rare CasX515_CD codons + 0.931207 0.042959 0.552488 0.153033 1134.52 S_47 original 02 0 43 07 4 -963.2 6 Mid CAI, no rare CasX515_CD codons + 0.981179 0.050801 0.597477 0.125766 1054.61 S_48 highcodonfreq 77 0 23 85 87 -1040.5 9 Mid CAI, no rare CasX515_CD codons + 0.981060 0.050119 0.597477 0.126448 S_49 highcodonfreq 82 0 33 85 53 -1038.8 1067.01 Mid CAI, no rare CasX515_CD codons + 0.887946 0.048755 0.542944 0.174505 1156.83 S_50 maxCAI 16 0 54 79 79 -960.5 8 Mid CAI, no rare CasX515_CD codons + 0.887838 0.048073 0.542944 0.175187 1145.73 S_51 maxCAI 52 0 64 79 46 -982.2 7 High CAI, no rare CasX515_CD codons + 0.936758 0.043300 0.553510 0.152010 S_52 original 68 0 38 57 91 -961.4 1130.61 High CAI, no rare CasX515_CD codons + 0.936080 0.044664 0.555555 0.152010 S_53 original 09 0 17 56 91 -953.3 1125.3 High CAI, no rare CasX515_CD codons + 0.986909 0.050460 0.598500 0.125426 1051.54 S_54 highcodonfreq 7 0 28 34 04 -1039.9 7 High CAI, no rare CasX515_CD codons + 0.986194 0.051824 0.600545 0.125426 1038.44 S_55 highcodonfreq 78 0 07 33 04 -1047.9 4 High CAI, no rare CasX515_CD codons + 0.892484 0.049778 0.546012 0.174164 1150.04 S_56 maxCAI 64 0 38 27 96 -983.3 2 87Attorney Docket No. SCRB-061 / 01WO 333322-2448 Δ CasX CDS CpG GC Uridine Mini DeCAI Rare codon mum frequenc frequenc frequenc free DegSco IDscriptionscore frequency y y y energy re (kcal / mol) CasX515_CD MaxCAI, no 0.880206 0.049437 0.538854 0.179618 1150.33 S_57 rare codons 3 0 44 81 27 -970.8 8 CasX515_CD MaxCAI, no 0.870139 0.047050 0.522494 0.191888 1186.41 S_58 rare codons 06 0 8 89 21 -954.4 6 CasX515_CD Max MFE, no 0.811096 0.045005 0.519086 0.194955 1175.20 S_59 rare codons 05 0 11 57 69 -1087.1 4 CasX515_CD Max MFE, no 0.808438 0.046368 0.513292 0.205180 1201.89 S_60 rare codons 5 0 91 43 64 -1055.2 2 CasX515_CD Max MFE, no 0.823749 0.041936 0.520109 0.199727 1192.69 S_61 rare codons 19 0 58 07 33 -1016.5 2 CasX515_CD Min MFE, no 0.770749 0.462167 0.214723 1288.38 S_62 rare codons 91 0 0.039209 69 93 -754.7 7 CasX515_CD Min MFE, no 0.769336 0.043300 0.470006 0.210633 1253.89 S_63 rare codons 55 0 38 82 95 -794.9 2 CasX515_CD Min MFE, no 0.775079 0.040572 0.472392 0.216428 1256.31 S_64 rare codons 7 0 79 64 08 -792.2 2

[0203] The coding sequence optimizations described above were performed using CasX variant 515. CasX variant protein 812 differs from 515 at a single position (amino acid residue 329). Specifically, CasX 515 has a glycine residue at position 329, where CasX 812 has a lysine residue. Since the coding sequences for the two proteins are so similar, a G329K mutation was introduced into all CasX 515 designs as described above to generate optimized CasX 812 sequences. It is anticipated that the optimized CasX 515 and CasX 812 coding sequences will share similar properties due to the very high degree of overlap between the sequences. The DNA and RNA sequences of the CasX 812 coding sequences are provided in Table 1H. Table 1H: DNA and RNA sequences of optimized CasX 812 coding sequences CasX CDS ID Description SEQ ID NO: (RNA) SEQ ID NO: (DNA) CasX812_CDS_1 Original sequence (“original”) 206 270 CasX812_CDS_2 Most frequently used human codons 207 271 (“highcodonfreq”) CasX812_CDS_3 Maximum CAI (“maxCAI”) 208 272 CasX812_CDS_4 Low MFE + original 209 273 CasX812_CDS_5 Low MFE + original 210 274 CasX812_CDS_6 Low MFE + highcodonfreq 211 275 CasX812_CDS_7 Low MFE + highcodonfreq 212 276 88Attorney Docket No. SCRB-061 / 01WO 333322-2448 CasX CDS ID Description SEQ ID NO: (RNA) SEQ ID NO: (DNA)89Attorney Docket No. SCRB-061 / 01WO 333322-2448 A)CasX812_CDS_49 Mid CAI, no rare codons + highcodonfreq 254 318 CasX812_CDS_50 Mid CAI, no rare codons + maxCAI 255 319 CasX812_CDS_51 Mid CAI, no rare codons + maxCAI 256 320 CasX812_CDS_52 High CAI, no rare codons + original 257 321 CasX812_CDS_53 High CAI, no rare codons + original 258 322 CasX812_CDS_54 High CAI, no rare codons + highcodonfreq 259 323 CasX812_CDS_55 High CAI, no rare codons + highcodonfreq 260 324 CasX812_CDS_56 High CAI, no rare codons + maxCAI 261 325 CasX812_CDS_57 MaxCAI, no rare codons 262 326 CasX812_CDS_58 MaxCAI, no rare codons 263 327 CasX812_CDS_59 Max MFE, no rare codons 264 328 CasX812_CDS_60 Max MFE, no rare codons 265 329 CasX812_CDS_61 Max MFE, no rare codons 266 330 CasX812_CDS_62 Min MFE, no rare codons 267 331 CasX812_CDS_63 Min MFE, no rare codons 268 332 90Attorney Docket No. SCRB-061 / 01WO 333322-2448 CasX CDS ID Description SEQ ID NO: (RNA) SEQ ID NO: (DNA) CasX812_CDS_64 Min MFE, no rare codons 269 333

[0204] Further, it is anticipated that other variants of the CasX protein that have an amino acid sequence that is highly similar to that of CasX 515 may be optimized in the same way, i.e., by introducing mutations into an optimized CasX 515 coding sequence. For example, only a single mutation distinguishes CasX variant 515 from variant 491, and only three mutations separate CasX variant 515 from CasX variant 676. Additional details regarding CasX variants and the specific mutations are described in WO2022120095A1, incorporated by reference in its entirety.

[0205] Finally, for the purpose of experimentation, the CasX coding sequences will include one of two variations of the C-terminal c-MYC NLS, which will remain constant for each CasX sequence. The DNA, RNA, and protein sequences of these two variations of the C-terminal c- MYC NLS are listed in Table 1I. Table 1I: DNA, RNA, and protein sequences of C-terminal c-MYC NLS C-terminal NLS RNA sequenceSEQ IDDNA sequenceSEQAA sequencSEQ ID NO: ID NO:eNO: CT_cMYC_NLS#1 GGAGGAGGAU 334 GGAGGAGGA 336 GGGSPAAKRV 338 CCCCAGCCGC TCCCCAGCCG KLD GAAGCGAGUG CGAAGCGAG AAACUGGAC TGAAACTGG AC CT_cMYC_NLS#2 GGAUCCCCAG 335 GGATCCCCAG 337 GSPAAKRVKL 339 CCGCGAAGCG CCGCGAAGC D AGUGAAACUG GAGTGAAAC GAC TGGAC Example 2: Design of 5’UTR sequences

[0206] The 5’ UTR of an mRNA molecule is a key determinant of both the stability of the mRNA and how efficiently it is translated into protein. Here, candidate 5’ UTR sequences for incorporation into the optimized mRNA were identified using the criteria described below. Design of candidate 5’UTR sequences:

[0207] To generate a diverse list of 5’ UTR sequences optimized for various parameters, as described in detail below, data sets from multiple published studies were analyzed and filtered based on different criteria to identify candidate 5’ UTR sequences. From one data set, candidate 91Attorney Docket No. SCRB-061 / 01WO 333322-2448 5’ UTRs (either naturally-occurring or randomly generated) that were expected to produce high protein expression levels were identified by sorting for UTRs with the highest mean ribosomal load (MRL) when part of a reporter transcript.

[0208] Using a second data set, candidate 5’ UTRs were identified from a group of naturally occurring UTRs that were experimentally validated, a group of naturally occurring and synthetic sequences that were selected based on >50% enrichment in the top two bins with the highest reporter expression, as well as a set of sequences that demonstrated positive enrichment in the bin with the third highest reporter expression levels.

[0209] From a third data set, candidate 5’ UTRs were identified by selecting 5’ UTRs with the highest predicted expression based on measured half-life and relative enrichment in polysome fractions.

[0210] Lastly, several UTRs commonly used in mRNA constructs were included, along with sequences identified as performing well in the HepG2 liver cell line from a fourth data set. The resulting list contains candidates expected to drive efficient expression but were identified by prioritization of different parameters, some of which include stability as a factor and others which do not.

[0211] In addition, several 5’ UTRs expected to have low efficacy were generated by scrambling sequences of 50 nucleotides with 50% GC content. These were generated for the purpose of determining whether the selected sequences generated substantial improvements over random sequences.

[0212] In total, 535’ UTR sequences were identified. The RNA and DNA sequences of the 5’ UTRs are provided in Table 2A, below. Table 2A: RNA and DNA sequences of 5’UTRs SEQ ID SEQ ID 5'UTR ID RNA sequence NO DNA sequence NO Description (DNA) (RNA) ACUCUUCUGGUCC ACTCTTCTGGTCCC 5UTR_opt_1 CCACAGACUCAGA 340 CACAGACTCAGAG 393 Human HBA GAGAACCCACC AGAACCCACC AGAAUAAACUAGU AUUCUUCUGGUCC AGAATAAACTAGT 5UTR_opt_2 CCACAGACUCAGA 341 ATTCTTCTGGTCCC 394 GAGAACCCGCCAC CACAGACTCAGAG C AGAACCCGCCACC 92Attorney Docket No. SCRB-061 / 01WO 333322-2448 SEQ ID SEQ ID 5'UTR ID RNA sequence NO DNA sequence NO Description (DNA) (RNA) GAAAUAAGAGAG GAAATAAGAGAGA AAAAGAAGAGUA AAAGAAGAGTAAGUTR_opt_3 AGAAGAAAUAUA 342 AAGAAATATAAGA 395 AGACCCCGGCGCC CCCCGGCGCCGCC GCCACC ACC CUAGCUUUUCUCU UC CTAGCTTTTCTCTTUTR_opt_4 UGUCAACCCCA CACGCCUUUGCCA 343 CTGTCAACCCCAC 396 Human ALB CC ACGCCTTTGCCACC CUCCCGGGCUGGC CTCCCGGGCTGGCUTR_opt_5 AGCAGGGCCCCAG 344 AGCAGGGCCCCAG 397 Human CYP2E1 CGCCACC CGCCACC ACUCCUCCCCAUC ACTCCTCCCCATCC CUCUCCCUCUGUC TCTCCCTCTGTCCCUTR_opt_6 CCUCUGUCCCUCU 345 TCTGTCCCTCTGAC 398 Human C3 GACCCUGCACUGU CCTGCACTGTCCCG CCCGCCACC CCACC ACAUUUGCUUCUG ACATTTGCTTCTGA ACACAACUGUGUU CACAACT Human HBB +UTR_opt_7 CACUAGCAACCUC 346 GTGTTCA CTAGCAACCTCAA 399 canonical Kozak AAACAGCCACC ACAGCCACC sequence AAAUAAGAGAGA AAATAAGAGAGAAUTR_opt_8 AAAGAAGAGUAA 347 AAGAAGAGTAAGA GAAGAAAUAUAA AGAAATATAAGAG 400 GAGCCACC CCACC AGCCCGGCUUCCG AGCCCGGCTTCCG ACGUGCAGCCUGG ACGTGCAGCCTGGUTR_opt_9 CAGUGCAGUGAGC 34 CAGTGCAGTGAGC UGUCUGGCCUUUU 8 TGTCTGGCCTTTTG 401 Human WDR61 GUCCUUGAUCCUU TCCTTGATCCTTGG GGUUAAGGAA TTAAGGAA GUUAGCUGAAUAG AUAUCAUCGACAG GTTAGCTGAATAG ATATCA TR_opt_10 ACA TCGACAGU UAGACCACUA UAGGUUUUCUUUC 349 ACATAGACCACTA 402 Human CHN1 CUUGUGAAUUCCA TAGGTTTTCTTTCC AA TTGTGAATTCCAAA GCGACCCCUGAGC GCGACCCCTGAGC AGUGUUCUCUGUG AGTGTTCTCTGTGCUTR_opt_11 CUGAGCGGCGGGA 350 TGAGCGGCGGGAC 403 Human PCP4 CUGAGCUGUUGAG TGAGCTGTTGAGTT UUAGAGCCAAC AGAGCCAACUTR_opt_12AUUACGCUUCCCU3 ATTACGCTTCCCTA AGGGACUGGGUGG51GGGACTGGGTGGG404 Human STXBP693Attorney Docket No. SCRB-061 / 01WO 333322-2448 SEQ ID SEQ ID 5'UTR ID RNA sequence NO DNA sequence NO Description (DNA) (RNA) GAGAGAAUAGGA AGAGAATAGGATC UCGAUUCACUGCG GATTCACTGCGTGC UGCUGUCCUCACU TGTCCTCACTTGTT UGUUCUACA CTACA UGAGAUACGUAGC AAGCAAUUGGAUU TGAGATACGTAGC UTR_opt_13 ACACUCCAAGAGA 352 AAGCAATTGGATTHumanACACTCCAAGA405CUUGGAUUACACU GAADAMTSL1C CTTGGATTACACTC AUCUGAGGCGUUU ATCTGAGGCGTTTG GUUGCAGCUACCU TTGCAGCTACCTGC UTR_opt_14 GCACUUCUAGAUU ACTTCTAGATTCAT CAUCUUCUUGUGA 353 CTTCTTGTGAGCCC 406 Human MVP GCCCUGGGCUUAG TGGGCTTAGGAGT GAGUCACC CACC GGAUGCUGACGCU GGATGCTGACGCT CUCUUCCUGUCUU CTCTTCCTGTCTTT UTR_opt_15 UGUGGCUCCGGAA 354 GTGGCTCCGGAAA 407 AGGCGUUUGGGAU GGCGTTTGGGATG CC CT CC CC TA GT CG GT TG TT AG C CG CA TT GA CT GT GA GA TG AT CA CA TG94Attorney Docket No. SCRB-061 / 01WO 333322-2448 AG CA TC CT GA AT CA TT CT AT GC CC GC AG AT CC TG CC AA GC AC GA A TT AC CT TA AT CT AC GC CC GA AT CT CG GC AA GA CT GG CT CC AC95Attorney Docket No. SCRB-061 / 01WO 333322-2448 AG TT AG GA TG AC AT GG AC TA TA CT CG GC CG CT AA AA GC CA GC AT TG CC AA TT GC GG TC AA GC AGGUUGGCCCGGCCA TG CC GG CUACAGAAACGAA AGAAAAAGUCUGU CT AUAAGCCAAAGGU AG GUUCGGGAAGAAA AT UTR_opt_34 AUAACCCCAUUGC 373 GT GUAG ACUUGAGUUU T GUGCCACUACUAC CTTGAGTTTGTAGG UCUGGAAAAGCCA TGCCACTACTACTC CC TGGAAAAGCCACC 96Attorney Docket No. SCRB-061 / 01WO 333322-2448 SEQ ID SEQ ID 'UTR ID RNA sequence NO DNA sequence NO Description (DNA) (RNA)97Attorney Docket No. SCRB-061 / 01WO 333322-2448AUAUAAGGGGAAC ATATAAGGGGAAC AACACCAAACAAG AACACCAAACAAG UTR_opt_41 CUGAAGGAGCUGA 380 CTGAAGGAGCTGA 433 Synthetic CCAGCCAGCUGAC CCAGCCAGCTGAC98Attorney Docket No. SCRB-061 / 01WO 333322-2448GAGCACAGCUUCU UUGCAGCUCCUUC CAGCTTCTTTGCAG GUUGCCGGUCCAC CTCCTTCGTTGCCG ACCCGCCACCAGU GTCCACACCCGCC UCGCCCC ACCAGTTCGCCCC CUUCCUUUUGUGA CTTCCTTTTGTGAC CUGGCGGUGAACG TGGCGGTGAACGA AGUGCGCAGUGCC GTGCGCAGTGCCA Human UTR_opt_49 ACAUUUGCUUCUG 388 CATTTGCTTCTGAC 441 MRPL18A + ACACAACUGUGUU ACAACTGTGTTCAC Human HBB CACUAGCAACCUC TAGCAACCTCAAA AAACAGACACC CAGACACC GGGAAAUAAGAG GGGAAATAAGAGA AGAAAAGAAGAG GAAAAGAAGAGTA UAAGAAGAAAUA AGAAGAAATATAA UAAGAGCCACCCG GAGCCACCCGCCT Tobacco etch UTR_opt_50 CCUCGCCGCCUCC 389 CGCCGCCTCCACA 442 virus CERT + ACAUUUGCUUCUG TTTGCTTCTGACAC HBB ACACAACUGUGUU AACTGTGTTCACTA CACUAGCAACCUC GCAACCTCAAACA AAACAGACACC GACACC CUUCCUUUUGUGA CTTCCTTTTGTGAC UTR_opt_51 CUGGCGGUGAACG 390 TGGCGGTGAACGA443 HumanAGUGCGCAGUGCC GTGCGCAGTGCCMRPL18AACAUUUGCUUCUG ACATTTGCTTCTGA UTR_opt_52 ACACAACUGUGUU 39 CACAACTGTGTTCA CACUAGCAACCUC 1 CTAGCAACCTCAA 444 Human HBB AAACAGACACC ACAGACACC ACUCUUCUGGUCC ACTCTTCTGGTCCC Human HBA TR_opt_53 CCACAG + U ACUCAGA GAGAACCCGCCAC 392 CACAGACTCAGAG 445 canonical Kozak C AGAACCCGCCACC sequence 99Attorney Docket No. SCRB-061 / 01WO 333322-2448 Example 3: Design of 3’UTR sequences

[0213] 3’ UTRs also have a significant impact on mRNA stability and translation efficiency and can determine both subcellular localization and tissue-specific expression. Efforts were conducted to identify candidate 3’ UTRs derived from naturally occurring sequences using the methodology described below. Design of 3’UTR sequences:

[0214] 3’ UTRs were selected using a similar approach to that outlined for the 5’ UTRs, above. Specifically, several published data sets were identified, and filtering strategies were used to generate a set of 3’ UTRs that are expected to be optimized for different parameters.

[0215] From one data set, candidate 3’ UTRs were selected for inclusion in the set of 3’ UTRs described herein by identifying 3’ UTRs with the highest predicted expression based on measured half-life and relative enrichment in polysome fractions.

[0216] From a second data set, candidate 3’ UTRs were selected for inclusion by identifying naturally occurring 3’ UTRs that potentially have high stability, as measured by their half-life in cells. Candidate 3’ UTRs were also selected for inclusion by identifying 3’ UTRs, when assayed, that yielded relatively high reporter expression.

[0217] Lastly, candidate 3’ UTRs were selected from a data set that examined and assayed stable RNA fragments as 3’ UTRs. Specifically, stable RNA fragments that were not necessarily from UTRs were identified, tested as 3’ UTRs, screened for stability, concatenated, and tested again. The resulting combinations with the highest determined half-life, excepting those containing the amino-terminal enhancer of split (AES) UTR, were selected for inclusion in the set of 3’ UTRs described herein.

[0218] As with the 5’ UTRs, several 3’UTR sequences commonly used in mRNA constructs or occurring in highly expressed liver genes were selected for inclusion in the set of 3’ UTRs.

[0219] In addition, a group of ENE (element for nuclear expression) sequences were identified and selected for inclusion as 3’UTR candidates. The ENE sequence is a unique RNA structure known to protect viral and eukaryotic noncoding RNAs from nuclear degradation and enhanced stability for the transcript through triple-helix formation with the poly(A) tail or 3’ terminal A- rich tract.

[0220] In addition, several 3’ UTRs expected to have low efficacy were generated by scrambling sequences of 200 nucleotides with 50% GC content. These were generated for the 100Attorney Docket No. SCRB-061 / 01WO 333322-2448 purpose of determining whether the selected sequences generated substantial improvements over random sequences.

[0221] Taken together, a set of 643’ UTRs selected either for optimal stability or optimal expression was generated. The RNA and DNA sequences of the 3’ UTRs are provided in Table 3A, below. Table 3A: RNA and DNA sequences of 3’UTRs SEQ ID SEQ ID 3'UTR ID RNA sequence NO DNA sequence NO Description (RNA) (DNA) GCUGGAGCCUCG GUGGCCAUGCUU GCTGGAGCCTCGGTG CUUGCCCCUUGG GCCATGCTTCTTGCC GCCUCCCCCCAGC CCTTGGGCCTCCCCC 3UTR_opt_1 CCCUCCUCCCCUU 446 CAGCCCCTCCTCCCCHumanTTCCTGCACC510CCUGCACCCGUAC CGTACHBA1CCCCGUGGUCUU CCCCGTGGTCTTTGA UGAAUAAAGUCU ATAAAGTCTGAGTGG GAGUGGGCGGCA GCGGCA GCUGGAGCCUCG GUGGCCUAGCUU GCTGGAGCCTCGGTG CUUGCCCCUUGG GCCTAGCTTCTTGCC GCCUCCCCCCAGC CCTTGGGCCTCCCCC CUU 447 CAGCCCCTCCTC Human 3UTR_opt_2 CCCUCCUCCC CCC TTCCTGCACCC 511 HBA CCUGCACCCGUAC GTAC (alter CGUGGUCUU CCCCGT nate) CCC GGTCTTTGA UGAAUAAAGUCU ATAAAGTCTGAGTGG GAGUGGGCGGC GCGGC CAUCACAUUUAA CATCACATTTAAAAG AAGCAUCUCAGC CATCTCAGCCTACCA CUACCAUGAGAA TGAGAATAAGAGAA UAAGAGAAAGAA AGAAAATGAAGATC AAUGAAGAUCAA AAAAGCTTATTCATC AAGCUUAUUCAU TGTTTTTCTTTTTCGT CUGUUUUUCUUU TGGTGTAAAGCCAAC UUCGUUGGUGUA ACCCTGTCTAAAAAA AAGCCAACACCC CATAAATTTCTTTAA 3UTR_opt_3 UGUCUAAAAAAC 448 TCATTTTGCCTCTTTT512HumanAUAAAUUUCUUU CTCTGTGCTTCAATTALBAAUCAUUUUGCC AATAAAAAATGGAA UCUUUUCUCUGU AGAATCTAATAGAGT GCUUCAAUUAAU GGTACAGCACTGTTA AAAAAAUGGAAA TTTTTCAAAGATGTG GAAUCUAAUAGA TTGCTATCCTGAAAA GUGGUACAGCAC TTCTGTAGGTTCTGT UGUUAUUUUUCA GGAAGTTCCAGTGTT AAGAUGUGUUGC CTCTCTTATTCCACTT UAUCCUGAAAAU CGGTAGAGGATTTCT 101Attorney Docket No. SCRB-061 / 01WO 333322-2448 SEQ ID SEQ ID 3'UTR ID RNA sequence NO DNA sequence NO Description (RNA) (DNA) UCUGUAGGUUCU AGTTTCTTGTGGGCT GUGGAAGUUCCA AATTAAATAAATCAT GUGUUCUCUCUU TAATACTCTTCTAAG AUUCCACUUCGG TTATGGATTATAAAC UAGAGGAUUUCU ATTCAAAATAATATT AGUUUCUUGUGG TTGACATTATGATAA GCUAAUUAAAUA TTCTGAATAAAAGAA AAUCAUUAAUAC CAAAAACCA UCUUCUAAGUUA UGGAUUAUAAAC AUUCAAAAUAAU AUUUUGACAUUA UGAUAAUUCUGA AUAAAAGAACAA AAACCA GCAUCACAUUUA AAAGCAUCUCAG CCUACCAUGAGA GCATCACATTTAAAA AUAAGAGAAAGA GCATCTCAGCCTACC AAAUGAAGAUCA ATGAGAATAAGAGA AUAGCUUAUUCA AAGAAAATGAAGAT UCUCUUUUUCUU CAATAGCTTATTCAT UUUCGUUGGUGU CTCTTTTTCTTTTTCG HumanUTR_opt_4 AAAGCCAACACC 449 TTGGTGTAAAGCCAA 513 ALB CUGUCUAAAAAA CACCCTGTCTAAAAA (truncated) CAUAAAUUUCUU ACATAAATTTCTTTA UAAUCAUUUUGC ATCATTTTGCCTCTTT CUCUUUUCUCUG TCTCTGTGCTTCAAT UGCUUCAAUUAA TAATAAAAAATGGA UAAAAAAUGGAA AAGAACCTAGATCT AGAACCUAGAUC U GCUCGCUUUCUU GCUGUCCAAUUU CUAUUAAAGGUU GCTCGCTTTCTTGCT CCUUUGUUCCCU GTCCAATTTCTATTA AAGUCCAACUAC AAGGTTCCTTTGTTC UAAA CCTAAGTCCAACTACUTR_opt_5 CUGGGGGA 450 TAAACTGGGGGATAT5HumanUAUUAUGAAGGG14AUCU TATHBBCCUUGAGC GAAGGGCCTTGA GGAUUCUGCCUA GCATCTGGATTCTGC AUAAAAAACAUU CTAATAAAAAACATT UAUUUUCAUUGC TATTTTCATTGCAA AA GCUGGAGCCUCG GCTGGAGCCTCGGTG GUGGCCAUGCUU GCCATGCTTCTTGCC CUUGCCCCUUGG CCTTGG HumanUTR_opt_6 45 GCCTCCCCC GCCUCCCCCCAGC 1 CAGCCCCTCCTCCCC 515 HBA + CCCUCCUCCCCUU TTCCTGCACCCGTAC human HBB CCUGCACCCGUAC CCCCGTGGTCTTTGA 102Attorney Docket No. SCRB-061 / 01WO 333322-2448 SEQ ID SEQ ID 3'UTR ID RNA sequence NO DNA sequence NO Description (RNA) (DNA) CCCCGUGGUCUU ATAAAGTCTGAGTGG UGAAUAAAGUCU GCGGCAGCTCGCTTT GAGUGGGCGGCA CTTGCTGTCCAATTT GCUCGCUUUCUU CTATTAAAGGTTCCT GCUGUCCAAUUU TTGTTCCCTAAGTCC CUAUUAAAGGUU AACTACTAAACTGGG CCUUUGUUCCCU GGATATTATGAAGG AAGUCCAACUAC GCCTTGAGCATCTGG UAAACUGGGGGA ATTCTGCCTAATAAA UAUUAUGAAGGG AAACATTTATTTTCA CCUUGAGCAUCU TTGCAA GGAUUCUGCCUA AUAAAAAACAUU UAUUUUCAUUGC AA GCUCGCUUUCUU GCUGUCCAAUUU CUAUUAAAGGUU GCTCGCTTTCTTGCT CCUUUGUUCCCU GTCCAATTTCTATTA AAGUCCAACUAC AAGGTTCCTTTGTTC UAAACUGGGGGA CCTAAGTCCAACTAC UAUUAUGAAGGG TAAACTGGGGGATAT CCUUGAGCAUCU TATGAAGGGCCTTGA GGAUUCUGCCUA GCATCTGGATTCTGC AUAAAAAACAUU CTAATAAAAAACATT HumanUTR_opt_7 UAUUUUCAUUGC 452 TATTTTCATTGCAAG 516 HBB + AAGCUGGAGCCU CTGGAGCCTCGGTGG human HBA CGGUGGCCAUGC CCATGCTTCTTGCCC UUCUUGCCCCUU CTTGGGCCTCCCCCC GGGCCUCCCCCCA AGCCCCTCCTCCCCT GCCCCUCCUCCCC TCCTGCACCCGTACC UUCCUGCACCCG CCCGTGGTCTTTGAA UACCCCCGUGGU TAAAGTCTGAGTGGG CUUUGAAUAAAG CGGCA UCUGAGUGGGCG GCA CAAGCACGCAGC CAAGCACGCAGCAA AAUGCAGCUCAA TGCAGCTCAAAACGC AACGCUUAGCCU TTAGCCTAGCCACAC AGCCACACCCCCA CCCCACGGGAAACA CGGGAAACAGCA GCAGTGATTAACCTT GUGAUUAACCUU TAGCAATAAACGAA UAGCAAUAAACG AGTTTAACTAAGCTA Human BGUTR_opt_8 AAAGUUUAACUA 453 TACTAACCCCAGGGT 517 + MTRNR1 AGCUAUACUAAC TGGTCAATTTCGTGC (alternate) CCCAGGGUUGGU CAGCCACACCGCTCG CAAUUUCGUGCC CTTTCTTGCTGTCCA AGCCACACCGCUC ATTTCTATTAAAGGT GCUUUCUUGCUG TCCTTTGTTCCCTAA UCCAAUUUCUAU GTCCAACTACTAAAC UAAAGGUUCCUU TGGGGGATATTATGA 103Attorney Docket No. SCRB-061 / 01WO 333322-2448 SEQ ID SEQ ID 3'UTR ID RNA sequence NO DNA sequence NO Description (RNA) (DNA) UGUUCCCUAAGU AGGGCCTTGAGCATC CCAACUACUAAA TGGATTCTGCCTAAT CUGGGGGAUAUU AAAAAACATTTATTT AUGAAGGGCCUU TCATTGCAA GAGCAUCUGGAU UCUGCCUAAUAA AAAACAUUUAUU UUCAUUGCAA GCUGCCUUCUGC GGGGCUUGCCUU GCTGCCTTCTGCGGG CUGGCCAUGCCC GCTTGCCTTCTGGCC ATGCCCTTCTT opt_9 UUCUUC CTCTUTR_ UCUCCC UUGCACCUGUAC 454 CCCTTGCACCTGTAC 518 Mouse HBA CUCUUGGUCUUU CTCTTGGTCTTTGAA GAAUAAAGCCUG TAAAGCCTGAGTAG AGUAGGAAG GAAG AGCAGCUGCCUG GAGCCUAUUCAC UAUGUUUAUUGC AGCAGCTGCCTGGA AAAAUCCUUUCG GCCTATTCACTATGT AAAUAAACAGUU TTATTGCAAAATCCT UCCUUGCACGGU TTCGAAATAAACAGT UUCUUGUUCC TTCCTTGCACGGTTTUTR_opt_10 CU GUGCUUGA 455 CHumanCUGA TTGTTCCCTCTGAG 519 TGCTTGATUBA1GCUUCUGCUGCC GCTTCTGC UUCCCCUUCAUG TGCCTTCCCCTTCAT CUGCUGCUGCUC GCTGCTGCTGCTCTG UGUUUGCUGUUC TTTGCTGTTCATGAC AUGACCCUUAUC CCTTATCCCTT CCUU UACCAUACUUAU AUACCUGCAACU AAGUGUUUAAAA TACCATACTTATATA UUAUGCUCUGUA CCTGCAACTAAGTGT ACUCUGUACUGC TTAAAATTATGCTCT UAGUAUUAGAAC GTAACTCTGTACTGC TAGTATTAUTR_opt_11 UAAAAAUCUUAA GAACTAA 456 AAATCHumanAAUACAGCCAGU TTAAAATACA520GCCAGTGCTTSFRS10GCUUAAUGCUUA AATGC UAUCAAUGUGGA TTATATCAATGTGGA UUUGUCGGCUUU TTTGTCGGCTTTTAT UAUGUAAUCUGU GTAATCTGTAATATG AAUAUGUAUAGC TATAGCAGGA AGGA AUUGCACAGCCG ATTGCACAGCCGTCG UCGACUUCUUCC ACTTCTTCCTTACATUTR_opt_12 UUACAUUCCCAC 457 TCCCACAGCTCCCTG521 HumanAGCUCCCUGGCC GCCGGGACCCAGCCTCF7L2GGGACCCAGCCCC CCAGCCGCTGTCGCT 104Attorney Docket No. SCRB-061 / 01WO 333322-2448 SEQ ID SEQ ID 3'UTR ID RNA sequence NO DNA sequence NO Description (RNA) (DNA) AGCCGCUGUCGC CGTCACCAAGTCTTT UCGUCACCAAGU AGAATAGCTTTAGCG CUUUAGAAUAGC TCGTGAACCCCGCTG UUUAGCGUCGUG CTTTGTTTATGGTTTT AACCCCGCUGCU GTTTCACTTTTCTTA UUGUUUAUGGUU ATTTGCCCCC UUGUUUCACUUU UCUUAAUUUGCC CCC AGAAUGCUAGUG UGUAUCUAUCAU GUAUGCAAUACU AGAATGCTAGTGTGT UUCCCCCUUUUU ATCTATCATGTATGC GCUUUGCUAACC AATACTTTCCCCCTT AAAGAGCAUAUA TTTGCTTTGCTAACC R_opt_13 UUUUACUG AAAGAGCATATATTTUT UCAGHumanUUGUCUCAACUC 458 TACTGTCAGTTGTCT522AUGAAUCCAUGU CAACTCP3B1U TTGAATCCA GGCGUUUUCUCU TGTGGCGTTTTCTCT GUCCUGCUGCUU GTCCTGCTGCTTCTT CUUUUGGCCUCC TTGGCCTCCTCGTTT UCGUUUUCCUUC TCCTTCTCTT UCUU AAGCUGUCGUUG AGAACUUAGGUU GGCACGUAGCGU AAGCTGTCGTTGAGA CUCAAGGUAUGC ACTTAGGTTGGCACG GUUCUCUCAAAG TAGCGTCTCAAGGTA GAAAGCUAUGCA TGCGTTCTCTCAAAG UCGCUG GAAAGCTATGCATCGUTR_opt_14 CUUCGU 459 CTGCTTCGTTGTCTG523 HumanUGUCUGAUUUUGFAM171A1CUUAGAUUUUGC ATTTTGCTTAGATTT UUUGGUUAGGUU TGCTTTGGTTAGGTT GCGUUUUGGGGU GCGTTTTGGGGTTTG UUGCCUUUUUUU CCTTTTTTTGTTGTCG GUUGUCGCUUAA CTTAAATGC AUGC UUUGCCUUAGCA UCACUGUUUAUG TTTGCCTTAGCATCA UUCUUCUUUUCU CTGTTTATGTTCTTCT GUCCUUUUUGGU TTTCTGTCCTTTTTGG AAAAAAAAACGC TAAAAAAAAACGCA AGGUUGUAUUCC GGTTGTATTCCAAAGUTR_opt_15 AAAGCUGUAUUA 460 CTGTATTAACACAAC524HumanACACAACAUUCU ATTCTCTTCCCCACTAKAP2CUUCCCCACUCCU CCTACCCAGCCTTCT ACCCAGCCUUCU TCAGAGCTGTGAATT UCAGAGCUGUGA TCATTACAGGAGCAT AUUUCAUUACAG TGAGGCAGA GAGCAUUGAGGC 105Attorney Docket No. SCRB-061 / 01WO 333322-2448 SEQ ID SEQ ID 3'UTR ID RNA sequence NO DNA sequence NO Description (RNA) (DNA) AGA UUCCUAUGUAUG CUUUUUUCCUUU UUUGCCCAGUUU TTCCTATGTATGCTT CCUUAUCACUUG TTTTCCTTTTTTGCCC CUGUAGAUGGCU AGTTTCCTTATCACT UAUUUUGCAUUC TGCTGTAGATGGCTT ATTTTGCATUTR_opt_16 AUGCAGACUAUG TCATGC 461 AGACTAHumanUUGCAAGUCUGU TGTTGCAAG 525 TCTGTTTCATCTAGARF6UUCAUCUAGUAA T ACUGAAAAUUAU AAACTGAAAATTATT UGCUUAAUCAAA GCTTAATCAAACTGC CUGCCGUUUGUC CGTTTGTCTTTTATAT UUUUAUAUUUAA TTAAGGCC GGCC GUCAGAAGCGAG UGUCCUUUUCUU UUGCUUCAGGCU GTCAGAAGCGAGTG AAGAGCUGCCUC TCCTTTTCTTTTGCTT GCUCUUUGUCCC CAGGCTAAGAGCTG CCCAUUAGGAUU CCTCGCTCTTTGTCC CUAUUAC CCCCATTAGGATTCTUTR_opt_17 AUAUG 4HumanCAAUUGUAGGUU 62 ATTACATATGCAATT526GTASH3KBP1CAACCUGUCCCU GGTTCAACCTGT UUCCCUGCCAGC CCCTTTCCCTGCCAG AAACCCCACCACC CAAACCCCACCACCC CUAAGAGAAAUU TAAGAGAAATTTTAG UUAGCUUAUAUA CTTATATATGA UGA GCCCUGUUUAGU UUGUUAUUGCUG CUUUUCUUUUUU GCCCTGTTTAGTTTG CUUUCUGUAUCU TTATTGCTGCTTTTCT AUGCCUUUUUUU TTTTTCTTTCTGTATC CACAGUAGUCCU TATGCCTTTTTTTCAC UGGCUCUGCA AGTAGTCCTTGGCTCUTR_opt_18 CG AAUGAUA 463 THumanGAAUA GCACGGAATAAAT527GATACCCSEPT11CCCUCAAAUCUA TCAAATCT AUUGGAUGUGCU AATTGGATGTGCTTT UUCGCCUUUGCA CGCCTTTGCATGTAA UGUAAGUACGGU GTACGGTAGTAAGA AGUAAGAAACCU AACCTTTGA UUGA GUUUAACAGUUU GTTTAACAGTTTTTG UUGUUACUAAGC TTACTAAGCTAATGTUTR_opt_19 UAAUGUAAAAUU AAAATTCAGCTATTAHumanCAGCUAUUAGAA 464 GAAAATTTATTGTCT 528DGKBAAUUUAUUGUCU CAGTTTTTATAGGCA CAGUUUUUAUAG TCTTTGCATGAAGAA 106Attorney Docket No. SCRB-061 / 01WO 333322-2448 SEQ ID SEQ ID 3'UTR ID RNA sequence NO DNA sequence NO Description (RNA) (DNA) GCAUCUUUGCAU AGCAGAAGTTTACCT GAAGAAAGCAGA GAAGTGATACTGCAT AGUUUACCUGAA ATTTTTGGTGCATGC GUGAUACUGCAU ATTCCCATAGATTTT AUUUUUGGUGCA TACATCTCCC UGCAUUCCCAUA GAUUUUUACAUC UCCC UAUUGAAAAACA AAAAAGGAAAAA TATTGAAAAACAAA AAAAAUGAGAUA AAAGGAAAAAAAAA AUGGGGGAAAAA ATGAGATAATGGGG AAGGACGCUGCU GAAAAAAAGGACGC UCACACCAAAGG TGCTTCACACCAAAGUTR_opt_20 UUUAAAAAAAUA GTTTAAAAAAATATTHumanUUUUCCAAAUGC 465 TTCCAAATGCACTAG529GABRB2ACUAGGAACAUU GAACATTGTAAAAA GUAAAAACCAGA CCAGAAAGGAGTGA AAGGAGUGAACA ACAGAATGCTAATG GAAUGCUAAUGA AAGATACTGCCCTAA AGAUACUGCCCU AG AAAG ACCUCCUUAGCU GCGACCACAUUC UUUCGUUUUGUU ACCTCCTTAGCTGCG UUGUUUUGUUUU ACCACATTCTTTCGT UAAACACCUGCU TTTGTTTTGTTTTGTT UACCCCUUAAAU TTTAAACACCTGCTTUTR_opt_21 GCAAUUUAUUUA ACCCCTTAAATGCAA CACUG 466 TTTATHumanCUUUUAC TTACTTTTACC530ACTGTCACAGATPM1UCACAGAAACAU AAC CCACAAGAUACC ATCCACAAGATACCA AGCUAGGUCAGG GCTAGGTCAGGGGG GGGUGGGGAAAA TGGGGAAAACACAT CACAUACAAAAA ACAAAAAGGCA GGCA AACCGUGGUACC CCCCCUUUUCUCC CCACGAUGAUAU AACCGTGGTACCCCC CUAUAUAUGUAU CCTTTTCTCCCCACG CUACAAUACAUA ATGATATCTATATAT UAUCUACACAUA GTATCTACAATACAT ATATCTACACAUTR_opt_22 CAGAAAGAAGCA TACA 467 GAAAGAAHumanGUUCUCACAAUG GCAGTTC531TCACAATGTTGCTASTC1UUGCUAGUUUUU G UGCUUCUCUUUC TTTTTTGCTTCTCTTT CCCCACCCUACUC CCCCCACCCTACTCC CCUCCAAUUCCCC CTCCAATTCCCCCTT CUUAAACUUCCA AAACTTCCAA A 107Attorney Docket No. SCRB-061 / 01WO 333322-2448 SEQ ID SEQ ID 3'UTR ID RNA sequence NO DNA sequence NO Description (RNA) (DNA) AUACUAUGAAGC CAGCCACAAGAU ACUAACAAAUGU ATACTATGAAGCCAG AUAAAUUGAAAA CCACAAGATACTAAC GUACCAAUACUU AAATGTATAAATTGA UUUGCACAUAGA AAAGTACCAATACTT UCAAAAUAGAG TTTGCACATAGATCAUTR_opt_23 U UGGG 468 AAAHumanGGAGAAUU TAGAGTGGAGA 532 ATTTGGGCTCCAAWDR26CUCCAAAAAUCA AA GGUUAGUAGGCU ATCAGGTTAGTAGGC CAUUUCCAUGUG TCATTTCCATGTGCT CUUAAAAAUGUU TAAAAATGTTTTTGT UUUGUUGUUAUG TGTTATGCATT CAUU CAGAUUAUGUAC AUAGUUUUCUAA AAAGCCUUUCUU CAGATTATGTACATA CUAAAAACGAAA GTTTTCTAAAAAGCC AAAGACCCCCCAC TTTCTTCTAAAAACG CCAAAAUGUUUC AAAAAAGACCCCCCUTR_opt_24 GAGUCAACAAAU ACCCAAAATGTTTCG 469 AGTCAACAHumanUUAAGAGACAGA AATTTAA533AUUUUCUC GAGACAGSOX1GCCC AGCCCAT CAUAAAUUUGUA TTTCTCCATAAATTT ACAUGCUAUUUU GTAACATGCTATTTT UAUGUGCAUGUU TATGTGCATGTTTTA UUAUGAGUUCAA TGAGTTCAAAAT AAU UAAAAUGAGGUC UAAAAAAUAAUU ACUAGUCGAGAC TAAAATGAGGTCTAA UAUUAUUCUUUA AAAATAATTACTAGT AACAGAACUGCC CGAGACTATTATTCT UUUUUCUACUCU TTAAACAGAACTGCC TTTTTCTACT HumanUTR_opt_25 UUAUGUAAACUC CTTTAT 470 GTAAACTC ZNRF3 UUUCUAUUGUGU TTTCTAT 534 TGTGTTGGTCTA (AL021393. UGGUCUAACAAG ACA AGGCA 1) GCACUAUUUUAA CTATTTTAAA AAUUUUUUAAUU ATTTTTTAATTTTTCC UUUCCCAUAGCA CATAGCACTTAAAAG CUUAAAAGAGAU AGATTTTG UUUG GAAUGGGGAGCU GAATGGGGAGCTAG AGGGUUCUUGUU GGTTCTTGTTGCCTT GCCUUCAGGGCC CAGGGCCAGGGCCA 26 AGGGCCAACAUC HumanUTR_opt_ ACATCTGGGGGAGG UGGGGGAGGGGG 471 GGGTCCATCCTGGAA 535 SYNGAP1 UCCAUCCUGGAA ACCAGTGGATCTTCA (ZBTB9) ACCAGUGGAUCU TGGGAATGAAATCCT UCAUGGGAAUGA GTCAGGGGGTGGAG 108Attorney Docket No. SCRB-061 / 01WO 333322-2448 SEQ ID SEQ ID 3'UTR ID RNA sequence NO DNA sequence NO Description (RNA) (DNA) AAUCCUGUCAGG GACCTGGGGGAGCA GGGUGGAGGACC GGCCAGGCCGTGCAT UGGGGGAGCAGG GGGCCTGTGAAGCTA CCAGGCCGUGCA UGGGCCUGUGAA GCUA AUACAGUUUUGU AUUCUGUCAAUG CAAAUAUAAGAC ATACAGTTTTGTATT AGGUUGAGCCUU CTGTCAATGCAAATA AAUCAUGUAACA TAAGACAGGTTGAG AAAUAUUUUGUA CCTTAATCATGTAAC GAUUAC AAAATATTTTGTAGAUTR_opt_27 AUAUUG 472 TTACATATTGATTTT53 HumanAUUUUUCAAAAA6GTF2A1LUUAAAAAUGUAU TCAAAAATTAAAAAT UUCAAACUAUUC GTATTTCAAACTATT UUACUGUGCUGU CTTACTGTGCTGTGA GAAUUUAUAUCU ATTTATATCTTCTTCT UCUUCUGCAAGA GCAAGACCTG CCUG CAGAGUAGGUUU UUUUUCGUGGGU UGGGGGGUAACU CAGAGTAGGTTTTTT UUACUACAAUUA TTCGTGGGTTGGGGG GUAAGUAUGGUG GTAACTTTACTACAA CAGAAUUUCAUG TTAGTAAGTATGGTG _28 CAA CAGAATTTCATGCAAUTR_opt AUGAGGAGUHumanGCCAGCAGUGUG 473 ATGAGGAGTGCCAG 537 CCSDE1AUAAUUUAAACA AGTGTGATAATTTA UAUUUAAACAAA AACATATTTAAACAA AACAAAAAAAAU AAACAAAAAAAATG GAAUGCACAAAC AATGCACAAACTTGC UUGCUGCUGCUU TGCTGCTTAGAT AGAU CUUUCUAUUGAA UCAUUUGAGUAC CAAGAUAAACUU CTTTCTATTGAATCA ACACCACAUACU TTTGAGTACCAAGAT UGGUGGGUGAAU AAACTTACACCACAT CCAAUUUUGUAG ACTTGGTGGGTGAAT AAUU CCAATTTTGTAGAATUTR_opt_29 CCUACACA 474 TCCTACACAGGCAAAHumanGGCAAAAUAGCA538AGCAG ASYT1UGAUCUG TAGCATGATCTGAG CAGCAUCCAGGC CAGCAGCATCCAGG UGACCUCAAGGA CTGACCTCAAGGAA AGCAUAGCCACA GCATAGCCACAAAA AAACAGAAUAGC CAGAATAGCACCT ACCUUTR_opt_30 AUUCUGAUCAAU 475 ATTCTGATCAATTGG 539 Human 109Attorney Docket No. SCRB-061 / 01WO 333322-2448 SEQ ID SEQ ID 3'UTR ID RNA sequence NO DNA sequence NO Description (RNA) (DNA) UGGCAAGAAAGG CAAGAAAGGGAGAT RBM39 GAGAUUUCAAAA TTCAAAATTATATTT UUAUAUUUCUUG CTTGATGGTATCTTT AUGGUAUCUUUU TCAATTAATGTATCT CAAUUAAUGUAU GTAAAAGTTTCTTTG CUGUAAAAGUUU TAAATACTATGTGTT CUUUGUAAAUAC CTGGTGTGTCTTAAA UAUGUGUUCUGG ATTCCAAACAAAATG UGUGUCUUAAAA ATCCCTGCATTTCCT UUCCAAACAAAA GAAGATGTTTA UGAUCCCUGCAU UUCCUGAAGAUG UUUA CGAGGACAAGCU GAGGAAGAGUCA CCUUCACAGGAA CGAGGACAAGCTGA GAGACAGUUGCU GGAAGAGTCACCTTC CCUGGGGAAGCU ACAGGAAGAGACAG GUCCAGGGCUCU TTGCTCCTGGGGAAG GUGUCC CTGTCCAGGGCTCTGUTR_opt_31 ACCACUHumanGGGCCAGACUGA 476 TGTCCACCACTGGGC540G CAGAZBTB37AACAUCCAGGG CTGAAACATCC GAGGGGGCUGAC AGGGGGAGGGGGCT CCUCUUCCCCUAU GACCCTCTTCCCCTA GAGCCAUAAGCA TGAGCCATAAGCAG GCCAGCAUCAGA CCAGCATCAGAGCC GCC AUCCAGAAUCAC AUGCACAAGUUU UUACCUAGCCAG ATCCAGAATCACATG UUGUUUCUGAGG CACAAGTTTTTACCT ACCAGGCAACUU AGCCAGTTGTTTCTG UUGAACUCCUGU AGGACCAGGCAACT TTTGAACTCCTGTCTUTR_opt_32 CUCUGUGAGAAU 477 CTGTGAGAATGTAHumanGUAUACUUUAUG TA541CUGAAAUG CTTTATGCIGF2BP3CUCU TCTCTGA UAUGACACCCAG AATGTATGACACCCA CUUUAAAACAAA GCTTTAAAACAAACA CAAACAAACAAA AACAAACAAACAAA CAAAAAAAGGGU AAAAGGGTGGGG GGGG UCGUCGCUCUGC TCGTCGCTCTGCAAC AACUUUUCUAUC TTTTCTATCTGAGCG UGAGCGCAUCUA CATCTATCAACCCAA UCAACCCAAUCC TCCTCTACAACCTCAUTR_opt_33 UCUACAACCUCA 478 TTTCAAAGAAGTACA542HumanUUUCAAAGAAGU GAGCGGCGGCCTTTAMLNRACAGAGCGGCGG AACTGCTGCTCGCAA CCUUUAAACUGC GGAAGTCCAGGCCG UGCUCGCAAGGA AGAGGCTTCCACAG 110Attorney Docket No. SCRB-061 / 01WO 333322-2448 SEQ ID SEQ ID 3'UTR ID RNA sequence NO DNA sequence NO Description (RNA) (DNA) AGUCCAGGCCGA AAGCAGGGACACTG GAGGCUUCCACA CGGGGGAAGTTGC GAAGCAGGGACA CUGCGGGGGAAG UUGC CAAGCACGCAGC AAUGCAGCUCAA AACGCUUAGCCU CAAGCACGCAGCAA AGCCACACCCCCA TGCAGCTCAAAACGC CGGGAAACAGCA TTAGCCTAGCCACAC GUGAUUAACCUU CCCCACGGGAAACA UAGCAAUAAACG GCAGTGATTAACCTT AAAGUUUAACUA TAGCAATAAACGAA AGCUAUACUAAC AGTTTAACTAAGCTA CCCAGGGUUGGU TACTAACCCCAGGGT pt_34 CAAUUUCGUGG HumanUTR_o C TGGTCAATTTCGTGG UCGCUUUCUUGC 479 CTCGCTTTCTTGCTG 543 MTRNR1 + UGUCCAAUUUCU TCCAATTTCTATTAA human Bg AUUAAAGGUUCC AGGTTCCTTTGTTCC UUUGUUCCCUAA CTAAGTCCAACTACT GUCCAACUACUA AAACTGGGGGATATT AACUGGGGGAUA ATGAAGGGCCTTGA UUAUGAAGGGCC GCATCTGGATTCTGC UUGAGCAUCUGG CTAATAAAAAACATT AUUCUGCCUAAU TATTTTCATTGCAA AAAAAACAUUUA UUUUCAUUGCAA GCUCGCUUUCUU GCUGUCCAAUUU CUAUUAAAGGUU GCTCGCTTTCTTGCT CCUUUGUUCCCU GTCCAATTTCTATTA AAGUCCAACUAC AAGGTTCCTTTGTTC UAAACUGGGGGA CCTAAGTCCAACTAC UAUUAUGAAGGG TAAACTGGGGGATAT CCUUGAGCAUCU TATGAAGGGCCTTGA GGAUUCUGCCUA GCATCTGGATTCTGC AUAAAAAACAUU CTAATAAAAAACATT UAUUUUCAUUGC TATTTTCATTGCAAC Human BGUTR_opt_35 AACAAGCACGCA 480 AAGCACGCAGCAAT 544 + human GCAAUGCAGCUC GCAGCTCAAAACGCT MTRNR1 AAAACGCUUAGC TAGCCTAGCCACACC CUAGCCACACCCC CCCACGGGAAACAG CACGGGAAACAG CAGTGATTAAC CAGUGAUUAAC CTTTAGCAATAAACG CUUUAGCAAUAA AAAGTTTAACTAAGC ACGAAAGUUUAA TATACTAACCCCAGG CUAAGCUAUACU GTTGGTCAATTTCGT AACCCCAGGGUU GCCAGCCACACC GGUCAAUUUCGU GCCAGCCACACC 111Attorney Docket No. SCRB-061 / 01WO 333322-2448 SEQ ID SEQ ID 3'UTR ID RNA sequence NO DNA sequence NO Description (RNA) (DNA) CAAGCACGCAGC AAUGCAGCUCAA AACGCUUAGCCU CAAGCACGCAGCAA AGCCACACCCCCA TGCAGCTCAAAACGC CGGGAAACAGCA TTAGCCTAGCCACAC GUGAUUAACCUU CCCCACGGGAAACA UAGCAAUAAACG GCAGTGATTAACCTT AAAGUUUAACUA TAGCAATAAACGAA AGCUAUACUAAC AGTTTAACTAAGCTA CCCAGGGUUGGU TACTAACCCCAGGGT CAAUUUCGUGCC TGGTCAATTTCGTGC HumanUTR_opt_36 AGCCACACCCAA CAGCCACACCCAAG MTRNR1 + GCACGCAGCAAU 481 CACGCAGCAATGCA 545 human GCAGCUCAAAAC GCTCAAAACGCTTAG MTRNR1 GCUUAGCCUAGC CCTAGCCACACCCCC CACACCCCCACGG ACGGGAAACAGCAG GAAACAGCAGUG TGATTAAC AUUAAC CTTTAGCAATAAACG CUUUAGCAAUAA AAAGTTTAACTAAGC ACGAAAGUUUAA TATACTAACCCCAGG CUAAGCUAUACU GTTGGTCAATTTCGT AACCCCAGGGUU GCCAGCCACACC GGUCAAUUUCGU GCCAGCCACACC CAAGCACGCAGC AAUGCAGCUCAA CAAGCACGCAGCAA AACGCUUAGCCU TGCAGCTCAAAACGC AGCCACACCCCCA TTAGCCTAGCCACAC CGGGAAACAGCA CCCCACGGGAAACAUTR_opt_37 GUGAUUAACCUU GCAGTGATTAACCTTHumanUAGCAAUAAACG 482 TAGCAATAAACGAA546MTRNR1AAAGUUUAACUA AGTTTAACTAAGCTA AGCUAUACUAAC TACTAACCCCAGGGT CCCAGGGUUGGU TGGTCAATTTCGTGC CAAUUUCGUGCC CAGCCACACC AGCCACACC CAAGCACGCAGC CAAGCACGCAGCAA AAUGCAGCUCAA TGCAGCTCAAAACGC AACGCUUAGCCU TTAGCCTAGCCACAC AGCCACACCCCCA CCCCACGGGAAACA CGGGAAACAGCA GCAGTGATTAACCTT GUGAUUAACCUU TAGCAATAAACGAA UAGCAAUAAACG AGTTTAACTAAGCTA HumanUTR_opt_38 AAAGUUUAACUA 483 TACTAACCCCAGGGT 547 MTRNR1 + AGCUAUACUAAC TGGTCAATTTCGTGC LSP1 CCCAGGGUUGGU CAGCCACACCTTCCA CAAUUUCGUGCC GCCAGACACCCGCCC AGCCACACCUUCC CCCGGCCCTGGCTAA AGCCAGACACCC GAAGTTGCTTCCTGT GCCCCCCGGCCCU TGCCAGCATGACCTA GGCUAAGAAGUU CCCTCGCCTCTTTGA 112Attorney Docket No. SCRB-061 / 01WO 333322-2448 SEQ ID SEQ ID 3'UTR ID RNA sequence NO DNA sequence NO Description (RNA) (DNA) GCUUCCUGUUGC TGCCATCCGCTGCCA CAGCAUGACCUA CCTCCTTTTGCTCCT CCCUCGCCUCUUU GGACCCTTTAGCCTC GAUGCCAUCCGC TCTGCCCTTCCACTC UGCCACCUCCUU TCTGACCCC UUGCUCCUGGAC CCUUUAGCCUCU CUGCCCUUCCACU CUCUGACCCC CAAGCACGCAGC AAUGCAGCUCAA AACGCUUAGCCU CAAGCACGCAGCAA AGCCACACCCCCA TGCAGCTCAAAACGC CGGGAAACAGCA TTAGCCTAGCCACAC GUGAUUAACCUU CCCCACGGGAAACA UAGCAAUAAACG GCAGTGATTAACCTT AAAGUUUAACUA TAGCAATAAACGAA AGCUAUACUAAC AGTTTAACTAAGCTA CCCAGGGUUGGU TACTAACCCCAGGGT CAAUUUCGUGCC TGGTCAATTTCGTGC HumanUTR_opt_39 AGCCACACCUGCC CAGCCACACCTGCCC MTRNR1 + CGUCCUCACCAA 484 GTCCTCACCAAGACT 548 human GACUGACUGCCU GACTGCCTGCTGCTT FCGRT GCUGCUUUGCUA TGCTACTGCCCGGGC CUGCCCGGGCCCA CCATGAGACTGACTT UGAGACUGACUU CCCACTGCTCTGCCT CCCACUGCUCUGC GCCTCTCCCCACTGC CUGCCUCUCCCCA ACTGGCACAGCCCCG CUGCACUGGCAC CCTTGCCGCTGCTGA AGCCCCGCCUUGC TCCATTGCCGGTGTG CGCUGCUGAUCC ACC AUUGCCGGUGUG ACC CAAGCACGCAGC CAAGCACGCAGCAA AAUGCAGCUCAA TGCAGCTCAAAACGC AACGCUUAGCCU TTAGCCTAGCCACAC AGCCACACCCCCA CCCCACGGGAAACA CGGGAAACAGCA GCAGTGATTAACCTT GUGAUUAACCUU TAGCAATAAACGAA UAGCAAUAAACG AGTTTAACTAAGCTA AAAGUUUAACUA TACTAACCCCAGGGT HumanUTR_opt_40 AGCUAUACUAAC TGGTCAATTTCGTGC MTRNR1 + CCCAGGGUUGGU 485 CAGCCACACCCTTTG 549 human CAAUUUCGUGCC CAGGATGAAACACTT HLA-DRB4 AGCCACACCCUU CCCCGCTTGGCTCTC UGCAGGAUGAAA ATTCTTCCACAAGAG CACUUCCCCGCUU AGACCTTTCTCCGGA GGCUCUCAUUCU CCTGGTTGCTACTGG UCCACAAGAGAG TTCAGCAACTCTGCA ACCUUUCUCCGG GAAAATGTCCTCCCC ACCUGGUUGCUA TGTGGCTGCCTCAGC 113Attorney Docket No. SCRB-061 / 01WO 333322-2448 SEQ ID SEQ ID 3'UTR ID RNA sequence NO DNA sequence NO Description (RNA) (DNA) CUGGUUCAGCAA TCATGCCTTTGGCCT CUCUGCAGAAAA GAAGTCCCAGCATTG UGUCCUCCCCUG ATGGCAGCCCCTCAT UGGCUGCCUCAG CTTCCAAGTTTTGTG CUCAUGCCUUUG CTCCCCTTTACCTAA GCCUGAAGUCCC CGCTTCCTGCCTCCC AGCAUUGAUGGC ATGCATCTGTACTCC AGCCCCUCAUCU TCC UCCAAGUUUUGU GCUCCCCUUUACC UAACGCUUCCUG CCUCCCAUGCAUC UGUACUCCUCC GCUCGCUUUCUU GCUGUCCAAUUU CUAUUAAAGGUU CCUUUGUUCCCU GCTCGCTTTCTTGCT AAGUCCAACUAC GTCCAATTTCTATTA UAAACUGGGGGA AAGGTTCCTTTGTTC UAUUAUGAAGGG CCTAAGTCCAACTAC CCUUGAGCAUCU TAAACTGGGGGATAT GGAUUCUGCCUA TATGAAGGGCCTTGA AUAAAAAACAUU GCATCTGGATTCTGC UAUUUUCAUUGC CTAATAAAAAACATT TR_opt_41 AAGCUCGCUUUC 486 TATTT Human BGU TCATTGCAAG CTCGCTTTCTT 550 + human UUGCUGUCCAAU GCTG GG TCC BG UUCUAUUAAA AATTTCTATTAA UUCCUUUGUUCC AGGTTCCTTTGTTCC CUAAGUCCAACU CTAAGTCCAACTACT ACUAAACUGGGG AAACTGGGGGATATT GAUAUUAUGAAG ATGAAGGGCCTTGA GGCCUUGAGCAU GCATCTGGATTCTGC CUGGAUUCUGCC CTAATAAAAAACATT UAAUAAAAAACA TATTTTCATTGCAA UUUAUUUUCAUU GCAA CAAGCACGCAGC CAAGCACGCAGCAA AAUGCAGCUCAA TGCAGCTCAAAACGC AACGCUUAGCCU TTAGCCTAGCCACAC AGCCACACCCCCA CCCCACGGGAAACA CGGGAAACAGCA GCAGTGATTAACCTT GUGAUUAACCUU TAGCAATAAACGAA UAGCAAUAAACG AGTTTAACTAAGCTA HumanUTR_opt_42 AAAGUUUAACUA 487 TACTAACCCCAGGGT 551 MTRNR1 + AGCUAUACUAAC TGGTCAATTTCGTGC human CCCAGGGUUGGU CAGCCACACCCTGAC PLD3 CAAUUUCGUGCC AGCGTGGGCAACGC AGCCACACCCUG CTGCCGCCTGCTCTG ACAGCGUGGGCA AGGCCCGATCCAGTG ACGCCUGCCGCCU GGCAGGCCAAGGCC GCUCUGAGGCCC TGCTGGGCCCCCGCG 114Attorney Docket No. SCRB-061 / 01WO 333322-2448 SEQ ID SEQ ID 3'UTR ID RNA sequence NO DNA sequence NO Description (RNA) (DNA) GAUCCAGUGGGC GACCCAGGTGCTCTG AGGCCAAGGCCU GGTCACGGTCCCTGT GCUGGGCCCCCGC CCCCGCACCCCCGCT GGACCCAGGUGC TCTGTCTGCCCCATT UCUGGGUCACGG GTGGCTCCTCAGGCT UCCCUGUCCCCGC CTCTCCCCTGCTCTC ACCCCCGCUUCUG CCACCTCTACCTCCA UCUGCCCCAUUG CCCCCAC UGGCUCCUCAGG CUCUCUCCCCUGC UCUCCCACCUCUA CCUCCACCCCCAC UUCCAGCCAGAC ACCCGCCCCCCGG CCCUGGCUAAGA TTCCAGCCAGACACC AGUUGCUUCCUG CGCCCCCCGGCCCTG UUGCCAGCAUGA GCTAAGAAGTTGCTT CCUACCCUCGCCU CCTGTTGCCAGCATG CUUUGAUGCCAU ACCTACCCTCGCCTC CCGCUGCCACCUC TTTGATGCCATCCGC CUUUUGCUCCUG TGCCACCTCCTTTTG GACCCUUUAGCC CTCCTGGACCCTTTA UCUCUGCCCUUCC GCCTCTCTGCCCTTC HumanUTR_opt_43 ACUCUCUGACCCC 488 CACTCTCTGACCCCC LSP1 + CAAGCACGCAGC AAGCACGCAGCAAT 552 human AAUGCAGCUCAA GCAGCTCAAAACGCT MTRNR1 AACGCUUAGCCU TAGCCTAGCCACACC AGCCACACCCCCA CCCACGGGAAACAG CGGGAAACAGCA CAGTGATTAACCTTT GUGAUUAACCUU AGCAATAAACGAAA UAGCAAUAAACG GTTTAACTAAGCTAT AAAGUUUAACUA ACTAACCCCAGGGTT AGCUAUACUAAC GGTCAATTTCGTGCC CCCAGGGUUGGU AGCCACACC CAAUUUCGUGCC AGCCACACC UACGGUAAUAGU TACGGTAATAGTGTA GUAGUCUUCUCA GTCTTCTCATCTTAG UCUUAGUAGUUA TAGTTAGCTCTCTCT GCUCUCUCUUAU TATATTAAGAAAAG AUUAAGAAAAGA AAAACAAAAACCCC AAACAAAAACCC CAGGTCGCTTTATTT CCAGGUCGCUUU TGACCTGTGTTAGGGUTR_opt_44 AUUUUGACCUGU 489 ACCAAAAACGGTGG553 TurnipGUUAGGGACCAA CAGCACTGTCTAGCTcrinkle virusAAACGGUGGCAG GCGGGCATTAGACTG CACUGUCUAGCU GAAAACTAGTGCTCT GCGGGCAUUAGA TTGGGTAACCACTAA CUGGAAAACUAG AATCCCGAAAGGGT UGCUCUUUGGGU GGGCTGTGGTGACCT AACCACUAAAAU TCCGAACTAAAAGAT 115Attorney Docket No. SCRB-061 / 01WO 333322-2448 SEQ ID SEQ ID 3'UTR ID RNA sequence NO DNA sequence NO Description (RNA) (DNA) CCCGAAAGGGUG AGCCTCCCTCCTCGC GGCUGUGGUGAC GCGGGGGGGGGGCC CUUCCGAACUAA TGCCC AAGAUAGCCUCC CUCCUCGCGCGG GGGGGGGGCCUG CCC UGCCCGUCCUCAC CAAGACUGACUG CCUGCUGCUUUG TGCCCGTCCTCACCA CUACUGCCCGGG AGACTGACTGCCTGC CCCAUGAGACUG TGCTTTGCTACTGCC ACUUCCCACUGC CGGGCCCATGAGACT UCUGCCUGCCUC GACTTCCCACTGCTC UCCCCACUGCACU TGCCTGCCTCTCCCC GGCACAGCCCCGC ACTGCACTGGCACAG CUUGCCGCUGCU CCCCGCCTTGCCGCT GAUCCAUUGCCG GCTGATCCATTGCCG HumanUTR_opt_45 GUGUGACCCAAG GTGTGACCCAAGCAC FCGRT + CACGCAGCAAUG 490 GCAGCAATGCAGCTC 554 human CAGCUCAAAACG AAAACGCTTAGCCTA MTRNR1 CUUAGCCUAGCC GCCACACCCCCACGG ACACCCCCACGGG GAAACAGCAGTGAT AAACAGCAGUGA TAAC UUAAC CTTTAGCAATAAACG CUUUAGCAAUAA AAAGTTTAACTAAGC ACGAAAGUUUAA TATACTAACCCCAGG CUAAGCUAUACU GTTGGTCAATTTCGT AACCCCAGGGUU GCCAGCCACACC GGUCAAUUUCGU GCCAGCCACACC CAAGCACGCAGC CAAGCACGCAGCAA AAUGCAGCUCAA TGCAGCTCAAAACGC AACGCUUAGCCU TTAGCCTAGCCACAC AGCCACACCCCCA CCCCACGGGAAACA CGGGAAACAGCA GCAGTGATTAACCTT GUGAUUAACCUU TAGCAATAAACGAA UAGCAAUAAACG AGTTTAACTAAGCTA AAAGUUUAACUA TACTAACCCCAGGGT AGCUAUACUAAC TGGTCAATTTCGTGC HumanUTR_opt_46 CCCAGGGUUGGU 491 CAGCCACACCGCCTT 555 MTRNR1 + CAAUUUCGUGCC GGCTCCTCCAGGAAG human AGCCACACCGCCU GCTCAGGAGCCCTAC CCL22 UGGCUCCUCCAG CTCCCTGCCATTATA GAAGGCUCAGGA GCTGCTCCCCGCCAG GCCCUACCUCCCU AAGCCTGTGCCAACT GCCAUUAUAGCU CTCTGCATTCCCTGA GCUCCCCGCCAGA TCTCCATCCCTGTGG AGCCUGUGCCAA CTGTCACCCTTGGTC CUCUCUGCAUUC ACCTCCGTGCTGTCA CCUGAUCUCCAU CTGCCATCTCCCCCC 116Attorney Docket No. SCRB-061 / 01WO 333322-2448 SEQ ID SEQ ID 3'UTR ID RNA sequence NO DNA sequence NO Description (RNA) (DNA) CCCUGUGGCUGU CACCCUUGGUCA CCUCCGUGCUGU CACUGCCAUCUCC CCCC CGUCUGCAUAAC UUUUAUUAUUUC CGTCTGCATAACTTT SindbisUTR_opt_47 UUUUAUUAAUCA 492 TATTATTTCTTTTATT virus U-rich U AATCAACAAAAT 556 ACAAAAUUUUG TTT element UUUUAACAUUUC GTTTTTAACATTTC (URE) GUCUUAGCAAGC UCUGAGCCAGGA GAUGGACAUAAA GTCTTAGCAAGCTCT CCAUAGCAAUCC GAGCCAGGAGATGG AACGUGUAACCG ACATAAACCATAGC CAAUGGGGCAAA AATCCAACGTGTAAC CAACAGGUGAAC CGCAATGGGGCAAA CGUGUCCACGGG CAACAGGTGAACCG UACCGA 493 TGTCCACGGG BarleyUTR_opt_48 CCUGGU CCTGG AG TTA 557 yellow AAGGAAAGCC CCGAAAGGAAA GCCAGTATC dwarf virus UAUCCAACACAG CAACAC CAAUGUGUUGGG AGCAATGTGTTGGGG GGUCACACCUUC GTCACACCTTCGGGG GGGGUACUCUUA TACTCTTAACGCTGA ACGCUGACACUC CACTCGAAAGAGCA GAAAGAGCAGUU GTTCGGCAACCC CGGCAACCC CCUCGCCCCGGAC CUGCCCUCCCGCC CCTCGCCCCGGACCTUTR_opt_49 AGGUGCACCCAC GCCCTCCCGCCAGGTHumaUG 494 GCACCCAnCUGCAAUAAA CCTGCAAT 558 AGCGAAGCCGG AAATCYBAC GCAGCGAAGC GA CGGGA UAACCCUACCUC AGUCGAAUUGGA TAACCCTACCTCAGT UUGGGUCAUACU CGAATTGGATTGGGTUTR_opt_50 GUUGUAGGGGUA 495 CATACTGTTGTAGGG 559 Poliovirus AAUUUUUCUUUA GTAAATTTTTCTTTA AUUCGGAG ATTCGGAG CCUCGCCCCGGAC CUGCCCUCCCGCC CCTCGCCCCGGACCT AGGUGCACCCAC GCCCTCCCGCCAGGT CUGCAAUAAAUG GCACCCACCTGCAATUTR_opt_51 CAGCGAAGCCGG 496 AAATGCAGCGAAGCHumanCGG CGGGACCTCGCCC560GACCUCGCCC CGCYBA, 1.5xACCUGCCCUCCCG GACCTGCCCTCCCGC CCAGGUGCACCC CAGGTGCACCCACCT ACCUGCAAUAAA GCAATAAATGC 117Attorney Docket No. SCRB-061 / 01WO 333322-2448 SEQ ID SEQ ID 3'UTR ID RNA sequence NO DNA sequence NO Description (RNA) (DNA) UGC GGUGCCUUUGAG AGUCUACUUUUG GGTGCCTTTGAGAGT CUCUCUUCGGAA CTACTTTTGCTCTCTT GAACCCUUAGGG CGGAAGAACCCTTA GUUCGUGCAUGG GGGGTTCGTGCATGGUTR_opt_52 GCUUGCAUAGCA 497 GCTTGCATAGCAAGT561 BromeAGUCUAGAUGCG CTAGATGCGGGTACCmosaic virusGGUACCGUACAG GTACAGTGTTGAAAA UGUUGAAAAACA ACACTGTAAATCTCT CUGUAAAUCUCU AAAAGAGACCA AAAAGAGACCA GACCACACAAGG CAGAUGGGCUAU AUAAACGUUUUC GCUUUUCCGUUU GACCACACAAGGCA ACGAUAUAUAGU GATGGGCTATATAAA CUACUCUUGUGC CGTTTTCGCTTTTCC AGAAUGAAUUCU GTTTACGATATATAG CGUAACUACAUA TCTACTCTTGTGCAG GCACAAGUAGAU AATGAATTCTCGTAA GUAGUUAACUUU CTACATAGCACAAGT AAUCUCACAUAG AGATGTAGTTAACTT CAAUCUUUAAUC TAATCTCACATAGCA AGUGUGUAACAU ATCTTTAATCAGTGTUTR_opt_53 UAGGGAGGACUU GTAACATTAGGGAG 498 GACTTGASARS-GAAAGAGCCACC AAGAGCC562AUUUUCACCG ACCACACoV2AC TTTTCACCG AGGCCACGCGGA AGGCCACGCGGAGT GUACGAUCGAGU ACGATCGAGTGTACA GUACAGUGAACA GTGAACAATGCTAG AUGCUAGGGAGA GGAGAGCTGCCTATA GCUGCCUAUAUG TGGAACAGCCCTAAT GAACAGCCCUAA GTGTAAAATTAATTT UGUGUAAAAUUA TAGTAGTGCTATCCC AUUUUAGUAGUG CATGTGATTTTAATA CUAUCCCCAUGU GCTTCTTAGGAGAAT GAUUUUAAUAGC GAC UUCUUAGGAGAA UGAC AUUAGUCAUUGG ATTAGTCATTGGTAA UAAUUCAAUGCC TTCAATGCCAACCTC AACCUCCACUGG CACTGGGTGGGTTAA GUGGGUUAAGGU GGTTGAGGTATAGAUTR_opt_54 UGAGGUAUAGAA 49 ATCCTATTCGCTCCTAlfalfaUCCUAUUCGCUC 9 GATAGGAGAAATTCT563mosaic virusCUGAUAGGAGAA ATATTGCTTATATAC AUUCUAUAUUGC GTGCTTATGCACGTA UUAUAUACGUGC TATAAATGCTCATGC UUAUGCACGUAU TAAATTGCATGAATG 118Attorney Docket No. SCRB-061 / 01WO 333322-2448 SEQ ID SEQ ID 3'UTR ID RNA sequence NO DNA sequence NO Description (RNA) (DNA) AUAAAUGCUCAU CCCCTAAGGGATGC GCUAAAUUGCAU GAAUGCCCCUAA GGGAUGC GAAGGUUUUUCU UUUCCUGAGAAA GAAGGTTTTTCTTTT ACAACACGUAUU CCTGAGAAAACAAC GUU ACGTATTGTTTTCTCUTR_opt_55 UUCUCAGGU 500 AGGTTTTGCTTTTMALAT1UUUGCUUUUUGG TG564 ENECCUUUUUCUAGC GCCTTTTTCTAGCTT UUAAAAAAAAAA AAAAAAAAAAAAAG AAAGCAAAA CAAAA AAAGCAAAACUA ACAUGAAACAAG GCUAGAAGUCAG GUCGGAUUAAGC AAAGCAAAACTAAC CAUAGUACGGAA ATGAAACAAGGCTA AAAACUAUGCUA GAAGTCAGGTCGGA CCUGUGAGCCCC TTAAGCCATAGTACG GUCCAAGGACGU GAAAAAACTATGCT UAAAAGAAGUCA ACCTGTGAGCCCCGT GGCCAUCAUAAA CCAAGGACGTTAAA UGCCAUAGCUUG AGAAGTCAGGCCAT AGUAAACUAUGC CATAAATGCCATAGC AGCCUGUAGCUC TTGAGTAAACTATGC CACCUGAGAAGG AGCCTGTAGCTCCAC UGUAAAAAAUCC CTGAGAAGGTGTAA GGGAGGCCACAA AAAATCCGGGAGGC ACCAUGGAAGCU CACAAACCATGGAA GUACGCAUGGCG GCTGTACGCATGGCG UAGUGGAC DengueUTR_opt_56 UAGC TAGTGGACTAGCGGT GGUUAGAGGAGA 501 TAGAGGAGACCCCTC 565 virus CCCCUCCCUUACA CCTTACAAATCGCAG (DEN2) AAUCGCAGCAAC CAACAATGGGGGCC AAUGGGGGCCCA CAAGGCGAGATGAA AGGCGAGAUGAA GCTGTAGTCTCGCTG GCUGUAGUCUCG GAAGGACTAGAGGT CUGGAAGGACUA TAGAGGAGACCCCC GAGGUUAGAGGA CCGAAACAAAAAAC GACCCCCCCGAAA AGCATATTGACGCTG CAAAAAACAGCA GGAAAGACCAGAGA UAUUGACGCUGG TCCTGCTGTCTCCTC GAAAGACCAGAG AGCATCATTCCAGGC AUCCUGCUGUCU ACAGAACGCCAGAA CCUCAGCAUCAU AATGGAATGGTGCTG UCCAGGCACAGA TTGAATCAACAGGTT ACGCCAGAAAAU CT GGAAUGGUGCUG UUGAAUCAACAG GUUCU 119Attorney Docket No. SCRB-061 / 01WO 333322-2448 SEQ ID SEQ ID 3'UTR ID RNA sequence NO DNA sequence NO Description (RNA) (DNA) GCUGGGUUUUUC CUUGUUCGCACC GCTGGGTTTTTCCTT GGA GTTCGCACCGGACACUTR_opt_57 CACCUCCAG UGACCAGACGGC 502 CTCCAGTGACCAGAC 566 PAN ENE AAGGUUUUUAUC GGCAAGGTTTTTATC CCAGU CCAGT AGGUGUUUCUUU UACUGAGUGCAG AGGTGTTTCTTTTAC CCCAUGGCCGCAC TGAGTGCAGCCCATG UCAGGU GCCGCACTCAGGTTTUTR_opt_58 UUUGCU UUUCACCUUCCC 503 TGCTTTTCACCTTCC 567 MENΒ ENE AUCUGUGAAAGA CATCTGTGAAAGAGT GUGAGCAGGAAA GAGCAGGAAAAAGC AAGCAAAA AAAA AGUGUAACUCAA CAGUUUUUCCUA AGTGTAACTCAACAG ACCACGC TTTTTCCTAACCACGUTR_opt_59 GUCGC 504 CGTCGCGTGGCAGGT568McDIVGUGGCAGGUUUU G TTTTTENEUUAAUCUGAGA AATCTGAGAG AUUACAUUC ATTACATTC GCUGUACUCUUU UCUUUGUCAUGG GCTGTACTCTTTTCTT UUUUCUC TGTCATGGTTTTCTCUTR_opt_60 AAAUA 505 AAATATGAGTTTTTA56TWIFB1UGAGUUUUUACA9UUU CATGENEUGACAAAGU ACAAAGTTTTT UAACGAGGCAGC AACGAGGCAGC GUGCUGUACUCU UUUUCCUCACUA GTGCTGTACTCTTTT UGGUUUUGUCCC TCCTCACTATGGTTTUTR_opt_61 GAAAGGGUUUUC 506 TGTCCCGAAAGGGTT570ATCOPIA2CUAGUAAGGUUU TTCCTAGTAAGGTTT7 ENEUAAUGAGGCAGC TAATGAGGCAGCAT AU GGCUGUACUCUU UUUUCCUGUCUA GGCTGTACTCTTTTT GGGUUUCUCACA TCCTGTCTAGGGTTTUTR_opt_62 AGGGUGAGUUUU 507 CTCACAAGGGTGAGT TTTACCTA 571 TCUP ENE ACCUAGACAGGU GACAGGT UUUUAACGAGGC TTTTAACGAGGCAAC AACC C CGUUUGUGUUGG UUUUUAUGACCA CGTTTGTGTTGGTTT pt_63 GCUUGG TTATGACCAGCTTGGUTR_o UACAAA ACCUGCUGGUGA 508 TACAAAACCTGCTGG 572 RRV ENE UUUUUUACCCAA TGATTTTTTACCCAA CAAA CAAA 120Attorney Docket No. SCRB-061 / 01WO 333322-2448 SEQ ID SEQ ID 3'UTR ID RNA sequence NO DNA sequence NO Description (RNA) (DNA) CUCAAAGAAUAU UUUUAAAGACUU CTCAAAGAATATTTT UUUUCCCCAACC TAAAGACTTTTTTCC 3UTR_opt_64 UCUGGGUUGGGU 509 CCAACCTCTGGGTTG 573 EHV2 ENE UUUUUCUCUUUA GGTTTTTTCTCTTTAA AAAUAUUCAAUA AATATTCAATAAAA AAA Example 4: Design of 3’ poly(A) tail sequences

[0222] The 3’ poly(A) tail is crucial for mRNA stability and translation efficiency, such that longer poly(A) tails are associated with increased mRNA stability, thereby allowing their translation and promoting high protein expression. While in vitro polyadenylation is possible using a poly(A) polymerase in a post-transcriptional enzymatic reaction, this process would result in variable lengths of poly(A) tails. Therefore, poly(A) tails can be encoded from plasmid DNA templates during in vitro transcription. Candidate poly(A) tail sequences for use were identified as described below.

[0223] Candidate poly(A) tails were selected to have a range of poly(A) tail lengths. While plasmid constructs containing a template 120 nucleotide A-tail were able to produce CasX mRNAs that resulted in effective editing, these plasmids often resulted in recombination during E.coli propagation, prompting the design of shorter poly(A) tails. In addition, split variants of the 120 nucleotide A-tail were selected as candidates, such that a single nucleotide or a restriction site was included in between two 60-A segments, since inclusion of split poly(A) tail variants was previously reported to reduce recombination during E.coli propagation significantly (Trepotec Z. et al., Segmented poly(A) tails significantly reduce recombination of plasmid DNA without affecting mRNA translation efficiency or half-life. RNA.25: 507-518 (2019)). Additionally, sequences encoding a 60A-tail and a 30A-tail, which represent the median and lower bounds of poly(A) tail length of human genes respectively (Chang H. et al., TAIL-seq: Genome-wide Determination of Poly(A) Tail Length and 3′ End Modifications. Mol Cell.53(6): 1044-1052 (2014)), were also included as candidate poly(A) tails. A 185 nucleotide A-tail was also included as a candidate poly(A) tail.

[0224] Furthermore, two ENE sequences, MALAT1 ENE and MENβ ENE, which also contain a genomically encoded A-rich tract, were selected for inclusion as poly(A) tail candidates as it was previously shown that incorporating these two ENE structures downstream of an open reading 121Attorney Docket No. SCRB-061 / 01WO 333322-2448 frame resulted in efficient translation of the transcript and protein expression, despite the absence of a poly(A) tail, indicating that an ENE sequence could serve as an alternative to a poly(A) tail (Wilusz J.E. et al., A triple helix stabilizes the 3’ ends of long noncoding RNAs that lack poly(A) tails. Genes & Dev.26: 2392-2407 (2012)).

[0225] Sequences of candidate poly(A) tails are provided in Table 4A. Table 4A: RNA and DNA sequences of poly(A) tails SEQ ID SEQ ID Poly(A) ID RNA sequence NO: DNA sequence NO: Description (RNA) (DNA) PolyAtail_opt_1 AAAAAAAAAAAA 574 AAAAAAAAAAAA 574 120 polyA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA PolyAtail_opt_2 AAAAAAAAAAAA 575 AAAAAAAAAAAA 575 120 split polyA AAAAAAAAAAAA AAAAAAAAAAAA G AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA GAAAAAAAAAAA GAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA A A PolyAtail_opt_3 AAAAAAAAAAAA 576 AAAAAAAAAAAA 580 120 split polyA AAAAAAAAAAAA AAAAAAAAAAAA RE Site AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA GCAUGCAAAAAAA GCATGCAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAA AAAAA122Attorney Docket No. SCRB-061 / 01WO 333322-2448PolyAtail_opt_7 AGGUGUUUCUUUU 503 AGGTGTTTCTTTTA 567 MENΒ ENE ACUGAGUGCAGCC CTGAGTGCAGCCC CAUGGCCGCACUC ATGGCCGCACTCA AGGUUUUGCUUUU GGTTTTGCTTTTCA CACCUUCCCAUCU CCTTCCCATCTGTG GUGAAAGAGUGA AAAGAGTGAGCAG GCAGGAAAAAGCA GAAAAAGCAAAA AAA PolyAtail_opt_8 AAAAAAAAAAAA 579 AAAAAAAAAAAA 579 AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAA AAAAA Example 5: In silico assessment of engineered mRNAs encoding CasX

[0226] In silico assessments were performed to evaluate the CasX coding sequence, 5’ UTR, and 3’ UTR described above in Examples 1, 2, and 3. Specifically, 5’ UTR, the CasX coding, C-terminal c-MYC NLS, and 3’ UTR sequences were combined to generate full-length mRNA sequences in silico (without poly(A) tails), and the mRNA half-life and mean ribosome load were predicted for each sequence. 123Attorney Docket No. SCRB-061 / 01WO 333322-2448 Methods:

[0227] Generation of full-length mRNA sequences: All possible combinations of 5’ UTR, CasX variant 515, and 3’ UTR RNA sequences listed in Tables 1F, 2A, and 3A were concatenated to generate “full-length” sequences of mRNAs encoding CasX. The full-length sequences included, from 5’ to 3’, a 5’ UTR, a start codon, a CasX 515 coding sequence, a C-terminal c-MYC NLS, a stop codon, and a 3’ UTR. For the purpose of these in silico assessments, the full-length mRNAs did not include a poly(A) tail. The CT_cMYC_NLS#1 C-terminal c-MYC NLS was used, with a DNA sequence of GGAGGAGGATCCCCAGCCGCGAAGCGAGTGAAACTGGAC (SEQ ID NO: 336), as shown in Table 1I. A total of 217,088 unique full-length sequences were generated, of which a representative subset of sequences are SEQ ID NOS: 823-112758.

[0228] In silico predictions: The Saluki model was used to predict the stability of the 217,088 unique sequences (see Agarwal, V. and Kelley, D. The genetic and biochemical determinants of mRNA degradation rates in mammals. bioRxiv 2022.03.18.484474). Specifically, 50 iterations of the model were performed for each unique sequence, and the results of the 50 iterations were averaged to generate one score for each unique sequence. The score indicates the predicted half- life of each mRNA sequence. Saluki scores were then aggregated for each element by grouping full-length sequences that contained each unique 5’UTR, coding sequence, or 3’UTR and calculating the minimum, median, and maximum Saluki scores in each group. While the Saluki model predicts stability of an entire mRNA sequence, this method allows inference of the effect of a specific component by summarizing the effect across many contexts.

[0229] Further, the mean ribosome loads of the 505’ UTR sequences were predicted using Optimus 5-prime (see Sample P.J., et al., Human 5’ UTR design and variant effect prediction from a massively parallel translation assay. Nat Biotechnol.2019; 37(7):803-809). Results:

[0230] The minimum, median, and maximum Saluki scores for each 5’ UTR, coding sequence, and 3’ UTR are provided in Table 5A, Table 5B, and Table 5C, respectively. Table 5A also provides the predicted mean ribosome loads of the 5’ UTRs. 124Attorney Docket No. SCRB-061 / 01WO 333322-2448 Table 5A: Minimum, median, and maximum Saluki scores, and predicted mean ribosomal load of 5’ UTR sequences 5'UTR ID MinimumMedian Saluki Maximum Saluki Predicted Mean Saluki ScoreScore Score Ribosomal Load 5UTR_opt_1 -1.58422744 -0.74769011 -0.186678683 6.07731628 5UTR_opt_2 -1.57569639 -0.737604464 -0.180700499 6.29265499 5UTR_opt_3 -1.567012461 -0.731931593 -0.174759532 5.73082876 5UTR_opt_4 -1.57960667 -0.744417866 -0.184041649 6.13810015 5UTR_opt_5 -1.578660222 -0.745102577 -0.185712759 5.09164476 5UTR_opt_6 -1.57325258 -0.729349455 -0.164771424 5.98160982 5UTR_opt_7 -1.57865912 -0.732558822 -0.184105474 6.4866457 5UTR_opt_8 -1.583337372 -0.736924557 -0.186123379 6.1625371 5UTR_opt_9 -1.587025907 -0.739709381 -0.167861728 6.61982346 5UTR_opt_10 -1.545003243 -0.718362716 -0.167476858 6.7498579 5UTR_opt_11 -1.584270356 -0.742138625 -0.178698065 6.46167278 5UTR_opt_12 -1.584991802 -0.736334933 -0.167479067 6.64097023 5UTR_opt_13 -1.578655898 -0.744705508 -0.18862524 6.40954876 5UTR_opt_14 -1.565393682 -0.721222232 -0.154008329 6.35987711 5UTR_opt_15 -1.59208614 -0.752806251 -0.189953679 6.07733965 5UTR_opt_16 -1.570016028 -0.716826523 -0.162977512 6.5754447 5UTR_opt_17 -1.613070106 -0.738435977 -0.170896214 6.56170321 5UTR_opt_18 -1.576272346 -0.739360811 -0.183063334 6.71501017 5UTR_opt_19 -1.595541786 -0.736149926 -0.182974292 6.62017918 5UTR_opt_20 -1.576077892 -0.723139592 -0.177060095 6.66853809 5UTR_opt_21 -1.58863473 -0.741829059 -0.190005941 6.62966824 5UTR_opt_22 -1.565392696 -0.734956635 -0.179715721 6.83887196 5UTR_opt_23 -1.582330202 -0.715403735 -0.152367629 6.59544849 5UTR_opt_24 -1.552570168 -0.723047794 -0.175313412 6.78724194 5UTR_opt_25 -1.551034702 -0.71071737 -0.151441152 6.84141254 5UTR_opt_26 -1.574964824 -0.726330448 -0.158579699 6.64828682 5UTR_opt_27 -1.606700726 -0.737811733 -0.174122987 6.77492714 5UTR_opt_28 -1.57901382 -0.723762503 -0.174122987 6.04942369 5UTR_opt_29 -1.606700726 -0.742888905 -0.184055026 6.44919205 5UTR_opt_30 -1.595017126 -0.736309503 -0.179384124 7.22303629 5UTR_opt_31 -1.561834396 -0.721559519 -0.16519898 6.53949118 5UTR_opt_32 -1.533095206 -0.665575993 -0.111538584 6.24772549 5UTR_opt_33 -1.664530532 -0.761174573 -0.145462007 6.21314621 5UTR_opt_34 -1.577602706 -0.711930722 -0.147210704 6.69931126 5UTR_opt_35 -1.64369445 -0.757201706 -0.16490564 6.23007441 5UTR_opt_36 -1.685786576 -0.774169409 -0.137649843 5.48898077 5UTR_opt_37 -1.671706658 -0.756704971 -0.130775585 5.92092514 125Attorney Docket No. SCRB-061 / 01WO 333322-2448 5'UTR ID MinimumMedian Saluki Maximum Saluki Predicted Mean Saluki ScoreScore Score Ribosomal Load 5UTR_opt_38 -1.64202425 -0.718884042 -0.096432822 5.69128609 5UTR_opt_39 -1.571405712 -0.706643429 -0.146395834 6.60515785 5UTR_opt_40 -1.629921038 -0.728996736 -0.131998279 6.16056633 5UTR_opt_41 -1.520504271 -0.663339045 -0.115901411 6.48897552 5UTR_opt_42 -1.562455712 -0.736555612 -0.18147448 6.13241386 5UTR_opt_43 -1.571847702 -0.712995813 -0.157506811 6.57987881 5UTR_opt_44 -1.565156842 -0.726369368 -0.171944938 6.64424801 5UTR_opt_45 -1.527794792 -0.688274064 -0.143828715 6.64293146 5UTR_opt_46 -1.536454049 -0.672773414 -0.121012352 6.68412971 5UTR_opt_47 -1.574500936 -0.694056421 -0.121012352 6.66480303 5UTR_opt_48 -1.70010409 -0.81481015 -0.163588105 5.99199152 5UTR_opt_49 -1.556966974 -0.704040034 -0.138870492 6.80749512 5UTR_opt_50 -1.535035784 -0.665248505 -0.107918238 6.58909607 5UTR_opt_51 -1.580890756 -0.750825848 -0.192879088 6.12803984 5UTR_opt_52 -1.579063562 -0.733363184 -0.185560152 6.55147266 5UTR_opt_53 -1.590676988 -0.738812866 -0.188814194 5.9850359 Table 5B: Minimum, median, and maximum Saluki scores of optimized CasX 515 coding sequences CasX CDS ID Minimum Saluki Score Median Saluki ScoreMaximum SalukiScoreCasX515_CDS_1 -1.276270583 -0.80565013 -0.390844832 CasX515_CDS_2 -0.876703868 -0.455582089 -0.096432822 CasX515_CDS_3 -1.211748978 -0.733429175 -0.329434389 CasX515_CDS_4 -1.361078026 -0.899259922 -0.449753925CasX515_CDS_12 -0.94956755 -0.515534692 -0.14570045 CasX515_CDS_13 -0.964657184 -0.537691315 -0.160798998 CasX515_CDS_14 -1.252129858 -0.755334392 -0.352953869 CasX515_CDS_15 -1.270761241 -0.786693741 -0.378518912 CasX515_CDS_16 -1.291767574 -0.852653872 -0.432825074 CasX515_CDS_17 -1.338042915 -0.859024128 -0.435093688 CasX515_CDS_18 -0.893450371 -0.491207323 -0.135711417 CasX515_CDS_19 -0.943841506 -0.501260824 -0.134829511 126Attorney Docket No. SCRB-061 / 01WO 333322-2448 CasX CDS ID Minimum Saluki Score Median Saluki ScoreMaximum SalukiScoreCasX515_CDS_20 -1.199148761 -0.730674855 -0.338585187 CasX515_CDS_21 -1.243276708 -0.736941194 -0.341646833 CasX515_CDS_22 -1.292638085 -0.830364447 -0.424572998 CasX515_CDS_23 -1.359647126 -0.872513599 -0.453113628 CasX515_CDS_24 -0.904045052 -0.485924223 -0.138373796 CasX515_CDS_25 -0.977293906 -0.518949465 -0.160125456 CasX515_CDS_26 -1.201853578 -0.718150145 -0.339953854 CasX515_CDS_27 -1.270635426 -0.757034657 -0.364939054 CasX515_CDS_28 -1.325505372 -0.866024976 -0.43203492 CasX515_CDS_29 -1.352023306 -0.884323071 -0.453901316 CasX515_CDS_30 -0.927938543 -0.504048193 -0.129813723 CasX515_CDS_31 -0.959258901 -0.521058356 -0.146759954 CasX515_CDS_32 -1.234992233 -0.746930089 -0.340845203 CasX515_CDS_33 -1.260038022 -0.766283625 -0.362492927 CasX515_CDS_34 -1.350439322 -0.913284819 -0.467113161 CasX515_CDS_35 -1.376202745 -0.948854251 -0.505408013 CasX515_CDS_36 -0.950635609 -0.532236439 -0.146584473 CasX515_CDS_37 -0.97826658 -0.559371973 -0.169712357 CasX515_CDS_38 -1.256820509 -0.786053682 -0.367529404 CasX515_CDS_39 -1.283190565 -0.819102584 -0.398520522 CasX515_CDS_40 -1.382411629 -0.914165848 -0.474372409 CasX515_CDS_41 -1.375032577 -0.910344739 -0.474009297 CasX515_CDS_42 -0.991888955 -0.545365368 -0.161672086 CasX515_CDS_43 -0.97757295 -0.538241333 -0.165169003 CasX515_CDS_44 -1.290917202 -0.792406938 -0.380275434 CasX515_CDS_45 -1.281035333 -0.784428907 -0.379257943 CasX515_CDS_46 -1.307984202 -0.846096545 -0.430669123 CasX515_CDS_47 -1.320033345 -0.875515231 -0.439583653 CasX515_CDS_48 -0.911838059 -0.489905457 -0.130505067 CasX515_CDS_49 -0.923016139 -0.51380438 -0.132251059 CasX515_CDS_50 -1.220055876 -0.731269742 -0.338857176 CasX515_CDS_51 -1.2295872 -0.757140256 -0.346471277 CasX515_CDS_52 -1.301823693 -0.853162909 -0.424430574 CasX515_CDS_53 -1.331644612 -0.893684444 -0.458315659 CasX515_CDS_54 -0.905622207 -0.491223801 -0.112802784 CasX515_CDS_55 -0.929578435 -0.512175291 -0.127307636 CasX515_CDS_56 -1.238255232 -0.76669424 -0.357324354 CasX515_CDS_57 -1.186819407 -0.744459083 -0.339841257 CasX515_CDS_58 -1.124544193 -0.68508431 -0.28822632 CasX515_CDS_59 -1.382360366 -0.910686856 -0.464845707 127Attorney Docket No. SCRB-061 / 01WO 333322-2448 CasX CDS ID Minimum Saluki Score Median Saluki ScoreMaximum SalukiScoreCasX515_CDS_60 -1.427170208 -0.97794161 -0.53927127 CasX515_CDS_61 -1.386955653 -0.950025069 -0.513806516 CasX515_CDS_62 -1.70010409 -1.296943221 -0.867329784 CasX515_CDS_63 -1.58658934 -1.164325231 -0.736781587 CasX515_CDS_64 -1.613899845 -1.201039946 -0.766761946 Table 5C: Minimum, median, and maximum Saluki scores of 3’ UTR sequences 3'UTR ID Minimum Saluki Score Median Saluki Score Maximum Saluki Score 3UTR_opt_1 -1.317539584 -0.642858423 -0.292736647 3UTR_opt_2 -1.296611167 -0.62211844 -0.275452487 3UTR_opt_3 -1.063853435 -0.437120967 -0.096432822 3UTR_opt_4 -1.144445734 -0.498608612 -0.159120376 3UTR_opt_5 -1.412255272 -0.738056765 -0.367505577 3UTR_opt_6 -1.155803867 -0.498870468 -0.149261757 3UTR_opt_7 -1.264531212 -0.588659126 -0.233549502 3UTR_opt_8 -1.130787783 -0.49429568 -0.154850209 3UTR_opt_9 -1.471560684 -0.808761974 -0.435894491 3UTR_opt_10 -1.370154278 -0.715674001 -0.343979925 3UTR_opt_11 -1.409351153 -0.730089139 -0.352064828 3UTR_opt_12 -1.327134124 -0.680032901 -0.318429057 3UTR_opt_13 -1.364250382 -0.685558797 -0.319590036 3UTR_opt_14 -1.463343602 -0.820467055 -0.430148382 3UTR_opt_15 -1.45647518 -0.804542607 -0.4341208 3UTR_opt_16 -1.43744171 -0.765664196 -0.377463834 3UTR_opt_17 -1.254342019 -0.593687301 -0.241743509 3UTR_opt_18 -1.453592006 -0.788362384 -0.411986616 3UTR_opt_19 -1.465972233 -0.793321602 -0.407944598 3UTR_opt_20 -1.579013669 -1.005101293 -0.636999913 3UTR_opt_21 -1.529343436 -0.917273526 -0.529597733 3UTR_opt_22 -1.282087585 -0.638906226 -0.293865836 3UTR_opt_23 -1.456185327 -0.821658156 -0.42892667 3UTR_opt_24 -1.574151682 -0.995769572 -0.589627523 3UTR_opt_25 -1.518914062 -0.852941129 -0.448912629 3UTR_opt_26 -1.52439632 -0.919919565 -0.558184792 3UTR_opt_27 -1.493287161 -0.830740986 -0.442725158 3UTR_opt_28 -1.43271665 -0.761920846 -0.388383057 3UTR_opt_29 -1.420718731 -0.792782307 -0.44464529 3UTR_opt_30 -1.70010409 -1.147198452 -0.734005353 3UTR_opt_31 -1.541417334 -0.969179061 -0.606522108 128Attorney Docket No. SCRB-061 / 01WO 333322-2448 3'UTR ID Minimum Saluki Score Median Saluki Score Maximum Saluki Score 3UTR_opt_32 -1.472386309 -0.84515231 -0.469368671 3UTR_opt_33 -1.505076486 -0.901025767 -0.548470304 3UTR_opt_34 -1.160729764 -0.516698048 -0.170923136 3UTR_opt_35 -1.229545346 -0.569438525 -0.229158096 3UTR_opt_36 -1.102728251 -0.475294364 -0.150547278 3UTR_opt_37 -1.259227712 -0.605794573 -0.264395838 3UTR_opt_38 -1.102789551 -0.471982745 -0.143954637 3UTR_opt_39 -1.281948486 -0.649313037 -0.313773287 3UTR_opt_40 -1.188768748 -0.558159484 -0.229956435 3UTR_opt_41 -1.241464232 -0.573000774 -0.21780836 3UTR_opt_42 -1.054724889 -0.437280599 -0.116078661 3UTR_opt_43 -1.120199561 -0.494018512 -0.165439554 3UTR_opt_44 -1.374700411 -0.709881086 -0.353099548 3UTR_opt_45 -1.325711153 -0.719006223 -0.373245444 3UTR_opt_46 -1.086604752 -0.459455626 -0.129574628 3UTR_opt_47 -1.564922455 -0.919441048 -0.535565052 3UTR_opt_48 -1.540429197 -0.974448101 -0.617872605 3UTR_opt_49 -1.497007553 -0.875378059 -0.515579011 3UTR_opt_50 -1.60227444 -0.98625274 -0.595239038 3UTR_opt_51 -1.365183658 -0.732701106 -0.392696773 3UTR_opt_52 -1.611643618 -1.035607534 -0.655521603 3UTR_opt_53 -1.301253467 -0.64405208 -0.285399406 3UTR_opt_54 -1.304863403 -0.643048085 -0.293164506 3UTR_opt_55 -1.529854352 -0.872183178 -0.479662008 3UTR_opt_56 -1.537900777 -0.980461242 -0.630345322 3UTR_opt_57 -1.553293988 -0.934722856 -0.559897931 3UTR_opt_58 -1.537760431 -0.905257375 -0.522770485 3UTR_opt_59 -1.485879825 -0.838879502 -0.472002565 3UTR_opt_60 -1.540772017 -0.890053805 -0.509574554 3UTR_opt_61 -1.538830841 -0.893873174 -0.511516764 3UTR_opt_62 -1.520633739 -0.867359772 -0.48925792 3UTR_opt_63 -1.613019892 -1.007049178 -0.618263639 3UTR_opt_64 -1.695025986 -1.113230815 -0.695101928 Example 6: Experimental methods to assess candidate engineered mRNAs encoding CasX

[0231] Candidate CasX mRNAs will be evaluated using various experimental screening assays to assess mRNA stability, protein translation efficiency and expression, and editing potency. Experimental evaluation of these key properties will further inform the selection of high- 129Attorney Docket No. SCRB-061 / 01WO 333322-2448 performing mRNA constructs to be delivered to the target cell or tissue for effective CasX expression and editing of the target nucleic acid.

[0232] Experimental design:

[0233] Using the screening assays described below, three sets of candidates CasX mRNAs will be experimentally evaluated. For these experiments, the CasX coding sequences will include one of two variations of the C-terminal c-MYC NLS (listed in Table 1I), which will remain constant across the CasX sequences. In screening assays involving the incorporation of a split FP “tag” (described below), the CT_cMYC_NLS#1, which includes two additional amino acids serving as a spatial buffer, will be used. The three sets of candidate CasX mRNAs are described as follows:

[0234] A) Set #1: The candidate sequences identified for each mRNA element (5’UTR, CasX coding sequence, 3’UTR, or poly(A) tail), as described in Examples 1-4, will be individually assessed. Specifically, these candidate sequences of the mRNA element of interest will be tested in the context of a full-length CasX mRNA construct, where the remaining mRNA components will be held constant. The corresponding experimental controls for each mRNA element will be a set of randomly generated sequences to determine whether the candidate sequences will demonstrate substantial improvement over scrambled controls.

[0235] B) Set #2: Candidate sequences identified for each mRNA element (5’UTR, CasX coding sequence, 3’UTR, or poly(A) tail) will be combined in every possible combination using a custom script to generate the full-length mRNA sequences, and the sequences will be scored using metrics that evaluate protein expression and mRNA stability. Scores will be aggregated for each mRNA element, and representative full-length sequences will be identified for subsequent screening assays. The corresponding negative control will be a set of randomly generated full- length CasX mRNA sequences, and a benchmark CasX mRNA sequence (e.g., the original, non- optimized mRNA sequence) previously demonstrated to induce editing will serve as a positive control.

[0236] C) Set #3: Candidate CasX mRNA-encoding constructs will be assessed with or without the incorporation of an optimized c-MYC NLS coding sequence (SEQ ID NO: 8-24; Table 1B) on the N-terminus of the CasX coding sequence to determine the functional effects of appending an optimized N-terminal NLS coding sequence on CasX expression and activity. The corresponding negative control will be a set of randomly generated sequences, while a 130Attorney Docket No. SCRB-061 / 01WO 333322-2448 benchmark CasX mRNA sequence (e.g., the original non-optimized mRNA sequence) previously demonstrated to induce editing will serve as a positive control.

[0237] The results obtained from screening each set of CasX mRNAs described above will inform the identification of combinations of mRNA elements to generate candidate CasX mRNA sequences with improved properties. These properties include use of sequences of key mRNA elements that induce enhanced protein expression, increased stability, and / or improved editing over experimental controls (e.g., benchmark mRNA). The candidate CasX mRNA sequences that contain combinations of optimized elements will also be assessed experimentally.

[0238] Production of candidate CasX mRNAs:

[0239] For some assays, candidate engineered CasX mRNAs will be generated by in vitro transcription (IVT) using a PCR product or linearized plasmid DNA template and a T7 RNA polymerase. If using a PCR product, DNA sequences encoding candidate mRNAs will be cloned into a plasmid containing a T7 promoter. Through PCR amplification, a poly(A) tail and barcode will be added. If using a plasmid, DNA sequences encoding candidate mRNAs will be cloned into a plasmid containing a T7 promoter, a unique barcode, and a poly(A) region. The plasmid DNA template will be linearized using the SapI restriction enzyme and then used to perform IVT reactions by incubating at 37oC for 2.5 hours. The N1-methyl-pseudouridine will be used to substitute the uridine ribonucleoside for all IVT reactions. Subsequently, DNase will be added to each IVT reaction to digest template DNA.

[0240] Poly(A)-tailed mRNAs will be purified from the IVT reaction mix containing DNase using Oligo d(T) chromatography with standard buffers, followed by RNA precipitation with lithium chloride. RNA pellets will be resuspended in Storage Solution and snap frozen in liquid nitrogen prior to storage at -80oC. These resulting candidate mRNAs will be tested in the various screening assays described below in an arrayed or pooled manner.

[0241] Methods to evaluate mRNA stability:

[0242] The following methods described below are alternative assays that can be performed to evaluate mRNA stability.

[0243] An initial high-throughput screen for candidate mRNAs with components that result in enhanced mRNA stability can be performed by long-read sequencing of the full-length CasX mRNA using PacBio® Sequel II sequencing.

[0244] To screen candidate mRNAs with components that result in enhanced mRNA stability, an in-solution RNA degradation time course experiment can be performed. Briefly, a pool of a 131Attorney Docket No. SCRB-061 / 01WO 333322-2448 fixed number of barcoded CasX mRNAs will be incubated in degradation buffer (50mM N- Cyclohexyl-2-aminoethanesulfonic acid (CHES) at pH 10 and 10 mM MgCl2) and subsequently quenched in Tris ethylenediaminetetraacetic acid (EDTA) at multiple timepoints (0, 0.5, 1, 2, 3, 4, 5, 6, 16 and 24 hours). The treated RNA will be reverse transcribed into cDNA, which will be subjected to PCR amplification and next-generation sequencing (NGS) to detect barcoded amplicons. For each candidate mRNA, barcoded counts at each timepoint will be calculated relative to the counts at the initial timepoint to determine the degradation rate of the candidate mRNA over time.

[0245] An arrayed screen in cells can also be performed in parallel to screen candidate mRNAs with components that result in enhanced mRNA stability. Briefly, individual barcoded CasX mRNAs will be transfected into a cell line, which will be generated to contain a constitutively expressed reporter gene with a unique barcode. The use of the barcoded reporter will enable the normalization of readouts across samples in an arrayed screen. Following transfection, cells will be harvested at the following time points: 1, 7, 12, and 24 hours post-transfection. RNA will be extracted and reverse transcribed into cDNA, which will be subjected to PCR amplification and NGS to detect barcoded amplicons. For each timepoint, barcoded counts from the candidate mRNA will be normalized by barcoded counts from the reporter gene to calculate the degradation rate of the candidate mRNA over time.

[0246] Furthermore, a pooled screen in cells can be performed to assess candidate mRNAs with components that result in enhanced mRNA stability. Briefly, a pool of barcoded CasX mRNAs will be transfected into a cell line containing a barcoded reporter, which was briefly described above. Cells will be harvested at 1, 7, 12, and 24 hours post transfection, and RNA will be extracted and reverse transcribed into cDNA, which will be subjected to PCR amplification and NGS to detect barcoded amplicons. For each timepoint, barcoded counts from each candidate mRNA in the pool will be normalized by barcoded counts from the reporter gene to calculate the relative degradation rate of each candidate mRNA over time. Methods to evaluate protein expression:

[0247] The following methods described below are alternative assays that may be performed to evaluate protein expression.

[0248] To screen candidate mRNAs with components that result in increased expression of the CasX protein, individual candidate CasX mRNAs will be transfected into HEK293T cells, which will be harvested two days post-transfection. Harvested cells will be fixed, permeabilized, and 132Attorney Docket No. SCRB-061 / 01WO 333322-2448 blocked prior to incubation with an in-house primary CasX antibody and subsequent incubation with an appropriate secondary antibody. Stained cells will be loaded onto an Attune™ NxT flow cytometer to determine CasX expression. Alternatively, transfected cells may be grown on coverslips that will be similarly processed for analysis using a standard protocol for immunocytochemistry.

[0249] An arrayed screen can be performed to screen candidate mRNAs with components that result in increased speed and efficiency of protein expression. Here, a variation of the split fluorescent protein (splitFP) technique may be employed. Briefly, a cell line will be generated to express the larger “detection” component of the FP, which will not express fluorescence until the smaller “tag” component of the FP is expressed in the cell to reconstitute with the larger component to generate a functional FP. Individual barcoded CasX mRNA candidates, which will include the “tag” of the splitFP, will be transfected into cells in 96-well plates. Upon expression of the “tagged” CasX protein, the “tag” will complement the “detection” component to produce a functional FP, enabling the quantification of FP+ cells at multiple timepoints (e.g., 2, 4, 12, and 24 hours) via flow cytometry to determine CasX expression over time. The relative change in CasX expression over time determined for each mRNA candidate will be used to compare and identify the high-performing candidate mRNAs.

[0250] Similarly, a pooled screen using the splitFP system can be performed to assess candidate mRNAs with components that result in increased speed and efficiency of protein expression. Briefly, a pool of barcoded CasX mRNA candidates, which will include the “tag” of the splitFP, will be transfected into cells containing the “detection” component. Upon expression of the “tagged” CasX protein, the “tag” will complement the “detection” component to produce a functional FP, enabling the isolation of FP+ cells at multiple timepoints (e.g., 2, 4, 12, and 24 hours) via fluorescence activated cell sorting (FACS). RNA will be extracted from harvested cells and reverse transcribed into cDNA. The resulting cDNA will be subjected to PCR amplification and NGS to detect barcoded amplicons to identify candidate mRNAs whose delivery resulted in efficient protein expression over time. Methods to evaluate ribosomal loading:

[0251] The following methods described below are alternative assays that may be performed to evaluate ribosomal loading.

[0252] To screen candidate mRNAs with components that result in increased ribosomal loading in an arrayed format, individual barcoded CasX mRNAs will be transfected into cells containing 133Attorney Docket No. SCRB-061 / 01WO 333322-2448 a reporter gene that will be uniquely barcoded. Transfected cells will be treated with cycloheximide at 1, 7, 12, and 24 hours post-transfection to halt ribosomal processing of mRNAs. Ribosomes will be purified with an antibody targeting rRNA to isolate associating mRNAs being actively translated. Isolated RNA from the ribosomal-enriched fraction will be reverse transcribed to cDNA. The resulting cDNA will be subjected to PCR amplification and NGS to detect barcoded amplicons. For each timepoint, barcoded reads from CasX mRNA candidates and the reporter gene will yield normalized measurements indicative of ribosomal engagement for each candidate mRNA. Alternatively, long-read sequencing of the isolated mRNAs may be performed using PacBio® Sequel II sequencing to identify candidate CasX mRNAs.

[0253] Similarly, a pooled screen can be performed to screen candidate mRNAs with components that result in increased ribosomal loading. Briefly, a pool of barcoded CasX mRNAs will be transfected into cells containing a barcoded reporter. Transfected cells will be treated with cycloheximide at 1, 7, 12, and 24 hours post-transfection to halt ribosomal processing of mRNAs. Ribosomes will be purified from cells with an antibody targeting rRNA to isolate associating mRNAs being actively translated. Isolated RNA from the ribosomal- enriched fraction will be reverse transcribed to cDNA. The resulting cDNA will be subjected to PCR amplification and NGS to detect barcoded amplicons. From each timepoint, barcoded reads from CasX mRNA candidates and the reporter gene will yield normalized measurements indicative of ribosomal engagement for each candidate mRNA. Enrichment of the percentage of reads from each mRNA candidate in the ribosomal enriched pool relative to the input sample will yield the candidates with high ribosomal loading. Alternatively, long-read sequencing of the isolated mRNAs may be performed using PacBio® Sequel II sequencing to identify candidate CasX mRNAs.

[0254] An alternative method to assess ribosomal loading is Translating Ribosome Affinity Purification (TRAP)-seq, which will involve generating a cell line via integration of a lentiviral construct encoding for a tagged ribosomal subunit. Using affinity purification, tagged ribosomes will be purified to isolate mRNAs being actively translated. Alternative forms of this assay use an rRNA antibody to purify ribosomes from a wild-type line as described above. Methods to evaluate editing potency:

[0255] To screen candidate mRNAs with components that result in increased editing potency and efficiency, individual candidate CasX mRNAs with a gRNA having a targeting spacer, 134Attorney Docket No. SCRB-061 / 01WO 333322-2448 either to an AAVS1 safe harbor site or the endogenous beta-2 microglobulin (B2M) locus, will be transfected into HEK293T cells. The gRNA with a non-targeting spacer will serve as an experimental control. Editing assessment will depend on the targeting spacer used: e.g., if targeting the AAVS1 locus, editing will be determined as indel rate detected by NGS; if targeting the B2M locus, editing will be measured by the loss of surface presentation of the B2M-dependent HLA complex, as detected by flow cytometry.

[0256] The results of these experiments are expected to identify candidate CasX mRNAs with enhanced mRNA stability, protein translation efficiency and expression, and editing potency. Example 7: Demonstration that altering the UTR sequences of the engineered CasX mRNA can affect CasX-mediated editing

[0257] 5’ and 3’UTRs are essential and required for efficient translation. Here, experiments were performed to demonstrate that altering the 5’ and 3’ UTR sequences of the engineered CasX mRNA affects CasX-mediated editing at a target locus when CasX mRNA and targeting gRNAs were delivered in vitro via transfection. Materials and Methods: IVT of CasX mRNA:

[0258] CasX 676 mRNA was generated by IVT via a third-party. Briefly, constructs encoding for a 5’UTR region, CasX 676 with flanking c-MYC NLSes, and a 3’UTR region were cloned into a plasmid containing a T7 promoter and 80-nucleotide poly(A) tail. The resulting plasmid was linearized prior to use for IVT reactions, which were carried out with CleanCap® AG and N1-methyl-pseudouridine. For the 5’ cap, the CleanCap® AG contains a m7G(5’)ppp(5’)mAG structure, where “m7G” denotes N7-methylguanosine, “mA” denotes 2’O-methyladenosine, and (5’)ppp(5’) denotes a 5’ to 5’ triphosphate bridge. An extra guanine nucleotide was incorporated following the CleanCap® AG to enhance transcription initiation, resulting in the incorporation of m7G(5’)ppp(5’)mAGG as the full 5’ cap structure.

[0259] IVT reactions were subsequently subjected to DNase digestion to remove template DNA and purification using an oligo-dT column. In this example, two configurations of CasX 676 mRNAs were generated for assessment in vitro. The encoding sequences of the two CasX mRNA configurations are detailed in Table 7A. Full-length RNA sequences encoding the CasX mRNA with the chemical modifications are listed in Table 7B. 135Attorney Docket No. SCRB-061 / 01WO 333322-2448 Table 7A: Encoding sequences of the two CasX mRNA molecules assessed in this example*. CasX mRNAComponent (ID) Description DNA sequenceSEQ IDIDNO:5’UTR Human HBA ACTCTTCTGGTCCCCACAGACTCAG 445 (5UTR_opt_53) AGAGAACCCGCCACC START codon + ATGGCCCCTGCTGCCAAGAGAGTGA 581 c-MYC NLS AGCTGGATAGCAGA CasX 676 CAGGAGATCAAGCGGATTAATAAA ATTCGGAGAAGACTGGTGAAGGATT CTAACACAAAGAAGGCTGGCAAGA CACGGGGCCCTATGAAGACACTGCT GGTGAGAGTGATGACACCCGACCTG AGAGAAAGACTGGAAAACCTGAGA AAGAAGCCTGAGAATATCCCCCAGC CCATCAGCAACACAAGCCGGGCCAA CCTGAATAAGCTGCTGACCGACTAC ACCGAAATGAAGAAGGCCATCCTGC ACGTGTATTGGGAAGAGTTCCAGAA AGACCCAGTCGGCCTGATGAGCAGA GTGGCTCAGCCTGCCAGCAAGAAGA TCGATCAGAACAAGCTGAAGCCCGA AATGGACGAGAAGGGGAACCTGAC AACCGCCGGCTTTGCCTGTAGCCAG TGCGGCCAGCCCCTGTTTGTGTACA CasX 676 AACTGGAACAGGTGAGCGAAAAGG mRNA GCAAGGCTTACACGAATTACTTCGG #1 CAGATGCAACGTGGCCGAGCACGA GAAGCTGATCAAGCTGGCCCAGCTG AAGCCTGAGAAGGATAGCGATGAG 582 GCAGTGACATATTCCCTGGGCAAGT TCGGACAGCGGGCCCTGGATTTTTA TTCCATTCATGTGACCAAGGAATCC ACCCACCCCGTCAAGCCTCTTGCCC AAATTGCCGGCAACAGATACGCCTC CAGCCCCGTGGGCAAGGCCCTGAGC GACGCCTGTATGGGCACCATCGCCA GCTTCCTGTCTAAGTACCAGGACAT TATCATCGAGCACCAGAAGGTGGTG AAGGGCAACCAGAAGAGACTGGAG AGCCTGCGCGAGCTGGCCGGCAAGG AAAACCTGGAGTATCCTAGCGTGAC CCTGCCTCCTCAGCCTCATACAAAG GAGGGCGTGGATGCCTACAACGAA GTGATCGCCCGGGTGCGGATGTGGG TGAACCTGAATCTGTGGCAGAAGCT GAAGCTGTCTAGAGACGACGCCAAG CCCCTGCTGAGACTGAAGGGCTTCC CCAGCTTCCCTCTGGTGGAGAGACA GGCAAATGAAGTGGACTGGTGGGA CATGGTGTGTAACGTGAAGAAGCTG 136Attorney Docket No. SCRB-061 / 01WO 333322-2448 CasX mRNAComponent (ID) Description DNA sequenceSEQ IDIDNO:ATCAATGAGAAGAAGGAGGACGGC AAAGTGTTCTGGCAGAATCTGGCCG GCTACAAGCGTCAGGAGGCCCTGCG GCCCTACCTGAGCAGCGAGGAAGAC AGAAAGAAGGGCAAGAAGTTCGCC CGGTATCAGCTGGGGGACCTGCTGC TGCACCTCGAGAAGAAGCACGGCG AAGACTGGGGGAAGGTGTACGATG AGGCCTGGGAGCGGATCGATAAGA AGGTGGAGGGCCTGAGCAAGCACA TCAAGCTGGAGGAGGAACGGAGAT CTGAGGACGCCCAGAGCAAGGCCG CCCTGACCGACTGGCTGAGAGCCAA GGCCAGCTTCGTCATCGAGGGGCTG AAGGAGGCCGACAAGGACGAGTTC TGCCGGTGCGAACTGAAGCTGCAGA AGTGGTACGGAGATCTGAGAGGCA AACCTTTCGCCATCGAGGCCGAGAA CAGCATCCTGGACATCAGCGGCTTC AGCAAGCAGTACAACTGCGCCTTTA TTTGGCAGAAGGACGGAGTGAAGA AGCTGAACCTGTACCTGATCATCAA CTATTTCAAGGGCGGCAAGCTGAGA TTCAAGAAGATCAAGCCTGAAGCCT TCGAGGCCAACAGATTCTACACCGT GATTAACAAGAAAAGCGGAGAGAT CGTGCCAATGGAAGTGAACTTCAAC TTCGACGACCCTAACCTGATCATCC TGCCCCTGGCATTTGGCAAGCGGCA GGGCAGAGAGTTCATCTGGAACGAC CTGCTGTCTCTGGAGACCGGCAGCC TGAAGCTGGCCAACGGCAGAGTGAT CGAGAAGACACTGTACAACAGACG AACCAGACAAGACGAGCCCGCCCTG TTTGTGGCCCTGACCTTCGAGAGAA GAGAGGTGCTGGACAGCAGCAATAT CAAGCCTATGAACCTGATCGGCGTG GACCGGGGCGAGAACATCCCTGCCG TGATCGCCCTTACCGACCCCGAGGG ATGCCCTCTGAGCCGGTTTAAAGAC AGCCTGGGCAACCCTACCCACATCC TGAGAATTGGCGAGTCCTACAAGGA GAAGCAGAGAACCATCCAGGCCAA GAAGGAGGTGGAGCAGCGGCGGGC TGGCGGCTACTCCCGGAAGTACGCC AGCAAGGCCAAGAACCTGGCCGAC GACATGGTTAGAAATACCGCCAGAG ACCTCCTGTACTACGCTGTGACCCA GGACGCCATGCTGATCTTCGAGAAC CTGAGCAGAGGCTTCGGCAGACAGG GCAAGAGAACCTTCATGGCCGAGAG ACAGTACACCCGGATGGAGGACTGG CTGACCGCCAAGCTGGCCTACGAGG GCCTGCCCTCTAAGACCTACCTGTC 137Attorney Docket No. SCRB-061 / 01WO 333322-2448 CasX mRNAComponent (ID) Description DNA sequenceSEQ IDIDNO:CAAGACCTTGGCACAGTACACCAGC AAGACATGCTCTAACTGCGGCTTCA CAATCACGAGCGCCGACTACGACCG GGTGCTGGAGAAACTGAAGAAGAC CGCCACAGGCTGGATGACCACCATT AACGGCAAGGAGCTGAAGGTGGAG GGCCAGATCACCTACTACAACAGGT ACAAACGGCAGAACGTGGTGAAGG ACCTGAGCGTGGAACTGGATAGACT GAGCGAGGAAAGCGTAAACAATGA CATCAGCAGCTGGACCAAGGGCCGG AGCGGCGAGGCCCTGAGCCTGCTGA AGAAGAGATTCTCCCACAGACCAGT GCAGGAGAAGTTCGTGTGTCTGAAC TGCGGCTTCGAGACCCACGCCGACG AGCAAGCCGCCCTGAACATCGCCCG GTCTTGGCTTTTCCTGCGGAGCCAG GAGTACAAGAAGTACCAGACAAAC AAGACCACAGGCAACACAGACAAG AGAGCCTTCGTCGAGACCTGGCAGA GCTTCTACAGAAAGAAGCTGAAGGA GGTGTGGAAGCCTGCCGTG c-MYC NLS + GGAAGCCCCGCTGCCAAGAGAGTG 583 STOP codons AAGCTGGACTAATAGATAA 3’UTR Human HBA GCTGGAGCCTCGGTGGCCATGCTTC (3UTR_opt_1) TTGCCCCTTGGGCCTCCCCCCAGCCC CTCCTCCCCTTCCTGCACCCGTACCC 510 CCGTGGTCTTTGAATAAAGTCTGAG TGGGCGGCA Poly(A) tail AAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAA 584 AAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAA 5’UTR TriLink AAATAAGAGAGAAAAGAAGAGTAA 400 (5UTR_opt_8) GAAGAAATATAAGAGCCACC START codon + ATGGCCCCTGCTGCCAAGAGAGTGA 581 c-MYC NLS AGCTGGATAGCAGA CasX 676 CAGGAGATCAAGCGGATTAATAAA ATTCGGAGAAGACTGGTGAAGGATT CasX 676 CTAACACAAAGAAGGCTGGCAAGA mRNA CACGGGGCCCTATGAAGACACTGCT #2 GGTGAGAGTGATGACACCCGACCTG AGAGAAAGACTGGAAAACCTGAGA AAGAAGCCTGAGAATATCCCCCAGC 582 CCATCAGCAACACAAGCCGGGCCAA CCTGAATAAGCTGCTGACCGACTAC ACCGAAATGAAGAAGGCCATCCTGC ACGTGTATTGGGAAGAGTTCCAGAA AGACCCAGTCGGCCTGATGAGCAGA GTGGCTCAGCCTGCCAGCAAGAAGA 138Attorney Docket No. SCRB-061 / 01WO 333322-2448 CasX mRNAComponent (ID) Description DNA sequenceSEQ IDIDNO:TCGATCAGAACAAGCTGAAGCCCGA AATGGACGAGAAGGGGAACCTGAC AACCGCCGGCTTTGCCTGTAGCCAG TGCGGCCAGCCCCTGTTTGTGTACA AACTGGAACAGGTGAGCGAAAAGG GCAAGGCTTACACGAATTACTTCGG CAGATGCAACGTGGCCGAGCACGA GAAGCTGATCAAGCTGGCCCAGCTG AAGCCTGAGAAGGATAGCGATGAG GCAGTGACATATTCCCTGGGCAAGT TCGGACAGCGGGCCCTGGATTTTTA TTCCATTCATGTGACCAAGGAATCC ACCCACCCCGTCAAGCCTCTTGCCC AAATTGCCGGCAACAGATACGCCTC CAGCCCCGTGGGCAAGGCCCTGAGC GACGCCTGTATGGGCACCATCGCCA GCTTCCTGTCTAAGTACCAGGACAT TATCATCGAGCACCAGAAGGTGGTG AAGGGCAACCAGAAGAGACTGGAG AGCCTGCGCGAGCTGGCCGGCAAGG AAAACCTGGAGTATCCTAGCGTGAC CCTGCCTCCTCAGCCTCATACAAAG GAGGGCGTGGATGCCTACAACGAA GTGATCGCCCGGGTGCGGATGTGGG TGAACCTGAATCTGTGGCAGAAGCT GAAGCTGTCTAGAGACGACGCCAAG CCCCTGCTGAGACTGAAGGGCTTCC CCAGCTTCCCTCTGGTGGAGAGACA GGCAAATGAAGTGGACTGGTGGGA CATGGTGTGTAACGTGAAGAAGCTG ATCAATGAGAAGAAGGAGGACGGC AAAGTGTTCTGGCAGAATCTGGCCG GCTACAAGCGTCAGGAGGCCCTGCG GCCCTACCTGAGCAGCGAGGAAGAC AGAAAGAAGGGCAAGAAGTTCGCC CGGTATCAGCTGGGGGACCTGCTGC TGCACCTCGAGAAGAAGCACGGCG AAGACTGGGGGAAGGTGTACGATG AGGCCTGGGAGCGGATCGATAAGA AGGTGGAGGGCCTGAGCAAGCACA TCAAGCTGGAGGAGGAACGGAGAT CTGAGGACGCCCAGAGCAAGGCCG CCCTGACCGACTGGCTGAGAGCCAA GGCCAGCTTCGTCATCGAGGGGCTG AAGGAGGCCGACAAGGACGAGTTC TGCCGGTGCGAACTGAAGCTGCAGA AGTGGTACGGAGATCTGAGAGGCA AACCTTTCGCCATCGAGGCCGAGAA CAGCATCCTGGACATCAGCGGCTTC AGCAAGCAGTACAACTGCGCCTTTA TTTGGCAGAAGGACGGAGTGAAGA AGCTGAACCTGTACCTGATCATCAA CTATTTCAAGGGCGGCAAGCTGAGA TTCAAGAAGATCAAGCCTGAAGCCT 139Attorney Docket No. SCRB-061 / 01WO 333322-2448 CasX mRNAComponent (ID) Description DNA sequenceSEQ IDIDNO:TCGAGGCCAACAGATTCTACACCGT GATTAACAAGAAAAGCGGAGAGAT CGTGCCAATGGAAGTGAACTTCAAC TTCGACGACCCTAACCTGATCATCC TGCCCCTGGCATTTGGCAAGCGGCA GGGCAGAGAGTTCATCTGGAACGAC CTGCTGTCTCTGGAGACCGGCAGCC TGAAGCTGGCCAACGGCAGAGTGAT CGAGAAGACACTGTACAACAGACG AACCAGACAAGACGAGCCCGCCCTG TTTGTGGCCCTGACCTTCGAGAGAA GAGAGGTGCTGGACAGCAGCAATAT CAAGCCTATGAACCTGATCGGCGTG GACCGGGGCGAGAACATCCCTGCCG TGATCGCCCTTACCGACCCCGAGGG ATGCCCTCTGAGCCGGTTTAAAGAC AGCCTGGGCAACCCTACCCACATCC TGAGAATTGGCGAGTCCTACAAGGA GAAGCAGAGAACCATCCAGGCCAA GAAGGAGGTGGAGCAGCGGCGGGC TGGCGGCTACTCCCGGAAGTACGCC AGCAAGGCCAAGAACCTGGCCGAC GACATGGTTAGAAATACCGCCAGAG ACCTCCTGTACTACGCTGTGACCCA GGACGCCATGCTGATCTTCGAGAAC CTGAGCAGAGGCTTCGGCAGACAGG GCAAGAGAACCTTCATGGCCGAGAG ACAGTACACCCGGATGGAGGACTGG CTGACCGCCAAGCTGGCCTACGAGG GCCTGCCCTCTAAGACCTACCTGTC CAAGACCTTGGCACAGTACACCAGC AAGACATGCTCTAACTGCGGCTTCA CAATCACGAGCGCCGACTACGACCG GGTGCTGGAGAAACTGAAGAAGAC CGCCACAGGCTGGATGACCACCATT AACGGCAAGGAGCTGAAGGTGGAG GGCCAGATCACCTACTACAACAGGT ACAAACGGCAGAACGTGGTGAAGG ACCTGAGCGTGGAACTGGATAGACT GAGCGAGGAAAGCGTAAACAATGA CATCAGCAGCTGGACCAAGGGCCGG AGCGGCGAGGCCCTGAGCCTGCTGA AGAAGAGATTCTCCCACAGACCAGT GCAGGAGAAGTTCGTGTGTCTGAAC TGCGGCTTCGAGACCCACGCCGACG AGCAAGCCGCCCTGAACATCGCCCG GTCTTGGCTTTTCCTGCGGAGCCAG GAGTACAAGAAGTACCAGACAAAC AAGACCACAGGCAACACAGACAAG AGAGCCTTCGTCGAGACCTGGCAGA GCTTCTACAGAAAGAAGCTGAAGGA GGTGTGGAAGCCTGCCGTG 140Attorney Docket No. SCRB-061 / 01WO 333322-2448 CasX mRNAComponent (ID) Description DNA sequenceSEQ IDIDNO:c-MYC NLS + GGAAGCCCCGCTGCCAAGAGAGTG 583 STOP codons AAGCTGGACTAATAGATAA 3’UTR Mouse HBA GCTGCCTTCTGCGGGGCTTGCCTTCT (3UTR_opt_9) GGCCATGCCCTTCTTCTCTCCCTTGC 518 ACCTGTACCTCTTGGTCTTTGAATAA AGCCTGAGTAGGAAG XbaI restriction site (partial)TCTAGPoly(A) tail AAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAA 584 AAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAA *Components are listed in a 5’ to 3’ order within the constructs Table 7B: Full-length RNA sequences of CasX mRNA molecules assessed in this example. The 5’ cap (m7G(5’)ppp(5’)mAG), discussed in the example herein, is not shown in the table. Modification ‘mψ’ = N1-methyl-pseudouridine CasX mRNARNA sequenceCasX 676 ACmψCmψmψCmψGGmψCCCCACAGACmψCAGAGAGAACCCGCCACCAmψGGCCC mRNA #1 (SEQ CmψGCmψGCCAAGAGAGmψGAAGCmψGGAmψAGCAGACAGGAGAmψCAAGCGG ID NO: 585) AmψmψAAmψAAAAmψmψCGGAGAAGACmψGGmψGAAGGAmψmψCmψAACACAA AGAAGGCmψGGCAAGACACGGGGCCCmψAmψGAAGACACmψGCmψGGmψGAGA GmψGAmψGACACCCGACCmψGAGAGAAAGACmψGGAAAACCmψGAGAAAGAAG CCmψGAGAAmψAmψCCCCCAGCCCAmψCAGCAACACAAGCCGGGCCAACCmψGA AmψAAGCmψGCmψGACCGACmψACACCGAAAmψGAAGAAGGCCAmψCCmψGCA CGmψGmψAmψmψGGGAAGAGmψmψCCAGAAAGACCCAGmψCGGCCmψGAmψGA GCAGAGmψGGCmψCAGCCmψGCCAGCAAGAAGAmψCGAmψCAGAACAAGCmψG AAGCCCGAAAmψGGACGAGAAGGGGAACCmψGACAACCGCCGGCmψmψmψGCC mψGmψAGCCAGmψGCGGCCAGCCCCmψGmψmψmψGmψGmψACAAACmψGGAAC AGGmψGAGCGAAAAGGGCAAGGCmψmψACACGAAmψmψACmψmψCGGCAGAmψ GCAACGmψGGCCGAGCACGAGAAGCmψGAmψCAAGCmψGGCCCAGCmψGAAGC CmψGAGAAGGAmψAGCGAmψGAGGCAGmψGACAmψAmψmψCCCmψGGGCAAGm ψmψCGGACAGCGGGCCCmψGGAmψmψmψmψmψAmψmψCCAmψmψCAmψGmψGA CCAAGGAAmψCCACCCACCCCGmψCAAGCCmψCmψmψGCCCAAAmψmψGCCGGC AACAGAmψACGCCmψCCAGCCCCGmψGGGCAAGGCCCmψGAGCGACGCCmψGmψ AmψGGGCACCAmψCGCCAGCmψmψCCmψGmψCmψAAGmψACCAGGACAmψmψA mψCAmψCGAGCACCAGAAGGmψGGmψGAAGGGCAACCAGAAGAGACmψGGAGA GCCmψGCGCGAGCmψGGCCGGCAAGGAAAACCmψGGAGmψAmψCCmψAGCGmψ GACCCmψGCCmψCCmψCAGCCmψCAmψACAAAGGAGGGCGmψGGAmψGCCmψA CAACGAAGmψGAmψCGCCCGGGmψGCGGAmψGmψGGGmψGAACCmψGAAmψCm ψGmψGGCAGAAGCmψGAAGCmψGmψCmψAGAGACGACGCCAAGCCCCmψGCmψ GAGACmψGAAGGGCmψmψCCCCAGCmψmψCCCmψCmψGGmψGGAGAGACAGGC AAAmψGAAGmψGGACmψGGmψGGGACAmψGGmψGmψGmψAACGmψGAAGAAGC mψGAmψCAAmψGAGAAGAAGGAGGACGGCAAAGmψGmψmψCmψGGCAGAAmψC mψGGCCGGCmψACAAGCGmψCAGGAGGCCCmψGCGGCCCmψACCmψGAGCAGC 141Attorney Docket No. SCRB-061 / 01WO 333322-2448 CasX mRNARNA sequenceGAGGAAGACAGAAAGAAGGGCAAGAAGmψmψCGCCCGGmψAmψCAGCmψGGGG GACCmψGCmψGCmψGCACCmψCGAGAAGAAGCACGGCGAAGACmψGGGGGAAG GmψGmψACGAmψGAGGCCmψGGGAGCGGAmψCGAmψAAGAAGGmψGGAGGGCC mψGAGCAAGCACAmψCAAGCmψGGAGGAGGAACGGAGAmψCmψGAGGACGCCC AGAGCAAGGCCGCCCmψGACCGACmψGGCmψGAGAGCCAAGGCCAGCmψmψCG mψCAmψCGAGGGGCmψGAAGGAGGCCGACAAGGACGAGmψmψCmψGCCGGmψG CGAACmψGAAGCmψGCAGAAGmψGGmψACGGAGAmψCmψGAGAGGCAAACCmψ mψmψCGCCAmψCGAGGCCGAGAACAGCAmψCCmψGGACAmψCAGCGGCmψmψC AGCAAGCAGmψACAACmψGCGCCmψmψmψAmψmψmψGGCAGAAGGACGGAGmψ GAAGAAGCmψGAACCmψGmψACCmψGAmψCAmψCAACmψAmψmψmψCAAGGGC GGCAAGCmψGAGAmψmψCAAGAAGAmψCAAGCCmψGAAGCCmψmψCGAGGCCA ACAGAmψmψCmψACACCGmψGAmψmψAACAAGAAAAGCGGAGAGAmψCGmψGC CAAmψGGAAGmψGAACmψmψCAACmψmψCGACGACCCmψAACCmψGAmψCAmψ CCmψGCCCCmψGGCAmψmψmψGGCAAGCGGCAGGGCAGAGAGmψmψCAmψCmψ GGAACGACCmψGCmψGmψCmψCmψGGAGACCGGCAGCCmψGAAGCmψGGCCAA CGGCAGAGmψGAmψCGAGAAGACACmψGmψACAACAGACGAACCAGACAAGAC GAGCCCGCCCmψGmψmψmψGmψGGCCCmψGACCmψmψCGAGAGAAGAGAGGmψ GCmψGGACAGCAGCAAmψAmψCAAGCCmψAmψGAACCmψGAmψCGGCGmψGGA CCGGGGCGAGAACAmψCCCmψGCCGmψGAmψCGCCCmψmψACCGACCCCGAGGG AmψGCCCmψCmψGAGCCGGmψmψmψAAAGACAGCCmψGGGCAACCCmψACCCAC AmψCCmψGAGAAmψmψGGCGAGmψCCmψACAAGGAGAAGCAGAGAACCAmψCC AGGCCAAGAAGGAGGmψGGAGCAGCGGCGGGCmψGGCGGCmψACmψCCCGGAA GmψACGCCAGCAAGGCCAAGAACCmψGGCCGACGACAmψGGmψmψAGAAAmψA CCGCCAGAGACCmψCCmψGmψACmψACGCmψGmψGACCCAGGACGCCAmψGCmψ GAmψCmψmψCGAGAACCmψGAGCAGAGGCmψmψCGGCAGACAGGGCAAGAGAA CCmψmψCAmψGGCCGAGAGACAGmψACACCCGGAmψGGAGGACmψGGCmψGAC CGCCAAGCmψGGCCmψACGAGGGCCmψGCCCmψCmψAAGACCmψACCmψGmψCC AAGACCmψmψGGCACAGmψACACCAGCAAGACAmψGCmψCmψAACmψGCGGCm ψmψCACAAmψCACGAGCGCCGACmψACGACCGGGmψGCmψGGAGAAACmψGAA GAAGACCGCCACAGGCmψGGAmψGACCACCAmψmψAACGGCAAGGAGCmψGAA GGmψGGAGGGCCAGAmψCACCmψACmψACAACAGGmψACAAACGGCAGAACGm ψGGmψGAAGGACCmψGAGCGmψGGAACmψGGAmψAGACmψGAGCGAGGAAAGC GmψAAACAAmψGACAmψCAGCAGCmψGGACCAAGGGCCGGAGCGGCGAGGCCC mψGAGCCmψGCmψGAAGAAGAGAmψmψCmψCCCACAGACCAGmψGCAGGAGAA GmψmψCGmψGmψGmψCmψGAACmψGCGGCmψmψCGAGACCCACGCCGACGAGC AAGCCGCCCmψGAACAmψCGCCCGGmψCmψmψGGCmψmψmψmψCCmψGCGGAGC CAGGAGmψACAAGAAGmψACCAGACAAACAAGACCACAGGCAACACAGACAAG AGAGCCmψmψCGmψCGAGACCmψGGCAGAGCmψmψCmψACAGAAAGAAGCmψG AAGGAGGmψGmψGGAAGCCmψGCCGmψGGGAAGCCCCGCmψGCCAAGAGAGmψ GAAGCmψGGACmψAAmψAGAmψAAGCmψGGAGCCmψCGGmψGGCCAmψGCmψm ψCmψmψGCCCCmψmψGGGCCmψCCCCCCAGCCCCmψCCmψCCCCmψmψCCmψGC ACCCGmψACCCCCGmψGGmψCmψmψmψGAAmψAAAGmψCmψGAGmψGGGCGGC AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAA CasX 676 AAAmψAAGAGAGAAAAGAAGAGmψAAGAAGAAAmψAmψAAGAGCCACCAmψG mRNA #2 (SEQ GCCCCmψGCmψGCCAAGAGAGmψGAAGCmψGGAmψAGCAGACAGGAGAmψCAA ID NO: 586) GCGGAmψmψAAmψAAAAmψmψCGGAGAAGACmψGGmψGAAGGAmψmψCmψAAC ACAAAGAAGGCmψGGCAAGACACGGGGCCCmψAmψGAAGACACmψGCmψGGmψ GAGAGmψGAmψGACACCCGACCmψGAGAGAAAGACmψGGAAAACCmψGAGAAA GAAGCCmψGAGAAmψAmψCCCCCAGCCCAmψCAGCAACACAAGCCGGGCCAACC mψGAAmψAAGCmψGCmψGACCGACmψACACCGAAAmψGAAGAAGGCCAmψCCm ψGCACGmψGmψAmψmψGGGAAGAGmψmψCCAGAAAGACCCAGmψCGGCCmψGA mψGAGCAGAGmψGGCmψCAGCCmψGCCAGCAAGAAGAmψCGAmψCAGAACAAG 142Attorney Docket No. SCRB-061 / 01WO 333322-2448CasX mRNARNA sequenceCmψGAAGCCCGAAAmψGGACGAGAAGGGGAACCmψGACAACCGCCGGCmψmψm ψGCCmψGmψAGCCAGmψGCGGCCAGCCCCmψGmψmψmψGmψGmψACAAACmψG GAACAGGmψGAGCGAAAAGGGCAAGGCmψmψACACGAAmψmψACmψmψCGGCA GAmψGCAACGmψGGCCGAGCACGAGAAGCmψGAmψCAAGCmψGGCCCAGCmψG AAGCCmψGAGAAGGAmψAGCGAmψGAGGCAGmψGACAmψAmψmψCCCmψGGGC AAGmψmψCGGACAGCGGGCCCmψGGAmψmψmψmψmψAmψmψCCAmψmψCAmψG mψGACCAAGGAAmψCCACCCACCCCGmψCAAGCCmψCmψmψGCCCAAAmψmψGC CGGCAACAGAmψACGCCmψCCAGCCCCGmψGGGCAAGGCCCmψGAGCGACGCC mψGmψAmψGGGCACCAmψCGCCAGCmψmψCCmψGmψCmψAAGmψACCAGGACA mψmψAmψCAmψCGAGCACCAGAAGGmψGGmψGAAGGGCAACCAGAAGAGACmψ GGAGAGCCmψGCGCGAGCmψGGCCGGCAAGGAAAACCmψGGAGmψAmψCCmψA GCGmψGACCCmψGCCmψCCmψCAGCCmψCAmψACAAAGGAGGGCGmψGGAmψG CCmψACAACGAAGmψGAmψCGCCCGGGmψGCGGAmψGmψGGGmψGAACCmψGA AmψCmψGmψGGCAGAAGCmψGAAGCmψGmψCmψAGAGACGACGCCAAGCCCCm ψGCmψGAGACmψGAAGGGCmψmψCCCCAGCmψmψCCCmψCmψGGmψGGAGAGA CAGGCAAAmψGAAGmψGGACmψGGmψGGGACAmψGGmψGmψGmψAACGmψGAA GAAGCmψGAmψCAAmψGAGAAGAAGGAGGACGGCAAAGmψGmψmψCmψGGCAG AAmψCmψGGCCGGCmψACAAGCGmψCAGGAGGCCCmψGCGGCCCmψACCmψGA GCAGCGAGGAAGACAGAAAGAAGGGCAAGAAGmψmψCGCCCGGmψAmψCAGCm ψGGGGGACCmψGCmψGCmψGCACCmψCGAGAAGAAGCACGGCGAAGACmψGGG GGAAGGmψGmψACGAmψGAGGCCmψGGGAGCGGAmψCGAmψAAGAAGGmψGGA GGGCCmψGAGCAAGCACAmψCAAGCmψGGAGGAGGAACGGAGAmψCmψGAGGA CGCCCAGAGCAAGGCCGCCCmψGACCGACmψGGCmψGAGAGCCAAGGCCAGCm ψmψCGmψCAmψCGAGGGGCmψGAAGGAGGCCGACAAGGACGAGmψmψCmψGCC GGmψGCGAACmψGAAGCmψGCAGAAGmψGGmψACGGAGAmψCmψGAGAGGCAA ACCmψmψmψCGCCAmψCGAGGCCGAGAACAGCAmψCCmψGGACAmψCAGCGGC mψmψCAGCAAGCAGmψACAACmψGCGCCmψmψmψAmψmψmψGGCAGAAGGACG GAGmψGAAGAAGCmψGAACCmψGmψACCmψGAmψCAmψCAACmψAmψmψmψCA AGGGCGGCAAGCmψGAGAmψmψCAAGAAGAmψCAAGCCmψGAAGCCmψmψCGA GGCCAACAGAmψmψCmψACACCGmψGAmψmψAACAAGAAAAGCGGAGAGAmψC GmψGCCAAmψGGAAGmψGAACmψmψCAACmψmψCGACGACCCmψAACCmψGAm ψCAmψCCmψGCCCCmψGGCAmψmψmψGGCAAGCGGCAGGGCAGAGAGmψmψCA mψCmψGGAACGACCmψGCmψGmψCmψCmψGGAGACCGGCAGCCmψGAAGCmψG GCCAACGGCAGAGmψGAmψCGAGAAGACACmψGmψACAACAGACGAACCAGAC AAGACGAGCCCGCCCmψGmψmψmψGmψGGCCCmψGACCmψmψCGAGAGAAGAG AGGmψGCmψGGACAGCAGCAAmψAmψCAAGCCmψAmψGAACCmψGAmψCGGCG mψGGACCGGGGCGAGAACAmψCCCmψGCCGmψGAmψCGCCCmψmψACCGACCCC GAGGGAmψGCCCmψCmψGAGCCGGmψmψmψAAAGACAGCCmψGGGCAACCCmψ ACCCACAmψCCmψGAGAAmψmψGGCGAGmψCCmψACAAGGAGAAGCAGAGAAC CAmψCCAGGCCAAGAAGGAGGmψGGAGCAGCGGCGGGCmψGGCGGCmψACmψC CCGGAAGmψACGCCAGCAAGGCCAAGAACCmψGGCCGACGACAmψGGmψmψAG AAAmψACCGCCAGAGACCmψCCmψGmψACmψACGCmψGmψGACCCAGGACGCC AmψGCmψGAmψCmψmψCGAGAACCmψGAGCAGAGGCmψmψCGGCAGACAGGGC AAGAGAACCmψmψCAmψGGCCGAGAGACAGmψACACCCGGAmψGGAGGACmψG GCmψGACCGCCAAGCmψGGCCmψACGAGGGCCmψGCCCmψCmψAAGACCmψACC mψGmψCCAAGACCmψmψGGCACAGmψACACCAGCAAGACAmψGCmψCmψAACm ψGCGGCmψmψCACAAmψCACGAGCGCCGACmψACGACCGGGmψGCmψGGAGAA ACmψGAAGAAGACCGCCACAGGCmψGGAmψGACCACCAmψmψAACGGCAAGGA GCmψGAAGGmψGGAGGGCCAGAmψCACCmψACmψACAACAGGmψACAAACGGC AGAACGmψGGmψGAAGGACCmψGAGCGmψGGAACmψGGAmψAGACmψGAGCGA GGAAAGCGmψAAACAAmψGACAmψCAGCAGCmψGGACCAAGGGCCGGAGCGGC GAGGCCCmψGAGCCmψGCmψGAAGAAGAGAmψmψCmψCCCACAGACCAGmψGC AGGAGAAGmψmψCGmψGmψGmψCmψGAACmψGCGGCmψmψCGAGACCCACGCC GACGAGCAAGCCGCCCmψGAACAmψCGCCCGGmψCmψmψGGCmψmψmψmψCCmψ 143Attorney Docket No. SCRB-061 / 01WO 333322-2448 CasX mRNARNA sequenceGCGGAGCCAGGAGmψACAAGAAGmψACCAGACAAACAAGACCACAGGCAACAC AGACAAGAGAGCCmψmψCGmψCGAGACCmψGGCAGAGCmψmψCmψACAGAAAG AAGCmψGAAGGAGGmψGmψGGAAGCCmψGCCGmψGGGAAGCCCCGCmψGCCAA GAGAGmψGAAGCmψGGACmψAAmψAGAmψAAGCmψGCCmψmψCmψGCGGGGCm ψmψGCCmψmψCmψGGCCAmψGCCCmψmψCmψmψCmψCmψCCCmψmψGCACCmψG mψACCmψCmψmψGGmψCmψmψmψGAAmψAAAGCCmψGAGmψAGGAAGmψCmψA GAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAA Synthesis of gRNAs:

[0260] In this example, gRNAs targeting the mouse PCSK9 locus were designed using gRNA scaffold 174 and chemically synthesized. The sequences of the PCSK9-targeting spacers are listed in Table 7C. Table 7C: Sequences of spacers targeting the mouse PCSK9 locus assayed in this example. Spacer ID Target RNA sequence SEQ ID NO: 27.103 mousePCSK9UAAUCUCCAUCCUCGUCCUG 58727.105 mousePCSK9CCAAGAAGCCAGGGAAGAGG 58827.106 mousePCSK9ACAUAUCUUUUAUGACCUCU 58927.107 mousePCSK9CUGGCUUCUUGGUGAAGAUG 59027.108 mousePCSK9UGGUGAAGAUGAGCAGUGAC 59127.116 mousePCSK9GCCGUUGCUCCAAGGUAUGG 59227.117 mousePCSK9UUCUUGGGGAUCAGGAGGCC 593Transfection of CasX mRNA and gRNA into mouse Hepa1-6 cells in vitro:

[0261] Editing at the mouse PCSK9 locus was assessed by delivering in vitro transcribed CasX mRNA (CasX mRNA #1 or CasX mRNA #2; see Table 7A) and synthesized gRNAs targeting PCSK9 into Hepa1-6 cells via transfection. Briefly, each well of 20,000 Hepa1-6 cells were lipofected with in vitro transcribed mRNA coding for CasX 676 and a PCSK9-targeting gRNA. 144Attorney Docket No. SCRB-061 / 01WO 333322-2448 After a media change, transfected cells were harvested at 20 hours post-transfection for editing assessment at the PCSK9 locus by NGS. As experimental controls, individual transfections of CasX mRNA #1 and CasX mRNA #2 without gRNAs were performed. NGS processing and analysis:

[0262] Genomic DNA (gDNA) from harvested cells were extracted using the Zymo Quick- DNA™ Miniprep Plus kit following the manufacturer’s instructions. Target amplicons were formed by amplifying regions of interest from extracted gDNA with a set of primers targeting the mouse PCSK9 locus. These gene-specific primers contain an additional sequence at the 5′ end to introduce an Illumina™ adapter and a 16-nucleotide unique molecular identifier. Amplified DNA products were purified with the Ampure XP DNA cleanup kit. Quality and quantification of the amplicon were assessed using a Fragment Analyzer DNA Analysis kit (Agilent Technologies, dsDNA 35-1500bp). Amplicons were sequenced on the Illumina™ MiSeq™ sequencer according to the manufacturer’s instructions. Raw fastq files from sequencing were quality-controlled and processed using cutadapt v2.1, flash2 v2.2.00, and CRISPResso2 v2.0.29. Each sequence was quantified for containing an insertion or deletion (indel) relative to the reference sequence, in a window around the 3’ end of the spacer. CasX editing activity was quantified as the total percent of reads that contain insertions, substitutions, and / or deletions anywhere within this window for each sample. Results:

[0263] CasX-mediated editing at the mouse PCSK9 locus was used to evaluate the effects of incorporating different 5’ and 3’ UTRs into the engineered CasX mRNA. The plot in FIG.2 shows the quantification of percent editing measured as indel rate at the PCSK9 locus in mouse Hepa1-6 cells transfected with CasX 676 mRNA #1 or CasX 676 mRNA #2 with the indicated PCSK9-targeting gRNAs. The data demonstrate that for all targeting spacers tested in this i t C X RNA #2 i t tl hibit d hi h diti levels at the mouse PCSK9 cifically, the highest level of RNA #2 resulted in ~35% #1 (FIG.2). sequences of the CasX cell-based assay. These n and engineering and justify145Attorney Docket No. SCRB-061 / 01WO 333322-2448 TR sequences as discussed in ated as 3’UTR sequences in nt into the 3’UTR of a ranscript from degradation. In ENE element can be used as a express the CasX mRNA. adenylation following the RNA. tes. Briefly, PCR was carlet with a forward primer ollowing: 1) a 100-nucleotide encoding these key elements IVT reactions. In addition, in Example 7) and N1- e digestion to remove earch RNA miniprep kit. coli PolyA Polymerase (New wed by column-based RNA sequences (without the Table 8B. assessed in this example*. SEQ e ID NO: 594TGAAGCTGGACTCT 595146Attorney Docket No. SCRB-061 / 01WO 333322-2448 SEQ e ID NO: AGATCAGAAGGAGA AGAAGGCCGGCAAG TCGTCAGAGTGATGA GGAAAACCTGAGAA GCCTATCAGCAACAC CTGCTGACCGACTA CTGCACGTGTACTGG TGGGCCTGATGAGC AGAAGATCGACCAG ACGAGAAGGGCAAT CTCTCAGTGTGGCC GAACAGGTGTCCGA TACTTCGGCAGATGT TGATTCTGCTGGCCC TGATGAGGCCGTGA ACAGAGAGCCCTGGA AAGAAAGCACACAC TCGCCGGCAATAGAT AGCCCTGTCCGATGC TTCCTGAGCAAGTAC AGAAGGTGGTCAAG GCCTGAGGGAGCTG ACCCCAGCGTGACC AGAAGGCGTGGACG AGTGAGAATGTGGG GCTGAAACTGTCCAG AGACTGAAGGGCTTC 596 ACAGGCCAATGAAG CAACGTGAAGAAGCT GGCAAGGTTTTCTG AGACAAGAAGCCCT GAGGACCGGAAGAA CAGCTGGGCGACCT CACGGCGAGGACTG TGGGAGAGAATCGA AAGCACATTAAGCT GGACGCCCAATCTAA GAGCCAAGGCCAGC AGGCCGACAAGGAC AGCTGCAGAAGTGGT CTTCGCCATTGAGGC AGCGGCTTCAGCAA GGCAGAAAGACGGC TGATCATCAATTACT CAAGAAGATCAAAC ATTCTACACCGTGAT GTGCCCATGGAAGT ACCTGATTATCCTG AGGGCAGAGAGTTCA GAAACCGGCTCTCT ATCGAGAAAACCCT GACGAGCCTGCTCT GAAGAGAGGTGCTG147Attorney Docket No. SCRB-061 / 01WO 333322-2448 SEQ e ID NO: TGAACCTGATCGGC CTGCTGTGATCGCCC ACTGAGCAGATTCAA CACATCCTGAGAATCCAGAGGACAATCCAG GCCAAGAAAGAGGTGGAACAGCGCAGAGCCGGCGGA TACTCTAGGAAGTACGCCAGCAAGGCCAAGAATCTG GCCGACGACATGGTCCGAAACACCGCCAGAGATCTG CTGTACTACGCCGTGACACAGGACGCCATGCTGATCT TCGAGAATCTGAGCAGAGGCTTCGGCCGGCAGGGCA AGAGAACCTTTATGGCCGAGAGGCAGTACACCAGAA TGGAAGATTGGCTCACAGCTAAACTGGCCTACGAGGG ACTGAGCAAGACCTACCTGTCCAAAACACTGGCCCAG TATACCTCCAAGACCTGCAGCAATTGCGGCTTCACCA TCACCAGCGCCGACTACGACAGAGTGCTGGAAAAGC TCAAGAAAACCGCCACCGGCTGGATGACCACCATCA ACGGCAAAGAGCTGAAGGTTGAGGGCCAGATCACCT ACTACAACAGGTACAAGAGGCAGAACGTCGTGAAGG ATCTGAGCGTGGAACTGGACAGACTGAGCGAAGAGA GCGTGAACAACGACATCAGCAGCTGGACAAAGGGCA GATCAGGCGAGGCTCTGAGCCTGCTGAAGAAGAGGT TTAGCCACAGACCTGTGCAAGAGAAGTTCGTGTGCCT GAACTGCGGCTTCGAGACACACGCCGATGAACAGGC TGCCCTGAACATTGCCAGAAGCTGGCTGTTCCTGAGA AGCCAAGAGTACAAGAAGTACCAGACCAACAAGACC ACCGGCAACACCGACAAGAGGGCCTTTGTGGAAACC TGGCAGAGCTTCTACAGAAAAAAGCTGAAAGAAGTC TGGAAGCCCGCCGTG Linker + c-MYC GGCTCCCCCGCCGCGAAGCGAGTGAAACTGGACGGA NLSTCCGGC597P2A mScarlet + GCAACAAACTTCTCTCTGCTGAAACAAGCCGGAGATG STOP codon TCGAAGAGAATCCTGGACCGATGGTATCAAAGGGTG AGGCGGTCATTAAGGAATTCATGCGGTTCAAAGTTCA CATGGAGGGGAGTATGAATGGTCACGAATTTGAGATC GAGGGTGAGGGAGAGGGTAGACCTTACGAAGGCACC CAGACAGCAAAACTCAAAGTCACTAAGGGCGGCCCA CTCCCTTTCTCATGGGATATACTGAGTCCTCAATTTAT GTATGGCTCCCGGGCCTTTATCAAGCATCCAGCCGAC ATACCCGATTACTATAAACAGTCATTCCCCGAGGGAT TCAAATGGGAGAGAGTGATGAATTTTGAAGACGGGG GTGCCGTGACCGTAACTCAAGATACCTCTTTGGAAGA 598 TGGTACCCTCATTTACAAGGTTAAGCTTAGAGGAACT AATTTCCCACCAGACGGACCCGTTATGCAGAAAAAAA CCATGGGTTGGGAGGCTAGCACCGAACGGCTGTATCC AGAGGATGGAGTTTTGAAAGGTGATATTAAGATGGCC CTGCGCCTGAAAGACGGAGGGCGGTACCTCGCAGATT TCAAAACTACCTACAAAGCTAAGAAACCGGTCCAAAT GCCGGGTGCTTATAACGTCGATAGGAAGCTGGATATA ACATCACACAACGAAGACTATACGGTTGTCGAACAGT ATGAGCGATCTGAGGGGAGGCACAGCACGGGCGGGA TGGATGAGCTTTACAAGTGA 148Attorney Docket No. SCRB-061 / 01WO 333322-2448 CasX 491 SEQ mRNA IDComponent (ID) DNA sequenceID NO:149Attorney Docket No. SCRB-061 / 01WO 333322-2448AACCCmΨACACACAmΨCCmΨGAGAAmΨCGGCGAGAGCmΨACAAAGAGAAGCAG AGGACAAmΨCCAGGCCAAGAAAGAGGmΨGGAACAGCGCAGAGCCGGCGGAmΨA CmΨCmΨAGGAAGmΨACGCCAGCAAGGCCAAGAAmΨCmΨGGCCGACGACAmΨGG mΨCCGAAACACCGCCAGAGAmΨCmΨGCmΨGmΨACmΨACGCCGmΨGACACAGGA CGCCAmΨGCmΨGAmΨCmΨmΨCGAGAAmΨCmΨGAGCAGAGGCmΨmΨCGGCCGGC AGGGCAAGAGAACCmΨmΨmΨAmΨGGCCGAGAGGCAGmΨACACCAGAAmΨGGAA GAmΨmΨGGCmΨCACAGCmΨAAACmΨGGCCmΨACGAGGGACmΨGAGCAAGACCm ΨACCmΨGmΨCCAAAACACmΨGGCCCAGmΨAmΨACCmΨCCAAGACCmΨGCAGCA150Attorney Docket No. SCRB-061 / 01WO 333322-2448GCCAAmΨGAAGmΨGGAmΨmΨGGmΨGGGACAmΨGGmΨCmΨGCAACGmΨGAAGA AGCmΨGAmΨCAACGAGAAGAAAGAGGAmΨGGCAAGGmΨmΨmΨmΨCmΨGGCAG AACCmΨGGCCGGCmΨACAAGAGACAAGAAGCCCmΨGAGGCCmΨmΨACCmΨGAG CAGCGAAGAGGACCGGAAGAAGGGCAAGAAGmΨmΨCGCCAGAmΨACCAGCmΨG GGCGACCmΨGCmΨGCmΨGCACCmΨGGAAAAGAAGCACGGCGAGGACmΨGGGGC AAAGmΨGmΨACGAmΨGAGGCCmΨGGGAGAGAAmΨCGACAAGAAGGmΨGGAAG GCCmΨGAGCAAGCACAmΨmΨAAGCmΨGGAAGAGGAAAGAAGGAGCGAGGACGC CCAAmΨCmΨAAAGCCGCmΨCmΨGACCGAmΨmΨGGCmΨGAGAGCCAAGGCCAGC mΨmΨmΨGmΨGAmΨCGAGGGCCmΨGAAAGAGGCCGACAAGGACGAGmΨmΨCmΨ GCAGAmΨGCGAGCmΨGAAGCmΨGCAGAAGmΨGGmΨACGGCGAmΨCmΨGAGAGG CAAGCCCmΨmΨCGCCAmΨmΨGAGGCCGAGAACAGCAmΨCCmΨGGACAmΨCAGC GGCmΨmΨCAGCAAGCAGmΨACAACmΨGCGCCmΨmΨCAmΨmΨmΨGGCAGAAAGA CGGCGmΨCAAGAAACmΨGAACCmΨGmΨACCmΨGAmΨCAmΨCAAmΨmΨACmΨm ΨCAAAGGCGGCAAGCmΨGCGGmΨmΨCAAGAAGAmΨCAAACCCGAGGCCmΨmΨC GAGGCmΨAACAGAmΨmΨCmΨACACCGmΨGAmΨCAACAAAAAGmΨCCGGCGAGA mΨCGmΨGCCCAmΨGGAAGmΨGAACmΨmΨCAACmΨmΨCGACGACCCCAACCmΨG AmΨmΨAmΨCCmΨGCCmΨCmΨGGCCmΨmΨCGGCAAGAGACAGGGCAGAGAGmΨm ΨCAmΨCmΨGGAACGAmΨCmΨGCmΨGAGCCmΨGGAAACCGGCmΨCmΨCmΨGAAG CmΨGGCCAAmΨGGCAGAGmΨGAmΨCGAGAAAACCCmΨGmΨACAACAGGAGAAC CAGACAGGACGAGCCmΨGCmΨCmΨGmΨmΨmΨGmΨGGCCCmΨGACCmΨmΨCGAG AGAAGAGAGGmΨGCmΨGGACAGCAGCAACAmΨCAAGCCCAmΨGAACCmΨGAmΨ CGGCGmΨGGACCGGGGCGAGAAmΨAmΨCCCmΨGCmΨGmΨGAmΨCGCCCmΨGAC AGACCCmΨGAAGGAmΨGCCCACmΨGAGCAGAmΨmΨCAAGGACmΨCCCmΨGGGC AACCCmΨACACACAmΨCCmΨGAGAAmΨCGGCGAGAGCmΨACAAAGAGAAGCAG AGGACAAmΨCCAGGCCAAGAAAGAGGmΨGGAACAGCGCAGAGCCGGCGGAmΨA CmΨCmΨAGGAAGmΨACGCCAGCAAGGCCAAGAAmΨCmΨGGCCGACGACAmΨGG mΨCCGAAACACCGCCAGAGAmΨCmΨGCmΨGmΨACmΨACGCCGmΨGACACAGGA 151Attorney Docket No. SCRB-061 / 01WO 333322-2448 CasX 491 mRNA IDRNA sequenceCGCCAmΨGCmΨGAmΨCmΨmΨCGAGAAmΨCmΨGAGCAGAGGCmΨmΨCGGCCGGC AGGGCAAGAGAACCmΨmΨmΨAmΨGGCCGAGAGGCAGmΨACACCAGAAmΨGGAA GAmΨmΨGGCmΨCACAGCmΨAAACmΨGGCCmΨACGAGGGACmΨGAGCAAGACCm ΨACCmΨGmΨCCAAAACACmΨGGCCCAGmΨAmΨACCmΨCCAAGACCmΨGCAGCA AmΨmΨGCGGCmΨmΨCACCAmΨCACCAGCGCCGACmΨACGACAGAGmΨGCmΨGG AAAAGC ΨCAAGAAAACCGCCACCGGC ΨGGA ΨGACCACCA ΨCAACGGCAAAΨCCmΨGCACGmΨGmΨACmΨGGGAAGAGmΨmΨCCAGAAAGACCCCGmΨGGGCCm ΨGAmΨGAGCAGAGmΨmΨGCmΨCAGCCmΨGCCAGCAAGAAGAmΨCGACCAGAAC AAGCmΨGAAGCCCGAGAmΨGGACGAGAAGGGCAAmΨCmΨGACCACAGCCGGCm ΨmΨmΨGCCmΨGCmΨCmΨCAGmΨGmΨGGCCAGCCmΨCmΨGmΨmΨCGmΨGmΨAC AAGCmΨGGAACAGGmΨGmΨCCGAGAAAGGCAAGGCCmΨACACCAACmΨACmΨm ΨCGGCAGAmΨGmΨAACGmΨGGCCGAGCACGAGAAGCmΨGAmΨmΨCmΨGCmΨGG CCCAGCmΨGAAACCmΨGAGAAGGACmΨCmΨGAmΨGAGGCCGmΨGACCmΨACAG CCmΨGGGCAAGmΨmΨmΨGGACAGAGAGCCCmΨGGACmΨmΨCmΨACAGCAmΨCC ACGmΨGACCAAAGAAAGCACACACCCCGmΨGAAGCCCCmΨGGCmΨCAGAmΨCGC CGGCAAmΨAGAmΨACGCCmΨCmΨGGACCmΨGmΨGGGCAAAGCCCmΨGmΨCCGA mΨGCCmΨGCAmΨGGGAACAAmΨCGCCAGCmΨmΨCCmΨGAGCAAGmΨACCAGGA 152Attorney Docket No. SCRB-061 / 01WO 333322-2448 CasX 491 mRNA IDRNA sequenceΨGCmΨGAAACAAGCCGGAGAmΨGmΨCGAAGAGAAmΨCCmΨGGACCGAmΨGGmΨ AmΨCAAAGGGmΨGAGGCGGmΨCAmΨmΨAAGGAAmΨmΨCAmΨGCGGmΨmΨCAA AGmΨmΨCACAmΨGGAGGGGAGmΨAmΨGAAmΨGGmΨCACGAAmΨmΨmΨGAGAm ΨCGAGGGmΨGAGGGAGAGGGmΨAGACCmΨmΨACGAAGGCACCCAGACAGCAAA ACmΨCAAAGmΨCACmΨAAGGGCGGCCCACmΨCCCmΨmΨmΨCmΨCAmΨGGGAmΨ 153Attorney Docket No. SCRB-061 / 01WO 333322-2448 CasX 491 mRNA IDRNA sequenceAmΨACmΨGAGmΨCCmΨCAAmΨmΨmΨAmΨGmΨAmΨGGCmΨCCCGGGCCmΨmΨm ΨAmΨCAAGCAmΨCCAGCCGACAmΨACCCGAmΨmΨACmΨAmΨAAACAGmΨCAmΨ mΨCCCCGAGGGAmΨmΨCAAAmΨGGGAGAGAGmΨGAmΨGAAmΨmΨmΨmΨGAAG ACGGGGGmΨGCCGmΨGACCGmΨAACmΨCAAGAmΨACCmΨCmΨmΨmΨGGAAGA mΨGGmΨACCCmΨCAmΨmΨmΨACAAGGmΨmΨAAGCmΨmΨAGAGGAACmΨAAmΨ mΨmΨCCCACCAGACGGACCCGmΨmΨAmΨGCAGAAAAAAACCAmΨGGGmΨmΨGG GAGGCmΨAGCACCGAACGGCmΨGmΨAmΨCCAGAGGAmΨGGAGmΨmΨmΨmΨGA AAGGmΨGAmΨAmΨmΨAAGAmΨGGCCCmΨGCGCCmΨGAAAGACGGAGGGCGGmΨ ACCmΨCGCAGAmΨmΨmΨCAAAACmΨACCmΨACAAAGCmΨAAGAAACCGGmΨCC AAAmΨGCCGGGmΨGCmΨmΨAmΨAACGmΨCGAmΨAGGAAGCmΨGGAmΨAmΨAA CAmΨCACACAACGAAGACmΨAmΨACGGmΨmΨGmΨCGAACAGmΨAmΨGAGCGAm ΨCmΨGAGGGGAGGCACAGCACGGGCGGGAmΨGGAmΨGAGCmΨmΨmΨACAAGmΨ GAAGGmΨGmΨmΨmΨCmΨmΨmΨmΨACmΨGAGmΨGCAGCCCAmΨGGCCGCACmΨ CAGGmΨmΨmΨmΨGCmΨmΨmΨmΨCACCmΨmΨCCCAmΨCmΨGmΨGAAAGAGmΨG AGCAGGAAAAAGCAAAA Transfection of CasX mRNA into HEK293T cells in vitro:

[0267] To assess the efficiency of CasX expression, HEK293T cells were seeded in a 96-well plate at ~30,000 cells per well in DMEM / F12 medium supplemented with 10% fetal bovine serum. The following day, cells were lipofected with three in vitro transcribed CasX 491 mRNA molecules with or without the additional enzymatic polyadenylation (sequences listed in Tables 8A and 8B). Two days post-transfection, cells were harvested to determine CasX expression, 154Attorney Docket No. SCRB-061 / 01WO 333322-2448155Attorney Docket No. SCRB-061 / 01WO 333322-2448System. After formulation, the LNPs were diluted 5-fold in PBS, pH 7.4, to decrease the ethanol 156Attorney Docket No. SCRB-061 / 01WO 333322-2448 concentration and increase the pH, which increases the stability of the particles. Buffer exchange of the mRNA / sgRNA-LNPs was achieved by overnight dialysis into PBS, pH 7.4, at 4°C using157

[0203] The coding sequence optimizations described above were performed using CasX variant 515. CasX variant protein 812 differs from 515 at a single position (amino acid residue 329). Specifically, CasX 515 has a glycine residue at position 329, where CasX 812 has a lysine residue. Since the coding sequences for the two proteins are so similar, a G329K mutation was introduced into all CasX 515 designs as described above to generate optimized CasX 812 sequences. It is anticipated that the optimized CasX 515 and CasX 812 coding sequences will share similar properties due to the very high degree of overlap between the sequences. The DNA and RNA sequences of the CasX 812 coding sequences are provided in Table 1H.Table 1H: DNA and RNA sequences of optimized CasX 812 coding sequences

[0204] Further, it is anticipated that other variants of the CasX protein that have an amino acid sequence that is highly similar to that of CasX 515 may be optimized in the same way, / .< ., by introducing mutations into an optimized CasX 515 coding sequence. For example, only a single mutation distinguishes CasX variant 515 from variant 491, and only three mutations separate CasX variant 515 from CasX variant 676. Additional details regarding CasX variants and the specific mutations are described in W02022120095A1, incorporated by reference in its entirety.

[0205] Finally, for the purpose of experimentation, the CasX coding sequences will include one of two variations of the C-terminal c-MYC NLS, which will remain constant for each CasX sequence. The DNA, RNA, and protein sequences of these two variations of the C-terminal c- MYC NLS are listed in Table II.Table II: DNA, RNA, and protein sequences of C-terminal c-MYC NLSExample 2: Design of 5’UTR sequences

[0206] The 5’ UTR of an mRNA molecule is a key determinant of both the stability of the mRNA and how efficiently it is translated into protein. Here, candidate 5’ UTR sequences for incorporation into the optimized mRNA were identified using the criteria described below. Design of candidate 5’UTR sequences:

[0207] To generate a diverse list of 5’ UTR sequences optimized for various parameters, as described in detail below, data sets from multiple published studies were analyzed and filtered based on different criteria to identify candidate 5’ UTR sequences. From one data set, candidate5’ UTRs (either naturally-occurring or randomly generated) that were expected to produce high protein expression levels were identified by sorting for UTRs with the highest mean ribosomal load (MRL) when part of a reporter transcript.

[0208] Using a second data set, candidate 5’ UTRs were identified from a group of naturally occurring UTRs that were experimentally validated, a group of naturally occurring and synthetic sequences that were selected based on >50% enrichment in the top two bins with the highest reporter expression, as well as a set of sequences that demonstrated positive enrichment in the bin with the third highest reporter expression levels.

[0209] From a third data set, candidate 5’ UTRs were identified by selecting 5’ UTRs with the highest predicted expression based on measured half-life and relative enrichment in polysome fractions.

[0210] Lastly, several UTRs commonly used in mRNA constructs were included, along with sequences identified as performing well in the HepG2 liver cell line from a fourth data set. The resulting list contains candidates expected to drive efficient expression but were identified by prioritization of different parameters, some of which include stability as a factor and others which do not.

[0211] In addition, several 5’ UTRs expected to have low efficacy were generated by scrambling sequences of 50 nucleotides with 50% GC content. These were generated for the purpose of determining whether the selected sequences generated substantial improvements over random sequences.

[0212] In total, 53 5’ UTR sequences were identified. The RNA and DNA sequences of the 5’ UTRs are provided in Table 2A, below.Table 2A: RNA and DNA sequences of 5’UTRsExample 3: Design of 3’UTR sequences

[0213] 3’ UTRs also have a significant impact on mRNA stability and translation efficiency and can determine both subcellular localization and tissue-specific expression. Efforts were conducted to identify candidate 3’ UTRs derived from naturally occurring sequences using the methodology described below.Design of 3’UTR sequences:

[0214] 3’ UTRs were selected using a similar approach to that outlined for the 5’ UTRs, above. Specifically, several published data sets were identified, and filtering strategies were used to generate a set of 3’ UTRs that are expected to be optimized for different parameters.

[0215] From one data set, candidate 3’ UTRs were selected for inclusion in the set of 3’ UTRs described herein by identifying 3’ UTRs with the highest predicted expression based on measured half-life and relative enrichment in polysome fractions.

[0216] From a second data set, candidate 3’ UTRs were selected for inclusion by identifying naturally occurring 3’ UTRs that potentially have high stability, as measured by their half-life in cells. Candidate 3’ UTRs were also selected for inclusion by identifying 3’ UTRs, when assayed, that yielded relatively high reporter expression.

[0217] Lastly, candidate 3’ UTRs were selected from a data set that examined and assayed stable RNA fragments as 3’ UTRs. Specifically, stable RNA fragments that were not necessarily from UTRs were identified, tested as 3’ UTRs, screened for stability, concatenated, and tested again. The resulting combinations with the highest determined half-life, excepting those containing the amino-terminal enhancer of split (AES) UTR, were selected for inclusion in the set of 3’ UTRs described herein.

[0218] As with the 5’ UTRs, several 3’UTR sequences commonly used in mRNA constructs or occurring in highly expressed liver genes were selected for inclusion in the set of 3’ UTRs.

[0219] In addition, a group of ENE (element for nuclear expression) sequences were identified and selected for inclusion as 3’UTR candidates. The ENE sequence is a unique RNA structure known to protect viral and eukaryotic noncoding RNAs from nuclear degradation and enhanced stability for the transcript through triple-helix formation with the poly(A) tail or 3’ terminal A- rich tract.

[0220] In addition, several 3 ’ UTRs expected to have low efficacy were generated by scrambling sequences of 200 nucleotides with 50% GC content. These were generated for thepurpose of determining whether the selected sequences generated substantial improvements over random sequences.

[0221] Taken together, a set of 64 3’ UTRs selected either for optimal stability or optimal expression was generated. The RNA and DNA sequences of the 3’ UTRs are provided in Table 3A, below.Table 3A: RNA and DNA sequences of 3’UTRsIllExample 4: Design of 3’ poly(A) tail sequences

[0222] The 3’ poly(A) tail is crucial for mRNA stability and translation efficiency, such that longer poly(A) tails are associated with increased mRNA stability, thereby allowing their translation and promoting high protein expression. While in vitro polyadenylation is possible using a poly(A) polymerase in a post-transcriptional enzymatic reaction, this process would result in variable lengths of poly(A) tails. Therefore, poly(A) tails can be encoded from plasmid DNA templates during in vitro transcription. Candidate poly(A) tail sequences for use were identified as described below.

[0223] Candidate poly(A) tails were selected to have a range of poly(A) tail lengths. While plasmid constructs containing a template 120 nucleotide A-tail were able to produce CasX mRNAs that resulted in effective editing, these plasmids often resulted in recombination during E.coli propagation, prompting the design of shorter poly(A) tails. In addition, split variants of the 120 nucleotide A-tail were selected as candidates, such that a single nucleotide or a restriction site was included in between two 60-A segments, since inclusion of split poly(A) tail variants was previously reported to reduce recombination during E.coli propagation significantly (Trepotec Z. et al., Segmented poly(A) tails significantly reduce recombination of plasmid DNA without affecting mRNA translation efficiency or half-life. RNA. 25: 507-518 (2019)). Additionally, sequences encoding a 60 A-tail and a 30 A-tail, which represent the median and lower bounds of poly(A) tail length of human genes respectively (Chang H. et al., TAIL-seq: Genome-wide Determination of Poly(A) Tail Length and 3' End Modifications. Mol Cell. 53(6): 1044-1052 (2014)), were also included as candidate poly(A) tails. A 185 nucleotide A-tail was also included as a candidate poly(A) tail.

[0224] Furthermore, two ENE sequences, MALAT1 ENE and MENP ENE, which also contain a genomically encoded A-rich tract, were selected for inclusion as poly(A) tail candidates as it was previously shown that incorporating these two ENE structures downstream of an open readingframe resulted in efficient translation of the transcript and protein expression, despite the absence of a poly(A) tail, indicating that an ENE sequence could serve as an alternative to a poly(A) tail (Wilusz J.E. et al., A triple helix stabilizes the 3’ ends of long noncoding RNAs that lack poly(A) tails. Genes & Dev. 26: 2392-2407 (2012)).

[0225] Sequences of candidate poly(A) tails are provided in Table 4A.Table 4A: RNA and DNA sequences of poly(A) tailsExample 5: In silica assessment of engineered mRNAs encoding CasX

[0226] In silico assessments were performed to evaluate the CasX coding sequence, 5’ UTR, and 3’ UTR described above in Examples 1, 2, and 3. Specifically, 5’ UTR, the CasX coding, C-terminal c-MYC NLS, and 3’ UTR sequences were combined to generate full-length mRNA sequences in silico (without poly(A) tails), and the mRNA half-life and mean ribosome load were predicted for each sequence.Methods:

[0227] Generation of full-length mRNA sequences: All possible combinations of 5’ UTR, CasX variant 515, and 3’ UTR RNA sequences listed in Tables IF, 2A, and 3A were concatenated to generate “full-length” sequences of mRNAs encoding CasX. The full-length sequences included, from 5’ to 3’, a 5’ UTR, a start codon, a CasX 515 coding sequence, a C-terminal c-MYC NLS, a stop codon, and a 3’ UTR. For the purpose of these in silico assessments, the full-length mRNAs did not include a poly(A) tail. The CT_cMYC_NLS#l C-terminal c-MYC NLS was used, with a DNA sequence of GGAGGAGGATCCCCAGCCGCGAAGCGAGTGAAACTGGAC (SEQ ID NO: 336), as shown in Table II. A total of 217,088 unique full-length sequences were generated, of which a representative subset of sequences are SEQ ID NOS: 823-112758.

[0228] In silico predictions: The Saluki model was used to predict the stability of the 217,088 unique sequences (see Agarwal, V. and Kelley, D. The genetic and biochemical determinants of mRNA degradation rates in mammals. bioRxiv 2022.03.18.484474). Specifically, 50 iterations of the model were performed for each unique sequence, and the results of the 50 iterations were averaged to generate one score for each unique sequence. The score indicates the predicted halflife of each mRNA sequence. Saluki scores were then aggregated for each element by grouping full-length sequences that contained each unique 5 ’UTR, coding sequence, or 3 ’UTR and calculating the minimum, median, and maximum Saluki scores in each group. While the Saluki model predicts stability of an entire mRNA sequence, this method allows inference of the effect of a specific component by summarizing the effect across many contexts.

[0229] Further, the mean ribosome loads of the 50 5’ UTR sequences were predicted using Optimus 5-prime (see Sample P.J., et al., Human 5’ UTR design and variant effect prediction from a massively parallel translation assay. Nat Biotechnol . 2019; 37(7):803-809).Results:

[0230] The minimum, median, and maximum Saluki scores for each 5’ UTR, coding sequence, and 3’ UTR are provided in Table 5A, Table 5B, and Table 5C, respectively. Table 5A also provides the predicted mean ribosome loads of the 5’ UTRs.Table 5A: Minimum, median, and maximum Saluki scores, and predicted mean ribosomal load of 5’ UTR sequencesTable 5B: Minimum, median, and maximum Saluki scores of optimized CasX 515 coding sequencesTable 5C: Minimum, median, and maximum Saluki scores of 3’ UTR sequencesExample 6: Experimental methods to assess candidate engineered mRNAs encoding CasX

[0231] Candidate CasX mRNAs will be evaluated using various experimental screening assays to assess mRNA stability, protein translation efficiency and expression, and editing potency. Experimental evaluation of these key properties will further inform the selection of high-performing mRNA constructs to be delivered to the target cell or tissue for effective CasX expression and editing of the target nucleic acid.

[0232] Experimental design:

[0233] Using the screening assays described below, three sets of candidates CasX mRNAs will be experimentally evaluated. For these experiments, the CasX coding sequences will include one of two variations of the C-terminal c-MYC NLS (listed in Table II), which will remain constant across the CasX sequences. In screening assays involving the incorporation of a split FP “tag” (described below), the CT_cMYC_NLS#l, which includes two additional amino acids serving as a spatial buffer, will be used. The three sets of candidate CasX mRNAs are described as follows:

[0234] A) Set #1 : The candidate sequences identified for each mRNA element (5’UTR, CasX coding sequence, 3’UTR, or poly(A) tail), as described in Examples 1-4, will be individually assessed. Specifically, these candidate sequences of the mRNA element of interest will be tested in the context of a full-length CasX mRNA construct, where the remaining mRNA components will be held constant. The corresponding experimental controls for each mRNA element will be a set of randomly generated sequences to determine whether the candidate sequences will demonstrate substantial improvement over scrambled controls.

[0235] B) Set #2: Candidate sequences identified for each mRNA element (5’UTR, CasX coding sequence, 3’UTR, or poly(A) tail) will be combined in every possible combination using a custom script to generate the full-length mRNA sequences, and the sequences will be scored using metrics that evaluate protein expression and mRNA stability. Scores will be aggregated for each mRNA element, and representative full-length sequences will be identified for subsequent screening assays. The corresponding negative control will be a set of randomly generated full- length CasX mRNA sequences, and a benchmark CasX mRNA sequence (e.g., the original, nonoptimized mRNA sequence) previously demonstrated to induce editing will serve as a positive control.

[0236] C) Set #3 : Candidate CasX mRNA-encoding constructs will be assessed with or without the incorporation of an optimized c-MYC NLS coding sequence (SEQ ID NO: 8-24; Table IB) on the N-terminus of the CasX coding sequence to determine the functional effects of appending an optimized N-terminal NLS coding sequence on CasX expression and activity. The corresponding negative control will be a set of randomly generated sequences, while abenchmark CasX mRNA sequence (e.g., the original non-optimized mRNA sequence) previously demonstrated to induce editing will serve as a positive control.

[0237] The results obtained from screening each set of CasX mRNAs described above will inform the identification of combinations of mRNA elements to generate candidate CasX mRNA sequences with improved properties. These properties include use of sequences of key mRNA elements that induce enhanced protein expression, increased stability, and / or improved editing over experimental controls (e.g., benchmark mRNA). The candidate CasX mRNA sequences that contain combinations of optimized elements will also be assessed experimentally.

[0238] Production of candidate CasX mRNAs:

[0239] For some assays, candidate engineered CasX mRNAs will be generated by in vitro transcription (IVT) using a PCR product or linearized plasmid DNA template and a T7 RNA polymerase. If using a PCR product, DNA sequences encoding candidate mRNAs will be cloned into a plasmid containing a T7 promoter. Through PCR amplification, a poly(A) tail and barcode will be added. If using a plasmid, DNA sequences encoding candidate mRNAs will be cloned into a plasmid containing a T7 promoter, a unique barcode, and a poly(A) region. The plasmid DNA template will be linearized using the SapI restriction enzyme and then used to perform IVT reactions by incubating at 37oC for 2.5 hours. The N1 -methyl -pseudouri dine will be used to substitute the uridine ribonucleoside for all IVT reactions. Subsequently, DNase will be added to each IVT reaction to digest template DNA.

[0240] Poly(A)-tailed mRNAs will be purified from the IVT reaction mix containing DNase using Oligo d(T) chromatography with standard buffers, followed by RNA precipitation with lithium chloride. RNA pellets will be resuspended in Storage Solution and snap frozen in liquid nitrogen prior to storage at -80oC. These resulting candidate mRNAs will be tested in the various screening assays described below in an arrayed or pooled manner.

[0241] Methods to evaluate mRNA stability:

[0242] The following methods described below are alternative assays that can be performed to evaluate mRNA stability.

[0243] An initial high-throughput screen for candidate mRNAs with components that result in enhanced mRNA stability can be performed by long-read sequencing of the full-length CasX mRNA using PacBio® Sequel II sequencing.

[0244] To screen candidate mRNAs with components that result in enhanced mRNA stability, an in-solution RNA degradation time course experiment can be performed. Briefly, a pool of afixed number of barcoded CasX mRNAs will be incubated in degradation buffer (50mM N- Cyclohexyl-2-aminoethanesulfonic acid (CHES) at pH 10 and 10 mM MgC12) and subsequently quenched in Tris ethylenediaminetetraacetic acid (EDTA) at multiple timepoints (0, 0.5, 1, 2, 3, 4, 5, 6, 16 and 24 hours). The treated RNA will be reverse transcribed into cDNA, which will be subjected to PCR amplification and next-generation sequencing (NGS) to detect barcoded amplicons. For each candidate mRNA, barcoded counts at each timepoint will be calculated relative to the counts at the initial timepoint to determine the degradation rate of the candidate mRNA over time.

[0245] An arrayed screen in cells can also be performed in parallel to screen candidate mRNAs with components that result in enhanced mRNA stability. Briefly, individual barcoded CasX mRNAs will be transfected into a cell line, which will be generated to contain a constitutively expressed reporter gene with a unique barcode. The use of the barcoded reporter will enable the normalization of readouts across samples in an arrayed screen. Following transfection, cells will be harvested at the following time points: 1, 7, 12, and 24 hours post-transfection. RNA will be extracted and reverse transcribed into cDNA, which will be subjected to PCR amplification and NGS to detect barcoded amplicons. For each timepoint, barcoded counts from the candidate mRNA will be normalized by barcoded counts from the reporter gene to calculate the degradation rate of the candidate mRNA over time.

[0246] Furthermore, a pooled screen in cells can be performed to assess candidate mRNAs with components that result in enhanced mRNA stability. Briefly, a pool of barcoded CasX mRNAs will be transfected into a cell line containing a barcoded reporter, which was briefly described above. Cells will be harvested at 1, 7, 12, and 24 hours post transfection, and RNA will be extracted and reverse transcribed into cDNA, which will be subjected to PCR amplification and NGS to detect barcoded amplicons. For each timepoint, barcoded counts from each candidate mRNA in the pool will be normalized by barcoded counts from the reporter gene to calculate the relative degradation rate of each candidate mRNA over time.Methods to evaluate protein expression:

[0247] The following methods described below are alternative assays that may be performed to evaluate protein expression.

[0248] To screen candidate mRNAs with components that result in increased expression of the CasX protein, individual candidate CasX mRNAs will be transfected into HEK293T cells, which will be harvested two days post-transfection. Harvested cells will be fixed, permeabilized, andblocked prior to incubation with an in-house primary CasX antibody and subsequent incubation with an appropriate secondary antibody. Stained cells will be loaded onto an Attune™ NxT flow cytometer to determine CasX expression. Alternatively, transfected cells may be grown on coverslips that will be similarly processed for analysis using a standard protocol for immunocytochemi stry .

[0249] An arrayed screen can be performed to screen candidate mRNAs with components that result in increased speed and efficiency of protein expression. Here, a variation of the split fluorescent protein (splitFP) technique may be employed. Briefly, a cell line will be generated to express the larger “detection” component of the FP, which will not express fluorescence until the smaller “tag” component of the FP is expressed in the cell to reconstitute with the larger component to generate a functional FP. Individual barcoded CasX mRNA candidates, which will include the “tag” of the splitFP, will be transfected into cells in 96-well plates. Upon expression of the “tagged” CasX protein, the “tag” will complement the “detection” component to produce a functional FP, enabling the quantification of FP+ cells at multiple timepoints (e.g., 2, 4, 12, and 24 hours) via flow cytometry to determine CasX expression over time. The relative change in CasX expression over time determined for each mRNA candidate will be used to compare and identify the high-performing candidate mRNAs.

[0250] Similarly, a pooled screen using the splitFP system can be performed to assess candidate mRNAs with components that result in increased speed and efficiency of protein expression. Briefly, a pool of barcoded CasX mRNA candidates, which will include the “tag” of the splitFP, will be transfected into cells containing the “detection” component. Upon expression of the “tagged” CasX protein, the “tag” will complement the “detection” component to produce a functional FP, enabling the isolation of FP+ cells at multiple timepoints (e.g., 2, 4, 12, and 24 hours) via fluorescence activated cell sorting (FACS). RNA will be extracted from harvested cells and reverse transcribed into cDNA. The resulting cDNA will be subjected to PCR amplification and NGS to detect barcoded amplicons to identify candidate mRNAs whose delivery resulted in efficient protein expression over time.Methods to evaluate ribosomal loading:

[0251] The following methods described below are alternative assays that may be performed to evaluate ribosomal loading.

[0252] To screen candidate mRNAs with components that result in increased ribosomal loading in an arrayed format, individual barcoded CasX mRNAs will be transfected into cells containinga reporter gene that will be uniquely barcoded. Transfected cells will be treated with cycloheximide at 1, 7, 12, and 24 hours post-transfection to halt ribosomal processing of mRNAs. Ribosomes will be purified with an antibody targeting rRNA to isolate associating mRNAs being actively translated. Isolated RNA from the ribosomal-enriched fraction will be reverse transcribed to cDNA. The resulting cDNA will be subjected to PCR amplification and NGS to detect barcoded amplicons. For each timepoint, barcoded reads from CasX mRNA candidates and the reporter gene will yield normalized measurements indicative of ribosomal engagement for each candidate mRNA. Alternatively, long-read sequencing of the isolated mRNAs may be performed using PacBio® Sequel II sequencing to identify candidate CasX mRNAs.

[0253] Similarly, a pooled screen can be performed to screen candidate mRNAs with components that result in increased ribosomal loading. Briefly, a pool of barcoded CasX mRNAs will be transfected into cells containing a barcoded reporter. Transfected cells will be treated with cycloheximide at 1, 7, 12, and 24 hours post-transfection to halt ribosomal processing of mRNAs. Ribosomes will be purified from cells with an antibody targeting rRNA to isolate associating mRNAs being actively translated. Isolated RNA from the ribosomal- enriched fraction will be reverse transcribed to cDNA. The resulting cDNA will be subjected to PCR amplification and NGS to detect barcoded amplicons. From each timepoint, barcoded reads from CasX mRNA candidates and the reporter gene will yield normalized measurements indicative of ribosomal engagement for each candidate mRNA. Enrichment of the percentage of reads from each mRNA candidate in the ribosomal enriched pool relative to the input sample will yield the candidates with high ribosomal loading. Alternatively, long-read sequencing of the isolated mRNAs may be performed using PacBio® Sequel II sequencing to identify candidate CasX mRNAs.

[0254] An alternative method to assess ribosomal loading is Translating Ribosome Affinity Purification (TRAP)-seq, which will involve generating a cell line via integration of a lentiviral construct encoding for a tagged ribosomal subunit. Using affinity purification, tagged ribosomes will be purified to isolate mRNAs being actively translated. Alternative forms of this assay use an rRNA antibody to purify ribosomes from a wild-type line as described above.Methods to evaluate editing potency:

[0255] To screen candidate mRNAs with components that result in increased editing potency and efficiency, individual candidate CasX mRNAs with a gRNA having a targeting spacer,either to an AAVS1 safe harbor site or the endogenous beta-2 microglobulin (B2M) locus, will be transfected into HEK293T cells. The gRNA with a non-targeting spacer will serve as an experimental control. Editing assessment will depend on the targeting spacer used: e.g., if targeting the AAVS1 locus, editing will be determined as indel rate detected by NGS; if targeting the B2M locus, editing will be measured by the loss of surface presentation of the B2M-dependent HLA complex, as detected by flow cytometry.

[0256] The results of these experiments are expected to identify candidate CasX mRNAs with enhanced mRNA stability, protein translation efficiency and expression, and editing potency.Example 7: Demonstration that altering the UTR sequences of the engineered CasX mRNA can affect CasX-mediated editing

[0257] 5’ and 3’UTRs are essential and required for efficient translation. Here, experiments were performed to demonstrate that altering the 5’ and 3’ UTR sequences of the engineered CasX mRNA affects CasX-mediated editing at a target locus when CasX mRNA and targeting gRNAs were delivered in vitro via transfection.Materials and Methods:IVT of CasX mRNA:

[0258] CasX 676 mRNA was generated by IVT via a third-party. Briefly, constructs encoding for a 5 ’UTR region, CasX 676 with flanking c-MYC NLSes, and a 3 ’UTR region were cloned into a plasmid containing a T7 promoter and 80-nucleotide poly(A) tail. The resulting plasmid was linearized prior to use for IVT reactions, which were carried out with CleanCap® AG and Nl-methyl-pseudouridine. For the 5’ cap, the CleanCap® AG contains a m7G(5’)ppp(5’)mAG structure, where “m7G” denotes N7-methylguanosine, “mA” denotes 2’0-methyladenosine, and (5’)ppp(5’) denotes a 5’ to 5’ triphosphate bridge. An extra guanine nucleotide was incorporated following the CleanCap® AG to enhance transcription initiation, resulting in the incorporation of m7G(5’)ppp(5’)mAGG as the full 5’ cap structure.

[0259] IVT reactions were subsequently subjected to DNase digestion to remove template DNA and purification using an oligo-dT column. In this example, two configurations of CasX 676 mRNAs were generated for assessment in vitro. The encoding sequences of the two CasX mRNA configurations are detailed in Table 7A. Full-length RNA sequences encoding the CasX mRNA with the chemical modifications are listed in Table 7B.Table 7A: Encoding sequences of the two CasX mRNA molecules assessed in this example*.Components are listed in a 5’ to 3’ order within the constructsTable 7B: Full-length RNA sequences of CasX mRNA molecules assessed in this example.The 5’ cap (m7G(5’)ppp(5’)mAG), discussed in the example herein, is not shown in the table. Modification ‘mq / ’ = Nl-methyl-pseudouridineSynthesis of gRNAs:

[0260] In this example, gRNAs targeting the mouse PCSK9 locus were designed using gRNA scaffold 174 and chemically synthesized. The sequences of the PCSK9-targeting spacers are listed in Table 7C.Table 7C: Sequences of spacers targeting the mouse PCSK9 locus assayed in this example.Transfection of CasX mRNA and gRNA into mouse Hepal-6 cells in vitro'.

[0261] Editing at the mouse PCSK9 locus was assessed by delivering in vitro transcribed CasX mRNA (CasX mRNA #1 or CasX mRNA #2; see Table 7A) and synthesized gRNAs targeting PCSK9 into Hepal-6 cells via transfection. Briefly, each well of 20,000 Hepal-6 cells were lipofected with in vitro transcribed mRNA coding for CasX 676 and a EGS' -targeting gRNA.After a media change, transfected cells were harvested at 20 hours post-transfection for editing assessment at the EGS' locus by NGS. As experimental controls, individual transfections of CasX mRNA #1 and CasX mRNA #2 without gRNAs were performed.NGS processing and analysis:

[0262] Genomic DNA (gDNA) from harvested cells were extracted using the Zymo Quick- DNA™ Miniprep Plus kit following the manufacturer’s instructions. Target amplicons were formed by amplifying regions of interest from extracted gDNA with a set of primers targeting the mouse PCSK9 locus. These gene-specific primers contain an additional sequence at the 5' end to introduce an Illumina™ adapter and a 16-nucleotide unique molecular identifier. Amplified DNA products were purified with the Ampure XP DNA cleanup kit. Quality and quantification of the amplicon were assessed using a Fragment Analyzer DNA Analysis kit (Agilent Technologies, dsDNA 35-1500bp). Amplicons were sequenced on the Illumina™ MiSeq™ sequencer according to the manufacturer’s instructions. Raw fastq files from sequencing were quality-controlled and processed using cutadapt v2.1, flash2 v2.2.00, and CRISPResso2 v2.0.29. Each sequence was quantified for containing an insertion or deletion (indel) relative to the reference sequence, in a window around the 3’ end of the spacer. CasX editing activity was quantified as the total percent of reads that contain insertions, substitutions, and / or deletions anywhere within this window for each sample.Results:

[0263] CasX-mediated editing at the mouse PCSK9 locus was used to evaluate the effects of incorporating different 5’ and 3’ UTRs into the engineered CasX mRNA. The plot in FIG. 2 shows the quantification of percent editing measured as indel rate at the PCSK9 locus in mouse Hepal-6 cells transfected with CasX 676 mRNA #1 or CasX 676 mRNA #2 with the indicated EGS’ -targeting gRNAs. The data demonstrate that for all targeting spacers tested in this experiment, CasX mRNA #2 consistently exhibited higher editing levels at the mouse PCSK9 locus compared to editing levels achieved by CasX mRNA #1. Specifically, the highest level of editing rate achieved was with spacer 27.116, where use of CasX mRNA #2 resulted in -35% editing efficiency compared to -20% editing level by CasX mRNA #1 (FIG. 2).

[0264] The results demonstrate that altering the 5’UTR and 3’UTR sequences of the CasX mRNA can affect the editing activity of CasX at a target locus in a cell-based assay. These findings indicate the importance of 5’ and 3 ’UTRs in mRNA design and engineering and justifyadditional screening studies to evaluate other candidate 5’ and 3’UTR sequences as discussed in previous examples.Example 8: Demonstration that ENE elements can be incorporated as 3’UTR sequences in the generation and expression of CasX mRNAs

[0265] As discussed in Example 3, incorporation of an ENE element into the 3’UTR of a reporter mRNA has been previously shown to protect the mRNA transcript from degradation. In this example, experiments were performed to demonstrate that an ENE element can be used as a 3’UTR sequence with the absence of a poly(A) tail to generate and express the CasX mRNA. Furthermore, experiments were performed to demonstrate that polyadenylation following the incorporated ENE element can improve expression of the CasX mRNA.Materials and Methods:IVT of CasX mRNA:

[0266] CasX 491 mRNA was generated by IVT using PCR templates. Briefly, PCR was performed on plasmids encoding CasX protein 491 and a P2A-mScarlet with a forward primer containing a T7 promoter and reverse primer encoding one of the following: 1) a 100-nucleotide poly(A) tail; 2) a MALAT1 ENE; or 3) a MENP ENE. Sequences encoding these key elements are listed in Table 8A. The resulting PCR templates were used for IVT reactions. In addition, the IVT reactions were carried out with CleanCap® AG (described in Example 7) and Nl- methyl-pseudouridine. IVT reactions were then subjected to DNase digestion to remove template DNA and column-based purification using the Zymo Research RNA miniprep kit. Additional polyadenylation to the mRNA was performed using E. coli PolyA Polymerase (New England Biolabs®) according to the manufacturer’s protocol, followed by column-based purification and elution in RNase-free water. Full-length DNA and RNA sequences (without the additional polyadenylation) encoding the CasX mRNA are listed in Table 8B.Table 8A: Encoding sequences of three CasX mRNA molecules assessed in this example*.Components are listed in a 5’ to 3’ order within the constructsTable 8B: Full-length sequences of CasX mRNA molecules assessed in this example. ListedRNA sequences do not show the 5’ cap (m7G(5’)ppp(5’)mAG) and do not contain additional polyadenylation. Modification ‘my’ = Nl-methyl-pseudouridineTransfection of CasX mRNA into HEK293T cells in vitro'.

[0267] To assess the efficiency of CasX expression, HEK293T cells were seeded in a 96-well plate at -30,000 cells per well in DMEM / F12 medium supplemented with 10% fetal bovine serum. The following day, cells were lipofected with three in vitro transcribed CasX 491 mRNA molecules with or without the additional enzymatic polyadenylation (sequences listed in Tables 8A and 8B). Two days post-transfection, cells were harvested to determine CasX expression efficiency by measuring for mScarlet fluorescence using an Attune™ NxT flow cytometer and analyzing the expression using Flow Jo™ software. Cells that were untransfected or transfected with CasX mRNA without a poly(A) tail served as negative controls.Results:

[0268] The effects of 1) using an ENE element as a 3’UTR sequence without an extended poly(A) tail, and 2) additional polyadenylation following incorporation of the ENE element, on the expression of CasX mRNA were examined. HEK293T cells were transfected with CasX 491 mRNA #l-#3, with or without the additional polyadenylation, and CasX expression was measured as the percentage of cells exhibiting mScarlet expression (FIG. 3). The data demonstrate that under the condition without the additional polyadenylation, CasX 491 mRNA #2 and #3, which contained the MALAT1 or MENp ENE sequence respectively, resulted in -60% of cells that are mScarlet+, indicating that -60% of total cells were expressing CasX 491 (FIG. 3). The findings also show that CasX expression increased by -33% upon treatment with the Poly(A) polymerase, suggesting that additional polyadenylation to the CasX mRNA containing either ENE element appeared to improve mRNA stability, and therefore enhancedCasX expression (FIG. 3). As anticipated, transfection of HEK293T cells with CasX mRNA without the poly(A) tail did not result in the detection of mScarlet+ cells, while the additional polyadenylation from treatment with the Poly(A) polymerase substantially increased the number of mScarlet+ cells (FIG. 3).

[0269] The results demonstrate that using an ENE element as a 3’UTR sequence, without an extended poly(A) tail, was sufficient to induce CasX expression in a cell-based assay. Furthermore, the data show that adding an extended poly(A) tail to the ENE element improved CasX expression, indicating that use of an ENE element with additional polyadenylation can enhance mRNA stability and expression.Example 9: Demonstration that CasX mRNA and targeting gRNA can be delivered via LNPs to achieve in vivo targeting

[0270] Experiments were performed to demonstrate that CasX mRNA and targeting gRNA can edit the ROSA26 locus in the liver in vivo when delivered via LNP.Materials and Methods:Mouse lines:

[0271] Mouse strains from Jackson Labs (C57BL / 6J) and Charles River Labs (C57BL / 6N) were used to assess CasX-mediated editing in vivo. Males and females between 4 and 8 weeks of age at the time of injection were used.LNP formulation:

[0272] LNP co-formulations were performed as described in Example 10. CasX 676 mRNA #1 (see Table 7A for sequences) and a gRNA using scaffold 174 with a spacer targeting ROSA26 (AGAAGAUGGGCGGGAGUCUU; SEQ ID NO: 660) were encapsulated within the same LNP using a 1 : 1 mass ratio for mRNA:gRNA. Formulated LNPs were buffer-exchanged to PBS for in vivo injection. At the time of injection, concentrated LNPs were mixed with sterile-filtered PBS and drawn into sterile syringes.In vivo administration of formulated LNPs:

[0273] Mice were anesthetized via isoflurane inhalation and anesthesia was verified by the loss of righting reflex and toe pinch. LNPs were administered intravenously, either through the retro- orbital sinus or lateral tail vein. Mice were observed for five minutes after injection to ensure recovery from anesthesia before being placed into their home cage. Mice were randomized intogroups and received injections of LNPs at the following doses: 0.3mg / kg, 1.0 mg / kg, or 3.0 mg / kg. Naive, uninjected animals served as experimental controls.

[0274] Mice were monitored daily for 7 days; after 7 days, mice were euthanized, and various tissues (liver, spleen, heart, and kidney) were harvested from each animal. Harvested tissues were processed for genomic DNA (gDNA) and RNA extraction using the Zymo Quick DNA / RNA Miniprep kit following the manufacturer’s instructions. Target amplicons were then amplified from the extracted gDNA with a set of primers targeting the mouse ROSA26 locus and processed as described earlier in Example 7 for editing assessment by NGS.Results:

[0275] FIG. 4 shows the editing results, as percent editing measured as indel rate, at the ROSA26 locus in mice treated with LNPs co-formulated with CasX 676 mRNA #1 and a ROSA26- targeting gRNA. As illustrated in FIG. 4, a dose-dependent effect was observed with editing at the ROSA26 locus, with the 3.0mg / kg and 1.0 mg / kg doses resulting in a statistically significant difference in editing compared to the editing level achieved with the naive control group.

[0276] The results of the experiment show that delivery of CasX mRNA and targeting gRNA via LNPs can induce editing in the liver. These findings demonstrate the efficacy of the CasX:gRNA system using mRNA delivery in LNPs to the liver, a target tissue of therapeutic relevance.Example 10: Formulation of lipid nanoparticles (LNPs) to deliver CasX mRNA and gRNA payloads to target cells

[0277] Experiments were performed to encapsulate CasX mRNA and gRNA into LNPs for delivery to target cells and tissue. Here, CasX mRNA and gRNA were encapsulated into LNPs using GenVoy-ILM™ lipids on the Precision NanoSystems Inc. (PNI) Ignite™ Benchtop System and following the manufacturer’s guidelines. GenVoy-ILM™ lipids are manufactured by PNI, with a proprietary composition of ionizable lipid:DSPC:cholesterol:stabilizer at 50: 10:37.5:2.5 mol%. Briefly, to formulate LNPs, equal mass ratios of CasX mRNA and gRNA were diluted in PNI Formulation Buffer, pH 4.0. GenVoy-ILM™ lipids were diluted 1 : 1 in anhydrous ethanol. mRNA / gRNA co-formulations were performed using either a 5: 1 or 6: 1 N / P ratio. The RNA and lipids were run through a PNI laminar flow cartridge at a flow rate ratio of 3 : 1 (RNA: Genvoy-ILM™ lipids) at a total flow rate of 12 mL / mi on the PNI Ignite™ Benchtop System. After formulation, the LNPs were diluted 5-fold in PBS, pH 7.4, to decrease the ethanolconcentration and increase the pH, which increases the stability of the particles. Buffer exchange of the mRNA / sgRNA-LNPs was achieved by overnight dialysis into PBS, pH 7.4, at 4°C using 10k Slide- A-Lyzer™ Dialysis Cassettes (Thermo Scientific™). Following dialysis, the mRNA / gRNA-LNPs were concentrated to > 0.5 mg / mL using 100 kDa Amicon®-Ultra Centrifugal Filters (Millipore) and then filter-sterilized. Formulated LNPs were analyzed on a Stunner (Unchained Labs) to determine their diameter and poly dispersity index (PDI). Encapsulation efficiency and RNA concentration was determined by RiboGreen™ assay using Invitrogen’s Quant-iT™ RiboGreen™ RNA assay kit. LNPs were used in various experiments as described herein to deliver CasX mRNA and gRNA to target cells and tissue.

Claims

Attorney Docket No. SCRB-061 / 01WO 333322-2448   What is claimed is:

1. An optimized messenger RNA (mRNA) encoding all or a portion of a CasX protein, wherein the optimized mRNA is optimized for expression in eukaryotic cells.

2. The optimized mRNA of claim 1, wherein the optimized mRNA comprises one or more of: a. a 5' cap; b. a 5' untranslated region (UTR) sequence; c. a sequence encoding a nuclear localization signal (NLS); d. a sequence encoding the CasX protein; e. a 3' UTR sequence; and f. a poly(A) sequence.

3. The optimized mRNA of claim 1 or claim 2, wherein the encoded CasX protein comprises a sequence selected from the group consisting of SEQ ID NOS: 1-7, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

4. The optimized mRNA of claim 1 or claim 2, wherein the encoded CasX protein comprises a sequence selected from the group consisting of SEQ ID NOS: 1-7.

5. The optimized mRNA of any one of claims 2-4, wherein the NLS is a c-MYC NLS or an SV40 NLS.

6. The optimized mRNA of any one of claims 3-5, wherein the optimized mRNA or a portion of the optimized mRNA is optimized for one or more of improved expression, reduced immunogenicity, increased stability, and enhanced manufacturability.

7. The optimized mRNA of claim 6, wherein the optimized mRNA or portion of the mRNA comprises a sequence encoding: a. the CasX protein; b. an N-terminal portion of the CasX protein comprising the first at least about 30, at least about 40, or at least about 50 amino acids of the CasX protein; c. the NLS; and / or d. the NLS and the CasX protein, wherein the NLS is linked N-terminal or C- terminal to the CasX protein.  Attorney Docket No. SCRB-061 / 01WO 333322-2448   8. The optimized mRNA of any one of claims 1-7, wherein the optimized mRNA or portion of the optimized mRNA comprises a sequence optimized utilizing parameters selected from one or more of: a. codon adaptation index (CAI); b. rare codon frequency; c. predicted mRNA half-life in a cell; d. predicted minimum free energy; e. GC content; f. CpG frequency; g. uridine frequency; and / or h. predicted mRNA degradation under hydrolytic conditions.

9. The optimized mRNA of claim 7 or claim 8, wherein the sequence encoding the CasX protein is selected from the group consisting of SEQ ID NOS: 78-141 and 206-269, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

10. The optimized mRNA of claim 7 or claim 8, wherein the sequence encoding the CasX protein is selected from the group consisting of SEQ ID NOS: 78-141 and 206-269.

11. The optimized mRNA of claim 7 or claim 8, wherein the sequence encoding the N- terminal portion of the CasX protein is selected from the group consisting of SEQ ID NOS: 42- 59, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

12. The optimized mRNA of claim 7 or claim 8, wherein the sequence encoding the N- terminal portion of the CasX protein is selected from the group consisting of SEQ ID NOS: 42- 59.

13. The optimized mRNA of claim 7 or claim 8, wherein the sequence encoding the NLS is selected from the group consisting of SEQ ID NOS: 8-24, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.  Attorney Docket No. SCRB-061 / 01WO 333322-2448   14. The optimized mRNA of claim 7 or claim 8, wherein the sequence encoding the NLS is selected from the group consisting of SEQ ID NOS: 8-24.

15. The optimized mRNA of claim 7 or claim 8, wherein the optimized mRNA is a sequence selected from the group consisting of SEQ ID NOS: 823-112758, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

16. The optimized mRNA of claim 7 or claim 8, wherein the optimized mRNA is a sequence selected from the group consisting of SEQ ID NOS: 823-112758.

17. The optimized mRNA of any one of claims 2-16, wherein the 5’ cap is linked to the 5' UTR in a 5'-5' linkage.

18. The optimized mRNA of any one of claims 2-17, wherein the 5' cap is a 7- methylguanylate cap.

19. The optimized mRNA of any one of claims 2-18, wherein the mRNA comprises one or more nucleoside analogs, chemically modified bases, biologically modified bases, intercalated bases, modified sugars, modified phosphate groups, and / or nonstandard nucleotide residues.

20. The optimized mRNA of any one of claims 2-18, wherein the 5' UTR sequence comprises a sequence selected from the group consisting of SEQ ID NO: 340-392, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 95% identity thereto.

21. The optimized mRNA of any one of claims 2-19, wherein the 3' UTR sequence comprises a sequence selected from the group consisting of SEQ ID NO: 446-509, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 95% identity thereto.

22. The optimized mRNA of any one of claims 2-21, wherein the poly(A) sequence comprises at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 185, or at least about 190 adenine nucleotides.

23. The optimized mRNA of any one of claims 2-21, wherein the poly(A) sequence comprises at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 95 adenine nucleotides linked to another sequence of at  Attorney Docket No. SCRB-061 / 01WO 333322-2448   least about 40, at least about 50, at least about 60, at least about 70 adenines, at least about 80, at least about 90, or at least about 95 adenine nucleotides by a spacer sequence having between 1 to 6 nucleotides other than adenine.

24. The optimized mRNA of any one of claims 2-23, wherein the poly(A) sequence comprises a sequence selected from the group consisting of SEQ ID NOS: 500, 503, and 574- 579, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

25. The optimized mRNA of any one of claims 2-23, wherein the poly(A) sequence comprises a sequence selected from the group consisting of SEQ ID NOS: 500, 503, and 574- 579.

26. The optimized mRNA of any one of claims 1-25, wherein the optimized mRNA comprises at least one nonstandard nucleotide, optionally wherein the nonstandard nucleotide is selected from 5-methyl-cytidine (“5 mC”), pseudouridine (“ψU”), and / or 2-thio-uridine (“2sU”)”.

27. The optimized mRNA of any one of claims 1-26, wherein the nucleic acid in encapsulated in a lipid nanoparticle (LNP).

28. The optimized mRNA of claim 27, wherein the LNP comprises one or more ionizable lipids, one or more helper phospholipids, one or more PEG-modified lipids, and / or cholesterol or a derivative thereof.

29. A vector comprising the optimized mRNA of any one of claims 1-26.

30. The vector of claim 29, wherein the vector is selected from the group consisting of an Adeno-Associated Viral (AAV) vector, a lentiviral vector, a herpes simplex virus (HSV) vector, a virus-like particle (VLP), a plasmid, a minicircle, a nanoplasmid, a DNA vector, and an RNA vector.

31. A system comprising the optimized mRNA of any one of claims 1-26 and a guide ribonucleic acid (gRNA) comprising a scaffold and a targeting sequence complementary to a target nucleic acid in a cell.

32. The system of claim 31, wherein the scaffold comprises a sequence selected from the group consisting of SEQ ID NOS: 820-822.

33. The system of claim 31 or claim 32, wherein the targeting sequence of the gRNA has 15, 16, 17, 18, 19, 20, 21, or 22 consecutive nucleotides linked 3' to the scaffold.  Attorney Docket No. SCRB-061 / 01WO 333322-2448   34. The system of any one of claims 31-33, wherein the optimized mRNA and the gRNA are encapsulated within an LNP.

35. The system of claim 34, wherein the LNP comprises one or more ionizable lipids, one or more helper phospholipids, one or more PEG-modified lipids, and / or cholesterol or a derivative thereof.

36. The system of any one of claims 31-35, for use in modifying a target nucleic acid of a cell.

37. A method of modifying a target nucleic acid in a cell, comprising introducing into the cell the system of any one of claims 31-36, resulting in the modification of the target nucleic acid.

38. The method of claim 37, wherein the CasX protein is expressed within the cell and is capable of forming a ribonucleoprotein (RNP) complex with the gRNA.

39. The method of claim 37, wherein the CasX protein is expressed within the cell and forms a ribonucleoprotein (RNP) complex with the gRNA.

40. The method of claim 38 or claim 39, wherein the modifying comprises binding of the RNP to the target nucleic acid and introducing a double-stranded break in the target nucleic acid.

41. The method of claim 40, wherein the modifying comprises introducing a mutation, an insertion, or a deletion in the target nucleic acid.

42. The method of any one of claims 37-41, wherein the modifying of the cell occurs in vitro or ex vivo.

43. The method of any one of claims 37-41, wherein modifying of the cell occurs in vivo in a subject.

44. The method of any one of claims 37-43, wherein the cell is a eukaryotic cell.

45. The method of claim 44, wherein the eukaryotic cell is selected from the group consisting of a rodent cell, a mouse cell, a rat cell, and a non-human primate cell.

46. The method of claim 45, wherein the eukaryotic cell is a human cell.

47. The method of any one of claims 37-46, wherein the cell is selected from the group consisting of an embryonic stem cell, an induced pluripotent stem cell, a germ cell, a fibroblast, an oligodendrocyte, a glial cell, a hematopoietic stem cell, a neuron progenitor cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell, a retinal cell, a cancer cell, a T-cell, a B- cell, an NK cell, a fetal cardiomyocyte, a myofibroblast, a mesenchymal stem cell, an autotransplanted expanded cardiomyocyte, an adipocyte, a totipotent cell, a pluripotent cell, a  Attorney Docket No. SCRB-061 / 01WO 333322-2448   blood stem cell, a myoblast, an adult stem cell, a bone marrow cell, a mesenchymal cell, a parenchymal cell, an epithelial cell, an endothelial cell, a mesothelial cell, a fibroblast cell, an osteoblast cell, a chondrocyte cell, an exogenous cell, an endogenous cell, a stem cell, a hematopoietic stem cell, a bone-marrow derived progenitor cell, a myocardial cell, a skeletal cell, a fetal cell, an undifferentiated cell, a multi-potent progenitor cell, a unipotent progenitor cell, a monocyte, a cardiac myoblast, a skeletal myoblast, a macrophage, a capillary endothelial cell, a xenogenic cell, an allogenic cell, an autologous cell, and a post-natal stem cell.

48. The method of any one of claims 43-47, wherein the subject is selected from the group consisting of a rodent, a mouse, a rat, and a non-human primate.

49. The method of any one of claims 43-47, wherein the subject is a human.

50. The method of claim 48 or claim 49, wherein the modifying occurs in the cells of the subject having a mutation in an allele of a gene wherein the mutation causes a disease or disorder in the subject.

51. The method of claim 50, wherein the modifying changes the mutation to a wild-type sequence of the gene or results in the expression of a functional gene product.

52. The method of claim 50, wherein the modifying knocks down or knocks out the gene causing the disease or disorder in the subject.

53. A pharmaceutical composition comprising the optimized mRNA of any one of claims 1- 28, the LNP of any one of claims 27-28, the vector of any one of claims 29-30, or the system of any one of claims 31-36, and a pharmaceutically acceptable excipient.

54. The pharmaceutical composition of claim 53, wherein the pharmaceutical composition is formulated for a route of administration selected from the group consisting of intravenous, intraportal vein injection, intraperitoneal, intramuscular, subcutaneous, intraarterial, intracerebroventricular, intracisternal, intrathecal, intracranial, intralumbar, intraperitoneal, intraocular, and oral routes.

55. The pharmaceutical composition of claim 53 or claim 54, wherein the pharmaceutical composition is in a liquid, lyophilized, or frozen form.

56. The pharmaceutical composition of any one of claims 53-55, wherein the pharmaceutical composition is in a pre-filled syringe for a single injection.

57. A pharmaceutical composition comprising the optimized mRNA of any one of claims 1- 28, the LNP of any one of claims 27-28, the vector of any one of claims 29-30, or the system of any one of claims 31-36 for use as a medicament for the treatment of a subject having a disease.  Attorney Docket No. SCRB-061 / 01WO 333322-2448   58. A kit comprising the optimized mRNA of any one of claims 1-28, the LNP of any one of claims 27-28, the vector of any one of claims 29-30, or the system of any one of claims 31-36, in a package.

59. The kit of claim 58, comprising a buffer, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, or any combination of the foregoing.

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