Effect of two mismatches as function of position and separation on Cas9 cleavage activity Raw data for figure 6 and supplementary figure 10 > Sp = SpCas9 Sa = SaCas9 HF1 = SpCas9 HF1 Hypa = HypaCas9 HiFi or IDT = IDT Alt-R HiFi Cas9 > 002 = pLibrary PS4 = KMlib002 003 = pLibrary EMX1 = KMlib003 ************************************************** pLibrary PS4 - supercoiled pool apparent rate of cleavage for target sequences with two misamtches separated by different number of nucleotides across the whole target length 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Sp 0.3162 0.3036 0.4378 0.4907 0.7487 0.383 0.3821 0.5219 0.3083 0.2902 0.3153 0.3306 0.3073 0.2624 0.231 0.2494 0.2151 0.05217 0.03976 Sa 0.2022 0.05615 0.1311 0.06415 0.1001 0.1064 0.16 0.1548 0.1745 0.2309 0.1899 0.1946 0.2067 0.09305 0.06218 0.1094 0.03225 0.05809 0.2294 HiFi 0.2394 0.2398 0.2227 0.243 0.3318 0.2722 0.2735 0.2494 0.2443 0.2648 0.2727 0.2697 0.2247 0.2194 0.1327 0.06295 0.09228 0.1633 0.1911 Hypa 0.1082 0.07861 0.1135 0.05477 0.1299 0.06608 0.05116 0.1078 0.1186 0.07143 0.05367 0.1328 0.1609 0.08382 0.05221 0.05496 0.0985 0.1691 0.2618 HF1 0.0473 0.04282 0.03991 0.04307 0.05049 0.0485 0.04987 0.04855 0.03541 0.03605 0.03153 0.02986 0.02452 0.02311 0.02855 0.01669 0.01498 0.01621 0.03939 apparent rate of cleavage for target sequences with two misamtches separated by different number of nucleotides across the PAM-distal region 0 1 2 3 4 5 6 7 8 Sp 0.2211 0.1675 0.1518 0.261 0.2584 0.3408 0.2684 0.6857 1.334 Sa 0.1228 0.03891 0.04556 0.02878 0.03827 0.1901 0.2266 0.2345 0.2884 HiFi 0.21 0.2003 0.1575 0.1673 0.1678 0.1453 0.02844 0.04095 0.1908 Hypa 0.08881 0.04862 0.04532 0.03609 0.02886 0.03178 0.02013 0.04379 0.03925 HF1 0.04832 0.04119 0.03328 0.02611 0.02753 0.02067 0.0254 0.03204 0.03076 apparent rate of cleavage for target sequences with two misamtches separated by different number of nucleotides across the seed region 0 1 2 3 4 5 6 7 8 Sp 0.8993 3.386 1.984 1.494 1.153 0.1906 0.3703 0.5436 0.04548 Sa 0.2479 0.07933 0.1568 0.1243 0.1013 0.04054 0.05371 0.1183 0.2295 HiFi 0.249 0.2558 0.3018 0.2112 0.3987 0.1804 0.246 0.25 0.212 Hypa 0.09586 0.1397 0.2114 0.1286 0.187 0.05077 0.1027 0.1613 0.226 HF1 0.03687 0.0316 0.03154 0.0435 0.02996 0.02939 0.03391 0.0876 0.09805 ************************************************** pLibrary EMX1 - supercoiled pool apparent rate of cleavage for target sequences with two misamtches separated by different number of nucleotides across the whole target length 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Sp 0.2055 0.1459 0.125 0.1286 0.2061 0.2314 0.2903 0.299 0.3632 0.3402 0.3549 0.3253 0.3128 0.3779 0.3084 0.2221 0.2242 0.173 0.2579 Sa 0.1669 0.2014 0.1643 0.1042 0.1582 0.1609 0.1312 0.1116 0.11 0.1221 0.1198 0.1317 0.1221 0.1786 0.1156 0.1744 0.04841 0.07514 0.1769 HiFi 0.1686 0.04864 0.04786 0.04809 0.1341 0.1872 0.2496 0.2819 0.3126 0.3343 0.3618 0.3155 0.3267 0.4035 0.2963 0.2457 0.2323 0.1979 0.3172 Hypa 0.1856 0.1434 0.1402 0.1253 0.1843 0.2141 0.3019 0.3141 0.3938 0.4908 0.5236 0.5364 0.706 0.9204 1.252 1.155 1.309 1.29 1.96 HF1 0.2286 0.1893 0.2043 0.1544 0.2181 0.2557 0.33 0.3104 0.3586 0.3883 0.3886 0.4709 0.5216 0.616 0.553 0.6078 0.6232 0.5967 0.9451 apparent rate of cleavage for target sequences with two misamtches separated by different number of nucleotides across the PAM-distal region 0 1 2 3 4 5 6 7 8 Sp 0.2556 0.2209 0.2226 0.2194 0.246 0.3208 0.2917 0.3235 0.2089 Sa 0.2292 0.264 0.2341 0.1583 0.2328 0.3021 0.2619 0.2154 0.224 HiFi 0.2884 0.2477 0.2466 0.2313 0.2612 0.2933 0.2935 0.3199 0.274 Hypa 0.1713 0.1301 0.1341 0.0778 0.218 0.2677 0.5996 1.208 0.5233 HF1 0.233 0.1856 0.201 0.1102 0.2691 0.2995 0.5756 0.3815 0.1919 apparent rate of cleavage for target sequences with two misamtches separated by different number of nucleotides across the seed region 0 1 2 3 4 5 6 7 8 Sp 0.2488 0.1661 0.08495 0.03527 0.1372 0.04137 0.1511 0.148 0.4157 Sa 0.1243 0.2041 0.1637 0.02723 0.1838 0.02367 0.04181 0.02817 0.02613 HiFi 0.1551 0.02988 0.02525 0.02527 0.02696 0.02176 0.07323 0.1552 0.3402 Hypa 0.2276 0.1411 0.108 0.1018 0.1399 0.06186 0.1875 0.2435 1.33 HF1 0.2712 0.2064 0.2223 0.169 0.1686 0.15 0.175 0.238 0.8576 ************************************************** ************************************************** ************************************************** pLibrary PS4 - nicked pool ΔRA and Nmax for target sequences with two misamtches separated by different number of nucleotides across the whole target length 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Sp ΔRA -0.014238097 -0.011879274 -0.012380094 -0.013379016 -0.019734557 -0.017085279 -0.014280615 -0.01185215 -0.006466626 -0.00739226 -0.006729899 -0.005497581 -0.004968352 -0.008067028 -0.00820008 -0.005762154 -0.007744717 -0.00244868 -0.01369476 Nmax 0.905910547 0.809345143 0.832001187 0.880178586 0.910853078 0.878582265 0.848553762 0.776408247 0.672053943 0.742047026 0.700402842 0.766760504 0.802426312 0.678245159 0.911386047 0.786644301 0.840602986 0.610923657 1 Sa ΔRA -0.015965301 -0.008942381 -0.013618251 -0.013912296 -0.009856128 -0.009911019 -0.015945895 -0.01898586 -0.020601602 -0.022956495 -0.015115277 -0.012364248 -0.010725743 -0.004515676 0.002697905 -0.001892029 0.001221715 -0.020764873 -0.031077398 Nmax 0.732812483 0.647638752 0.706169128 0.760777684 0.684190208 0.727711409 0.847076289 0.92965073 0.827144235 0.808937824 0.645207623 0.618729097 0.537567104 0.504891314 0.864479548 0.563012897 0.793073365 0.796407186 1 HF1 ΔRA 0.019597709 0.017240566 0.013508201 0.015090231 0.014396917 0.01417592 0.017296126 0.016550162 0.012123418 0.015122546 0.018146608 0.017799399 0.010762854 0.013402455 0.016664248 0.012399054 0.008121817 0.00826353 0.002547653 Nmax 0.814135497 0.792438529 0.834681216 0.92325383 0.868139917 0.970356714 0.978161405 0.870380762 0.836910654 0.873614489 0.90374119 0.843847707 0.726407587 0.762618005 1 0.738275244 0.714986718 0.671571518 0.742113032 Hypa ΔRA 0.029393688 0.027432092 0.026508277 0.025974003 0.025722622 0.029058305 0.028537404 0.024464042 0.022508777 0.022341813 0.024969102 0.026256095 0.021886315 0.021594522 0.031059557 0.028334563 0.024673947 0.015772071 0.019926445 Nmax 0.897721257 0.812209637 0.822842803 0.889735309 0.903273239 0.971619992 0.900885499 0.849036001 0.784945798 0.760734974 0.761511126 0.85038569 0.735001785 0.734490468 1 0.830841353 0.740246973 0.593599827 0.72423874 HiFi ΔRA 0.000474827 0.006393599 0.004607139 0.007164523 0.001374893 -8.89693E-05 -0.000427501 0.003248706 0.000937168 0.00494483 0.00533431 0.005517569 0.00822579 0.010707421 0.01813288 0.014106705 0.015576284 0.006719817 0.002010755 Nmax 0.951440223 0.840100313 0.788463538 0.855349337 0.791626233 0.816446079 0.775215711 0.808829403 0.719922485 0.784597716 0.789011756 0.809169381 0.798320636 0.903747285 1 0.883052524 0.827548051 0.659236073 0.647444507 ΔRA and Nmax for target sequences with two misamtches separated by different number of nucleotides across the PAM-distal region 0 1 2 3 4 5 6 7 8 Sp ΔRA -0.02815738 -0.014347023 -0.011803529 -0.015787566 -0.020428902 -0.015403038 -0.012765245 -0.018745941 -0.011486588 Nmax 1 0.859012289 0.821541845 0.885320267 0.8132646 0.713341466 0.646658781 0.705264246 0.568440104 Sa ΔRA -0.005814882 0.000353067 -0.001116414 0.000429767 -0.000723258 -0.001569913 -0.012929244 -0.024410881 -0.034821116 Nmax 0.316501435 0.279187138 0.255580204 0.348947982 0.405300907 0.403038272 0.536254853 0.795088044 1 HF1 ΔRA 0.026221057 0.022598992 0.020595796 0.018924065 0.017430209 0.009048446 0.008733584 0.007606054 0.006981521 Nmax 0.946068393 0.956690743 1 0.99990075 0.915443098 0.710834125 0.587549635 0.618724531 0.512218411 Hypa ΔRA 0.032201162 0.032221382 0.03422606 0.02712551 0.026756184 0.021431909 0.018503953 0.017418638 0.013602178 Nmax 0.890086146 0.835576591 0.895596608 0.855764155 1 0.748089558 0.721638023 0.615843611 0.599933027 HiFi ΔRA -0.003552983 0.013478287 0.011191383 0.016711877 0.008527757 0.010742541 0.008280883 0.014399908 0.000545253 Nmax 0.91131311 1 0.896357552 0.970331638 0.809637736 0.717795217 0.794536092 0.719010419 0.639491509 ΔRA and Nmax for target sequences with two misamtches separated by different number of nucleotides across the seed region 0 1 2 3 4 5 6 7 8 Sp ΔRA -0.000199017 -0.003771891 -0.005673667 -0.002706692 -0.006496802 -0.006319831 -0.003665951 -0.006818731 -0.00853925 Nmax 0.880137629 0.849376583 0.813477963 0.839450418 0.883720437 0.844223853 0.871428182 0.697949735 1 Sa ΔRA -0.011527994 -0.008519988 -0.014447329 -0.017154561 -0.009242489 -0.00643679 -0.004069163 -0.005184781 -0.032402205 Nmax 0.514549962 0.410551455 0.532410521 0.630071747 0.497180496 0.457784277 0.530157438 0.705267435 1 HF1 ΔRA 0.015823273 0.010026234 0.004714377 0.009381947 0.005567595 0.00048643 0.0123113 0.00638014 0.003662193 Nmax 0.829695435 0.687741992 0.679517624 0.71421188 0.699417785 0.737357339 0.891584867 0.842169362 1 Hypa ΔRA 0.030492716 0.017739467 0.015877092 0.019711939 0.022699404 0.022483518 0.025965993 0.018255481 0.019564779 Nmax 1 0.731957523 0.772438114 0.854907915 0.937050066 0.946168978 0.960750116 0.965131816 0.955040109 HiFi ΔRA 0.005261841 0.002441931 0.001452217 0.001703961 0.003198332 0.00421371 0.001837404 -0.00011837 -0.00252682 Nmax 1 0.621419249 0.572857581 0.653444215 0.741773085 0.915148695 0.817018793 0.655877641 0.770657874 ************************************************** pLibrary EMX1 - nicked pool ΔRA and Nmax for target sequences with two misamtches separated by different number of nucleotides across the whole target length 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Sp ΔRA -0.014198879 -0.010639855 -0.013974062 -0.012596828 -0.015844269 -0.016392771 -0.014816641 -0.017739063 -0.017387914 -0.017573245 -0.015299154 -0.017074258 -0.017905772 -0.019625968 -0.01957498 -0.014186017 -0.016074624 -0.01321939 -0.012123911 Nmax 0.865936269 0.906838783 0.970580791 0.867679732 0.931426338 0.956860471 0.882424254 0.916699404 0.931263211 0.94487464 0.981184083 0.979045534 0.975361719 0.97107155 0.987944489 0.939379088 1 0.926480995 0.896931966 Sa ΔRA 4.54472E-05 0.004062218 0.002688854 -0.000157752 0.003778051 0.000200589 0.00347057 0.003401531 0.002499182 0.000558258 0.00160804 -0.003013431 0.000402274 -0.001884514 -0.001115132 0.004116176 -0.002225092 -0.001468607 0.00135224 Nmax 0.946396824 0.981524793 0.958234587 0.863249759 1 0.933066234 0.9399267 0.995148802 0.975889256 0.883220316 0.88950586 0.839527031 0.872859195 0.848471118 0.831067987 0.826273821 0.820533545 0.810956817 0.922151629 HF1 ΔRA -4.01008E-05 0.003086839 0.001237369 -0.000286677 0.004398153 -0.004272576 -0.006550303 -0.004650736 -0.005621731 -0.00682584 -0.006532359 -0.00616658 -0.008914458 -0.011112014 -0.012694387 -0.010365771 -0.012734015 -0.019803442 -0.015903395 Nmax 0.920622322 0.910753744 0.941094187 0.842486155 1 0.91676537 0.933000104 0.869394272 0.885299149 0.866126402 0.941510098 0.90985885 0.941291259 0.953552953 0.992221885 0.928193442 0.790340499 0.829189218 0.712503827 Hypa ΔRA -0.009790696 -0.006089291 -0.008244537 -0.00866445 -0.007346098 -0.011740445 -0.015821269 -0.014674608 -0.01856957 -0.014964199 -0.018160114 -0.019617314 -0.023006634 -0.019342167 -0.017574545 -0.023793316 -0.018062728 -0.021723045 -0.025633176 Nmax 0.839413287 0.878124326 0.888528609 0.760845052 0.821306077 0.858179503 0.891968368 0.857415051 0.883311697 0.842007443 0.914099934 0.919831898 1 0.875845462 0.854376859 0.871810156 0.719666428 0.701210463 0.632291108 HiFi ΔRA -0.001559035 0.000760924 0.001079615 -0.000616703 -0.002900545 -0.005240841 -0.003086453 -0.004695262 -0.003476214 -0.001213415 -0.001904481 0.000443022 -0.003410655 -0.002763504 -0.003536079 -0.002532944 0.001138218 0.003159686 0.001631935 Nmax 0.863916612 0.981469253 1 0.842411493 0.880536637 0.831334654 0.764340005 0.790983778 0.773464701 0.785544371 0.808666259 0.847373958 0.850320976 0.741714769 0.709381181 0.678039491 0.757286216 0.704424708 0.770159891 ΔRA and Nmax for target sequences with two misamtches separated by different number of nucleotides across the PAM-distal region 0 1 2 3 4 5 6 7 8 Sp ΔRA -0.024451623 -0.023247359 -0.028416125 -0.026102136 -0.025919929 -0.029885216 -0.024962354 -0.029186007 -0.028025569 Nmax 0.903525805 0.972203449 1 0.927435506 0.802773405 0.854959292 0.702297323 0.814913177 0.817041292 Sa ΔRA -0.002663474 -0.000249262 2.58429E-05 -0.003821401 -0.000209502 -0.003114884 -0.002496662 0.002041043 0.004136734 Nmax 0.747302308 0.765390517 0.763475402 0.775251094 0.7543613 0.836565629 0.864555082 0.894169559 1 HF1 ΔRA 0.003398372 0.009339329 0.00949748 0.009065473 0.011754981 0.005221532 -0.002759248 -0.004482073 -0.008634952 Nmax 0.852666201 0.923436295 1 0.961813307 0.887197357 0.875308742 0.790681362 0.644361286 0.780854535 Hypa ΔRA -0.005792736 -0.002068577 -0.004362502 -0.005341208 -0.001194003 -0.007787003 -0.01500903 -0.020556013 -0.028546207 Nmax 0.796760841 0.982854591 1 0.858126279 0.821865627 0.743928479 0.787546374 0.674359076 0.78673459 HiFi ΔRA -0.02114306 -0.016417221 -0.021244076 -0.013371412 -0.020865755 -0.026467471 -0.022940921 -0.027700006 -0.022656988 Nmax 0.950567268 1 0.991441363 0.913489267 0.834672558 0.881511388 0.76112655 0.879303543 0.815195256 ΔRA and Nmax for target sequences with two misamtches separated by different number of nucleotides across the seed region 0 1 2 3 4 5 6 7 8 Sp ΔRA -0.005016177 -0.000845848 -0.000594825 -0.001512892 -0.001076514 -0.000371698 -0.001087879 -0.001326901 -0.010830083 Nmax 0.73452744 0.949536004 1 0.869647533 0.998346083 0.969849067 0.743427031 0.767726984 0.719252847 Sa ΔRA 0.003082784 0.001573274 0.002434189 0.003055632 -0.003755676 0.000201887 0.004914804 0.011097077 -0.002162845 Nmax 0.820650396 0.775409729 0.869066682 0.757379111 0.789558234 0.867447976 0.786118901 1 0.893990058 HF1 ΔRA -0.00280974 -0.001848423 -0.003597306 -0.00592812 0.003496805 -0.006073984 -0.008098513 -0.01116658 -0.017373394 Nmax 0.904607425 0.824849019 0.883208037 0.805970802 0.964544328 0.862239683 0.833315073 0.96466845 1 Hypa ΔRA -0.014573063 -0.007971209 -0.008279709 -0.006524793 -0.010602188 -0.008551159 -0.009911301 -0.013788205 -0.017644673 Nmax 0.982788976 0.911504914 0.947919236 0.83960499 0.929397137 0.914236256 0.865528279 0.951112307 1 HiFi ΔRA 0.00776281 0.007232795 0.009337439 0.006008204 0.008632499 0.007406352 0.006001399 0.00690925 0.003138876 Nmax 0.801979732 0.894880301 1 0.797631711 0.939593973 0.870114394 0.839754656 0.769278169 0.72202073