Methods and compositions for modification of protospacer adjacent motif specificity of cas12a

A CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) Cas12a polypeptide, wherein the Cas12a polypeptide comprises:

• (a) an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:
• 180 and a mutation located at one or more than one of the following positions of K135, S168, T246, N263, T307, L324, E330, S345, R359, D367, I369, E379, Y381, I417, F474, E479, E484, D489, L498, N590, K167, Q231, A454, F657, F337, L459, I507, M531, D654, E659, S170, V245, I425, V463, L492, K514, N112, E223, Y426, I455, S117, A239, T295, N100, K387, A404, D405, D423, E539, D572, N211, N527, Y553, Y554, L321, K192, I344 and/or E443, optionally a mutation located at position K135, S168, T246, N263, T307, L324, E330, S345, R359, D367, I369, E379, Y381, I417, F474, E479, E484, D489, L498, and/or N590, and/or a mutation comprising one or more of K135N, S168N, T246S, N263I, T307S, L324M, E330V, S345R, R359S, D367V, I369M, E379G, Y381H, I417F, F474L, E479V, E484V, D489Y, L498M, N590Y, K167I, Q231, A454V, F657, F337L, L459M, I507V, M531I, D654E, E659V, S170L, V245I, I425V, V463A, L492P, K514N, N112D, E223V, Y426D, 1455F, S117C, A239T, T295I, N100S, K387E, A404V, D405G, D423V, E539V, D572G, N211S, N527S, Y553H, Y554N, L321V, K192I, I344T and/or E443D, optionally a mutation comprising one or more of K135N, S168N, T246S, N263I, T307S, L324M, E330V, S345R, R359S, D367V, 1369M, E379G, Y381H, I417F, F474L, E479V, E484V, D489Y, L498M, and/or N590Y, optionally the mutation of D489Y, L324M, E484V and/or E330V, with reference to position numbering of SEQ ID NO:
• 180, wherein the polypeptide exhibits a reduced PAM stringency and increased recognition of new protospacer adjacent motifs (PAMs) as compared to the amino acid sequence of SEQ ID NO:
• 180,
• (b) an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:
• 51 and a mutation located at one or more than one of the following positions of E153, S186, R267, I285, Y336, L352, S358, E372, R386, E391, R393, K403, A405, A434, V486, E491, P494, R499, E508, A602, I185, Q252, E465, L698, F365, Q470, A517, A541, L695, P700, A188, S266, I442, L474, G502, K524, T114, P244, L443, I466, D119, Q260, K324, T101, R411, A427, G428, S440, E549, E584, S234, M537, Y563, Y564, A349, R210, L371, and/or K459, optionally a mutation at position E153, S186, R267, I285, Y336, L352, S358, E372, R386, E391, R393, K403, A405, A434, V486, E491, P494, R499, E508, and/or A602, optionally a mutation at position R499, L352, P494 and/or S358, with reference to position numbering of SEQ ID NO:
• 51, wherein the polypeptide exhibits a reduced PAM stringency and increased recognition of new protospacer adjacent motifs (PAMs) as compared to the amino acid sequence of SEQ ID NO:
• 51, or
• (c) an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:
• 55 and a mutation located at one or more than one of the following positions of I162, P196, V290, S308, Q358, L377, K385, T400, Q423, D431, P433, K443, T445, A480, I454, D550, E559, V564, A573, N666, I195, Q275, A525, F741, Y392, L529, I582, A606, G738, D743, S198, F289, I488, T533, E567, K589, S116, A267, F489, A525, N121, T283, E343, T104, L451, R467, D468, P486, E614, E648, T243, N602, Y628, Y629, L374, S220, L399, and/or Q507, optionally a mutation located at I162, P196, V290, S308, Q358, L377, K385, T400, Q423, D431, P433, K443, T445, A480, I454, D550, E559, V564, A573, and/or N666, optionally a mutation at position V564, L377, E559 and/or K385, with reference to position numbering of SEQ ID NO:
• 55, wherein the polypeptide exhibits a reduced PAM stringency and increased recognition of new protospacer adjacent motifs (PAMs) as compared to the amino acid sequence of SEQ ID NO:
• 55.