Targeted mutagenesis using base editors

A method of identifying an agronomically important phenotype in a cellular system, comprising the following steps:

• (a) selecting at least one nucleic acid sequence of interest in the genetic material of the cellular system;
• (b) providing at least one saturated targeted endogenous mutagenesis editor (STEME) complex, or a sequence encoding the same, wherein the at least one STEME complex comprises an array of guide RNAs, or a sequence encoding the same, targeting the at least one nucleic acid sequence of interest, wherein the at least one STEME complex comprises a STEME having an amino acid sequence with at least 96% identity to either SEQ ID NO:
• 176 or SEQ ID NO:
• 184;
• (c) introducing the at least one saturated targeted endogenous mutagenesis editor (STEME) complex, or the sequence encoding the same, into the genetic material of the cellular system;
• (d) cultivating the cellular system under conditions to obtain a M0 population of the cellular system;
• (e) crossing the M0 population of the cellular system with a wildtype population of the cellular system comprising the at least one nucleic acid sequence of interest to obtain a progeny population of the cellular system;
• (f) obtaining a progeny population of the cellular system having at least one modification in the at least one nucleic acid sequence of interest;
• (g) screening the progeny population of the cellular system for the agronomically important phenotype associated with at the least one modification in the at least one nucleic acid of interest;
• and
• (h) identifying and thereby selecting an agronomically important phenotype in the cellular system,
• wherein the array of guide RNAs of the at least one STEME complex comprises at least one guide RNA molecules, or a sequence encoding the same, targeting the at least one nucleic acid sequence of interest.

A method of generating a modified cellular system having an agronomically important phenotype, the method comprises the following steps:

• (a) selecting at least one nucleic acid sequence of interest in the genetic material of the cellular system;
• (b) providing at least one saturated targeted endogenous mutagenesis editor (STEME) complex, or a sequence encoding the same, wherein the at least one STEME complex comprises an array of guide RNAs, or a sequence encoding the same, targeting the at least one nucleic acid sequence of interest, wherein the at least one STEME complex comprises a STEME having an amino acid sequence with at least 96% identity to either SEQ ID NO:
• 176 or SEQ ID NO:
• 184;
• (c) introducing the at least one saturated targeted endogenous mutagenesis editor (STEME) complex, or a sequence encoding the same, into the cellular system;
• (d) obtaining a cellular system comprising at least one modification in the at least one nucleic acid sequence of interest;
• (e) cultivating the cellular system under conditions to obtain a M0 population of the cellular system;
• (f) screening the M0 population of the cellular system for the agronomically important phenotype associated with the at least one modification in the at least one nucleic acid sequence of interest;
• and
• (g) identifying and thereby selecting a cellular system from the M0 population having the agronomically important phenotype;
• and
• (h) obtaining a modified cellular system having the agronomically important phenotype,
• wherein the array of guide RNAs of the at least one STEME complex comprises at least one guide RNA molecules, or a sequence encoding the same, targeting the at least one nucleic acid sequence of interest.

A method of generating a progeny of a modified cellular system having an agronomically important phenotype, the method comprises the following steps:

• (a) selecting at least one nucleic acid sequence of interest in the genetic material of the cellular system;
• (b) providing at least one saturated targeted endogenous mutagenesis editor (STEME) complex, or a sequence encoding the same, wherein the at least one STEME complex comprises an array of guide RNAs, or a sequence encoding the same, targeting the at least one nucleic acid sequence of interest, wherein the at least one STEME complex comprises a STEME having an amino acid sequence with at least 96% identity to either SEQ ID NO:
• 176 or SEQ ID NO:
• 184;
• (c) introducing the at least one saturated targeted endogenous mutagenesis editor (STEME) complex, or a sequence encoding the same, into the genetic material of the cellular system;
• (d) cultivating the cellular system under conditions to obtain a M0 population of the cellular system;
• (e) crossing the M0 population of the cellular system with a wildtype population of the cellular system comprising the at least one nucleic acid sequence of interest to obtain a progeny population of the cellular system;
• (f) obtaining a progeny population of the cellular system having at least one modification in the at least one nucleic acid sequence of interest;
• (g) screening the progeny population of the cellular system for the agronomically important phenotype associated with at the least one modification in the at least one nucleic acid of interest;
• and
• (h) identifying and thereby selecting a cellular system from the progeny population having the agronomically important phenotype,
• (i) obtaining a progeny of a modified cellular system having the agronomically important phenotype,
• wherein the array of guide RNAs of the at least one STEME complex comprises at least one guide RNA molecules, or a sequence encoding the same, targeting the at least one nucleic acid sequence of interest.