Extended single guide RNA and use thereof
The present disclosure relates to: an extended guide RNA and a composition for base editing, comprising the same; and a method for base editing and a method for producing genetically modified animals or plants, both methods using the composition for base editing.
1. A method of producing a genetically modified eukaryotic cell having a base substitution, comprising:
(a) preparing a composition for base editing comprising
(i) an adenosine base editor (ABE) or cytosine base editor (CBE),
wherein the ABE is selected from the group consisting of apolipoprotein B editing complex 1 (APOBEC1) and activation-induced deaminase (AID), the CBE is tRNA-specific adenosine deaminase (tadA);
(ii) Cas 9 nickase (nCas9); and
(iii) an extended guide RNA (ggX20) hybridizable with a target sequence,
wherein the ggX20 comprises a sequence of 20 nucleotides of the target sequence, and two additional non-complementary guanines (G) in front of 20 nucleotides in the 5′ direction from protospacer adjacent motif (PAM),
wherein the target sequence is selected from HEK2, HBB, and FANCF;
(b) introducing the composition into a eukaryotic cell,
wherein the extended guide RNA hybridizes with the target sequence,
wherein the nCas9 makes a single stranded nick,
wherein the ABE converts an adenosine to a guanine at position 18 of the target sequence in the 5′ direction from the PAM;
wherein the CBE converts a cytosine to a thymine at position 20, 21, or 23 of the target sequence in the 5′ direction from the PAM, and
wherein the base substitution does not cause a double stranded break.
2. The method according to claim 1 , wherein the composition further comprises an uracil DNA glycosylase inhibitor (UGI).
3. The method according to claim 1 , wherein the composition further comprises a nuclear localization sequence (NLS).