Compositions for inducing modifications of target endogenous nucleic acid sequences in nucleuses of eukaryotic cells
The present disclosure relates to targeted genome editing in eukaryotic cells or organisms. More particularly, the present disclosure provides for compositions that may induce modifications in target endogenous nucleic acid sequences in nucleuses of eukaryotic cells. The composition may comprise a single-chain guide RNA (sgRNA) and a Streptococcus pyogenes Cas9 protein. In some embodiments, the sgRNA and the Cas9 protein may be present in a molar ratio ranging from 29:14.0 to 29:1.4.
1. A method of inducing a modification of a target endogenous nucleic acid sequence in a nucleus of a eukaryotic cell, comprising:
preparing a single-chain guide RNA (sgRNA), wherein the sgRNA comprises a crRNA and a tracrRNA;
preparing a Cas9 protein, wherein the Cas9 protein comprises a nuclear localization signal (NLS);
combining the sgRNA and the Cas9 protein in vitro to form a Cas9/sgRNA complex, wherein the sgRNA and the Cas9 protein are present in a molar ratio ranging from 29:14 to 29:1.4; and
introducing the Cas9/sgRNA complex into the eukaryotic cell, whereby the Cas9/sgRNA complex induces the modification of the target endogenous nucleic acid sequence in the nucleus of the eukaryotic cell.
2. The method of claim 1 , wherein the Cas9 protein is a Streptococcus pyogenes Cas9 protein.
3. The method of claim 2 , wherein the Streptococcus pyogenes Cas9 protein is a recombinant protein.
4. The method of claim 1 , wherein the Cas9 protein was expressed in E. coli.
5. The method of claim 1 , wherein the eukaryotic cell is a mammalian cell.
6. The method of claim 5 , wherein the mammalian cell is a human cell.
7. The method of claim 1 , wherein the Cas9/sgRNA complex is introduced into the eukaryotic cell by a method selected from the group consisting of electroporation, DEAE-dextran treatment, lipofection, nanoparticle-mediated transfection, and protein transduction domain mediated transduction.
8. The method of claim 1 , wherein the Cas9/sgRNA complex is introduced into the eukaryotic cell by transfection.
9. The method of claim 1 , wherein the Cas9/sgRNA complex is introduced into the eukaryotic cell by electroporation.
10. The method of claim 1 , wherein the target endogenous nucleic acid comprises a trinucleotide protospacer adjacent motif (PAM) recognized by the Cas9 protein, wherein the PAM consists of trinucleotide 5′-NGG-3′.
11. The method of claim 1 , wherein the NLS is disposed at the C-terminus of the Cas9 protein.
12. The method of claim 1 , wherein the modification includes any one of a deletion, insertion, or substitution of at least one nucleotide.
13. The method of claim 1 , wherein the method further comprises allowing the eukaryotic cell to divide into a plurality of cells, each of which comprises the modification to the target endogenous nucleic acid sequence.