	US 11,807,878 B2
	CRISPR-Cas systems for genome editing
Zhenglin Hou, Ankeny, IA (US); Tautvydas Karvelis, Paberze (LT); Virginijus Siksnys, Vilnius (LT); and Joshua K Young, Johnston, IA (US)
Assigned to PIONEER HI-BRED INTERNATIONAL, INC.
Filed by PIONEER HI-BRED INTERNATIONAL, INC., Johnston, IA (US)
Filed on Jan. 21, 2021, as Appl. No. 17/154,374.
Application 17/154,374 is a continuation of application No. 16/713,184, filed on Dec. 13, 2019, granted, now 10,934,536.
Claims priority of provisional application 62/913,492, filed on Oct. 10, 2019.
Claims priority of provisional application 62/852,788, filed on May 24, 2019.
Claims priority of provisional application 62/819,409, filed on Mar. 15, 2019.
Claims priority of provisional application 62/794,427, filed on Jan. 18, 2019.
Claims priority of provisional application 62/779,989, filed on Dec. 14, 2018.
Prior Publication US 2021/0139874 A1, May 13, 2021
Int. Cl. C12N 15/82 (2006.01); C12N 9/22 (2006.01); C12N 15/11 (2006.01); C12N 9/78 (2006.01); C12N 15/90 (2006.01)

CPC C12N 9/22 (2013.01) [C12N 9/78 (2013.01); C12N 15/11 (2013.01); C12N 15/8213 (2013.01); C12N 15/902 (2013.01); C12N 2310/20 (2017.05); C12N 2800/80 (2013.01)]

25 Claims

1. A synthetic composition comprising:

(a) a Cas-alpha endonuclease or a functional fragment thereof, comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 35 wherein the Cas-alpha endonuclease or the functional fragment comprises a tri-split RuvC domain, at least one zinc finger motif, and the following amino acid motifs: GxxxG, ExL, Cx_nC, and Cx_n(C or H); wherein G=Glycine, E=Glutamate, C=Cysteine, H=Histidine, L=Leucine, x=any amino acid and wherein n=an integer between 0 and 11;

(b) a target double-stranded DNA polynucleotide, wherein the target double-stranded DNA polynucleotide is heterologous to the source of the Cas-alpha endonuclease, and

(c) a guide polynucleotide comprising a variable targeting domain that comprises a region of complementarity to the target double-stranded DNA polynucleotide; wherein the Cas-alpha endonuclease or the functional fragment thereof recognizes a PAM sequence on the target double-stranded DNA polynucleotide, wherein the guide polynucleotide and the Cas-alpha endonuclease or the functional fragment thereof, form a complex that binds to the target double-stranded DNA polynucleotide, wherein the Cas-alpha endonuclease or the functional fragment thereof comprises DNA binding activity.