US 12,435,324 B2
Lachnospiraceae sp. Cas12a mutants with enhanced cleavage activity at non-canonical TTTT protospacer adjacent motifs
Liyang Zhang, North Liberty, IA (US); and Christopher Anthony Vakulskas, North Liberty, IA (US)
Assigned to INTEGRATED DNA TECHNOLOGIES, INC., Coralville, IA (US)
Filed by INTEGRATED DNA TECHNOLOGIES, INC., Coralville, IA (US)
Filed on Feb. 28, 2024, as Appl. No. 18/590,655.
Application 18/590,655 is a division of application No. 17/245,401, filed on Apr. 30, 2021, granted, now 11,999,979.
Claims priority of provisional application 63/090,912, filed on Oct. 13, 2020.
Claims priority of provisional application 63/018,592, filed on May 1, 2020.
Prior Publication US 2024/0368570 A1, Nov. 7, 2024
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 9/22 (2006.01)
CPC C12N 9/22 (2013.01) 8 Claims
OG exemplary drawing
 
1. A method of editing a gene in a cell having a non-canonical TTTT protospacer adjacent motif (PAM) with a CRISPR ribonucleoprotein complex, the method comprising:
contacting a cell with the CRISPR ribonucleoprotein complex comprising a guide RNA and a mutant Lachnospiraceae bacterium ND2006 Cas12a (“LbCas12a”) polypeptide comprising three substitutions: G146R, R182V, and E795Q, relative to a wild-type LbCas12a amino acid sequence of SEQ ID NO: 2, wherein the mutant LbCas12a polypeptide comprises at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 3984.