TY - JOUR
T1 - Computationally designed hyperactive Cas9 enzymes
AU - Vos, Pascal D.
AU - Rossetti, Giulia
AU - Mantegna, Jessica L.
AU - Siira, Stefan J.
AU - Gandadireja, Andrianto P.
AU - Bruce, Mitchell
AU - Raven, Samuel A.
AU - Khersonsky, Olga
AU - Fleishman, Sarel J.
AU - Filipovska, Aleksandra
AU - Rackham, Oliver
N1 - Funding Information:
Work in our laboratories is supported by fellowships and project grants from the National Health and Medical Research Council (APP1154646 to A.F. and APP1154932 to O.R.), the Australian Research Council (DP210103816 to A.F. and O.R.), the European Research Council (815379 to S.J.F.), the Israel Science Foundation (1884/19 to S.J.F.) and by the Dr. Barry Sherman Institute for Medicinal Chemistry (to S.J.F.). S.J.F., A.F., and O.R. are investigators of the ARC Centre of Excellence in Synthetic Biology (CE200100029). P.D.V. was supported by Colliers International. J.L.M. and A.P.G. are supported by Research Training Program (RTP) Scholarships. We thank David Chandler from the Australian Genomics Research Facility (AGRF), Perth, Western Australia, for assistance with amplicon sequencing and Jef Boeke, New York University, for the kind gift of the S. cerevisiae BY4738 strain.
Funding Information:
This work was supported by The Ministry of tribal affairs for NFST fellowships. Author Ms. Gugulothu Thara is grateful to NFST, New Delhi (Ref. no. 201819-NFST-TEL-00427) for providing fellowship. One of the authors D. Ashok thanks to University Grants Commission, New Delhi, India for the award of BSR-Faculty Fellowship (no. F.18-2011/(BSR). The author Ravinder Dharavath is grateful to CSIR, New Delhi for providing SRF Fellowship (File no. 09/132(0865)/2017-EMR-I). The authors are grateful to the Head, Department of Chemistry, Osmania University, and Hyderabad for laboratory facilities and CFRD, OU, Hyderabad.
Publisher Copyright:
© 2022, The Author(s).
PY - 2022/5/31
Y1 - 2022/5/31
N2 - The ability to alter the genomes of living cells is key to understanding how genes influence the functions of organisms and will be critical to modify living systems for useful purposes. However, this promise has long been limited by the technical challenges involved in genetic engineering. Recent advances in gene editing have bypassed some of these challenges but they are still far from ideal. Here we use FuncLib to computationally design Cas9 enzymes with substantially higher donor-independent editing activities. We use genetic circuits linked to cell survival in yeast to quantify Cas9 activity and discover synergistic interactions between engineered regions. These hyperactive Cas9 variants function efficiently in mammalian cells and introduce larger and more diverse pools of insertions and deletions into targeted genomic regions, providing tools to enhance and expand the possible applications of CRISPR-based gene editing.
AB - The ability to alter the genomes of living cells is key to understanding how genes influence the functions of organisms and will be critical to modify living systems for useful purposes. However, this promise has long been limited by the technical challenges involved in genetic engineering. Recent advances in gene editing have bypassed some of these challenges but they are still far from ideal. Here we use FuncLib to computationally design Cas9 enzymes with substantially higher donor-independent editing activities. We use genetic circuits linked to cell survival in yeast to quantify Cas9 activity and discover synergistic interactions between engineered regions. These hyperactive Cas9 variants function efficiently in mammalian cells and introduce larger and more diverse pools of insertions and deletions into targeted genomic regions, providing tools to enhance and expand the possible applications of CRISPR-based gene editing.
UR - http://www.scopus.com/inward/record.url?scp=85131011470&partnerID=8YFLogxK
U2 - 10.1038/s41467-022-30598-9
DO - 10.1038/s41467-022-30598-9
M3 - Article
C2 - 35641498
AN - SCOPUS:85131011470
SN - 2041-1723
VL - 13
JO - Nature Communications
JF - Nature Communications
IS - 1
M1 - 3023
ER -