High-throughput continuous evolution of compact Cas9 variants targeting single-nucleotide-pyrimidine PAMs

Tony P Huang; Zachary J Heins; Shannon M Miller; Brandon G Wong; Pallavi A Balivada; Tina Wang; Ahmad S Khalil; David R Liu

doi:10.1038/s41587-022-01410-2

High-throughput continuous evolution of compact Cas9 variants targeting single-nucleotide-pyrimidine PAMs

Nat Biotechnol. 2023 Jan;41(1):96-107. doi: 10.1038/s41587-022-01410-2. Epub 2022 Sep 8.

Authors

Tony P Huang^#^{1

2

3}, Zachary J Heins^#^{4

5}, Shannon M Miller^{1

2

3}, Brandon G Wong^{4

5}, Pallavi A Balivada^{4

5}, Tina Wang^{1

2

3

6}, Ahmad S Khalil^{7

8

9}, David R Liu^{10

11

12}

Affiliations

¹ Merkin Institute of Transformative Technologies in Healthcare, The Broad Institute of Harvard and MIT, Cambridge, MA, USA.
² Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA.
³ Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA.
⁴ Biological Design Center, Boston University, Boston, MA, USA.
⁵ Department of Biomedical Engineering, Boston University, Boston, MA, USA.
⁶ Department of Chemistry, University of Wisconsin-Madison, Madison, WI, USA.
⁷ Biological Design Center, Boston University, Boston, MA, USA. khalil@bu.edu.
⁸ Department of Biomedical Engineering, Boston University, Boston, MA, USA. khalil@bu.edu.
⁹ Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA. khalil@bu.edu.
¹⁰ Merkin Institute of Transformative Technologies in Healthcare, The Broad Institute of Harvard and MIT, Cambridge, MA, USA. drliu@fas.harvard.edu.
¹¹ Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA. drliu@fas.harvard.edu.
¹² Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA. drliu@fas.harvard.edu.

^# Contributed equally.

Abstract

Despite the availability of Cas9 variants with varied protospacer-adjacent motif (PAM) compatibilities, some genomic loci-especially those with pyrimidine-rich PAM sequences-remain inaccessible by high-activity Cas9 proteins. Moreover, broadening PAM sequence compatibility through engineering can increase off-target activity. With directed evolution, we generated four Cas9 variants that together enable targeting of most pyrimidine-rich PAM sequences in the human genome. Using phage-assisted noncontinuous evolution and eVOLVER-supported phage-assisted continuous evolution, we evolved Nme2Cas9, a compact Cas9 variant, into variants that recognize single-nucleotide pyrimidine-PAM sequences. We developed a general selection strategy that requires functional editing with fully specified target protospacers and PAMs. We applied this selection to evolve high-activity variants eNme2-T.1, eNme2-T.2, eNme2-C and eNme2-C.NR. Variants eNme2-T.1 and eNme2-T.2 offer access to N₄TN PAM sequences with comparable editing efficiencies as existing variants, while eNme2-C and eNme2-C.NR offer less restrictive PAM requirements, comparable or higher activity in a variety of human cell types and lower off-target activity at N₄CN PAM sequences.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

CRISPR-Associated Protein 9 / genetics
CRISPR-Associated Protein 9 / metabolism
CRISPR-Cas Systems*
Gene Editing*
Genome, Human / genetics
Humans
Pyrimidines

Substances

CRISPR-Associated Protein 9
Pyrimidines

Abstract

Publication types

MeSH terms

Substances

Grants and funding