Engineered Dual Selection for Directed Evolution of SpCas9 PAM Specificity

Overview

Journal Nat Commun

Specialty Biology

Date 2021 Jan 14

PMID 33441553

Citations 10

Authors

Gregory W Goldberg

Jeffrey M Spencer

David O Giganti

Brendan R Camellato

Neta Agmon

David M Ichikawa

Jef D Boeke

Marcus B Noyes

Affiliations

Soon will be listed here.

Abstract

The widely used Streptococcus pyogenes Cas9 (SpCas9) nuclease derives its DNA targeting specificity from protein-DNA contacts with protospacer adjacent motif (PAM) sequences, in addition to base-pairing interactions between its guide RNA and target DNA. Previous reports have established that the PAM specificity of SpCas9 can be altered via positive selection procedures for directed evolution or other protein engineering strategies. Here we exploit in vivo directed evolution systems that incorporate simultaneous positive and negative selection to evolve SpCas9 variants with commensurate or improved activity on NAG PAMs relative to wild type and reduced activity on NGG PAMs, particularly YGG PAMs. We also show that the PAM preferences of available evolutionary intermediates effectively determine whether similar counterselection PAMs elicit different selection stringencies, and demonstrate that negative selection can be specifically increased in a yeast selection system through the fusion of compensatory zinc fingers to SpCas9.

Citing Articles

Utilizing Target Sequences with Multiple Flanking Protospacer Adjacent Motif (PAM) Sites Reduces Off-Target Effects of the Cas9 Enzyme in Pineapple.

Shu H, Luan A, Ullah H, He J, Wang Y, Chen C Genes (Basel). 2025; 16(2).

PMID: 40004545 PMC: 11855603. DOI: 10.3390/genes16020217.

SPLICER: a highly efficient base editing toolbox that enables in vivo therapeutic exon skipping.

Miskalis A, Shirguppe S, Winter J, Elias G, Swami D, Nambiar A Nat Commun. 2024; 15(1):10354.

PMID: 39609418 PMC: 11604662. DOI: 10.1038/s41467-024-54529-y.

Engineered transcription-associated Cas9 targeting in eukaryotic cells.

Goldberg G, Kogenaru M, Keegan S, Haase M, Kagermazova L, Arias M Nat Commun. 2024; 15(1):10287.

PMID: 39604381 PMC: 11603292. DOI: 10.1038/s41467-024-54629-9.

Evolved cytidine and adenine base editors with high precision and minimized off-target activity by a continuous directed evolution system in mammalian cells.

Zhao N, Zhou J, Tao T, Wang Q, Tang J, Li D Nat Commun. 2024; 15(1):8140.

PMID: 39289397 PMC: 11408606. DOI: 10.1038/s41467-024-52483-3.

Base Editors-Mediated Gene Therapy in Hematopoietic Stem Cells for Hematologic Diseases.

Zhang C, Xu J, Wu Y, Xu C, Xu P Stem Cell Rev Rep. 2024; 20(6):1387-1405.

PMID: 38644403 PMC: 11319617. DOI: 10.1007/s12015-024-10715-5.

References

Fontana J, Dong C, Kiattisewee C, Chavali V, Tickman B, Carothers J . Effective CRISPRa-mediated control of gene expression in bacteria must overcome strict target site requirements. Nat Commun. 2020; 11(1):1618. PMC: 7113249. DOI: 10.1038/s41467-020-15454-y. View

Walton R, Christie K, Whittaker M, Kleinstiver B . Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants. Science. 2020; 368(6488):290-296. PMC: 7297043. DOI: 10.1126/science.aba8853. View

Carrico Z, Strobel K, Atreya M, Clark D, Francis M . Simultaneous selection and counter-selection for the directed evolution of proteases in E. coli using a cytoplasmic anchoring strategy. Biotechnol Bioeng. 2015; 113(6):1187-93. DOI: 10.1002/bit.25904. View

Hu J, Miller S, Geurts M, Tang W, Chen L, Sun N . Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature. 2018; 556(7699):57-63. PMC: 5951633. DOI: 10.1038/nature26155. View

Bhargava R, Onyango D, Stark J . Regulation of Single-Strand Annealing and its Role in Genome Maintenance. Trends Genet. 2016; 32(9):566-575. PMC: 4992407. DOI: 10.1016/j.tig.2016.06.007. View

Dorr B, Ham H, An C, Chaikof E, Liu D . Reprogramming the specificity of sortase enzymes. Proc Natl Acad Sci U S A. 2014; 111(37):13343-8. PMC: 4169943. DOI: 10.1073/pnas.1411179111. View

Kouprina N, Larionov V . Transformation-associated recombination (TAR) cloning for genomics studies and synthetic biology. Chromosoma. 2016; 125(4):621-32. PMC: 5025352. DOI: 10.1007/s00412-016-0588-3. View

Meng X, Wolfe S . Identifying DNA sequences recognized by a transcription factor using a bacterial one-hybrid system. Nat Protoc. 2007; 1(1):30-45. DOI: 10.1038/nprot.2006.6. View

Nussenzweig P, McGinn J, Marraffini L . Cas9 Cleavage of Viral Genomes Primes the Acquisition of New Immunological Memories. Cell Host Microbe. 2019; 26(4):515-526.e6. PMC: 7558852. DOI: 10.1016/j.chom.2019.09.002. View

10.

Oakes B, Xia D, Rowland E, Xu D, Ankoudinova I, Borchardt J . Multi-reporter selection for the design of active and more specific zinc-finger nucleases for genome editing. Nat Commun. 2016; 7:10194. PMC: 4729830. DOI: 10.1038/ncomms10194. View

11.

Meng X, Brodsky M, Wolfe S . A bacterial one-hybrid system for determining the DNA-binding specificity of transcription factors. Nat Biotechnol. 2005; 23(8):988-94. PMC: 1435991. DOI: 10.1038/nbt1120. View

12.

Yi L, Gebhard M, Li Q, Taft J, Georgiou G, Iverson B . Engineering of TEV protease variants by yeast ER sequestration screening (YESS) of combinatorial libraries. Proc Natl Acad Sci U S A. 2013; 110(18):7229-34. PMC: 3645551. DOI: 10.1073/pnas.1215994110. View

13.

van Houte S, Ekroth A, Broniewski J, Chabas H, Ashby B, Bondy-Denomy J . The diversity-generating benefits of a prokaryotic adaptive immune system. Nature. 2016; 532(7599):385-8. PMC: 4935084. DOI: 10.1038/nature17436. View

14.

Hirano S, Nishimasu H, Ishitani R, Nureki O . Structural Basis for the Altered PAM Specificities of Engineered CRISPR-Cas9. Mol Cell. 2016; 61(6):886-94. DOI: 10.1016/j.molcel.2016.02.018. View

15.

Simon A, dOelsnitz S, Ellington A . Synthetic evolution. Nat Biotechnol. 2019; 37(7):730-743. DOI: 10.1038/s41587-019-0157-4. View

16.

Deveau H, Barrangou R, Garneau J, Labonte J, Fremaux C, Boyaval P . Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. J Bacteriol. 2007; 190(4):1390-400. PMC: 2238228. DOI: 10.1128/JB.01412-07. View

17.

Ran F, Cong L, Yan W, Scott D, Gootenberg J, Kriz A . In vivo genome editing using Staphylococcus aureus Cas9. Nature. 2015; 520(7546):186-91. PMC: 4393360. DOI: 10.1038/nature14299. View

18.

Mojica F, Diez-Villasenor C, Garcia-Martinez J, Almendros C . Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology (Reading). 2009; 155(Pt 3):733-740. DOI: 10.1099/mic.0.023960-0. View

19.

Zetsche B, Gootenberg J, Abudayyeh O, Slaymaker I, Makarova K, Essletzbichler P . Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 2015; 163(3):759-71. PMC: 4638220. DOI: 10.1016/j.cell.2015.09.038. View

20.

Anders C, Niewoehner O, Duerst A, Jinek M . Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature. 2014; 513(7519):569-73. PMC: 4176945. DOI: 10.1038/nature13579. View