Crispr-guided dna polymerases enable diversification of all nucleotides in a tunable window

ABSTRACT The capacity to diversify genetic codes advances our ability to understand and engineer biological systems1,2. A method for continuously diversifying user-defined regions of a

genome would enable forward genetic approaches in systems that are not amenable to efficient homology-directed oligonucleotide integration. It would also facilitate the rapid evolution of

biotechnologically useful phenotypes through accelerated and parallelized rounds of mutagenesis and selection, as well as cell-lineage tracking through barcode mutagenesis. Here we present

EvolvR, a system that can continuously diversify all nucleotides within a tunable window length at user-defined loci. This is achieved by directly generating mutations using engineered DNA

polymerases targeted to loci via CRISPR-guided nickases. We identified nickase and polymerase variants that offer a range of targeted mutation rates that are up to 7,770,000-fold greater

than rates seen in wild-type cells, and editing windows with lengths of up to 350 nucleotides. We used EvolvR to identify novel ribosomal mutations that confer resistance to the antibiotic

spectinomycin. Our results demonstrate that CRISPR-guided DNA polymerases enable multiplexed and continuous diversification of user-defined genomic loci, which will be useful for a broad

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ACKNOWLEDGEMENTS We thank S. McDevitt at the University of California, Berkeley Vincent J. Coates Genomics Sequencing Laboratory for assistance with high-throughput sequencing, the Arkin

laboratory for supplying _E. coli_ strain RE1000, W. DeLoache for helping edit our manuscript, and the Innovative Genomics Institute for funding. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS

* Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA Shakked O. Halperin, Connor J. Tou, Eric B. Wong, Cyrus Modavi, David V. Schaffer & John E. Dueber *

University of California, Berkeley–University of California, San Francisco Graduate Program in Bioengineering, University of California, Berkeley, Berkeley, CA, USA Shakked O. Halperin 

& Cyrus Modavi * Innovative Genomics Institute, University of California Berkeley and San Francisco, Berkeley, CA, USA Shakked O. Halperin, David V. Schaffer & John E. Dueber *

Department of Chemical and Biomolecular Engineering, University of California Berkeley, Berkeley, CA, USA David V. Schaffer * Department of Molecular and Cell Biology, University of

California, Berkeley, Berkeley, CA, USA David V. Schaffer * Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, CA, USA David V. Schaffer * Biological Systems

& Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA John E. Dueber Authors * Shakked O. Halperin View author publications You can also search for this author

inPubMed Google Scholar * Connor J. Tou View author publications You can also search for this author inPubMed Google Scholar * Eric B. Wong View author publications You can also search for

this author inPubMed Google Scholar * Cyrus Modavi View author publications You can also search for this author inPubMed Google Scholar * David V. Schaffer View author publications You can

also search for this author inPubMed Google Scholar * John E. Dueber View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS S.O.H. conceived of

all designs, designed the study, contributed to the execution of all experiments, analysed all of the data and wrote the manuscript; C.J.T. contributed to plasmid construction and assay

execution for fluctuation analyses, and spectinomycin-resistance mutation identification; E.B.W. contributed to plasmid construction and assay execution for PolI3M mutant screening, Phi29

screening, multiplexing and spectinomycin-resistance identification; S.O.H., C.M., D.V.S. and J.E.D. contributed to assay design. The manuscript was read, edited and approved by all authors.

CORRESPONDING AUTHORS Correspondence to David V. Schaffer or John E. Dueber. ETHICS DECLARATIONS COMPETING INTERESTS The Regents of the University of California have filed a provisional

patent application (62/662,043 and 62/556,127) related to the technology described in this work to the United States Patent and Trademark Office; S.O.H. is listed as the inventor. ADDITIONAL

INFORMATION PUBLISHER’S NOTE: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. EXTENDED DATA FIGURES AND TABLES

EXTENDED DATA FIG. 1 BIAS OF CYTIDINE DEAMINASE-MEDIATED TARGETED DIVERSIFICATION. Previous tools enabling diversification of user-defined loci by substituting cytosines and guanines limit

the protein coding space that can be explored4,5. This chart shows which amino acids can (green) and cannot (red) be reached by mutating cytosines and guanines to any other base for each of

the 64 codons, highlighting that only 32% of missense mutations are achievable with targeted cytidine deaminases. The white area depicts the original amino acid identity. EXTENDED DATA FIG.

2 THE DIRECTION OF EVOLVR-MEDIATED MUTAGENESIS RELATIVE TO THE GRNA IS DEPENDENT ON WHICH STRAND IS NICKED. Our previous fluctuation analysis in Fig. 1e demonstrated that nCas9(D10A)–PolI3M

mutates a window 3′ of the nick site. Here we directly tested whether mutations are generated 5′ of the nick site using a different gRNA. Because DNA polymerases synthesize in the 5′-to-3′

direction, we anticipated that nCas9(D10A)–PolI3M would not provide an elevated mutation rate 5′ of the nick site. We indeed found that expressing a guide RNA which targeted

nCas9(D10A)–PolI3M to nick 16 nucleotides 3′ from the nonsense mutation (indicated by a red cross) did not show targeted mutagenesis. We hypothesized that we could induce targeted

mutagenesis using the same gRNA by using a Cas9 variant harbouring the H840A mutation, which nicks the DNA strand non-complementary to the gRNA, rather than the D10A mutation, which nicks

the strand complementary to the gRNA. nCas9(H840A)–PolI3M increased the mutation rate 16 nucleotides 3′ from the nick by 52-fold compared to the global mutation rate of cells expressing an

off-target gRNA. We used the D10A nCas9 variant for all subsequent experiments. Data are mean ± 95% confidence intervals from ten biologically independent samples. *_P_ < 0.0001;

two-sided Student’s _t_-test. EXTENDED DATA FIG. 3 POLI5M ELEVATES MUTATION RATES 1 NUCLEOTIDE, BUT NOT 11 NUCLEOTIDES, FROM THE NICK COMPARED TO POLI3M. PolI3M with additional F742Y and

P796H mutations (PolI5M) elevates the mutation rate 33-fold 1 nucleotide from the nick compared to PolI3M. PolI5M did not have a higher mutation rate than PolI3M 11 nucleotides from the

nick. Data are mean ± 95% confidence intervals from ten biologically independent samples. *_P_ < 0.0001; two-sided Student’s _t_-test. EXTENDED DATA FIG. 4 FUSING A HIGHLY PROCESSIVE DNA

POLYMERASE TO ENCAS9 INCREASES THE TARGET WINDOW LENGTH. PolI was exchanged for a more processive and higher-fidelity bacteriophage Phi29 DNA polymerase (Phi29). Owing to Phi29 not having a

flap endonuclease, residues 1–325 of PolI were inserted between enCas9 and Phi29. Using gRNAs targeting different distances from the nonsense mutation, we found that Phi29 with two

previously reported fidelity-reducing mutations (N62D and L384R) elevated the mutation rate 56 nucleotides from the nick compared to the global mutation rate28,29. When we expressed Phi29’s

single-stranded binding protein (ssb), which is known to improve the activity of Phi29, we observed an elevation in the targeted mutation rate30. Finally, because the activity of Phi29 is

known to decrease at temperatures above 30 °C and the fluctuation analysis was performed at 37 °C, we added mutations previously reported to improve the thermostability of Phi29 (iPhi29) and

observed a targeted mutation rate 347 nucleotides from the nick site that was significantly greater than the global mutation rate31. Unfortunately, mutations decreasing Phi29’s fidelity are

known to decrease its processivity explaining our inability to identify Phi29 variants that retain high processivity while offering as high of a mutation rate as PolI3M28. Data are mean ± 

95% confidence intervals from ten biologically independent samples. *_P_ < 0.0001; two-sided Student’s _t_-test. EXTENDED DATA FIG. 5 REMOVING INTERNAL RIBOSOME BINDING SEQUENCES

DECREASES EVOLVR-MEDIATED OFF-TARGET MUTAGENESIS. enCas9–PolI3M–TBD was codon optimized to remove strong ribosome binding sites in the EvolvR coding sequence that were predicted to produce

an untethered DNA polymerase. The off-target mutation rate decreased 4.14-fold when expressing enCas9–PolI3M–TBD-CO compared to enCas9–PolI3M–TBD (_P_ = 0.000482) whereas the on-target

mutation rate only decreased 1.23-fold. Data are mean ± 95% confidence intervals from ten biologically independent samples. *_P_ < 0.0001; two-sided student’s _t_-test. EXTENDED DATA FIG.

6 EVOLVR-MEDIATED MUTAGENESIS CAN BE COUPLED WITH A NON-SELECTABLE GENETIC SCREEN. A, To test the capability for coupling EvolvR-mediated mutagenesis with a non-selectable genetic screen,

we designed a target plasmid containing a GFP cassette with an early termination codon in the GFP coding sequence (pTarget-GFP*). After co-transforming pEvolvR with pTarget-GFP* and growing

for 24 h, we analysed and sorted the GFP-positive fraction. In the two replicates expressing an off-target gRNA, we did not detect or sort any GFP cells. By contrast, for the two replicates

expressing a gRNA nicking four nucleotides away from the chain-terminating mutation in the coding sequence of GFP, we found that 0.06% and 0.07% of the total cells were GFP positive. These

results agree with sequencing outcomes from Fig. 1b, which showed that expressing nCas9–PolI3M for 24 h produces substitutions in the target region at frequencies between 0.5% to 1%. B,

After culturing the sorted populations, both replicates expressing an off-target gRNA did not show growth, whereas both replicates expressing the on-target gRNA grew bright green. EXTENDED

DATA FIG. 7 EVOLVR ENABLES TARGETED GENOME DIVERSIFICATION WITHOUT AFFECTING VIABILITY OR GROWTH RATE. A, The viability of TG1 _E. coli_ expressing EvolvR targeted to the essential _rpsE_

gene was significantly higher than TG1 _E. coli_ transformed with the MP6 plasmid and induced with 25 mM arabinose and 25 mM glucose (a previously developed plasmid for continuous

non-targeted mutagenesis32, _P_ = 0.0108) as well as XL1-Red _E. coli_ (a previously developed strain for continuous non-targeted mutagenesis33, _P_ = 0.0105). Viability was measured

relative to TG1 _E. coli_ transformed with an empty control plasmid. Data are mean ± s.d. from three biologically independent samples. *_P_ < 0.05; two-tailed _t_-test. B, TG1 _E. coli_

transformed with an empty control plasmid and TG1 _E. coli_ transformed with pEvolvR targeting the _rpsE_ gene resulted in similar growth curves whereas XL1-Red _E. coli_ and TG1 _E. coli_

transformed with MP6 plasmid and induced with 25 mM arabinose and 25 mM glucose grew much slower and saturated at lower final optical densities. Shaded area represents mean ± s.d. from three

biologically independent samples. C, The spectinomycin-resistant CFUs per ml saturated culture of TG1 _E. coli_ targeting EvolvR to the _rpsE_ gene was significantly higher than XL1-Red _E.

coli_ (_P_ = 0.022) and TG1 _E. coli_ transformed with MP6 plasmid and induced with 25 mM arabinose and 25 mM glucose (_P_ = 0.0049). Data are mean ± s.d. from three biologically

independent samples. *_P_ < 0.05; two-tailed _t_-test. EXTENDED DATA FIG. 8 EVOLVR-MEDIATED MUTAGENESIS PERFORMS BETTER THAN A PREVIOUS NON-TARGETED DIVERSIFICATION TECHNIQUE. To compare

the performance of EvolvR and the previously developed non-targeted mutagenesis plasmid MP6 in screen-based directed evolution applications, we co-transformed pEvolvR (enCas9–PolI3M–TBD) or

MP6 with a target plasmid containing a GFP cassette with an early termination codon in the GFP coding sequence (pTarget-GFP*). The cultures expressing EvolvR were grown for 24 h and the MP6

cultures followed a two day growth–induction protocol as previously described. Flow cytometry revealed that cultures expressing EvolvR and an on-target gRNA resulted in 28-fold more

GFP-positive cells than MP6 cultures. EXTENDED DATA FIG. 9 LOCATIONS OF GRNA TARGETS RELATIVE TO THE _RPSE_ GENE AND MUTATIONS IN RIBOSOMAL PROTEIN S5 THAT CONFER SPECTINOMYCIN RESISTANCE.

A, enCas9–PolI3M–TBD was targeted to five dispersed loci in the endogenous _rpsE_ gene using gRNAs that nick after the 119th, 187th, 320th, 403rd or 492nd base pair of the 504-bp _rpsE_

coding sequence. The locations of the previously identified _rpsE_ mutations that provide spectinomycin resistance are coloured orange, and the region where we identified new

spectinomycin-resistance mutations is highlighted in red. B, The mutations that we discovered confer spectinomycin resistance would be expected to move Lys26 (which is predicted to hydrogen

bond with spectinomycin) relative to the spectinomycin-binding pocket. We hypothesized that mutations that move Lys26 relative to the spectinomycin-binding pocket remove that hydrogen bond

and destabilize the interaction of spectinomycin with the ribosome, thereby conferring spectinomycin resistance. C, Therefore, we tested whether deleting any single amino acid between

residues 16 and 35 confers spectinomycin resistance. We found that deleting residues 23, 24, 25, 26, 27 or 28 provides spectinomycin resistance whereas deleting any of the residues between

16 and 22 or 29 and 35 does not. These results support the hypothesis that one mechanism of resistance to spectinomycin is disruption of the interaction between Lys26 and spectinomycin. Data

are mean ± s.d. from three biologically independent samples. SUPPLEMENTARY INFORMATION SUPPLEMENTARY TABLE Supplementary Table 1: Oligonucleotides, gRNAs, plasmids, and amino acid sequences

used in this study. REPORTING SUMMARY RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Halperin, S.O., Tou, C.J., Wong, E.B. _et al._ CRISPR-guided DNA

polymerases enable diversification of all nucleotides in a tunable window. _Nature_ 560, 248–252 (2018). https://doi.org/10.1038/s41586-018-0384-8 Download citation * Received: 01 December

2017 * Accepted: 19 June 2018 * Published: 01 August 2018 * Issue Date: 09 August 2018 * DOI: https://doi.org/10.1038/s41586-018-0384-8 SHARE THIS ARTICLE Anyone you share the following link

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