Science - USA (2019-01-04)

expanded genomic targeting via the minimal
Cas12c2 PAM, high-fidelity genome editing using
Cas12i nicking ( 27 ), or sensitive and durable
nucleic acid detection via collateral cleavage by
the thermostable Cas12g1 ( 18 , 22 ). We antici-
pate that our discovery framework will yield
new CRISPR-Cas variants as genomic and meta-
genomic sequence databases grow, expanding the
understanding of CRISPR biology and the nucleic
acid manipulation toolbox.

REFERENCES AND NOTES

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ACKNOWLEDGMENTS

We thank the entire Arbor Biotechnologies team for support and

comments on this work.Funding:Arbor Biotechnologies is a

privately funded company. K.S.M. and E.V.K. are supported by

the intramural program of the U.S. Department of Health

and Human Services (to the National Library of Medicine).

Author contributions:W.X.Y. and D.A.S., with input from P.H.,

D.R.C., L.E.A., J.M.C., E.K.S., S.S., S.C., and A.J.G., conceived

and designed the study. D.R.C. and D.A.S. designed and

implemented the computational searches, with additional input

from K.S.M. and E.V.K., including phylogenetic analysis and

classification. W.X.Y., D.A.S., P.H., L.E.A., J.C., E.K.S., S.S., S.C.,

and A.J.G. performed all of the experimental work and analyzed

the data. W.X.Y. and D.A.S. wrote the manuscript with input

from E.V.K. and help from all authors.Competing interests:

W.X.Y., P.H., L.E.A., J.M.C., E.K.S., S.S., S.C., A.J.G., D.R.C.,

and D.A.S. are employees and shareholders of Arbor

Biotechnologies, Inc. W.X.Y., D.R.C., and D.A.S. are current or

former officers and D.R.C. is a director of Arbor Biotechnologies.

Arbor Biotechnologies has filed patents related to this work.

Data and materials availability:Alldataareavailableinthe

manuscript or the supplementary material. All reagents are

available to the academic community through Addgene.

Sequencing data are available on the NCBI Sequence Read

Archive under Bioproject ID PRJNA496291.

SUPPLEMENTARY MATERIALS

http://www.sciencemag.org/content/363/6422/88/suppl/DC1

Materials and Methods

Figs. S1 to S19

Tables S1 to S10

References ( 29 – 33 )

14 October 2018; accepted 20 November 2018

Published online 6 December 2018

10.1126/science.aav7271

Yanet al.,Science 363 ,88–91 (2019) 4 January 2019 4of4

Fig. 4. In vivo dsDNA interference by Cas12c.

(A) Evaluation of a minimal active system for Cas12c,

with heatmaps showing strongly depleted CRISPR

arrays from in vivo screening in different Cas12c

system compositions. Gray boxes indicate data not

available. (B) (Top) Distribution of bit scores for

all permutations of 1- to 3-nt motifs within the

target and 15-nt flanking sequences corresponding

to strongly depleted arrays. (Bottom) Web logos

from target-flanking sequences. (C) Overview

of minimal components and interference

mechanisms of Cas12g, -h, -i, and -c. Asterisks

denote putative mechanisms subject to

additional validation.

A B

C
Cas12h,i binary complex

5’ crRNA3’

Cas12h: 870 - 924aa;

Cas12i: 1033 - 1093aa

3’ 5’

ssRNA

ssDNA ssDNA

ssDNA?

5’3’ 3’5’

Cis Trans

DNA RNA

Cleavage:

3’5’ * 5’3’ Substrate:

ssDNA or ssRNA

cleavage

System

components

dsDNA cleavage

Cas12c ternary complex

crRNA

tracrRNA

5’

3’ 5’

3’

Cas12c: 1209 - 1330aa

3’ 5’

*?

Subtype V-C Subtype V-H, I

Cas12g ternary complex

3’

3’ 5’

5’ crRNA

tracrRNA

Cas12g: 720 - 830aa

ssRNA3’ 5’

?

Subtype V-G

Distance to 5’ target end

Bit score^0

1

2 2 2

-6

Bit score

0

4

3

2

1

Cas12c1 Cas12c2 OspCas12c

1nt 2nt 3nt

# nt in motif permutation

(log)

800

<5

Screen

Hits

Cas1

dCas12c

Cas12c (WT)

Noncoding

OspCas12c

Cas12c2

Cas12c1

top strand bottom strand

CRISPR array expression

RESEARCH | REPORT

on January 7, 2019^

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