Yanet al.,Science 363 ,88–91 (2019) 4 January 2019 2of4

Fig. 1. Discovery and screening of type V

CRISPR-Cas diversity.(A) Classification tree

of type V effectors (Cas12 proteins) with the

corresponding CRISPR-Cas loci organization

shown for each branch. Cas12 proteins analyzed

in this work are highlighted in red. (B) Design

of in vivo screen effector and noncoding

plasmids. CRISPR array libraries were

designed with spacers uniquely and uniformly

sampled from both strands of pACYC184 or

E. coliessential genes, then flanked by two

DRs and transcribed by a J23119 promoter.

(C) Workflow schematic of the in vivo

E. coliscreen.

AB

C

RBS + mH6

Effector [Accessory]

LacO

T7 lac

Effector

plasmid

in pET-28a(+)

LacO Concatenated NC sequences

T7 lac

Noncoding

(NC) plasmid

in pACYC184

[Accessory] Effector CRISPR array

Natural

Locus

CRISPR Array

Library

DR DR

J23119 J23119

DR DR

UMI reverse array direction

(spacers targeting pACYC184 and E. coli essential genes)

+

Readout

depleted arrays

mature crRNA

& tracrRNAs

small

RNAseq

output and

input plasmid

surviving PCR & NGS

E. coli

Assembly

transformation components

Effector plasmid

CRISPR array library

+ Noncoding plasmid

or pACYC

Reaction

antibiotic

selection

depletion of E. coli with

active CRISPR systems

E. coli with

CRISPR systems

V-C

V-D

V-A

V-E

V-F

2

V-H

V-U3

V-F

V-U4

V-U2

V-F

V-U1

V-G

V-U5

V-B

V-I

TnpB

Cas12c (C2c3)

Cas12d (CasY)

Cas12a (Cpf1)

Cas12e (CasX)

Cas12h

Cas14b

C2c10

Cas14a

C2c9

C2c8

Cas14c

C2c4

Cas12g

C2c5

Cas12b (C2c1)

Cas12i

Interference Cas effector RuvC-I, II, III Nuc

Adaptation Cas4 Cas1 Cas2

Noncoding CRISPR array tracrRNA

A

E F G H

15% TBE-Urea

cleaved

ssDNA

150

75

50

Cas12g1-WT

Mature-crRNA

tracrRNA

Target ssRNA

Cas12g1-D513A

Collateral ssDNA

37°C 50°C

SYBR-labeled collateral ssDNA IR800 5’ labeled target ssRNA

cleaved

ssRNA

(^150) 15% TBE-Urea
50
80
Cas12g1-WT
Mature-crRNA
tracrRNA
Target ssRNA
Cas12g1-D513A
37°C 50°C
B
D
C
<5 (log) 400
Screen Hits
top strand bottom strand
DNA strand
Spacer target
Cas12g1
CRISPR array expression
E. coli
EG
pACYC
AS
AS
S
S
WT WT
D513AA513D D513AA513D
0 102
0
40
80
nt
min. CRISPR array
Reads Mapped (×10
3 )
mature crRNA
(^00)
6
12
nt
Reads Mapped (×10
3 ) Cas12g1 Native
locus
Noncoding
plasmid
tracrRNA
CRISPR array
1230

top strand spacers^0

6
12
18
24
30

bottom strand spacers

30
6
12
18
24
0
RNA expression (count)
1 (log) 14000
fold depletion
3 (log) 10
ORI Te t R CamR
Cas12g1-WT
Mature-crRNA
tracrRNA
Nontarget ssRNA
Target ssRNA
Collateral ssDNA
15% TBE-Urea
cleaved
ssDNA
SYBR-labeled collateral ssDNA
150
75
50
37°C 50°C
IR800 5’ labeled
target/nontarget ssRNA
IR800 5’ labeled
collateral ssRNA
15%
cleaved TBE-Urea
ssRNA
Cas12g1-WT
Mature-crRNA
tracrRNA
Nontarget ssRNA
Target ssRNA
Collateral ssRNA
150
50
80
150
50
80
Fig. 2. Cas12g displays RNA-activated target cleavage of RNA and
collateral trans-cleavage of RNA and ssDNA.(A) Strongly depleted
CRISPR arrays from in vivo screening of Cas12g1 and its noncoding
plasmid mapped to pACYC184. (B) Heatmap showing strongly depleted
CRISPR arrays (screen hits) to evaluate RuvC and substrate strand
dependencies of Cas12g1 (S, sense; AS, antisense; EG, essential genes).
A513D was cloned from the D513A construct to rescue its activity. Strongly
depleted CRISPR arrays in negative control screens without the effector
were subtracted from this and similar analyses. (CandD) Mature crRNA
(C) and tracrRNA (D) identified from small RNA sequencing of in vivo
screen samples containing Cas12g1 and noncoding plasmid. The
schematic above tracrRNA shows construction of noncoding plasmid
from native locus sequences. (EandF) Target ssRNA activated collateral
ssDNA cleavage at 37°C and 50°C (E) and target and collateral ssRNA
cleavage at 37°C (F). (GandH) Cleavage assays targeting collateral
ssDNA (G) and ssRNA (H) with purified RuvC mutant dCas12g1 D513A.
RESEARCH | REPORT
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