Technology

A Rapid Method for Directed Gene Knockout for

Screening in G0 Zebrafish
Graphical Abstract Authors
Roland S. Wu, Ian I. Lam, Hilary Clay,
Daniel N. Duong, Rahul C. Deo,
Shaun R. Coughlin

Correspondence
shaun.coughlin@ucsf.edu

In Brief
Wu et al. describe a pipeline for CRISPR/
Cas9 genetic screening with optimized,
redundant gene targeting to produce
penetrant gene disruption in zebrafish.
The authors evaluate this system on
several genes and apply this strategy to
50 empirically identified zebrafish
cardiomyocyte transcription factors,
uncovering a role for zbtb16a in cardiac
development.

Highlights
d Redundant targeting of single genes generates nearly
complete gene disruption

d Effects are early and durable, complementing morpholino-

based approaches

d A genome-scale guide set provides a resource to aid

automated experimental design

d A transcription factor screen uncovers a role for zbtb16a in

heart development

Wu et al., 2018, Developmental Cell 46, 112–125

July 2, 2018 ª 2018 Elsevier Inc.
https://doi.org/10.1016/j.devcel.2018.06.003

A Rapid Method for Directed Gene Knockout
for Screening in G0 Zebrafish
Roland S. Wu,1,2 Ian I. Lam,1 Hilary Clay,1 Daniel N. Duong,1 Rahul C. Deo,1,2 and Shaun R. Coughlin1,2,3,4,*
1Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94158, USA
2Division of Cardiology, Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA
3Present address: Novartis Institutes for Biomedical Research, 22 Windsor Street, 4D-340, Cambridge, MA 02139, USA
4Lead Contact
*Correspondence: shaun.coughlin@ucsf.edu
https://doi.org/10.1016/j.devcel.2018.06.003
SUMMARY
Zebrafish is a powerful model for forward genetics.
Reverse genetic approaches are limited by the
time required to generate stable mutant lines.
We describe a system for gene knockout that
consistently produces null phenotypes in G0 zebra-
fish. Yolk injection of sets of four CRISPR/Cas9
ribonucleoprotein complexes redundantly targeting
a single gene recapitulated germline-transmitted
knockout phenotypes in >90% of G0 embryos for
each of 8 test genes. Early embryonic (6 hpf) and sta-
ble adult phenotypes were produced. Simultaneous
multi-gene knockout was feasible but associated
with toxicity in some cases. To facilitate use, we
generated a lookup table of four-guide sets for
21,386 zebrafish genes and validated several.
Using this resource, we targeted 50 cardiomyocyte
transcriptional regulators and uncovered a role of
zbtb16a in cardiac development. This system pro-
vides a platform for rapid screening of genes of inter-
est in development, physiology, and disease models
in zebrafish.
INTRODUCTION
Genetic screens in zebrafish are a powerful tool for probing
mechanisms governing vertebrate development, tissue biology,
physiology, and disease. Large clutch sizes, external develop-
ment, and optical transparency are intrinsic advantages (Strei-
singer et al., 1981). Advances in light microscopy and availability
of lines with fluorescent cell-lineage tags and signaling reporters
have enhanced the power of this system. Targeted gene editing
with techniques such as the clustered regularly interspaced
palindromic repeat (CRISPR)/Cas9 system has revolutionized
reverse genetic manipulation of zebrafish and other model or-
ganisms (Ablain et al., 2015; Auer et al., 2014; Burger et al.,
2016; Hoshijima et al., 2016; Hwang et al., 2013; Jao et al.,
2013; Moreno-Mateos et al., 2015; Shah et al., 2015; Varshney
et al., 2015), and reverse genetic screens that target specific
gene sets of interest can now be considered as a complement
to forward screening approaches and morpholinos. With stan-
112 Developmental Cell 46, 112–125, July 2, 2018 ª 2018 Elsevier Inc.
Developmental Cell
Technology
dard application of CRISPR/Cas9 technology in zebrafish, nulls
are often obtained and analyzed at the F2 generation, limiting
throughput. Progress toward methods for G0 knockouts has
been made (Burger et al., 2016; Moreno-Mateos et al., 2015;
Shah et al., 2015), but consistently penetrant biallelic gene
disruption in the G0 generation will be necessary to interrogate
genes of unknown function at moderate throughput.
We report that yolk injection of sets of four CRISPR/Cas9 ribo-
nucleoprotein complexes (Cas9 RNP) that redundantly target
a single gene reproduced homozygous null phenotypes with
penetrance approaching that of standard germline-transmitted
knockouts. We explore the toxicity of this system and its use
for simultaneous multi-gene knockouts, and we provide a
genome-scale guide set lookup table to aid the design of reverse
genetic screens in zebrafish. We demonstrate the utility of this
system by screening 50 transcription factors expressed in zebra-
fish cardiomyocytes to uncover a role for zbtb16a in cardiac
development.
DESIGN
We aimed to exploit CRISPR/Cas9 technology and the remark-
able fecundity and other features of zebrafish to enable rapid,
moderate-throughput reverse genetic screens in this vertebrate
system (Howe et al., 2013). We sought a system that delivers
rapid, consistent biallelic gene disruption such that gene
knockout could be assumed to have occurred in nearly all G0
embryos to allow meaningful preliminary screening of candidate
genes without genotyping. The criterion for success in testing
was reproduction of known null phenotypes in >90% of G0 em-
bryos. Additional desired features included ease of guide design
and CRISPR/Cas9 RNP production and compatibility with deliv-
ery by yolk sac injection to enhance throughput.
RESULTS
Simultaneous Use of Multiple Guides per Gene in Cas9
RNP Complexes Increases the Efficiency of Functional
Gene Disruption
Currently available algorithms for guide design produce guides
with variable targeting efficiency. Furthermore, even with 100%
efficiency of targeting a coding sequence, non-homologous
repair of an individual targeting event may or may not ablate
function of the encoded protein. An oversimplified but illustrative
model in which (1) a frameshift mutation occurs in two-thirds of
 (legend on next page)

Developmental Cell 46, 112–125, July 2, 2018 113

repairs, (2) a frameshift disrupts protein function, and (3) a repair
without a frameshift does not disrupt function predicts that tar-
geting a single site in a coding sequence once would yield bial-
lelic loss-of-function alleles only 44% of the time. By contrast,
targeting a coding sequence at two independent sites would
yield biallelic loss of function 79% of the time, targeting thrice
93%, and targeting four times 98%. To increase the probability
of including a guide(s) with efficient gene targeting and of obtain-
ing loss of protein function, we designed sets of four guides to
redundantly target coding sequences.
To first test multi-guide targeting, we chose genes with well-
characterized, easily scored homozygous null phenotypes but
lacking heterozygous phenotypes so that recapitulation of null
phenotypes in G0 embryos could be used as a functional readout
of biallelic disruption. These genes and the conventions used for
scoring phenotypes as Strong (recapitulating homozygous null)
or Intermediate (partial phenotypes) are described in Figure S1.
In addition, we also recorded instances of common dysmorphic
phenotypes, including tail and truncal morphologic abnormal-
ities, which were not specific for guide sequence and distinct
from the known homozygous null phenotype of the targeted
gene (Figure S1F). Given the reliability of CRISPR/Cas9 delivery
by cytoplasmic injection at the one-cell stage, this route was
used for initial studies of gene-targeting efficiency. Subsequent
studies routinely used yolk injections.
We first examined s1pr2 (miles apart, mil). s1pr2 nulls display
failed migration of the heart fields to the midline and cardia bifida
(Kupperman et al., 2000). s1pr2 heterozygotes have grossly
normal heart development. Cas9 RNPs were first cytoplasm-in-
jected carrying one single-guide RNA (sgRNA) targeting s1pr2.
Three of four single guides produced failed midline migration
of the heart fields in nearly all G0 embryos when analyzed at
1 day post fertilization (dpf), but at 4 dpf, cardia bifida was pre-
sent in only a minority of these embryos with most showing an
intermediate phenotype (Figure 1A). By contrast, s1pr2!/! em-
bryos derived from heterozygous crosses of stable mutant lines
have fully penetrant cardia bifida at 4 dpf (Kupperman et al.,
2000). Thus, mosaicism for s1pr2 knockout in G0 embryos in-
jected with single-guide Cas9 RNPs may have allowed compen-
sation by 4 dpf. The fourth single guide produced no phenotype
in the majority of embryos and an intermediate phenotype in the
remainder at both 1 and 4 dpf (Figure 1A), highlighting the unpre-
dictability of guide efficiency. In contrast to all of the single-guide
injections, injection of the same total quantity of Cas9 RNP car-
rying a mixture of the same four guides produced cardia bifida
with nearly complete penetrance at both 1 and 4 dpf (Figures
1A, 1B, and S1A).
To more directly examine the effects of redundant targeting on
the s1pr2 gene, we cloned and sequenced an amplicon from DNA
prepared from a clutch of embryos collected at 1 dpf after injection
with the four-guide Cas9 RNP mix at the one-cell stage. None
of 30 amplicons sequenced were wild-type (Figures 1C and 1D).
Eighty-three percent of alleles had a frameshift leading to a pre-
mature stop codon. The remaining 17% had insertions or dele-
tions coding for 12 or more amino acids (Figures 1C and 1D). All
alleles had been targeted at two or more sites. Guide number 1,
which was effective in generating the miles apart phenotype, tar-
geted 26/30 alleles, while ineffective guide 4 targeted only 10/30
(Figures 1A and 1C). Deletions spanning from one guide site to
another occurred in 18/30 alleles.
In addition to scoring for the specific s1pr2 cardia bifida pheno-
type, we scored the G0 embryos for a dysmorphic appearance
associated with toxic or off-target effects (Figure 1A). Single-
guide injections produced dysmorphic embryos at 1 and 4 dpf
at a rate of 0%–2% and 7%–10%, respectively. Four-guide injec-
tions produced dysmorphic embryos at 1 and 4 dpf at 7% and
17%, respectively. The total concentration of Cas9 RNP injected
was the same for the single- and four-guide injections. These and
subsequent experiments (see below) suggest that increasing
guide number increased toxicity. However, the absolute rate of
toxicity––17% or less in two replicate experiments in which the
null phenotype was reproduced in >90% of G0 embryos––would
be acceptable for screening applications.
Taken together, the results outlined above suggested that use
of a four-guide set to redundantly target s1pr2 produced more
effective functional disruption of the s1pr2 gene than use of sin-
gle guides, likely due to a combination of increased frequency of
gene targeting and more effective gene disruption by multiple
targeting events per allele. Recapitulation of the s1pr2 null
phenotype in nearly all G0 embryos at a level of toxicity accept-
able in most screening applications encouraged further study.
We next probed the effect of single- and multiple-guide
injections targeting two additional test genes with easily scored
homozygous null phenotypes: tnnt2a (silent heart, sih) and
slc24a5 (golden, gol) (Figures 2A and S2A). Homozygous null
tnnt2a mutants have no heartbeat due to failed sarcomeric
development (Sehnert et al., 2002). Heterozygotes lack a pheno-
type. Three of the four single guides targeting tnnt2a produced
the full null phenotype in less than 20% of embryos and a partial
phenotype in the remainder (Video S1 and Figure S2A). One of
the four guides produced the full null phenotype in "75% of
embryos. The same four guides in combination produced the
full null phenotype in almost all embryos (Figure S2A). Few dys-
morphic embryos were detected (Figure S2A).

Figure 1. G0 Mutation of s1pr2 by Single- or Four-Guide Cas9 RNP

(A) Embryos were cell-injected at the one-cell stage with single-guide or four-guide Cas9 RNP targeting s1pr2 and scored for phenotypes at 1 day post fertilization
(dpf) and 4 dpf. Embryos were first scored as dysmorphic or not (lower panel). Non-dysmorphic embryos were then scored for the miles apart cardia bifida
phenotype as defined in Figure S1. The percentage with strong (blue), intermediate (red), or no (green; wild-type [WT]) phenotype is indicated. The number of non-
dysmorphic embryos scored is shown at bottom. ****p < 0.0001; ns, not significant; Fisher’s exact test for Strong versus other phenotype. None of 76 embryos
injected with control sgRNA exhibited cardia bifida; 5% were dysmorphic. The figure represents pooled data from two independent experiments.
(B) Representative Tg(myl7:GFP) G0 embryo with a Strong cardia bifida phenotype (bottom) after injection of a four-guide Cas9 RNP mix. A control clutchmate is
shown at the top. Tg(myl7:GFP) labels the heart fields (green). Scale bar, 250 mm.
(C and D) Embryos injected with a four-guide Cas9 RNP mix targeting s1pr2 were collected at 1 dpf. DNA was prepared from two pools of 3 embryos (two
separate experiments); s1pr2 amplicons were cloned and 30 were sequenced. (C) Wild-type (top line) and ten representative mutant sequences from injected
embryos. Arrows denote predicted site of double-stranded breaks corresponding to each of the four guides used in (A). Sequences predicted to hybridize with
CRISPR sgRNA spacers are highlighted with alternating green and yellow shading. (D) Summary of characteristics of 30 s1pr2 alleles analyzed.

114 Developmental Cell 46, 112–125, July 2, 2018


Homozygous null mutants of slc24a5 lack pigmentation of
retinal pigment epithelium and melanophores in skin at 2 dpf
(Lamason et al., 2005). Heterozygotes retain pigmentation. The
slc24a5 phenotype is particularly sensitive for detecting mosai-
cism as indicated by pigmented cell patches in a non-pigmented
surround. We focused on the retinal pigment epithelium for the
purposes of scoring (Figure S1B). One of four individual guides
targeting slc24a5 produced complete depigmentation in about
50% of the embryos at 2 dpf (Figure 2A). The remainder pro-
duced complete depigmentation in 15% or less. By contrast,
four-guide injection produced complete depigmentation in
"80% of G0 embryos and nearly complete depigmentation in
the rest (Figure 2A). Few dysmorphic embryos were detected
under any condition (Figure 2A). We next compared single-,
double-, triple-, and four-guide injections. The two- and
three-guide combinations containing the most effective single
guide (#4) were most effective (Figure 2B). The four-guide set
was substantially more efficacious than any of the two- and
three-guide combinations, and the rate of dysmorphic embryos
was not higher than that seen with the three-guide sets and one
of the two-guide sets tested (Figure 2B).
The results described above highlighted the variability of sin-
gle guides for producing efficient biallelic gene disruption in G0
zebrafish embryos and suggested that injection of Cas9 RNP
with four-guide sets to redundantly target a single gene pro-
duced biallelic disruption with little mosaicism and efficient
reproduction of known homozygous null phenotypes for the
three genes examined. We went on to test the efficiency of
four-guide Cas9 RNP mixtures for three additional genes that
Figure 2. Recapitulation of Null Phenotypes
in G0 Embryos by Four-Guide Targeting of
Five Additional Test Genes
Embryos were cell-injected (A–C) or yolk-injected
(D) at the one-cell stage with single- or four-guide
Cas9 RNP targeting the indicated genes and
scored for phenotypes as described in Figure S1.
(A) Penetrance of gene-specific phenotype and
toxicity (as percent dysmorphic) in embryos after
single and four-guide Cas9 RNP injections target-
ing slc24a5.
(B) Penetrance of gene-specific phenotype and
toxicity (as percent dysmorphic) in embryos after
the indicated single-, double-, triple-, and four-
guide Cas9 RNP injections targeting slc24a5 using
the individual guides in (A). Scoring was performed
blinded to treatment.
(C and D) Penetrance of specific phenotypes and
toxicity in embryos after cell (C) or yolk (D) injection
of a four-guide mix of Cas9 RNP targeting the
indicated genes.
In all panels, the number of embryos scored for
the null phenotype associated with the indicated
gene is noted below each column. **p < 0.01,
****p < 0.0001; ns, not significant; Fisher’s exact
test for Strong phenotype. Each experiment was a
pool of at least two independent experiments.
produce easily scored phenotypes in ho-
mozygous nulls and not in heterozygotes:
tbx16 (spadetail, spt), which is required for
non-notochordal trunk mesoderm formation; tbx5a (heartstrings,
hst), which is required for fin development and proper cardiac
looping; and npas4l (cloche, clo), which is required for most he-
matopoietic and endothelial development (Garrity et al., 2002;
Griffin et al., 1998; Reischauer et al., 2016) (Figures S1C–S1E).
Injection of four-guide sets for each of these genes produced
the cognate homozygous null phenotypes in "100% of G0 em-
bryos (Figures S1C–S1E and 2C). No specific phenotypes were
induced by a control set of four scrambled guides, and dysmor-
phic embryos were detected at a rate of <15% for all conditions
examined (Figure 2C).
Optimizing sgRNA Production, Dose, and Delivery Route
of Cas9 RNP
Toward streamlining workflow, we adapted a previously
described cloning-free method to generate sgRNA templates
(Varshney et al., 2015). A single template elongation reaction us-
ing four primers, each containing a different ‘‘spacer’’ targeting
sequence, was used to generate a mixture of four-guide tem-
plates that was in turn used in a single in vitro transcription reac-
tion to produce a mixture of the four desired sgRNAs. Cas9 RNP
carrying a slc24a5 four-guide mix made in this manner was as
effective at producing the slc24a5 null phenotype in G0 embryos
as a Cas9 RNP set made by mixing the same guides produced
separately from individual templates (Figure S2B). Dysmorphic
embryos were present at <8% in both cases. To assess whether
each guide is being effectively synthesized by this pooled in vitro
transcription, we performed a single in vitro transcription reac-
tion with guides targeting four distinct genes. Injection of this
Developmental Cell 46, 112–125, July 2, 2018 115
guide mix produced the four expected phenotypes, confirming
effective transcription of each guide (Figure S2C). This technique
reduced cost and increased throughput for synthesizing four-
guide sets.
We next examined the effect of varying the dose of the four-
guide slc24a5 sgRNA mixture on effectiveness and toxicity (Fig-
ure S2D). Cas9 protein was mixed with varying amounts of the
sgRNA guide mix to generate preassembled Cas9 RNP such
that injections would contain 800 pg of Cas9 protein (Burger
et al., 2016) and 100, 250, 1,000 or 2,500 pg of sgRNA. The
rate of production of the slc24a5 depigmentation phenotype
increased from 78% at 100 pg to 98% at 1,000 and 2,500 pg
of sgRNA (Figure S2D). Dysmorphic embryos were present at
13% or less at 100, 250, and 1,000 pg of sgRNA but increased
to 47% at 2,500 pg (Figure S2D). Accordingly, we standardized
remaining injections to 1,000 pg of multi-guide sgRNA preas-
sembled with 800 pg of Cas9 protein, a 6:1 molar ratio of sgRNA
to Cas9 protein. The increase in effectiveness of Cas9 RNP pre-
pared with a sgRNA/Cas9 molar ratio above 1:1 may be due to
incomplete incorporation of sgRNA into RNP complexes or other
causes.
In the experiments described above, Cas9 RNPs were injected
into the cytoplasm of the fertilized egg at the one-cell stage. Yolk
injection affords about four times the throughput of cytoplasmic
cell injections in our hands. To test the possibility that Cas9 RNP,
which has a compact globular structure (Nishimasu et al., 2014),
might be efficiently transferred from the yolk to the cell, we pro-
duced a Cas9-GFP fusion protein (Burger et al., 2016) and in-
jected preassembled Cas9-GFP RNPs into the yolk at the one-
cell stage. By 2 hr post fertilization (hpf), GFP fluorescence had
translocated almost completely to the cell(s) with little residual
fluorescence in the yolk (Figure S2E). In addition, the fluores-
cence in cells had the expected nuclear localization (Figure S2E).
We next compared the efficacy of cell- and yolk-injected Cas9
RNP carrying four-guide sets for each of five test genes (Figures
2C and 2D). Yolk injections with Cas9 RNP targeting slc24a5
produced full depigmentation in 93% in G0 embryos (Figures
2D and S2F); tbx5a produced the expected bilateral pectoral
fin agenesis in 96% of G0 embryos and partial phenotypes in
the remaining 4% (Figures 2D and S1C); tbx16 produced the
expected spadetail phenotype in all G0 embryos (Figures 2D
and S1D); tnnt2a produced silent heart in all G0 embryos (Fig-
ure 2D and Video S1); and npas4l produced the cloche cardiac
phenotype and severely impaired endothelial and hematopoietic
development in nearly all G0 embryos (Figures 2D and S1E). Yolk
injection of Cas9 RNP with a set of four scrambled guides
produced no specific phenotype. Dysmorphic embryos were
present at 1%–11% for all guide sets tested (Figure 2D). Thus,
yolk injections of four-guide Cas9 RNP reproduced null pheno-
types for five test genes in >90% of G0 embryos––a rate not sub-
stantially less than that produced by cell injection (Figure 2C)––at
acceptable levels of toxicity.
Onset and Durability of G0 Phenotypes
To probe the temporal limit of onset of G0 phenotypes, we in-
jected four-guide Cas9 RNP targeting sox32 (casanova, cas),
which is necessary for expression of the early endoderm marker
sox17 and for endoderm development (Kikuchi et al., 2001).
When one-cell-stage embryos from a cross of hemizygous
116 Developmental Cell 46, 112–125, July 2, 2018
Tg(sox17:GFP) zebrafish were injected with Cas9 RNP with a
scrambled four-guide set, endodermal GFP expression was de-
tected in "75% of embryos at 10 hpf, the expected Mendelian
rate (Figures 3A and 3B). By contrast, when one-cell-stage em-
bryos were injected with four-guide Cas9 RNP targeting sox32,
no sox17:GFP reporter expression was detected in embryos
at 10 hpf (Figures 3A and 3B). qRT-PCR confirmed ablation of
sox17 mRNA expression in 10 hpf wild-type embryos injected
with sox32 Cas9 RNP (Figure 3C). Observation of sox32 G0 em-
bryos at 24 hpf and later revealed the expected morphologic
phenotypes (Figure S3A). Additional qRT-PCR of embryos
collected at various times after either cell or yolk injection of
sox32 Cas9 RNP revealed that sox17 mRNA expression was ab-
lated as early as 6 hpf (Figure 3D). These results imply that injec-
tion of four-guide set Cas9 RNP can lead to effective biallelic
gene disruption within a few hours of injection into the yolk or
cell of the embryo and may be useful in screens seeking early
developmental phenotypes. Injection of sox32 Cas9 RNP-tar-
geted embryos with sox32 mRNA increased sox17 expression
in control embryos and reversed ablation of sox17 expression
in sox32-targeted embryos (Figure S3B), suggesting that
mRNA rescue might be used as a follow-up step to assess the
specificity of new phenotypes found using G0 four-guide target-
ing in screens.
Single-guide Cas9 RNP injections targeting s1pr2 could
produce phenotypes at 1 dpf that were attenuated at 4 dpf,
perhaps due to mosaicism, while four-guide Cas9 RNP injection
produced a phenocopy of the germline nulls at 1 and 4 dpf
(Figure 1A). To further evaluate the durability of the effects of
four-guide Cas9 RNP injections in G0 zebrafish, we targeted
tyrosinase (tyr), which leads to the sandy (sdy) phenotype
of pigmentation loss and impaired vision (Page-McCaw
et al., 2004). Injection of four-guide Cas9 RNP targeting tyr pro-
duced the sandy phenotype in 32/32 G0 embryos. Adult survi-
vors showed persistence of the sandy pigmentation defect
at 2 months of age (Figure 3E).
Comparison of G0 Genetic Disruption to Morpholino
Knockdown
Although it often suffers from off-target effects (Kok et al., 2015),
knockdown of gene expression by injection of morpholino oligo-
mers (MOs) is an established approach for quickly assessing the
effect of gene loss of function and an obvious alternative to injec-
tion of Cas9 RNP for G0 analysis studies (Stainier et al., 2017).
The durable effects of Cas9 RNP offers a distinction from MOs,
as the activity of MOs usually does not persist beyond 72 hpf
due to dilution and clearance of the oligomer. How Cas9 RNP
toxicity compares with MOs will likely depend on the specific
sgRNAs and MOs used, and Cas9 RNP may be superior in
some cases. We compared the effectiveness and toxicity of
four-guide Cas9 RNP targeting s1pr2 with a well-characterized
s1pr2 MO (Fukui et al., 2014; Kai et al., 2008) that in our experi-
ence has a narrow efficacy to toxicity window. Injection of the
s1pr2 four-guide Cas9 RNP phenocopied the full miles apart car-
dia bifida phenotype in 98% of embryos as defined in Figure S1B;
6% of embryos were dysmorphic (Figure S3C). Injection of the
s1pr2 MO at 8 ng/embryo produced cardia bifida in only 52%
of embryos and the distance between the cardiac fields was
generally closer than that seen in the full miles apart phenotype
Figure 3. Onset and Durability of Phenotypes in G0 Embryos and Adults
Embryos were cell-injected at the one-cell stage with four-guide Cas9 RNP targeting casanova (sox32) (A–D) or tyr (E) or with scrambled guides as indicated.
(A–D) Impaired endoderm marker expression in G0 embryos after four-guide targeting of sox32. (A and B) Fish hemizygous for the endoderm marker
Tg(sox17:GFP) were intercrossed and the resulting embryos injected with the indicated four-guide Cas9 RNP and imaged at 10 hpf. (A) Representative images.
Scale bar, 250 mm. (B) Percentage of embryos expressing GFP (green) or not (blue). Dotted line indicates 75%, the percentage of fluorescent embryos expected
from the hemizygous transgenic cross. ****p < 0.0001; ns, not significant; Fisher’s exact test for presence or absence of GFP fluorescence. The number of
embryos analyzed is shown in each column. (C) Embryos injected with sox32 four-guide Cas9 RNP were collected at 10 hpf and sox17 expression was measured
by qPCR. Expression of sox17 mRNA in scrambled control-targeted and sox32-targeted embryos relative to uninjected controls is shown. Graph represents
mean ± SEM (n = 4). ***p < 0.001, unpaired two-tailed t test. (D) sox17 expression in embryos cell-injected (black) or yolk-injected (gray) with scrambled (scr)
or sox32 four-guide Cas9 RNP at the 1-cell stage and collected at 6 or 8 hpf. Expression is normalized to that in uninjected embryos. ****p < 0.0001; ns,
not significant by one-way ANOVA with Sidak’s multiple comparisons test compared with equivalent scr experiment in injection method and collection time.
(E) Phenotype durability. Embryos were injected with a four-guide Cas9 RNP mix targeting tyr and raised to adulthood. A representative 2-month-old G0 tyr-
targeted zebrafish is on the right; control is on the left. Scale bar, 2 mm. Note lack of dark stripes characteristic of tyr knockouts.

(Figures S3C and S3D); dysmorphic embryos were present at
4%. Increasing the dose of s1pr2 MO did not increase its effec-
tiveness but did increase toxicity (Figure S3B).
To evaluate phenotype durability, we compared Cas9 RNP
with morpholino knockdown of the tyr pigmentation allele. Dual
MOs targeting tyr mRNA were used as described by Pickart
et al. (2004); these MOs and four-guide Cas9 RNP were similarly
effective for suppressing retinal pigment epithelial pigmentation
at 3 dpf (Figure S3E). However, by 8 dpf, eye pigmentation
had substantially recovered in the MO group but remained
decreased in the Cas9 RNP group (Figure S3E). Our results sug-
gest that four-guide Cas9 RNP has the expected durability
advantage over MOs and can provide more effective gene loss
of function and/or less toxicity in at least some cases.
Simultaneous Disruption of Multiple Genes Using
Four-Guide Set Cas9 RNP Complexes
Redundant gene function can prevent phenotypes with single
gene knockout, and the teleost-specific genome duplication
may exacerbate this phenomenon (Howe et al., 2013). To deter-
mine whether four-guide Cas9 RNP injection can be used to
produce effective G0 knockout of two genes, we again targeted
genes with the easily scored phenotypes described in Figure S1.
Injection of Cas9 RNP generated with four-guide sets for
both tnnt2a and tbx16 produced their cognate silent heart and
spadetail phenotypes in >98% of G0 embryos without detect-
able toxicity (Figure 4A). Similarly, injection of Cas9 RNP gener-
ated with four-guide sets for both slc24a5 and tbx5a produced
their cognate depigmentation and bilateral pectoral fin agenesis
phenotypes in >91% of G0 embryos; 11% of embryos were dys-
morphic (Figure 4B). Thus, simultaneous dual-gene knockouts
can be generated with acceptable toxicity using combined guide
sets in at least some cases.
Toward probing the limits of this system, we attempted triple
and quadruple genetic disruption. Injection of Cas9 RNP gener-
ated with 3 four-guide sets targeting slc24a5, tbx16, and tnnt2a
or 4 four-guide sets targeting slc24a5, tbx16, tnnt2a, and
npas4l produced the multiple gene knockout phenotypes in G0
embryos, but dysmorphic embryos were frequently observed
(Figure S4A). To further assess the contribution of guide
mix complexity to toxicity, we targeted a single gene, tyr, with
increasing numbers of guides. Four different four-guide sets tar-
geting tyr produced the expected depigmentation phenotype in
virtually all G0 embryos and dysmorphic embryos at 6%–14%.
Injection of the same total quantity of Cas9 RNP generated with
combinations of these guide sets such that 8, 12, or 16 guides
were included again led to the expected depigmentation
phenotype in nearly all embryos but produced dysmorphic em-
bryos at rates of 40%–50% (Figure S4B). These data suggest
that increasing the diversity of the guide set may be associated

Developmental Cell 46, 112–125, July 2, 2018 117


Figure 4. Dual-Gene Disruption Using Four-Guide Set Cas9 RNP
(A) Penetrance of the indicated specific phenotypes and dysmorphic pheno-
type at 1 dpf in embryos cell-injected with Cas9 RNP carrying two four-guide
sets, one for tbx16 and one for tnnt2a.
(B) Penetrance of the indicated specific phenotypes and dysmorphic pheno-
type at 2 dpf in embryos cell-injected with Cas9 RNP carrying two four-guide
sets, one for tbx5a and one for slc24a5. The number of embryos analyzed is
shown at top. Guide sets were mixed and embryos were injected with the
same total amount of sgRNA and Cas9 protein as used single-gene targeting.
Data from two independent experiments were pooled.
with increased toxicity, perhaps due to additive off-target effects.
Toxicity appears to depend on the specific guides used, as
8-guide mixtures targeting tnnt2a/tbx16 or slc24a5/tbx5a pro-
duced little toxicity (Figures 4A and 4B) but an 8-guide mixture
targeting tyr produced significant toxicity (Figure S4B).
Genome-Scale Design of Four-Guide Sets
To facilitate experimental design, we sought to design four-guide
sets targeting all annotated coding sequences (CDS) in the
zebrafish genome. For each guide set, we parsed coding se-
quences into five equal segments and attempted to target
guides to each of the first four (Figures 5A and 5B). We then elim-
inated guides with multiple alignments in the genome or runs of
guanine nucleotides that could interfere with primer synthesis.
We ranked the guides according to stringent off-target predic-
tions, the degree of 50 modification necessary to accommodate
118 Developmental Cell 46, 112–125, July 2, 2018
T7-mediated in vitro transcription, and the predicted minimum
free energy of folded spacer structures (Horlbeck et al., 2016;
Lorenz et al., 2011; Moreno-Mateos et al., 2015).
Our algorithm detected 25,638 genes (44,487 transcripts) with
coding sequences. Four-guide sets satisfying our criteria were
identified for 21,386 genes (38,975 transcripts) (Figure 5C) and
are provided in Table S2. To assess the efficacy of guide sets
from this lookup table, we tested four such guide sets. Two
were new guide sets targeting tnnt2a and tbx5a. These produced
the expected silent heart and defective pectoral fin phenotypes,
respectively, in >97% of G0 embryos (Figure 5D). We also tested
four-guide sets for hand2 (hands off, han) and spns2 (ko157).
Injection of Cas9 RNP generated with these guide sets produced
the expected null phenotype of cardia bifida (Kawahara et al.,
2009; Yelon et al., 2000) in >92% of G0 embryos (Figure 5D).
These 4 four-guide sets produced dysmorphic embryos at a
rate of 2%–14%.
Sequencing of Alleles Generated by Multi-guide
Cas9 RNP
In most cases, the targeting strategy proposed in Figures 5A
and 5B would lead to longer DNA segments between sgRNA
sites than those employed in our initial characterization of
four-guide targeting of the s1pr2 gene (Figure 1). To explore
the effects of using the targeting strategy in Figure 5 and the re-
sulting guide sets (Table S2), we examined two additional genes
in detail. First, we yolk-injected embryos with a four-guide set
Cas9 RNP targeting the hand2 gene, prepared DNA at 2 dpf,
PCR-amplified the hand2 gene, and generated >40 TA clones
for hand2 alleles from each of three targeted embryos. Among
139 clones sequenced, 89 unique sequences were obtained.
Only 2% were wild-type (Figures 6A and 6B). The remainder
encoded truncated proteins with an average predicted length
of 38 (range 24–58) amino acids; the full-length protein contains
205 amino acids (Figure 6B). Individually all guides were
effective, with each targeted site disrupted in >86% of alleles
sequenced (Figure 6B). There was a high rate of site-spanning
deletions in one embryo (70% of alleles) but not in the other
two embryos (<10% of alleles). No deletions between the sites
targeted by guides 1 and 4, the most 50 and 30 sites, respec-
tively, were detected. Their sites are separated by 455 bp.
Deletions between guides 2 and 4, which are separated by
321 bp, were detected in 12% of alleles in one embryo but
not in the other two embryos.
We next sequenced a second, larger gene at higher
sequencing depth to obtain a more robust catalog of alleles
generated by this strategy. Commonly used, short-read
sequencing platforms are unsuited for uninterrupted sequencing
of large amplicons. Thus, we chose to utilize a long-read
sequencing platform (Pacific Biosciences) to sequence DNA
from embryos cell-injected with a four-guide Cas9 RNP targeting
sox32. The amplicon was 1,375 bp, which was within the length
constraints of the system, and included the coding sequence
shared by two exons and an intron. Multiplexed sequencing of
six embryos generated 8,480 reads representing 3,401 unique
alleles. The most common protein and DNA sequences found
in one representative embryo, and cumulative data for all six em-
bryos are shown in Figures 6C, 6D, and S6A. The majority of mu-
tations were localized to individual guide sites, suggesting that

guides usually acted independently (Figure S5A). Different
guides acted with variable effectiveness (Figure 6E). Mutations
were found at sites corresponding to guides 2 and 4 in >80%
of alleles in each of the six embryos, while mutations were found
at sites corresponding to guides 1 and 3 in <20% of alleles in
each of the six embryos. Site-spanning deletions occurred
in only 5.7% of alleles overall (Figure S5A). More complex muta-
tions such as duplications and large inversions occurred in <1%
of alleles (Figure S5A).
The frequency of site-spanning mutations decreased with
increasing distance between guide sites (Figure 6F). Deletion fre-
quency between more adjacent guide sites 1–2 and 3–4 was
3.2% and 2.1%, respectively. Deletion frequency between
more distant guide sites 2–3, 1–3, 2–4, and 1–4 was 0.4%,
0.16%, 0.16%, and 0.03%, respectively (Figure 6F). The lower
deletion frequency between ‘‘distant’’ sites 2–4 compared with
that between proximate sites 1–2 and 3–4 is notable given the
lower individual guide-targeting efficiency for guides 1 and 3
compared with 2 and 4 (Figure 6E).
In contrast to the hand2 guide set, the sox32 guide set
included two individual guides with <20% efficacy (Figures 6E
and S5A). Despite this, the sox32 guide set produced embryos
in which only 3% of alleles were wild-type and 94% (range of
90%–99% over the six embryos) encoded truncated proteins
averaging 152 amino acids in length; the wild-type protein con-
tains 307 amino acids (Figures 6C and S5A). These data illustrate
the variability of guide efficacy and the ability of a multi-guide tar-
geting strategy to overcome this phenomenon.
In one of six embryos, all sequenced alleles shared a splice
site mutation between sgRNA sites 2 and 3 (Figure 6D). We found
this surprising, as we would expect to find at least one more
mutated or wild-type allele at this site in a diploid zebrafish. It
is possible that a primer site was missing, perhaps due to a dele-
tion, translocation, or polymorphism. Alternatively, there may
have been a biological cause such as early mutation followed
by homology-directed repair events using the mutated allele as
a template.
Figure 5. Genome-Scale Design of Four-
Guide Sets for G0 Gene Disruption
(A and B) Outline of targeting strategy. Predicted
coding sequences (A) were divided into five equal
sections and sgRNA guides (arrows) were de-
signed to target each of the first four without regard
to whether they might map to the same or different
exons (B).
(C) Summary of genome-scale design results. For
the 44,487 predicted coding sequence-containing
transcripts annotated in the zebrafish genome,
38,975 four-guide sets with the above-described
distribution that met the criteria listed in STAR
Methods could be designed covering 21,386 of the
25,638 annotated genes.
(D) Testing of additional guide sets generated by
this method and drawn from Table S2. Embryos
were yolk-injected with four-guide Cas9 RNP for
the indicated gene. Percentage of embryos with
the gene-specific null phenotype and percent
dysmorphic is shown. The number of non-dys-
morphic embryos analyzed for gene-specific phe-
notypes is indicated below each column. Data
were pooled from two independent experiments.
One hundred and four reads revealed an identical insertion
mutation at sgRNA site 2 and the site 2–3 splice site mutation.
Examination of these sequences at the other guide sites re-
vealed that these 104 reads represented 36 unique sequences
(Figure S5B). Among these sequences, several carried identical
mutations at sgRNA site 4 but distinct or no mutations at sgRNA
site 1, demonstrating a diverging but related family of alleles (Fig-
ure S5B). These data suggest that alleles can undergo multiple
cycles of double-stranded break and repair segregated by cell
division, leading to sequential accumulation of mutations and a
heterogeneous mixture of related complex alleles. These data
also suggest the multiple targeting of single genes seen in our
studies may reflect single events occurring at distinct times.
This phenomenon may contribute to the relatively low frequency
of site-spanning deletions, which are presumed to require simul-
taneous Cas9-mediated double-stranded breaks.
A Screen of Cardiomyocyte Transcription Factors
Reveals a Role for zbtb16a in Cardiac Development
We used the guide set lookup table (Table S2) to design a model
reverse genetic screen for transcription factors that might partic-
ipate in cardiac development because transcription factor genes
known to cause defects in cardiac development should be redis-
covered and provide a robust set of positive controls (Staudt
and Stainier, 2012). We first generated a list of genes enriched
in zebrafish cardiomyocytes by performing RNA sequencing
(RNA-seq) on fluorescence-activated cell sorting (FACS)-puri-
fied cardiomyocytes from 20-hpf Tg(myl7:lifeact-GFP) embryos.
Cardiomyocyte markers were enriched in the sorted cells
as expected (Figure S6A). Transcripts of expressed genes en-
coding known or probable transcription factors were identified
(Table S3), and the 50 most abundantly expressed were selected
for targeting. This list included mef2c, tbx5a, tbx20, pbx4, hand2,
gata5, hif1al, and nkx2.5, which are known to contribute to car-
diac development in zebrafish and/or mammals and served as
positive controls (Staudt and Stainier, 2012). As negative con-
trols, we utilized 10 four-guide sets with random sequences.
Developmental Cell 46, 112–125, July 2, 2018 119
Figure 6. Uninterrupted Sequencing of Genes Targeted with Multi-guide Cas9 RNP
(A) Representative predicted Hand2 protein sequences from DNA collected from an embryo yolk-injected with hand2 Cas9 RNP. hand2 alleles were PCR-amplified
and TA-cloned. Guide sites are indicated at the top. The reference Hand2 protein sequence is in bold. Mutant sequences are listed in order of decreasing relative
frequency. Note that most of the encoded polypeptides were truncation mutants due to early stops. + denotes number of amino acids to end of protein.
(B) Aggregate data from three clutchmate embryos yolk-injected with hand2 Cas9 RNP and analyzed as in (A).
(C) Representative predicted Sox32 protein sequences from an embryo cell-injected with sox32 Cas9 RNP. DNA was sequenced by long-read, high-throughput
sequencing. Guide sites and a splice site are indicated at the top. The reference Sox32 protein sequence is in bold. Mutant sequences are listed in order of
decreasing relative frequency, which is indicated on the right. + denotes number of amino acids to start (left) or end (right) of protein.
(D) Genomic sox32 DNA sequence corresponding to the alleles shown in (C) highlighting mutations at the predicted sgRNA sites and at a splice junction between
sgRNA sites 2 and 3. Note that allele sequences 8 and 9 (red asterisks) share identical insertional mutations at site 2 but distinct sequences at sites 1 and 4, and
allele sequences 4 and 5 share identical site 2 mutations but distinct site 4 mutations. The sox32 reference sequence is at the top in bold with exons in uppercase
and the intron in lowercase.
(E) Percentage of alleles mutated at each sox32 sgRNA site in each of six embryos sequenced with high-throughput, long-read sequencing. A total of 8,480 reads
representing 3,401 unique sox32 alleles were analyzed.
(F) Frequency of site-spanning deletions between each combination of guide target sites for the six embryos sequenced with high-throughput, long-read
sequencing. Percentages of total sequenced alleles are plotted according to genomic distance between guide sites. ySites 2–4 combination with two guides with
highest individual targeting efficiency (E) but low rate of intersite deletions.

Changes in expression of nppb, which exhibits increased
expression in response to myocardial stretch associated with
heart failure in humans and in zebrafish embryos (Becker et al.,
2012), was used as an endpoint (Figure S6B). Targeting of
hand2 and hif1al was associated with increased expression as
might be expected (Figure S6B). Interestingly, targeting of other
genes known to contribute to cardiac development, e.g., tbx20,
pbx4, nkx2.5, tbx5a, and gata5, was associated with decreased
nppb expression at 1 dpf, perhaps due to decreased cardiomyo-
cyte differentiation or number (Figure S6B).
The largest increase in nppb expression was associated with
targeting zbtb16a, a gene encoding a Kruppel (C2H2-type) tran-
scription factor. ZBTB16 has been reported to mediate angio-
tensin II-induced hypertrophy and Gata4 expression, and altered

120 Developmental Cell 46, 112–125, July 2, 2018


(E) Quantification of nppb expression in AE phenotype embryos. AE phenotype embryos were selected from zbtb16a D55bp heterozygous incrosses and
compared with clutchmate controls not exhibiting a cardiac phenotype. **** p < 0.0001, unpaired two-tailed t test.
(F) Representative spinning-disk confocal image of heart from AE phenotype homozygous zbtb16a D55bp null Tg(myl7:GFP) embryo collected at 2 dpf. a, atrium;
v, ventricle.
ZBTB16 expression has been associated with congenital heart
disease in human studies (McKean et al., 2016; Wang et al.,
2012). In zebrafish, zbtb16a is expressed ubiquitously at
24 hpf and can be observed in the heart at 48 hpf (Thisse and
Thisse, 2004). However, its role in cardiac development has
not been directly examined. Accordingly, we selected zbtb16a
for further study.
To probe the specificity of the effects of zbtb16a targeting, we
first designed three additional independent guide sets targeting
zbtb16a, two using the standard strategy (Figures 5A and 5B)
and one limited to a single exon, specifically targeting the
C2H2 zinc finger DNA-binding domain to avoid large genetic
deletions. Injection of these guide sets was associated with
increased nppb expression as measured by qPCR (Figure S7A).
Next, an nppb expression reporter line carrying firefly luciferase
under control of the nppb promoter (nppb:F-Luc) (Becker et al.,
2012) was used to assess nppb expression at 2 dpf. Injection of
Cas9 RNP with each of the zbtb16a four-guide sets led to
increased nppb expression (Figure 7A). Scrambled sgRNA and
slc24a5 four-guide sets were without effect (Figure 7A). These
results suggest that increased nppb expression in the zbtb16a-
Figure 7. zbtb16a Disruption by Cas9 RNP
Impairs Cardiac Development
(A) Confirmation of increased nppb expression in
response to zbtb16a disruption. Tg(nppb:F-Luc)
embryos were yolk-injected with four distinct
four-guide sets targeting zbtb16a or with control
scrambled and slc24a5 guide sets, then collected
at 2 dpf for luciferase assay. zbtb16a guide sets 1–3
targeted the coding sequence as in Figures 6A
and 6B. zbtb16a guide set 4 specifically targeted
the C2H2 zinc finger repeat domain. *p < 0.05,
**p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not
significant compared with scrambled by one-way
ANOVA and Sidak’s multiple comparison test.
Mean ± SEM; n = 24 for each group.
(B) Representative spinning-disk confocal images
of hearts from Tg(kdrl:GFP) embryos collected
at 2 dpf after injection with Cas9 RNP with zbtb16a
four-guide set (right) or a scrambled control set
(left). Tg(kdrl:GFP) labels endocardium here. a,
atrium; v, ventricle.
(C) Representative images of zbtb16a wild-type
Tg(myl7:GFP) embryo (left), Cas9 RNP-injected
G0 embryo with atrial enlargement phenotype
(AE) (middle), and F3 zbtb16a homozygous null
embryo with AE phenotype (right) at 48 hpf. The
AE phenotype includes marked chamber enlarge-
ment, pericardial edema, and impaired circulation.
Fluorescent images at the bottom show myocar-
dium. Scale bar, 250 mm.
(D) Genotype and phenotype of F2 and F3 embryos
derived from F1 zbtb16a D55bp heterozygous in-
crosses, F1 zbtb16a ins2bp heterozygous incross,
F1 zbtb16a D7bp heterozygote/D55bp heterozy-
gote transcross, and incross of F2 zbtb16a D55bp
heterozygotes that had been outcrossed once with
wild-type. All embryos were scored for AE pheno-
type at 2 dpf and then genotyped.
targeted embryos was likely a result of loss of zbtb16a
expression.
We next evaluated cardiac chamber function and morphology
in more detail with an endothelial/endocardial labeled
Tg(kdrl:GFP) transgenic line. A fraction of G0 embryos injected
with zbtb16a four-guide set exhibited heart failure as indicated
by pericardial edema together with prominent atrial enlargement
(AE), impaired atrioventricular separation, and decreased heart
rate compared with a scrambled control guide-injected embryos
(Figures 7B, 7C, and S7C; Videos S2 and S3). In some cases,
these abnormalities were severe and associated with lack of
visible erythrocyte circulation. We refer to this severe phenotype
as AE hereafter.
We next determined whether the AE phenotype seen in
zbtb16a gene-targeted G0 embryos could be reproduced in
zbtb16a null embryos generated from stable mutant lines. Three
lines were generated using two distinct zbtb16a guide strategies.
One guide set generated a line carrying a 7-bp deletion (zbtb16a
D7bp) and a second line carrying a 2-bp insertion
(zbtb16a ins2bp); both alleles disrupted the BTB domain of the
Zbtb16a protein and led to premature translational termination
Developmental Cell 46, 112–125, July 2, 2018 121
(Figure S7B). The second guide set generated a zbtb16a D55bp
allele, which contained a 55-bp deletion and additional muta-
tions in the zbtb16a DNA-binding domain (Figure S7B); this line
was generated in a Tg(myl7:GFP) background. We examined
heterozygote incrosses within individual lines to generate homo-
zygous nulls as well as a transcross of heterozygotes from
different lines to generate compound heterozygotes. All embryos
in a clutch were scored for phenotype and then all were
genotyped.
In an incross of F1 zbtb16a D55bp heterozygotes, the AE
phenotype was detected in 44/128 (33%) zbtb16a nulls, 1/132
(0.7%) wild-types, and 5/265 (1.9%) heterozygotes. In an in-
cross of F1 zbtb16a ins2bp heterozygotes, the AE phenotype
was detected in 4/54 (7%) zbtb16a nulls, 0/81 (0%) wild-types,
and 0/135 (0%) heterozygotes. In a transcross of zbtb16a
D55bp and zbtb16a D7bp F1 heterozygotes, the AE phenotype
was detected in 9/65 (14%) compound heterozygotes, 0/58
(0%) wild-types, and 2/146 (1.3%) heterozygotes. Additionally,
in an incross of zbtb16a D55bp F2 heterozygotes that had
been outcrossed to wild-type fish, the AE phenotype was de-
tected in 11/52 (21%) zbtb16a homozygous null F3 embryos,
0/41 (0%) wild-types, and 0/103 (0%) heterozygotes (Figure 7D).
In additional experiments, 87%–100% of embryos with the AE
phenotype selected from incrosses and transcrosses of F1
zbtb16a heterozygotes were zbtb16a nulls, and 100% of F3 em-
bryos selected from an F2 incross of an outcrossed zbtb16a
mutant were zbtb16a nulls (Figures S7E and S7F). No F3 em-
bryos (0/177) from an incross of F2 zbtb16a wild-type clutch-
mates from the zbtb16a D55bp outcross used to generate the
parents for the zbtb16a D55bp F2 heterozygote incross showed
the AE phenotype (Figure S7F). Like G0 zbtb16a embryos with
the AE phenotype, AE embryos derived from incrosses of
zbtb16a heterozygotes showed elevated nppb expression (Fig-
ure 7E), prominent atrial enlargement, impaired atrioventricular
separation, and decreased heart rate at 2 dpf (Figures S7C
and S7D; Videos S3 and S4). Seventeen out of 17 embryos
with the AE phenotype identified at 2 dpf progressed to cardiac
atresia, did not regain circulation by 5 dpf, and were not viable.
Seventeen clutchmates that lacked the AE phenotype at 2 dpf
remained unaffected and viable at 5 dpf. Taken together, these
results provide strong evidence that the G0 zbtb16a knockout
phenotype is recapitulated in zbtb16a nulls derived from
stable mutant lines, that the AE phenotype is tightly linked to
the zbtb16a genotype, and that zbtb16a loss of function is asso-
ciated with a partially penetrant severe cardiac development
defect.
DISCUSSION
We report a Cas9 RNP-based method for efficient gene disrup-
tion that generates null phenotypes in G0 zebrafish embryos. We
show that use of mixes of four sgRNA guides to redundantly
target a single gene produces null phenotypes with nearly com-
plete penetrance at a level of toxicity (<17% dysmorphic em-
bryos in all cases examined) that would be acceptable for
many moderate-throughput reverse genetic screening applica-
tions. Early embryonic and stable adult phenotypes were pro-
duced. Dual-gene targeting was feasible with this approach, a
useful feature for addressing redundancy or searching for mod-
122 Developmental Cell 46, 112–125, July 2, 2018
ifiers. We provide a lookup table of four-guide sets for 21,386 ze-
brafish genes, 83% of the 25,638 predicted coding region-con-
taining genes currently annotated in the zebrafish genome
(Howe et al., 2013). In a focused reverse genetic screen of 50
cardiomyocyte transcription factors, we demonstrated that this
technique identified genes known to be involved in cardiac
development and also uncovered a role for zbtb16a in the
same. Thus, this system provides a framework for rapid, moder-
ate-throughput reverse genetic screening in G0 zebrafish em-
bryos and adults.
Several factors may contribute to the increased efficiency of
four-guide versus single-guide Cas9 RNP injections in our study.
The efficiency of targeting by sgRNAs designed for a given target
gene varies (Moreno-Mateos et al., 2015) perhaps due to chro-
matin structure, unannotated SNPs, alternative start sites, or
alternative splicing. Even after successful targeting, non-homol-
ogous repair will leave a fraction of mutants with functional or
hypomorphic alleles. Use of multiple guides should increase
the probability of including efficient guide(s) and enable multiple
targeting events per coding sequence. The latter increases the
probability of loss of protein function. High-throughput, long-
read sequencing of DNA from embryos targeted with a four-
guide CRISPR/Cas9 RNPs to sox32 revealed that most of the
sox32 alleles contained multiple mutations confined to individual
guide sites rather than deletions between sites. Targeting of
other ‘‘larger’’ genes using the strategy outlined in Figure 5 might
be expected to produce similar results. Despite variable effi-
ciency of the guides, the combined effect of sox32 mutations
at the four-guide sites led to premature stop codons in >90%
of sequenced alleles.
Long-read sequences of sox32 alleles from targeted embryos
also suggests that CRISPR/Cas9 action may occur over several
DNA replication cycles such that distinct mutations at inefficient
guide sites become superimposed upon a common mutation
that occurred earlier at an efficient guide site (Figure S5B).
Alternatively, mutation may occur repeatedly at a single site
with some guides but not others.
Several features of the effectiveness of four-guide Cas9 RNP
targeting in zebrafish embryos are worth noting. Its effectiveness
in generating gene loss of function in G0 embryos permits flex-
ible use of already developed zebrafish lines for screens, e.g.,
lines with reporters such as nppb:FF-luc or lines with fluores-
cently tagged cell types of interest. Lines with fixed mutations
might also be used for suppressor screens. The ability to detect
changes in nppb mRNA levels in pooled targeted embryos in the
cardiac transcription factor screen reported here suggests that
four-guide Cas9 RNP targeting can be efficient enough to permit
the use of biochemical assays of pooled embryos as an initial
screening endpoint.
Injection of zebrafish embryos with preassembled four-
guide Cas9 RNP can ablate target gene function rapidly. The
observation that sox32 targeting suppressed sox17 expression
within 6 hr of injection suggests that early phenotypes can be
interrogated with this method. Such applications will presum-
ably be limited for genes with maternally deposited mRNA,
and translation-blocking morpholinos may be preferred in
such cases.
Phenotypes generated in four-guide G0 embryos were stable.
This was not a preordained result in that single-guide injections
of s1pr2 Cas9 RNP (Figure 1) produced transient phenotypes,
presumably related to incomplete targeting and mosaicism.
The stability of phenotypes associated with use of four-guide
sets opens the possibility of screening for phenotypes that man-
ifest in juvenile and adult zebrafish. This capability is not afforded
by morpholino knockdown, which produces rapid but transient
loss of gene expression.
Injection of increasing numbers of guides was variably associ-
ated with increased toxicity as indicated by the presence of dys-
morphic embryos, presumably related to increased numbers of
off-target mutations. Indeed, use of more than four guides to
target a single gene (tyrosinase) increased toxicity despite equiv-
alent overall guide concentration. We envision use of multi-guide
G0 Cas9 RNP-mediated gene knockout for preliminary screens,
with ‘‘hits’’ requiring subsequent rigorous confirmation. Use of
specific screening endpoints (e.g., phenotypes with specific
anatomical or gene expression changes or reversion of a mutant
phenotype) might reduce the risk of false positives. Preliminary
confirmation of hits may be done by targeting candidate genes
with independent guides to be followed by characterization of
phenotypes generated using incrosses of outcrossed stable
mutant lines (as done here for zbtb16a). Use of four-guide injec-
tions reproduced null phenotypes in >90% of G0 embryos and
produced <17% dysmorphic embryos for all test genes studied,
levels of efficacy and toxicity that are likely acceptable for typical
initial screening applications.
Simultaneous dual-gene targeting using multi-guide Cas9
RNP mixes in G0 embryos is possible but will be limited by
toxicity on a case-by-case basis. Some eight-guide sets target-
ing two genes simultaneously produced the expected double
phenotypes with high penetrance without significant toxicity,
but other eight-guide sets produced dysmorphic embryos at
rates approaching 50%.
To facilitate screens, we provide a lookup table of four-
guide sets for most zebrafish genes (Table S2). Use of a
four-guide Cas9 RNP produced by one template elongation
reaction, one in vitro transcription, and one incubation with
Cas9 protein makes for convenient and inexpensive reagent
production. Yolk injection of the complexes makes for rapid
throughput. We typically are able to yolk inject more than
300 fertilized one-cell-stage embryos in about 30 min. Thus,
about 50 knockout embryos can be produced for each of six
genes from a single zebrafish clutch, and embryos from
multiple clutches can be injected at a given sitting. The initial
screen of 50 transcription factors described here was done
in 2 weeks.
The moderate throughput of this methodology may be best
applied to targeted candidate gene screens like that described
here. We used RNA-seq to identify a ‘‘parts list’’ of a class of mol-
ecules of interest (transcription factors) expressed in a cell type
of interest (cardiomyocytes) at a time point of interest (20 hpf),
taking advantage of zebrafish lines with cell-type fluorescent
tags for FACS. The list included transcription factors known
to be necessary for zebrafish cardiac development such as
pbx4, hand2, and tbx20, and the screen identified these genes
suggesting that gene knockout and initial endpoint chosen
(altered nppb expression) were effective. The screen also uncov-
ered a role for zbtb16a in cardiac development. Further experi-
mentation will be required to identify the mechanisms by which
zbtb16a contributes to zebrafish heart formation and its roles
in mammals.
In summary, the methods and lookup table reported here
provide a framework for moderate-throughput reverse genetic
interrogation in G0 zebrafish embryos and adults. The efficiency
of the system allows two operators to disrupt 30 or more genes in
a single injection session and rapid phenotyping. This approach
complements existing methods to more fully exploit the many
useful features of the zebrafish model.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
d KEY RESOURCES TABLE
d CONTACT FOR REAGENT AND RESOURCE SHARING
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
B Zebrafish Handling and Maintenance
d METHOD DETAILS
B sgRNA In Vitro Transcription
B Cas9 Ribonucleoprotein Complex Preparation and
Microinjection
B Preparation of Cas9-GFP Protein
B Morpholino Microinjection
B Rescue Injections
B Genomic DNA Extraction and Sequencing
B Genomic DNA Extraction and High-throughput
Sequencing and Analysis
B RNA Extraction and Quantitative PCR
B nppb Luciferase Assay
B sgRNA Design
B Cardiomyocyte Purification
B RNA-Seq Library Preparation and Sequencing
B RNA-Seq Data Processing and Transcription Factor
Annotation
B Cardiac Transcription Factor Screening Microin-
jections
B Assessment of Cardiac Function and Morphology
B Generation of Stable Knockout Lines
d QUANTIFICATION AND STATISTICAL ANALYSIS
d DATA AND SOFTWARE AVAILABILITY
SUPPLEMENTAL INFORMATION
Supplemental Information includes seven figures, three tables, and four videos
and can be found with this article online at https://doi.org/10.1016/j.devcel.
2018.06.003.
ACKNOWLEDGMENTS
We are grateful to Gary Moulder for aid in experimental preparation. Cell sort-
ing was performed at the Laboratory for Cell Analysis at UCSF. RNA-seq was
performed at the Institute of Human Genetics at UCSF. PacBio sequencing
was performed at the DNA Technologies and Expression Analysis Cores at
the UC Davis. This work was supported in part by the NIH (S.R.C., HL65590
and HL054737; R.S.W., 5F32HL116194).
AUTHOR CONTRIBUTIONS
Conceptualization, R.S.W.; Methodology, R.S.W. and S.R.C.; Formal Analysis,
R.S.W., S.R.C., and R.C.D.; Investigation, R.S.W., I.I.L., H.C., and D.N.D.;
Developmental Cell 46, 112–125, July 2, 2018 123
Software and Writing – Original Draft, R.S.W.; Writing – Review & Editing,
R.S.W. and S.R.C.; Visualization, R.S.W.; Supervision, S.R.C.
DECLARATION OF INTERESTS
The authors declare no competing interests.
Received: October 18, 2017
Revised: May 14, 2018
Accepted: June 5, 2018
Published: July 2, 2018
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STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Chemicals, Peptides, and Recombinant Proteins
Alt-R Cas9 nuclease Integrated DNA Technologies Cat# 1074181
Cas9-GFP nuclease This study N/A
Critical Commercial Assays
Promega Steady-Glo reagent Promega Cat# E2510
Thermo Scientific Expression Assays Life Technologies N/A
NuGen Ovation V2 RNA-Seq System NuGen Cat# 7102
NuGen Ultralow DR Library Prep NuGen Cat# 0030
Ambion T7 Megascript Kit Life Technologies Cat# AM1334
SMRTbell Template Prep Kit 1.0 Pacific Biosciences Cat# 100-259-100
Deposited Data
Genome-Scale Guide Sets This study N/A
Zebrafish Cardiomyocyte RNA-Seq This study N/A
Cardiomyocyte RNA-Seq GEO Database https://www.ncbi.nlm.nih.gov/geo/ GEO: GSE115263
sox32 long-read amplicon sequencing GEO Database https://www.ncbi.nlm.nih.gov/geo/ GEO: GSE115233
Experimental Models: Organisms/Strains
D. rerio strain: Tg(myl7:GFP) Provided by Didier Stainier (Max Planck Institute, RRID: ZFIN_ZDB-GENO-100618-3
Bad Nauheim, Germany)
D. rerio strain: Tg(myl7:lifeact-GFP) Provided by Didier Stainier (Max Planck Institute, (Reischauer et al., 2014)
Bad Nauheim, Germany)
D. rerio strain: Tg(kdrl:GFP) Provided by Didier Stainier (Max Planck Institute, RRID: ZFIN_ZDB-GENO-170727-1
Bad Nauheim, Germany)
D. rerio strain: Tg(kdrl:rasCherry) Provided by Didier Stainier (Max Planck Institute, RRID: ZFIN_ZDB-GENO-110511-4
Bad Nauheim, Germany)
D. rerio strain: Tg(nppb:F-luc) Provided by Calum MacRae (Brigham and (Becker et al., 2012)
Women’s Hospital, Boston, USA)
D. rerio strain: Tg(UAS:Dendra) Provided by Herwig Baier (Max Planck Institute, (Arrenberg et al., 2009)
Martinsried, Germany)
D. rerio strain: Tg(sox17:GFP) Provided by Didier Stainier (Max Planck Institute, (Sakaguchi et al., 2006)
Bad Nauheim, Germany)
D. rerio strain: zbtb16a +/- (D7bp) This study N/A
D. rerio strain: zbtb16a +/- (ins2bp) This study N/A
D. rerio strain: zbtb16a +/- (D55bp) This study N/A
Oligonucleotides
See Table S1 N/A N/A
Recombinant DNA
pCS2 sox32 Plasmid Provided by Orion Weiner (University of (Kikuchi et al., 2001)
California San Francisco, San Francisco, USA)
pCS2 GFP Provided by Orion Weiner (University of N/A
California San Francisco, San Francisco, USA)
pMJ922 Plasmid Provided by Martin Jinek (University of Zurich, Addgene# 78312
Zurich, Switzerland)
Software and Algorithms
Sickle 1.33 https://github.com/najoshi/sickle RRID: SCR_006800
Scythe 0.981 https://github.com/vsbuffalo/scythe RRID: SCR_011844
Tophat2 2.0.13 https://ccb.jhu.edu/software/tophat/index.shtml RRID: SCR_013035
Cufflinks 2.1.1 http://cole-trapnell-lab.github.io/cufflinks/ RRID: SCR_014597
(Continued on next page)

e1 Developmental Cell 46, 112–125.e1–e4, July 2, 2018

Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
Bowtie 1.1.2 http://bowtie-bio.sourceforge.net/index.shtml RRID: SCR_005476
Biopython http://biopython.org RRID: SCR_007173
Python https://www.python.org RRID: SCR_008394
Photoshop CS4 Adobe Systems RRID: SCR_014199
Prism 7 Graphpad Software RRID: SCR_005375
Fiji https://github.com/fiji/fiji RRID: SCR_002285
High Resolution Melt Software 2.0.2 Thermofisher Scientific Cat# 4397808
CRISP-ID http://crispid.gbiomed.kuleuven.be/ (Dehairs et al., 2016)
Ape http://biologylabs.utah.edu/jorgensen/wayned/ape/ RRID: SCR_014266
SMRT Link 5.0.1.9585 Pacific Biosciences RRID: SCR_002942
Micro-Manager https://micro-manager.org/ RRID: SCR_000415

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Shaun R.
Coughlin (shaun.coughlin@ucsf.edu).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Zebrafish Handling and Maintenance

All zebrafish studies were performed in accord with IACUC-approved protocols at the University of California, San Francisco.
Wild-type zebrafish were in the Ekkwill background. Embryos were raised at 28# C. Embryos were injected at the one-cell stage
and analyzed between 4 hour and 8 days post-fertilization. Adults were analyzed at 3 months post-fertilization. In all instances,
microinjections were performed on embryos from multiple simultaneously produced clutches.

METHOD DETAILS

sgRNA In Vitro Transcription

Templates for guide RNA production were generated by annealing and elongation (Phusion Flash High-Fidelity Master Mix, Life
Techologies) of a forward primer containing a T7 promoter and guide sequence and a reverse primer encoding the standard chimeric
sgRNA scaffold (Varshney et al., 2015). The sequences used are listed in Table S1. Oligonucleotides were obtained from Integrated
DNA Technologies. The DNA template was purified (MinElute PCR Purification, Qiagen) and in vitro transcribed (Megascript Kit,
Ambion). RNA was purified using RNA Clean and Concentrator-5 (Zymo Research).

Cas9 Ribonucleoprotein Complex Preparation and Microinjection

His-tagged Cas9 protein with one N-terminal and two C-terminal nuclear localization sequences was obtained from Integrated DNA
Technologies. The protein was diluted to yield a 10 mM Cas9 solution in 20 mM Tris-HCl, 600 mM KCl, 20% glycerol and stored
at -20# C. Unless otherwise specified, the Cas9 solution was mixed 1:1 with sgRNA for a 5 mM Cas9, 1 mg/mL (31mM) sgRNA solution
to generate Cas9 RNP complexes. "0.05% phenol red dye was added for visualization of injections. When guides were mixed,
from 1 to 16 guides were combined such that the total sgRNA concentration remained 1 mg/mL. The Cas9 protein sgRNA mixture
was incubated at 37# C for 5 minutes. Microinjection was performed by injecting 0.5-1 nL of the mixture into 1-cell stage embryos
in either the yolk or the cell cytoplasm as indicated.

Preparation of Cas9-GFP Protein

Cas9-GFP protein was produced using pMJ922, a gift from Martin Jinek. Briefly, a His6 and maltose-binding protein (MBP)/TEV site-
tagged Cas9-GFP with 2 nuclear localization sequences was expressed in Rosetta competent cells. Cells were lysed using sonicat-
ion in 20 mM HEPES pH 7.5, 300 mM KCl, 1 mM TCEP, 10 mM imidazole. Clarified lysate was applied to a HisTrap FF crude column
(GE Healthcare) on an ÄKTA Protein Purification System (GE Healthcare) and eluted with a step-wise gradient to 300 mM imidazole.
The imidazole was removed by overnight dialysis at 4# C (Slide-A-Lyzer, Thermo Scientific) in the presence of TEV protease to remove
the His-MBP affinity tag. A second purification step was performed with a HiTrap Heparin column (GE Healthcare) with a linear KCl
gradient of 300-1500 mM. The eluted fractions containing Cas9-GFP were pooled and dialyzed overnight (Slide-A-Lyzer, Thermo
Scientific) into 20 mM HEPES pH 7.5, 600 mM KCl, 20% glycerol followed by concentration (Centricon, EMD Millipore) and storage
at -20# C.

Developmental Cell 46, 112–125.e1–e4, July 2, 2018 e2

Morpholino Microinjection
Morpholinos were obtained from Gene Tools, LLC. The s1pr2 morpholino was used as previously described (Kai et al., 2008). For tyr,
two morpholinos were co-injected (Pickart et al., 2004).

Rescue Injections
sox32 or GFP mRNA was transcribed in vitro (mMessage Machine, Ambion) from the corresponding cDNA sequences in the pCS2
vector. For microinjections, scrambled control or sox32 sgRNA Cas9 RNP were assembled as detailed above. The Cas9 RNP was
mixed at a 1:1 volume ratio with mRNA for a final concentration of 100 ng of sox32 mRNA or 80ng of GFP mRNA. 2 nL of this mixture
were then yolk injected into 1-cell stage embryos.

Genomic DNA Extraction and Sequencing

Crude genomic DNA was extracted from whole zebrafish embryos using Sigma-Aldrich Tissue Prep Solution, Extraction Solution,
and Neutralization B Solution. The targeted s1pr2 and hand2 loci were amplified by PCR and cloned for sequencing (TOPO TA
Cloning, Life Technologies).

Genomic DNA Extraction and High-throughput Sequencing and Analysis

Genomic DNA was isolated from six clutchmate embryos cell-injected with sox32 Cas9 RNP collected at 2 dpf (DNeasy Blood
and Tissue Kit, Qiagen). The coding sequence was amplified by barcoded PCRs (Phusion Flash High-Fidelity Master Mix, Life
Technologies), pooled, and purified (MinElute PCR Purification, Qiagen). The pooled and barcoded amplicons were converted
into a circular library (SMRTbell Template Prep, Pacific Biosciences) and sequenced with MagBead loading on a PacBio RSII
platform (Pacific Biosciences) with P6C4 chemistry. The resulting ‘‘bax.h5’’ data were converted into bam files using the ‘‘bax2bam’’
program of the SMRT Link toolset (Pacific Biosciences). Circular consensus sequences were generated using the ‘‘ccs’’ program of
the SMRT Link tools without read lengths and minimum pass number restrictions and using the ‘‘–bystrand’’ option to generate a
consensus sequences for forward and reverse strands separately.
Reads that did not pass a minimum phred score threshold and that did not contain adapter sequences and sox32
sequence were removed. The sequences then underwent removal of potential sequencing errors specific to the platform by identi-
fication of sequences differing in length by one base pair following 3 or more consecutive, identical bases compared to wild-type
sox32 sequence with masking of 40 bp windows around the sgRNA cut sites. These sequences were then matched to the wild-
type sequence. These changes would be expected to decrease the rate of false positive protein truncation predictions while
decreasing sensitivity for bona fide single base pair mutations. There there did not appear to be significant contributions from other
types of sequencing errors. The analysis was performed in Python supported by the Biopython package.

RNA Extraction and Quantitative PCR

RNA was extracted from whole embryos in Trizol (Life Technologies) and purified (Direct-zol, Zymo Research). To aid in consistency,
embryos collected in the screening assay were additionally homogenized using vortex agitation with zirconium beads. cDNA was gener-
ated using either Superscript Vilo (Life Technologies) or SensiFast (Bioline) cDNA synthesis kits. Primers and probes were purchased
from Integrated DNA Technologies for nppb and the eef1a1l1 housekeeping gene. A Taqman Gene Expression Assay (Thermo Fisher
Scientific) was obtained to assess sox17 expression. qPCR was performed on the Applied Biosystems 7900HT platform.

nppb Luciferase Assay

For nppb luciferase assays, embryos from a Tg(nppb:FF-luc) line (a gift from Calum MacRae) were injected with Cas9 RNP
as described above. At 2 dpf, embryos carrying the luciferase transgene were identified using the associated eye marker,
transferred to a 96-well luminescence plate in egg water (Instant Ocean) and Promega Steady-Glo reagent was added. After incu-
bation for 1 hour, luminescence was detected on a Promega GloMax luminometer.

sgRNA Design
sgRNAs designed prior to construction of the Genome-Scale Guide Set were designed using the CRISPR Design website (http://
crispr.mit.edu). In experiments with s1pr2, tnnt2a, npas4l, slc24a5, tbx16, and tbx5a, dual guanine nucleotides were added to
the 5’ end of the guide if they were not present in the endogenous sequence.
For the Genome-Scale Guide Set, all annotated protein-coding sequences were extracted from the GRCz10.80 genome build.
Potential sgRNA spacers with a maximum length of 20 bp were identified and several parameters were calculated. Off-
target alignments were estimated using Bowtie with the following parameters: -n 3 -l 15 -e 39 (Horlbeck et al., 2016). A less stringent
alignment was applied using the identical options substituted with an ‘‘-e 50’’ parameter. RNA free energy was estimated using the
Vienna RNAfold Python package (Lorenz et al., 2011). Guides were also scored for the effect of 5’ guide alteration (necessary for T7
in vitro transcription) on guide efficiency (Moreno-Mateos et al., 2015).
Guides with >1 e39 Bowtie alignment or runs of more than 4 guanine nucleotides were eliminated. The guides were chosen by
sorting, in order of priority, number of e50 Bowtie alignments, degree of 5’ alterations, and spacer free energy (Horlbeck et al.,
2016). For each protein-coding transcript, the coding sequence was divided into 5 equal sections and the highest-ranking guide
for each of the first four sections was selected. If one guide without >1 e39 Bowtie alignment or a >4 guanine run could not be

e3 Developmental Cell 46, 112–125.e1–e4, July 2, 2018

identified for each of the first four sections, guides were not selected for the transcript. The scripts for this processing were written in
Python supported by the Biopython package.

Cardiomyocyte Purification
Approximately 200 Tg(myl7:lifeact-GFP) embryos were collected at 20 hpf and dechorionated with pronase (Sigma-Aldrich).
Embryos were anesthetized with tricaine in cold Embryo Water (10% Hank’s Buffered Salt Solution) for 5 minutes. Embryos were
washed with ice-cold calcium and magnesium-free Hank’s Buffered Salt Solution (HBSS) and transferred to digestion solution
(3.75mg/mL Collagenase 1A (Sigma-Aldrich), 2.5mg/mL Trypsin IX-S (Sigma-Aldrich) in HBSS) and triturated on ice with a
small-bore lime glass pipette (orifice custom sized by flame polishing to a 27 gauge needle outer diameter, VWR) intermittently
for 15 minutes. Fetal bovine serum was added to 10% to stop digestion. Cells were washed with Hank’s Buffered Salt Solution
with calcium and magnesium then filtered through a 40mm cell strainer. The cell suspension was sorted at 4# C on a FACSAria2
cell sorter (BD Biosciences) and GFP-positive cells collected in Trizol (Life Technologies). RNA was purified with DNase digestion
using the RNeasy Micro kit (Qiagen).

RNA-Seq Library Preparation and Sequencing

RNA was extracted from purified cardiomyocytes from 9 biological replicates. RNA quality was assessed using Bioanalyzer RNA Pico
kit (Agilent) to confirm RIN score of >8. cDNA production and amplification was performed using the NuGen RNA-Seq Ovation V2
System. cDNA was sheared using a Covaris S2 to a target size of 200-300 bp with further size selection and purification using Agen-
court RNA Clean XP beads (Beckman Coulter) and NuGen Ultralow DR Library preparation kit. Quality control was performed using
Bioanalyzer High Sensitivity DNA kit (Agilent). Sequencing was performed on the Illumina HiSeq platform.

RNA-Seq Data Processing and Transcription Factor Annotation

Illumina datasets were filtered and trimmed using the Sickle and Scythe software programs. Reads were aligned to GRCz10.80 using
Tophat2 and abundance was estimated using Cufflinks, averaging the FPKM measurements of each of the 9 replicates. Predicted
transcription factors were identified using Pfam and Superfamily annotations derived from the DBD transcription factor database,
which were referenced to GRCz10.80 genome annotations obtained through Ensembl.

Cardiac Transcription Factor Screening Microinjections

Guide sets were derived from the Genome-Scale guide resource (Table S2). Forty 1-cell stage Ekkwill (EKW) wild-type embryos
were injected with Cas9 RNP complexes programmed with each guide set, as described above. Embryos were incubated at
28# C. At 30 hpf, any dysmorphic embryos were excluded and RNA was recovered from 20 of the remaining embryos in each group.

Assessment of Cardiac Function and Morphology

Tg(kdrl:GFP) and Tg(myl7:GFP) embryos were imaged at 2 dpf on a Nikon spinning-disk confocal microscope equipped with an
Andor sCMOS camera. Videos were obtained at each Z-plane using a custom Micro-Manager script (Staudt et al., 2014).

Generation of Stable Knockout Lines

We analyzed lines with three different zbtb16a alleles. All were predicted null alleles. Two were generated using zbtb16a guide 1; one
featured a 7 bp deletion (D7bp) and the other a 2 bp insertion (ins2bp). The third allele, D55bp, was generated using zbtb16a guide
set 4 injected into a Tg(myl7:GFP) background; this allele contained a 55 bp deletion in the DNA-binding domain and additional
mutations. We analyzed both incrosses and intercrosses of these three alleles. Genotyping was performed using a combination
of high-resolution melt analysis (SensiFast HRM, Bioline), PCR amplicon sequencing with CRISP-ID analysis (Dehairs et al.,
2016), and gel electrophoresis.

QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical analysis was performed using PRISM 7 statistical analysis software (GraphPad Software). Numbers of embryos and sta-
tistical tests are indicated in the figure legends.
The scoring in the following figures was blinded to treatment: Figures 2B, S2C, S5B, S9C, and S9D. The scoring in the following
figures was not blinded to treatment: Figures 1A, 2A, S2A, S2B, S2D, S2F, 4, and 5D. All experiments involving phenotyping of
zbtb16a null lines required sacrifice of the embryo for genotyping and were thus, by necessity, scored blind to genotype.

DATA AND SOFTWARE AVAILABILITY

The genome-scale guide set resource is provided in Table S2. The software used for generating the guide sets is available upon
request. Cardiomyocyte RNA-Seq and genomic high-throughput sequencing data were uploaded to the NCBI Gene Expression
Omnibus (https://www.ncbi.nlm.nih.gov/geo/) under accession numbers GEO: GSE115263 and GSE115233, respectively.

Developmental Cell 46, 112–125.e1–e4, July 2, 2018 e4