RecV recombinase system for in vivo targeted optogenomic modifications of single cells or cell populations

RecV recombinase

system

for

in vivo

targeted optogenomic modifications of

single cells or cell

populations

Shenqin Yao

eng

Yuan

Ben Ouellette

Thomas Zhou

Marty Mortrud

Pooja Balaram

Soumya

Chatterjee

Yun Wang

Tanya L. Daigle

Bosiljka

Tasic

Xiuli Kuang

Hui Gong

Qingming Luo

Shaoqun

Zeng

Andrew Curtright,

Ajay Dhaka,

Anat Kahan

Viviana Gradinaru

Radosław Chrapkiewicz

Mark

Schnitzer

Hongkui Zeng

and

Ali Cetin

Allen Institute for Brain Science, Seattle, Washington, USA.

CNC program, Stanford University,

Palo

Alto,

California, USA.

School of Optometry and Ophthalmology, Wenzhou Medical

College, Wenzhou, Zhejiang, China.

Britton Chance Center for Biomedical Photonics, Wuhan

National Lab for Optoelectronics, Huazhong

University of Science and Technology, Wuhan, Hubei,

China.

Department of Biological structure, UW, Seattle, WA, USA.

Division of Biology and Biological Engineering

California Institute of Technology, Pasadena, California, USA.

*These authors contributed equally

to this work

Correspondence should be

addressed to A.C. (

alic@alleninstitute.org

Keywords:

Light

inducible,

optogenomic,

recombinase, recombination, RecV, Vivid, Cre, Dre, Flp

ABSTRACT

Brain circuits are composed of vast numbers of intricately interconnected neurons with diverse molecular,

anatomical and p

hysiological properties. To allow

highly specific “user

defined”

targeting of individual neurons

for structural and functional studies

, we modified three site

specific DNA recombinases, Cre, Dre and Flp, by

combining them with a fungal light

inducible prot

ein, Vivid,

to create light

inducible recombinases (named RecV)

We generated viral vectors to express these light

inducible recombinases and demonstrated that they can induce

genomic modifications in dense or sparse populations of neurons in

superficial a

s well as deep brain areas of

live mouse brains by one

photon o

r two

photon light induction.

These l

ight

inducible recombinases can produce

highly targeted, sparse and strong labeling of individual neurons

in multiple loci and species.

They

can be used

combination with other genetic strategies

to achieve

specific i

ntersectional targeting of

mouse cortical

layer

inhibitory somatostatin neurons.

n mouse cortex sparse light

induced recombination

allows whole

brain

morphological reconstruction

to iden

tify axonal projection specificity.

Furthermore these enzymes

allow

single

cell targeted

genetic modifications

via

soma

restricted

two

photon

light

stimulation

in individual cortical neurons

and can be used in combination with functional optical indicators

with minimal interference

In summary

RecVs

enable spatiotempo

rally

precise, targeted

opto

genomic modifications that

could

greatly facilitate detailed

analysis of

eural circuits at

the

single

cell level

linking genetic identity, morphology, connecti

vity and function.

INTRODUCTION

To understand how a biological system works it is often useful to

define

its building blocks and

understand

how

those building blocks work

together to generate its function. Mammalian brain is one of the most complex

biolo

gical systems. It is composed of millions to billions of cells

with diverse

characteristics. To understand this

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extraordinary complexity, it will be essential

to define cell types based on properties such as gene expression,

morphology and physiology at

the

single cell level. Furthermore, the unique properties of individual cells

need to

be related to their connectivity patterns and their activities in a behavio

ral context. Anatomical information

combined with genetic identity and functional properties at the single cell level will enable

better

analysis of brain

circuitry underlyi

ng complex behaviors in health and disease.

One of the most powerful approaches to

characterizing cell types and studying their functions relies on mouse

genetics

trategies that rely on transgenic or viral expression of recombinases allow a highly specific level of

genetic modification

A m

ultitude of cell type

specific Cre recombinase mouse lines has led to many

discoveries in neuroscience over the last few decades.

Further

improvements on

spatiotemporal control

can

allow much higher resolution

manipulations and

studies of biological systems

Current

finding

individual

cells/neurons

in vivo

;

characterizing

and

genetically manipulat

ing

them

for targeted

genomic modifications

as well as

express

ing

genes

in a targeted manner

is challenging

to execute in

an efficient

manner

. The state

the

art

approach

for introducing an exogenous gene to a specific neuron is eit

her by the

patch clamp

technique

or via

single

cell electroporation

7,8

technique

are

highly challenging and usually

result in low and variable yields.

Sparse neuro

nal labeling or manipulation can be achieved by controlling

transgene

recombination by lowering the dose of inducers (

e.g.

, tamoxifen, in the case of CreER) or

by employing

‘inefficient’ recombinase reporters

(

e.g.

, MADM

). However, the sparse genetic modification achieved using

these methods is random and cannot be easily directed to specific

or individual

cells of interest.

Light is a particu

larly powerful and versatile regulator with its tremendous adjustabil

ity in the dimensions of

spectrum, intensity, space (location and size) and time (timing and duration). Using photons to access and

genetically modify individual neurons will offer an imp

rovement over the current state

the

art. Multi

photon

additive cha

racteristics of light that can generate a spatiotemporally

restricted excitation

are

well suited

for

achieving fine

spatial control

. Thus, modifying current genomic manipulation enzymes to make them light

inducible could be an ideal approac

h to reach a high spatiotemporal resolution for targeted

single cell

manipulations.

everal light inducible

protein

based spatiotemporal control

systems

have been

develope

d to date

These

systems depend on various light sensing modules to co

ntrol protein states, protein localization, transcription

and genetic alterations.

So far optical manipulation of

genomes

optogenomics

of individually targeted single

cells within live intact tissues

has

not

been

demonstrated using such methods.

To leave

a permanent genetic

mark in individual cells in this study, we developed and validated light inducible

site

specific

recombinase

systems and its associated set of viral tools via comparing and optimizing various light inducible systems with

the cr

iteria t

hat they induce robust genomic modifications with no or minimally detectable background under

light conditions at different genomic locations and species. Our work resulted in the generation of highly

efficient light inducible versions of the mo

st commo

nly used site

specific recombinases

Cre, Dre and Flp

that

allow tight population level or target

specific single cell level optogenomic modifications

in vivo

RESULTS

A Split Vivid

Cre enables efficient light

inducible

site

specific DNA modification

To spatiotemporally regulate site

specific recombination, we generated a light

inducible genetic switch based on

a fungal light sensitive protein

–

Vivid (VVD)

chose VVD mainly because it is the smallest (450 bp) of all

Light Oxygen or Voltage (LOV) domain

containing proteins, and therefore suitabl

e for fitting into viral vectors

with limited genomic capacity. In addition, the spectral proper

ties of VVD

its excitation and emission drops

sharply to

near

with light

above ~520 nm

would allow us to use other fluorescent proteins, activity indicators

or optogenetic molecules that work at longer wavelengths

20,35

Upon light illumination, VVD forms a homodimer

due to

the

conformational changes induced by Flavin Adenine

Dinucleotide (FAD) cofactor within the LOV domain. The FAD cofactor within the LOV domain has

a peak single

photon (1P)

excitation at 450 nm and

peak two

photon (2P) activation at 900 nm wavelength

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generate

a precise spatiotemporal

control of gene expression, we used a split

Cre recombinase system

20,37

and

converted

it into a light

inducible system using the VVD protein (

Fig. 1

). For Cre recombinase to function

roperly

the N

termin

and the C

termin

al portion

of the protein need to come in close proximity

form the

correct tertiary structure. The crystal structure of Cre

is known

and so

VVD

35,41

. In the dimer form, the N

termin

of one VVD

monomer

gets into close proximity to the C

terminus of the other (

Fig. 1a

Guided by this

information o

design

attempted to bring together the N

and C

portions

of Cre in the correct orientation upon

light

induced conformational change and

dimerization of VVD. To split the Cre protein, we relied on

a previous

split Cre system, since

was shown to generate stable dimers

fused the inactive N

terminal segment of

Cre to the N terminus of one VVD

mon

omer

and inactive C

terminal

segment of Cre to the C terminus of another

VVD monomer, which we codon diversified to reduce the sequence similarity

between the two VVD monomers

and

minimize the risk of recombination.

cloned each

of these

into recomb

inan

t adeno

associated virus

(rAAV) expression vector

(

Fig. 1

)

The resulting NCreV and CCreV constructs along with

fluo

rescent

Cre

reporter construct

were co

transfected into

mammalian

cells and robust light

inducible recombination

was

observed with 458 nm

LED

light induction compared to the no

light condition

(

Fig.

d, e

We named these new

proteins CreV, and the

general light

inducible Vivid

recombinase system RecV.

Vivid

based light

inducible system

for Dre recombinase

broaden the capabilities of the RecV approach

we modified

another site

specific DNA recombinase

Dre

which is a relative of Cre, and is likewise derived from a bacterial phage

. Dre

recombinase recognizes a

sequence

called Rox

, which is different from LoxP.

This is especially important since it would allow us to utilize

already existing Cre driver mouse lines to study Cre

defined popu

lations

and to develop intersectional strategies

to further refine cell type

specific genetic manipulation.

We reasoned that the sequences surrounding the homology region within Dre N

terminus, where we split Cre

into two non

functional

compartments

could also

be targeted to generate a split Dre

(

Fig.

)

After generation

of the DreV constructs based on CreV designs

we tested

the

light inducibility of DreV constructs using fluorescent

Dre reporter plasmids

(

Fig.

and

Supp.

Fig. 1

). Our results indicate that t

he light

induced DreV recombination

is efficient

using 1P

excitation

and is similar to CreV (

Fig.

d, e

Spatiotemporal regulation of gene expression can be further fine

tuned using 2P excitation since it has a much

narrower

point spread function in the

axial direction. We tested if the VVD based recombination can take place

using 2P light under cell culture conditions. Our results indicate that DreV

mediated light

inducible recombination

can be achieved by application of 900

nm wavelength of 2P light (

pp.

Fig.

Single

RecV

expression constructs

and comparison to other existing light inducible

recombinase

systems

To implement the RecV strategy efficiently and reduce the number of viruses or transgenic mouse lines

needed

use this system

, it is pre

ferable to co

express the two halves of RecV

in a single construct. To achieve this,

we tested a variety of ways to link the N and C components of CreV or DreV that would provide the highest

efficiency of recombination

with the least amount of background

recombination

IRES, 2A, single open reading

frame with permutations of the open reading frames

; see methods section for more details

(

Supp. Fig. 3

)

mong these constructs

highest

efficienc

ies

ere

observed

when

optimized

elements from

Cre

Magnet

(

mutant

VVD

)

were used

which

named

iCreV and iDreV

iCreV and iDreV are composed of

and C

terminal

portions of the Cre and Dre split recombinases

fused to nuclear localized

wild

type

VVD and co

expressed with

optimized linkers and

2A elements that wer

e used in Cre

Mag

nets

(

Supp. Fig. 1

)

compared

iCreV and iDreV,

with

Magnet

ased

ersions

In cell culture

Magnet

based constructs

induced

significant amount of background recombination in the absence of li

ght

reV and i

reV exhibited

control levels of

recombination in the absence of light and retained high levels of inducibility

–

~1.6 fold higher

for iCreV as compared to split CreV and ~0.6 fold lower for iDreV compa

red to

split DreV in relative fluorescence

intensities

(

Supp. Fig. 3

)

For both iCreV and Cre

Magnet

efficient

recombination was observed

in vivo

after

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light stimulation

please see the details of the

in vivo

experiments below

However,

in no ligh

t control cases with

Cre

Magnet

back

ground

recombination was observed

throughout the

brain structures

~50 cells per 100

μm

coronal slice

whereas

CreV

did not lead to

significant

recombination

(

Supp. Fig. 4

)

We also

compared

the

cryptochrome

sed light inducible Cre recombination system

CRY2

with iCreV.

The

omparison

in cell culture conditions

revealed that

iCreV

performs ~4 fold better after 60 minutes of light

stimulation than

the

2 system

hereas

the

2 based Cre recombination system allows only a slight

increase over baseline recombination

(

~1.2 fold

)

(

Supp.

Fig.

We were not able to test the Cry2 based

constructs

in vivo

since the

genes

were too

large

to fit into the

rAAV

viral vectors

we used.

Our results

suggest

that under the assayed conditions,

VVD

based optogenomic control

tighter and more

suitable for

certain

downstream applications

compared to the previously described systems

Design and screening

light

inducible Flp rec

ombinase

increase intersectional genomic modification

possibilities,

we turned into a third wi

dely used site

specific DNA

recombinase: Flp

here

isn’t enough

protein

sequence

similarity

between Flp,

a recombinase from yeast, and

Cre or Dre, recombinases from bacterial phages

to guide a split based on homology

Thus, we resorted to

structure

based

de novo

design

and screening for split

sites based on c

rystal structure of the Holliday junction

bound Fl

. Using

a more efficient

codon

optimized variant of Flp

(FlpO)

; we made Flp splits at 21 loop

locations that

correspond to

transitions

between

alpha helices and

/or

beta sheets with hopes to not alter overall

functionality within the dimer form

We generated

these

VVD

fused split

Flp

V constructs using the iCreV

backbone by replacing the N

and C

termini of Cre with the 21 split Flp variants (

Supp.

Fig.

After gene

synthesis,

these constructs were cloned into CMV promoter

driven mammalian expression plasmids

and tested for light

induced recombination in

cell culture

via transient transfections

together

with

a Flp

dependent

fluorescent

reporter plasmid. Amo

ng the variants,

FlpV2, 19 and 20 gave the most significant light

induced

recombination

(

Supp. Fig. 5b

ark conditions

for

ll these cases were almost identical to the controls indicating

that,

for

iCreV and iDreV, the background recombination

was

almost non

existent.

n an effort to

further improve the efficiency of light

inducible

Flpase

activity

fter

initial scre

en, we

designed

62 more iFlpV

variants

to scan

Flp

split sites surrounding those of iFlpV2, 19 and 20

(

Supp. Fig. 5c

)

Among all these

constructs

FlpV2 still yielded the highest

efficiency.

ight

inducible recombination with RecVs

with

whole

brain infect

ions

in vi

Recently

a new blood brain barrier permeable rAAV serotype, PHP.eB, was reported to be effectively infecting

large proportions of the entire nervous system in mice after retroorbital delivery

Thus,

we reasoned

this

technique

could be a

good and

rapid

approach

for ex

amining RecV constructs in the entire mouse brain

examined background

recombination in the absence of light in

any part of

the brain

as well as

spatial restriction

light inducib

recombination.

For this

purpose,

we generated

EF1

promoter

driven Cre

expressing rAAV as well as RecV rAAVs

of the

PHP.eB serotype.

found

that

PHP.eB EF1a

Cre virus, when injected

either

intracerebroventricularly (ICV)

retroorbitally into the

fluorescence Cre

reporter mice

efficiently

infect

the entire brain

relatively

homogenously

(

Fig.

, Supp. Fig. 6a

To our knowledge

this is the first demonstration of effective PHP.eB

mediated gene delivery into the

brain

using the ICV route.

This

method may help

further

restrict

the infection

nervous system and may

help

overcome obstacles related to intravenous delivery of

genes for both basic

research as well as gene therapy field applications.

We next tested the light

inducible recombination using RecVs with whole brain infections. To do

first we

injected a mixture of

PHP.eB

NDreV and CDreV virus

into the right ventricle of

the

Dre

dependent

fluorescent

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reporter

mouse

, Ai66R.

fter

an incubation period of two weeks,

the left hemisphere

exposed to light.

gradient of recombination

could be observed from the top of the left hemisphere to deeper

structures

(

Fig.

eper brain structures and sites far

away

from the light stimulation did not have any fluorescently labeled cell

except for

ICV needle tract

where

recombination took

place

likely due to multiple copies of infection.

In addition,

we tested the iCreV

construct

with PHP.eB serotype

delivered by ICV or RO routes,

in a Cre

dependent nuclear

reporter mouse line

, Ai75

. Very similarly

we observed substantial recombination in

the hemisphere

exposed to

light regardless of the

viral delivery route

(

Fig.

and Supp. Fig. 6b

Equivalent tests of the

iDreV

construct

a Dre reporter line yielded a gradient of recombination from the light stimulated hemisphere

(

Fig.

As an

ditional control iCreV

injected Cre

dependent repo

rter mice were maintained without light stimulation for four

weeks after initial injection. No significant amount of recombination was observed, indicating tight optogenomic

control under these conditions (

Supp. Fig. 6

). Finally, equivalent tests of

the iFlpV construct in the Flp reporter

mouse line resulted in efficient and specific light

induced recombination

(

Fig.

These results

indicat

that

the technique allows highly specific light

inducible reco

mbination.

Thus far all the recombinase repor

ter mouse lines we used were generated via targeted insertions into the

Rosa26

locus

. To test if a different locus

could be modified by the RecV system, we RO injected PHP.eB iCreV

rAAVs into the Ai167 ChrimsonR reporter mouse line, which is inserted into the Tigre

locus

. We

again

observe

efficient recombination

shaped

as a gradient

from the

light

stimulated

left

hemisphere

confirming that other

genomic

loci

in mice

can be modified by the RecV system

(

Supp. Fig. 6

also

tested

the

feasibility of light inducible recombination within a

deeper brain area

striatum

. Deep

brain

imaging

or recording is

more challenging due to tissue damage and blood clotting

;

both create

obstacles

for

light

passing

through

the tissue

By utilizing 1P

light through an optical fiber we were

able

to induce loc

recombination

via

iCreV in

the

striatum of

Ai162

GCaMP6s Cre

reporter mice

and record

changes in

fluorescence due to calcium concentration

dynamics

befo

re and after light stimulation (

Supp.

Fig

)

ue to

the localized

illumination

GCaMP

was expressed

directly

under the

fiber

in comparison

to the

wide

expression of

red fluorescence coming from the control virus

(

Supp

Fig

)

This

expression pattern

beneficial

for

calcium

imaging and recording, as

it allows expression directly at the desired location

Our results

show

that light

mediated genomic modifications can be efficiently spatiotemporally regulated

in vivo

also

with

deep

brain

targets

therefore

providing a tool for

restricted

reporter expre

ssio

n under the optical device

Cell

class specific targeting by intersection of viral RecV

and transgenic recombinases

Versatile and refined cell type targeting can be achieved by combined use of two or more

recombinases with

distinct

recombination ac

tivit

ies

For example,

new RecV tools could be combined with existing transgenic

recombinase lines and intersectional reporters.

To test the feasibility of this approach

injected PHP.eB iFlpV viruse

toge

ther with a

PHP.eB

Cre/Flp

dependent

fluorescent

reporter

virus

into

the cortex of mice containing the

Rbp4

Cre

100 transgene, which

drives Cre expression mostly in L5 excitatory cortical cells

. After unilateral light stimulation we observe

layer

spe

cific reporter gene

expression

within the light

stimulated hemisphere

in these mice

(

Fig.

)

, suggesting high

degree of intersectional specificity under the tested conditions

To provide further evidence that light

mediated intersectional targeting can b

e achieved in other

neuron types,

we used the Sst

IRES

FlpO

mouse line, which expresses FlpO recombinase selectively in inhibitory somatostatin

(Sst) neurons, in conjunction with iCreV. We crossed this mouse line to a Cre/Flp double dependent fluorescent

eporter mouse line

Ai65. The resulting double

positive mice were retro

orbitally injected with PHP.eB iCreV

viruses to infect the entire brain relatively homogenously. Reporter expression was observed in sparsely

distributed neurons close to the light stim

ulation site across

multiple cortical layers (

Fig

). To ascertain that

the recombination we observed within the light induced hemisphere is indeed specific to the Sst

positive neurons,

we performed immunohistochemistry and found that all the fluorescent

ly labeled neurons

were also immune

reactive to the SST antibody (

Fig

)

. These results confirm that tight intersectional control can be achieved by

combining virally delivered iFlpV or iCreV with transgenic class

specific Cre or FlpO, respectively.

(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint

http://dx.doi.org/10.1101/553271

doi:

bioRxiv preprint first posted online Feb. 18, 2019;

eV induces

tight

light

dependent recombination in zebrafish

embryos

in man

y tissue types.

To determine if RecVs could effectively induce light

dependent recombination in

other loci and organisms other

than mice we

tested it in

another model organism: Dani

o rerio

(zebrafish)

. W

e co

injected plasmids containing

either

CreV or C

CreV under the control of the ubiquitous ubiquitin (

ubi

) promoter into single cell

zebrafish

embryos of transgenic zebrabow strain tg

(

ubi:Zebrabow

)

and reared embryos either in the light or the dark

for 72 hours. In

this line within

the default state, RFP is exp

ressed in all cells. After Cre

dependent

recombination

YFP and/or CFP

are expected to be

expressed. Of the 24 viable embryos reared in the dark

1/24 displayed any recombination as measured by

the presence of YFP or CFP. Of the 37 viable embryos

reared in

the light nearly all

(

32/37

)

had YFP or CFP expression, confirming that

CreV

induced recombination

was light dependent. We observed recombination in many tissues including muscle, skin, heart,

spinal cord

neurons and non

neuronal cells,

hindbrain, trigeminal ganglion, Rohon

Beard sensory neurons

and

hair cells of

the lateral line (

Fig

. 3

), suggesting that Cre

is effective at inducing recombination across many cell types

and tissues

RecV

diated sparse labeling enables

whole

brain reconstruction of single neuron morphologies

The development and

in vivo

validation of the light

inducible recombinase system (RecV)

opens

doors to many

applications. Here we present

several of

such application

mainly using

light to control the precise location and

number of neurons labeled to achieve the desired sparsity of targeted neuronal populations for the

visualization

functional imaging

as well as

reconstruction of single neuron morphologies across th

e entire bra

in to

understand

their axonal projection specificity.

Cre

mediated

in vivo

reporter expression using a Cre reporter mouse line Ai139 result

in strong expression of

GFP in multiple cortical layers. Ai139 is a

TIGRE

2.0

reporter line expressing

very

high level of GFP (via tTA

mediated transcriptional amplification) allowing visualization

of detailed cellular morphologies

We tested

if light

applied to the tissue could

sparsely

induce

CreV, to result in

sparse yet strong expression

of the reporter at the

single cell level, which would enable

whole

brain reconstruction of single neuron morphologies.

For this purpose, we injected the cortex of Ai139 mice with the 1:1 mixture of NCreV and CCreV rAAVs in ser

ial

dilutions and applied various durations of light across the skull

induce recombinat

ion in a sparse population of

cortical neurons. We then imaged the whole brains using the fluorescence micro

optical sectioning tomography

(fMOST) technique

Lower dose of CreV virus

es and shorter duration of light induction led to sparse and strong

labeling of

individual neurons (

Supp.

Fig.

). Sparseness was low enough that axons from many neurons could

be traced. In the example brain shown in

Figure

(also see

Supp.

Movies 1 and 2

), 8 primary somatosensory

cortical neurons were manually reconstructed. These n

eurons include 3

ayer 2/3 pyramidal cells (PCs) with

ipsilateral cortico

cortical projections, 2

ayer 2/3 PCs with contralateral cortico

cortical projections, and 3

ayer

5 t

hick tufted PCs with ipsilateral cortico

subcortical projections, revealing dist

inct axonal projection patterns.

many

cases

where

restricted

genetic access is not possible

with conventional methods

desirable to further

confine

the region of r

eco

mbination by precise

induction

through localized

illumination

demonstrate this

ability

we performed

headpost craniotomy and

two

photon (

)

ssisted local laser stimulation experiments

trained head

fixed

Ai139 reporter mice

To achieve sparseness,

injected

a 1:5 mixture of rAAV iCreV and

rAAV tdTomato

expressing viruses

into the

primary visual cortex.

The

tdTomato

virus

was

included

to guide our

light

stimulation

as well as to serve as a control of overall infection

breadth

. Our results indic

ate that local

and

sparse labeling of neurons is achievable using this method. Furthermore, due to the strength of the

Ai139 reporter

expression

individual axons and boutons can be readily visualized without immunohistochemical enhancement

at sites far aw

ay from neuron

al cell

bodies

, suggesting that

these brains could be subjected to

whole

neuron

reconstruction

(

Supp.

Fig.

)

RecVs

enable

mediated single cell specific targeted

optogenomic modifications in combination with

functional imaging

in vivo

(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint

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doi:

bioRxiv preprint first posted online Feb. 18, 2019;

powerful application for the light

inducible iCreV system is to identify

e.g.

functionally

genetically

individual

cells of interests and then activate the Cre

dependent gene expression in these

defined cells at will. However,

illumination is not s

uitable for this application, since it inevitably activates the iCreV in cells

within

the

light

path in addi

tion to the target. One approach to achieve single

cell specific targeting is

through 2P

activat

ion

iCreV

which minimizes

unspecific targeting. T

o investigate the feasibility of this approach

in vivo

at single cell

level

, we next tested the iCreV system

using

induction in m

ouse

somatosensory

cortex (

Fig

We co

injected iCre

and GFP

expressing viruses in the

somatosensory

cortices

of a

dependent red

fluorescent reporter mouse line (Ai14)

. The GFP fluorescence served as a landmark for target cells. After

identifying the target cell, we focused the laser in the middle of the cell soma (potentially nuclear region), and

then

laser

scanned

within this small region

as a

window of

~6x8

under various conditions of

laser power

and time. Seven to ten days after this initial induction session, we re

imaged the target cells to examine the

expression of tdTomato. In order to reveal all the ind

ucible cells in the region, we then exposed the neu

rons

LED

light

for 30 minutes, and then counted all the tdTomato

exp

ressing cells seven days later.

We showed that

induction of iCreV led to

dependent gene expression in target cell with singl

cell

precision. In many cases, we observed potent

tdTomato expression in the target cell but not in the inducible cell

right

next to it (

Fig

and Supp

Fig.

), demonstrating the high spatial specificity. We tested various

induction protocols and

plotted the probability of iCreV activation against the distance to the target cell in each

condition (

Fig

). The results showed that target cell induction rate increased as more laser power and scan

time was applied during the

induction. However, power

ful induction in some cases also led to

nspecific

induction within 10

radius of the target cell (as high as 18%), which may

result

from the activation of the

iCreV constructs in the neural processes surrounding the target cell

, or from micron

level mo

vement of the mouse

brain during the induction window. Cells within

the

radius from the target cell showed a baseline

induction rate of 6

7%. Since the induction rate observed in session 1 was very low (~3% on average,

Supp

Fig.

), this non

tar

geted induction was likely due to the non

specific activation of iCreV by ambient

light during

mouse handling, by the two

photon stimulation during acquiring the Z

stack images

or by spurious recombination

due to

very

high multiplicity of infection with th

e viral vectors

We next tested the feasibility of combining calcium imaging with

the iCreV system, by measuring the induction

probability of iCreV after 30 minutes

calcium imaging. We co

injected iCreV and GCaMP7f

viral constructs

Ai14 mice cortex. 920 nm excitation provided good quality jGCaMP7f signal, however

was also potent in

activating the iCreV system (close to 10

0%).

reduce

iCreV

induction during the imaging session, we tested

longer wavelengt

h excitation of jGCaMP7f at 1000, 1010 and 1040 nm. At these wavelengths, the induction of

iCreV was significantly reduced, with moderately reduced jGCaMP7f signals (

Fig.

DISCUSSION

this

study, we

used

wild

type VVD

to create three light

induci

ble recombinases Cre,

Dre, and Flp, thus

expanding the repertoire of

optogenomic

manipulation tools even further. We also showed that these light

inducible recombinases work highly efficiently and intersectionally in the

mouse

brain

to label specific cell

classes

or types. We further demonstrated that RecVs allow effective light induc

ed optogenomic modifications in multiple

loci within the mouse genome and zebrafish.

provide

proof

principle

experiments showing

that RecV mediated light

inducible site

specific DNA

modification

are possible

in the mammalian nervous system at sing

le cell level.

e provide

examples of single

cell

soma

targeted

mediated

optogenomic modifications

and established imaging and conversion parameters

to induce

such

modifica

tions with unprecedented

spatial

resolution.

Our data also provide a quantitativ

description of the induction specificity of iCreV at different 2P wavelengths and support the feasibility of

combining calcium imaging with iCreV system in mice

in vivo

Ligh

inducible Cre

recombinases were

reported previously

32,33,54

, and they were mainly based on two types of

light

inducible protein dimerization systems: CRY2

CIB1

and

Magnets

A direct comparison of

improved

CRY2

based

and the

VVD

based system presented in this study shows that the CreV system

may be preferable

ue to its higher level of inducibility under the conditions tested

in vitro

Additionally, our results indica

te that

the

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doi:

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heterodimeric

Magnet

based light

inducible Cre recombination is efficient, but it is also relatively leaky compared

our

VVD

based counterpart

both

in vitro

and

in vivo

incorporating the improved designs reported

in the

Magnet

system

we were able to produce efficient RecV co

expression constructs

, thereby decreasing the

number of independent components that need to be delivered to the experimental animal

The demonstration

the

ICV route for efficient whole

brain infection

usin

g PHP

.eB AAV viruses

could

valuable

in many scenarios. For example, it could

infect the brain

of embry

s in which RO

vein

is too small to

inject

or to avoid damage to the eye of the

embryo since in mice RO route requires penetrating a needle next to

eye

This approach

may

further

help

focus infection

the nervous system and

overcome obstacles related

to intravenous delivery of genes for basic research

a multitude of mammalian species. Genetic modifications

of larger animal models such as prima

tes

are expensive. Intravenous delivery in the adult primate may require

substantial number of viral particles.

Thus,

the PHP.eB

viruses are

delivered by the ICV route during adulthood

or development, it may help

infect majority of the nervous system

th much less virus

. This

approach

may also

overcome immune

response related issues

that interfere with infections

. Finally

this

method

may be useful in

clinical

applications

for gene therapy purposes

The spatiotemporally selective targeting of cell popul

ations or individual cells using light could allow fine

scale

combinatorial functional, anatomical and circuit

level studies of cell type

specific networks.

In addition to

combining

opsin expression with

CreV,

and

achieving

efficient GCaMP expression usin

CreV

, t

he RecV viral

vectors and Cre/Flp

dependent

DreV or

FlpV mouse lines can be combined with existing cell type

specific

genetic tools, and RecVs can also be used to generat

e new driver lines.

New RecV versions incorporating drug

inducible (e.g. t

amoxifen

or trimethoprim (TMP) inducible

) recombinases can be developed to further reduce

background

recombination

and enhance tempor

al specificity.

With this approach

one can envision performing

a variety of combinatorial experiments including

single

cell activity imaging using genetically encoded voltage

57,58

or calcium indicators

; single cell monosynaptic rabies traci

6,7

or targeted optogenetic

and chemogenetic

protein expression

, all in a noninvasive or minimally invasive manner. The sp

ectral and temporal separation

well as drug

inducible versions could enable sequential investigations of funct

ionally relevant cell populations

followed by light

mediated DNA recombination to selectively activate effector gene expression in specific cells of

interest

to examine their structure and connectivity and/or to perturb their function.

This technology c

ould also enable

loss/gain

function studies

by swi

tching genes off or on with much refined

regional and cell type specificity

, followed by monitoring of

effects on physiology or animal behavior

. One

could

also

employ this method to

study development

switching genes

on or off

with

high

temporal precisio

n during

the rapid cascade of events throughout the proliferation and differentiation processes. The half

life of the VVD

homodimer is ~2 hours after brief light activation

. One

can

try to apply targeted light at very precise time points

using standard or gentler

in utero

techniqu

es. This

may be

advantageous over the current CreER

based genetic

fate mapping studies

as it would eliminate

the

tamoxifen

side effects

as well as provide

spatially and temporally

more precise

recombination

. Fur

thermore, this approach

could

allow highly targeted

neuronal

network

reconstructions by selectively labeling neurons in particular locations or having

particular activity patterns or

functional properties (

e.g.

, using 2P induction). Sparse expression of re

porter genes using diluted Cre viruses or

low doses of tamoxifen in CreER mice already exist. However, with RecV

one

can

perform even more

spatiotempo

rally restricted induction, and it can be combined with prior functional characterization using 2P

imagin

and

optical indicators in an intersectional manner.

The RecV approach can also be used in other species of interest in which light could be used to

access individual

cells of interest. For model systems in which germline genetic modification is feasible

, the experiments can be

designed to integrate multiple components of the described systems into the germline

. In model systems in

which germline modification is not available

or is prohibitively expensive

, these

experiments can be performed

by using viral vectors in which a certain level of cell

type specificity may be achieved by using short cell type

specific promoters or enhancers

63,64

, or target

defined specificity ma

y be achieved with highly efficient

retrogradely infecting designer viruses

Overall, the broad range of potential applications show that the light

inducible recombinase system should

enable much improved spatio

temporal precision and multiple combinatorial st

rategies

for

the micro

and macro

level analys

s of neural circuits

s well as many other biological systems

in a multitude of organisms

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doi:

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METHODS

Plasmid and virus construction for

RecVs

Sequences of NCre

VVD, VVD

CCre, NCre

VVD

IRES

VVD

CCre, VVD

CCre

IRES

NCre

VVD, NCre

VVD

PQR

VVD

CCre, VVD

CCre

PQR

NCre

VVD, VVD

CDre

IRES

NDre

VVD, NCre

Magnets

NLS

P2A

NLS

Magnets

CCre, NCre

VVD

NLS

P2A

NLS

VVD

CCre,

NDre

Magnets

NLS

P2A

NLS

Magnets

CDre, NDre

VVD

NLS

P2A

NLS

VVD

CDre, and all

FlpV versions were chemically

synthesized (GenScript, Piscataway, NJ).

59 amino acid N terminus

and

343

C terminus of Cre were used

in all CreV cases.

To screen poly

cist

ronic cassettes wi

th the best light

inducible recombinase activity, N and C

parts of RecV, as well as IRES

mediated, PQR

mediated and P2A

mediated RecV poly

cistronic cassettes were

cloned into pCDNA3.1 with the CMV promoter (

Supp.

Fig. 1

To generate re

combinant AAV viru

ses expressing split VVD

Cre (CreV) or VVD

Dre (DreV), the N or C part of

CreV and DreV were cloned after the human EF1a promoter, followed by WPRE and hGH

polyA signal (

Supp.

Fig. 1

). The Cre reporters, pAAV

EF1a

Flex

dTomato or EGFP

WPR

hGHpA, used pair

s of double inverted

LoxP and Lox2272 sites to flank the reporter dTomato or EGFP sequence. The Dre reporter, pAAV

EF1a

Frex

dTomato

WPRE

hGHpA, was generated by inserting an inverted dTomato sequence flanked with Rox sites after

the huma

n EF1a promoter, f

ollowed by WPRE and hGH

polyA signal (

Supp.

Fig. 1

FlpV variants were generated with custom gene synthesis as follows:

FlpV1: 11 amino acids (aa) N and 412

aa C;

FlpV2: 27 aa N and 396 aa C;

FlpV3: 49 aa N and 374 aa C;

FlpV4: 67 aa N and 356 aa

FlpV5 72 aa

N and 351 aa C;

FlpV6: 85 aa N and 338 aa C;

FlpV7: 95 aa N and 328 aa C;

FlpV8: 114 aa N and 309 aa C;

FlpV9: 129 aa N and 294 aa C;

FlpV10: 151 aa N and 272 aa C;

FlpV11: 169 a

a N and 254 aa C;

FlpV12: 197

aa N and 226 aa C;

FlpV1

3: 208 aa N and 215 aa C;

FlpV14: 237 aa N and 186 aa C;

FlpV15: 251 aa N and

172 aa C;

FlpV16: 290 aa N and 133 aa C;

FlpV17: 318 aa N and 105 aa C;

FlpV18: 343 aa N and 80 aa C;

FlpV19: 374 aa

N and 49 aa C;

FlpV20: 388 aa N and 35 aa C;

FlpV21:

408 aa N and 15 aa C.

Additional

iFlpV2 variants were generated spanning amino acids

and 366

405

covering the entire region

leading to

61 additional constructs

onstruct

was generated based

on iFlpV2 with an addition of the linker GGSGG

present b

etween the C terminus VVD and FlpV to also N terminus FlpV and VVD.

These constructs were cloned

in pcDNA3.1 mammalian expression plasmids.

AAV1, AAV DJ and AAV PHP.eB serotype viruses were

produced in house with titers of AAV1

EF1a

NCreV,

1.05 x 10

gen

ome copies

(GC)

; AAV1

EF1a

CCreV, 5.16 x 10

; AAV1

EF1a

NDreV, 4.20 x 10

; AAV1

EF1a

CDreV, 5.40 x 10

; AAV

EF1a

Cre, 2.00 x 10

; AAV1

CAG

Flex

EGFP, 1.34 x 10

; AAV

EF1a

Frex

dTom

ato, 1.90 x 10

; 7.7 x 10

; 1.6 x 10

; AAV

PHP.eB

EF1a

Cre, 5.

8 x10

; AAV

PHP.eB

Syn

NDreV, 4.2 x

; AAV

PHP.eB

EF1a

CDreV, 3.9 x 10

;

PHP.eB iCreV, 2.6 x 10

;

PHP.eB iDreV, 3.3 x 10

PHP.eB iFlpV,

2.7 x 10

AAV

PHP.eB

EF1a

eGFP

2.03E+13

and

AAV

Cre

Magnets

3.00E+13

per ml.

AAV5.CAG.tdTomato

(1.0 x 10

C/mL) were purchased

from

UNC Vector core.

Light activation in cultured cells

HEK293T cells were seeded into 6

well plates one day before transfection and reached 80% confluency on the

day of transfection. Cells were co

transfected with a reporter express

ing dTomato

–

for Cre and Flp

or dTomato

for Dre

for Cre, Dre or Flp mediated

recombination and various constructs of split

RecVs

. Cells in the control

groups were transfected with reporters alone. Each condition contained 4 replicates. Plates were kept i

n dark

immediately after transfection. Twenty

four hours later, cells were expo

sed to blue light, and were then kept in

dark immediately after light exposure. Cells were imaged for fluorescent reporter expression 48 hours after light

induction, using an in

verted fluorescence microscope.

RecV

activated dTomato expression in each condi

tion

was quantified using Image J.

Surface/Cortical

In vivo

1P optogenomic modifications

Stereotaxic injections were made into adult C57BL/6J (stock no. 00064, The Jackson Lab

oratory, Bar Harbour,

ME) or transgenic reporter mice with a 1:1:1 or 1:1 mixtu

re of three or two different rAAVs. For all experiments,

animals were anesthetized with isoflurane (5% induction, 1.5% maintenance) and placed on a stereotaxic frame

(model no.

1900, David Kopf Instruments, Tujunga, CA). An incision was made to expose the

skull, including

(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint

http://dx.doi.org/10.1101/553271

doi:

bioRxiv preprint first posted online Feb. 18, 2019;

regma,

da, and the target sites. Stereotaxic coordinates were measured from Bregma and were based on

The Mouse Brain in Stereotaxic Coordinates

66,67

. A hole was made above the target by thinning the skull using

a small drill bit until only a very thin layer remained. An opening was then made using a micro

probe, and the

remaining thinned skull was gently pulled away

. All animals were injected at each target with 500 nl of virus at

a rate of ~150 nl/min using a Nanoject II microinjector (Drummond Scientific, Broomall, PA). Intraventricular

injection of AAV

PHP.eB viruses was conducted by injecting 2 μl of virus into

the lateral ventricle using a Nanoject

II microinjectior. The glass pipettes had inner diameters between 10

20 μm.

Two weeks following AAV injection, animals were anesthetized and returned to the

stereotaxic frame. An incision

was made in the previous location to once again reveal the location of the injection sites. An LED light source

(LED

64s, Amscope, Irvine, CA) was mounted to the surgical microscope and positioned 3

4 inches directly

above

the animal’s skull. The amount of time the animal was exposed to light was varied by experiments. Small

amounts of sterile PBS were periodically applied to the scalp and skull to prevent drying.

Two weeks following light exposure, animals were perfused

with 4% paraformaldehyde (PFA). Brains were

dissected and post

fixed in 4% PFA at room temperature for 3

–

6 h and then overnight at 4°C. Brains were then

rinsed briefly with PBS and stored in PBS with 10% sucrose solution. Brains were then sectioned at a t

hickness

of 100 μm while frozen on a sliding microtome (Leica SM2010 R, Nussloch, Germany). Brain sections were

mounted on 1x3 in. Plus slides and coverslipped with Vectashield with DAPI (H

1500, Vector Laboratories,

Burlingame, CA). Slides were then image

d using a 10x objective on a Leica TCS SP8 confocal microscope

(Leica Microsystems, Buffalo Grove, IL).

For fMOST imaging, two weeks or longer following light exposure, animals were perfused with 4%

paraformaldehyde (PFA). Brains were dissected and post

ixed in 4% PFA at room temperature for 3

–

6 h and

then overnight at 4°C. Brains were then transferred to PBS with 0.1% sodium azide for storage at 4°C until

embedding.

Surface/Cortical

n vivo

population

optogenomic modifications

A titanium head

plate

was attached to the skull of mice to allow positioning and restraint of the animal during

imaging. The hole of the head plate was positioned over visual cortical areas, approximately 2.9 mm posterior

and 2.7 mm lateral from Bregma. A 5 mm craniotomy

was cu

t into the skull using a dental drill. The dura was

then removed, and a multilayer glass coverslip was positioned above the craniotomy. The head plate and

coverslips were secured using cyanoacrylate glue and metabond. After a period of at least one w

eek, a

dental

drill was used to remove the cement and metabond holding the coverslip in place, and the coverslip was

removed. A Dumont Nanoject II was then

used to inject 500 nL of viruses

into visual cortex. A new coverslip was

placed and adhered. The ar

ea abo

ve the coverslip was blocked from light using a combination of dental cement

and

Kwik

cast, both mixed with black acrylic paint powder.

After at least 3 weeks following viral injection, the animal received two

photon laser stimulation. Under dark

conditi

ons, the

wik

cast was

removed,

and the animal’s head plate was mounted in position. The injection area

was identified by the presence of

the EGFP labelled cells. Laser output was set to 900 nm to optimally induce

recombination. A 600 x 600 μm area was sti

mulated at three depths (100 μm, 150 μm, and 200 μm) for 15

minutes each. After stimulation, black

Kwik

cast was reapplied. Two weeks foll

owing stimulation, mice were

perfused.

eep

brain

in vivo

stimulation/imaging experiments

For deep brain optogenomic m

odification and imaging experiments stereotaxic

injections were made into

Ai162

GC6s (Stock No. 031562, The Jackson Laboratory, Bar Harbour, ME)

Cre dependent

GCaMP6s reporter

mice with a 1:1 mixture of PHP

eB.iCreV and a

control

AAV

unconditionally expres

sing red fluorescent protein

(AAV5.CAG.tdTomato)

into the striatum

. For all experiments, animals were anesthetized with isoflurane (5%

induction, 1.5% maintenance) and placed on a

stereotaxic

frame

(

942

, David Kopf Instruments, CA, USA). An

incision was ma

de to expose the skull, including

regma,

da, and the target sites. Stereotaxic coordinates

were measured from Bregma and were based on The Mouse Brain Atlas

66,67

full

hole was made above the

target. All ani

mals were injected with 400 nl X 2 of virus mixture, at two

dorsoventral positions

, 300um apart,

at a rate of ~80 nl/min using

UltraMicroPump

(

UMP3

4, World Precision Instrument

Sarasota

FL)

Following

(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint

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doi:

bioRxiv preprint first posted online Feb. 18, 2019;

virus

injecti

, an optical fiber with cut length of

5 mm and diameter of 400

m (NA 0.48, Doric lenses, Quebec,

QC, Canada) for

stimulation

was firmly mounted to a stereotaxic holder.

The

optical fiber

was then inserted

to the

triatum (AP

+1.0

mm, ML ± 1.3 mm, DV

3.5 mm, from either left or right sid

e) through a

craniotomy

and

positioned

300

m above the deeper viral injection site. A thin layer of

metabond

was applied on the skull

surface to

secure

the fiber.

In addition,

a thick layer of black dental cement was applied

to secure

fiber implant

for 1P

illumination

to allow positioning and restraint of the animal.

One week following AAV injection of 1:1 virus mixture of PHP.eB.iCreV

and

AAV5.CAG.tdTomato into

GCaMP6s reporter

mice, animals’ baseline signals were recorded

with a

fiberphotometry rig for 1

0 minutes

the

home

cage

. Fiberphotometry is a method

for

measuring population

calcium

dependent

fluorescence from

genetically defined cell types in deep brain structures usin

g a single optical fiber for both excitation and emission

in freely moving mice

. Detailed description of the system can be found elsewher

After recording, mice were

connected to a 447nm laser (Opto Engine LLC, UT) using a 200

μ

m optical fiber, illuminated with 5m

, 100ms

pulses, 1Hz for 3

0 min

utes

(

TTL

controlled by

OTPG_4, Doric lenses, Qu

ebec, QC, Canada)

, in the home

cage

. A week following light exposure, fiberphotometry signal was recorded again

for 10 minutes

Fiberphotometry peak detection was performed with MATLAB (R2018a), using ‘f

indpeaks’ function, using a

prominence of 2.5.

Mice were perfused 4 weeks after illumination.

Animals were perfused with 4% paraformaldehyde (PFA). Brains were dissected and post

fixed in 4% PFA

overnight at 4°C. Brains were then rinsed briefly with PBS a

nd then sectioned at a thickness of 100 μm on

vibr

tome (VT1200 Leica Biosystems, IL, USA). Brain sections were incubated in a blocking solution, containing

1x PBS solution

with

0.1% Triton X

100 and 10% normal donkey serum (NDS; Jackson ImmunoResearch,

PA,

USA) for at least an hour, washed, and fur

ther incubated with blocking buffer containing primary antibody (see

below for details) at 4C overnight. Afterward, sections were thoroughly washed three times (15 min each) in 1x

PBS and then transferred into

the

blocking solution with secondary antibody

(see below for details) for 2h at

room temperature.

Finally,

sections were again washed by 1x PBS solution four times (15 min each), mounted

on glass microscope slides (Adhesion Superfrost Plus Glass Slides, #5

075

Plus, Brain Research Laboratories,

MA, USA

), dried, and coverslipped with mounting media (Prolong Diamond, P36965, Thermo

Fischer, CA, USA).

For primary antibody: chicken

anti

herry (1:1000; ab205402, Abcam, Cambridge, UK

)

was used

. For

secondary ant

ibody, anti

chicken Alexa Fluor 594 (1:500; 70

585

155, Jackson ImmunoResearch) was used.

Fluorescent images from brain tissue were acquired

with

LSM 880 confocal microscope (Carl Zeiss, Jena,

Germany). We used a 10x Plan Apochromat air objective (NA

0.45), 25x Plan Apochromat water immersion

obj

ective (NA 1.2) and

three

laser wavelengths (488 nm, 561 nm, and 633 nm). Image acquisition was

controlled by Zen 2011 software (Zeiss), which also allowed automated tiling, and maximum intensity

projection. Im

ages were not further processed.

Expression co

unts were done by summation of the values of

the fluorescence within 1mmX1mm below the fiber tip subtracted with the same area at the opposite

hemisphere, line by line, and normalized to the maximal value.

brafish experiments

CreV and C

CreV were

PCR amplified from pAAV

Ef1a NCreV and pAAV

Ef1a CCreV, and cloned into the

pDest

ubi vector (Addgene plasmid # 27323) by Gibson assembly with the following primers: 5' N

Cre overlap

pdest

UBI: attcgacccaagtttgtac

aaaaaagcaggctggacgccaccatgacgagtgatgaggtt;

5' C

Cre overlap pdest

UBI:

tcgacccaagtttgtacaaaaaagcaggctggacgccaccatgcatacactgtatgcccc; 3' polyA (used for both constructs):

actgctcccttccctgtccttctgcatcg

atgatgatccagacatgataagatacattga

The pDest

ubi:N

vCre

pDes

t and pDest

ubi:C

vCre mixture containing e

qual amounts of each plasmid (25pg

each) and tol2 transposase RNA (25 pg) was injected into one

cell stage tg(ubi:Zebrabow

M) zebrafish embryos.

Embryos were either light or dark reared for 72 hrs. At 3dpf injecte

d embryos were anesthetized with Mesab,

mou

nted in 2% agarose, and imaged on a Zeiss LSM 880 confocal microscope.

fMOST imaging

All tissue preparation has been described previously

. Following fixation, each intact brain was rinsed three

times (6 h for two washes and 12 h for the third wash) at 4°C in a 0.01 M PBS solution (Sigma

Aldrich Inc

., St.

Louis, US). Then the brain was subse

quently dehydrated via immersion in a graded series of ethanol mixtures

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(50%, 70%, and 95% (vol/vol) ethanol solutions in distilled water) and the absolute ethanol solution three times

for 2 h each at 4°C. After d

ehydration, the whole brain was impregnated

with Lowicryl HM20 Resin Kits (Electron

Microscopy Sciences, cat.no. 14340) by sequential immersions in 50, 75, 100 and 100% embedding medium in

ethanol, 2 h each for the first three solutions and 72 h for the fi

nal solution. Finally, each whole brain was

embedded in a gelatin capsule that had been filled with HM20 and polymerized at 50°C for 24 h.

The whole brain imaging is

performed

using a fluorescence microscopic optical sectioning tomography (fMOST)

system. T

he basic structure of the imaging

system is the combination of a wide

field upright epi

fluorescence

microscopy with a mechanic sectioning system. This system runs in a wide

field block

face mode but updated

with a new principle to get better image contras

t and speed and thus enables high

throughput imaging of the

fluorescence protein labeled sample (manuscript in preparation). Each time we do a block

face fluorescence

imaging across the whole coronal plane (X

Y axes), then remove the top layer (Z axis) by

a diamond knife, and

then expose n

ext layer, and image again. The thickness of each layer is 1.0 micron. In each layer imaging, we

used a strip scanning (X axis) model combined with a montage in Y axis to cover the whole coronal plane

. The

fluorescence, collected using a microscope o

bjective, passes a bandpass filter

and is recorded with a TDI

CCD

camera. We repeat these procedures across the whole sample volume to get the required dataset.

The objective used is 40X WI with numerical aperture (NA) 0.8 to provide a designed optical res

olution (at 520

nm) of 0.37 μm in

XY axes. The imaging gives a sample voxel of 0.30 x 0.30 x 1.0 μm to provide proper resolution

to trace the neural process. The voxel size can be varied upon difference Objective. Other imaging parameters

for GFP imaging i

nclude an excitation wavelength of

488 nm, and emission filter with passing band 510

550 nm.

Cortical targeted in vivo 2P stimulation of single cells

For

in vivo

targeted single cell optogenomic modifications as well as

simultaneous

GCaMP

imaging

experim

ents Stanford

Administrative Panel on Laboratory Animal Care (APLAC) approved all animal procedures.

Ai14 mice (The Jackson Laboratory, 07908) of two to four months age were used for experiments. For viral

vector injection, mice were anesthetized with isof

lurane. iCreV

virus used: AAV2/PHP.eB

EF1a

Cre

; GFP virus

used: AAV2/PHP.B

CAG

GFP; GCaMP virus used: AAV2/9

CamkIIa

jGCaMP7f. Viral vectors were loaded into

a glass pipette and injected into cortex with picospritzer (Paker Hannifin). ~500 nL was deliver

ed over 15 min

utes

and then a 4 mm craniotomy was made with the injection site at the center, 30 minutes after the injection. Dura

was removed before cover glass was installed and sealed. A custom

made head bar and cover were secured

with dental cement on

mouse skull. I

maging experiments started one month after the surgery to allow gene

expression.

Mouse was mounted on a running wheel with head fixation and was remained awake during the whole

experiment. Before imaging, the head mount cover was removed. In

order to opera

te the mouse and the

microscope, a red LED light was used for illumination (WAYLLSHINE). Ultrasound gel (Parker, Aquasonic) was

put on the cover glass for the water immersion objective lens. The mouse was aligned manually without checking

the

focal plane w

ith eye piece to avoid iCreV induction during this process.

For induction experiment, a femtosecond Ti:sapphire laser (Spectra

Physics, Mai Tai) was tuned to 920 nm

wavelength. The scanning and image acquisition were achieved with a Prairie (

Bruker) two

oton microscope,

through a 20X 0.95 N.A. water immersion lens (Olympus XLUMPLFLN

W 0.95 NA 20×). For all the imaging

sessions, laser power at a specimen was kept at 25 mW and it was monitored (Thorlabs, PM100D and S130C)

at an additional outp

ut of the opti

cal path before entering the microscope, whose splitting ratio was calibrated

prior the measurements. During the first imaging session, a starting point with unique vessel pattern was

identified and recorded as the starting position. All targ

et cells’ rela

tive coordinates to the starting position were

recorded and used for relocation in later sessions. Images were acquired with 4 μs pixel dwell time at 1024 x

1024 pixel frame size (field of view 450 micron x 450 micron), with 3 μm Z

axis step

size. After id

entifying a target

cell, an induction scanning was carried out using the ROI function to limit the scanning the middle of the target

cell’s somata region. The scanning pixel dwell time is increased to 10 μs and various scanning conditions wer

used in each

mouse tested. After the first induction session, the head cover was re

installed to seal the

craniotomy from ambient light. Seven to ten days after the first session, all the cells were re

imaged to check

tdTomato expression and at the end u

nderwent a 30

minutes blue LED exposure (5 mW, 470 nm, Thorlabs,

M470L3). And the third imaging session was carried out seven to ten days after the LED exposure.

(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint

http://dx.doi.org/10.1101/553271

doi:

bioRxiv preprint first posted online Feb. 18, 2019;

For calcium imaging experiment, a custom

made two

photon microscope was used with an objective

lens

Olympus

XLUMPLFLN

W 0.95 NA 20×, 8 kHz resonant scanner head (Cambridge Technologies,

CRS8K/

6215H). Fluorescence light have been captured using H11706

40 and H10770PA

40 photomultiplier

tubes equipped with low noise amplifiers (Femto, DHPCA

100), wh

ose analog sig

nal was subsequently digitized

(National Instruments, NI

5732) and formed into images using field programmable gate array (National

Instruments, FPGA NI

7961R) and ScanImage software (Vidrio Technologies) [Pologruto et al., Biomedical

Enginee

ring Online 2:

13, 2003]. GCaMP signal was collected at 30 Hz frame repetition rate for 30 minutes in

each mouse.

Data availability

All relevant plasmids will be deposited to Addgene. Data are available from the corresponding author upon

request.

ACKNOWLE

DGMENTS

We are grateful to the Structured Science teams at the Allen Institute for their technical support in stereotaxic

injections and mouse colony management. The

work was funded by the Allen Institute for Brain Science, NIMH

BRAIN

Initiative grant

RF1M

H114106

to A.C.,

NSFC Science Fund for Creative Research Group of China

(Grant No. 61421064) to H.G., Q.L. and S.Z

., NIH Director's New Innovator award IDP20D017782

and

NIH/NIA 1R01AG047664

to V.G.

and

Colvin divisional fellowship

Division of Biology a

Biological

Engineering

California Institute of Technology

A.K.

NIH Brain Initiative

U01NS107610

grant to Mark

Schnitzer.

mmunohistochemistry

experiments

igure 8

ere

performed in the Biological Imaging Facility,

with the support of the

Califor

nia Institute of Technology

Beckman

Institute and the Arnold and Mabel Beckman

Foundation.

The cre

ation of Ai139

mouse line was supported by the NIH grant R01DA036909 to B.T.

The

authors thank Sevi Durdu

, Bilal Kerman

and Keisuke Yonehara for critical read

ing and feedback.

The authors

wish

to thank the Allen Institute founder, Paul G. Allen, for his vision, encourag

ement, and support.

AUTHOR CONTRIBUTIONS

A.C. concept

ualized

the light

inducible recombinase system. S.Y. performed cloning and characterizati

on of the

construct

as well

participated in image acquisition. B.O. performed all the surgeries and image acquisition.

T.Z. Performed cloning. M.M. performed some of the surgeries and light stimulations. T.D. performed some of

the initial cloning exper

iments. B.T. and H.Z. contribute

d to the generation of the Ai139 transgenic mice. H.G.,

Q.L. and S.Z. acquired fMOST data. X.K. and Y.W. performed Neurolucida reconstructions. V.G. and A.K.

performed deep brain imaging experiments.

S.C. and P.B. performed

2P induced

recombination experim

ents.

A.CT. and A.D. performed zebrafish experiments.

R.C., P.Y and M.S. performed the targeted single cell 2P

experiments and combinatorial

cortical

GCamp

calcium imaging experiments.

A.C. and H.Z. designed and

coordinate

d the study as well as wrote the manuscript, with inputs from all coauthors.

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint

http://dx.doi.org/10.1101/553271

doi:

bioRxiv preprint first posted online Feb. 18, 2019;