Delivery Issues

Dana CarrollUniversity of Utah/IGI

IGI CRISPR WorkshopJuly 12, 2017

Cas9

Donor DNA

sgRNA

How do we get these

into those?

Delivery of genome editing materials

Cell propertiestransformabilitysensitivityrepair pathways

DNA deliveryregulatory sequencestoxicitymutagenesispersistence

Viral deliveryregulatory sequencestropismcapacity

RNA deliverystabilitychemical modifications

Protein deliverycell entrystability

Considerations

In vivo vs. ex vivo

Somatic vs. germline

Various organisms

Viral vectorPlasmid DNAmRNA, sgRNA

RNP

LipofectionElectroporation

Manual injection

Delivery to cultured cells

Nanoparticles

In vitro transcribed mRNA

RNA delivery

In vitro transcribed sgRNA

Synthetic sgRNA

Electroporation, lipofection, etc.

NLS 1326..1346

FLAG 1275..1295

CAG promoter (from AAV vector) 714..1141

BbsI 260..265

Cas9 1371..5471

f1 origin 6001..6307

BGH polyA 5550..5753NLS (nucleoplasmin) 5472..5519

AmpR gene 6826..7686

pBR322 origin 7841..8460

pX330

8506 bp

FLAG 1296..1319

FLAG 1254..1274

CAG enhancer 444..713gRNA body 268..349

Stuffer 251..267BbsI 252..257

hU6 promoter 10..242

pX330Zhang lab

Plasmid vector

lentiCRISPRv2Zhang lab

RV primer 3129..3150BsmBI 2858..2863hU6 promoter 2617..2849

Stuffer 2857..4737

BsmBI 4731..4736gRNA body 4738..4819

EF1a promoter 4862..5110

Cas9 5111..9214NLS 9215..9262

lentiCRISPRv2

14873 bpBGH polyA 10887..11114

HIV PPT/LTR 10608..10858

WPRE 9967..10542

PuroR 9353..9946

FLAG 9263..9286P2A 9287..9352

U6 promoter Stuffer Cas9

EF1a promoter

BsmBI BsmBI

sgRNA body

Viral vector

http://depts.washington.edu/dwrlab/research/transduction-raav/

https://www.addgene.org/viral-vectors/aav/aav-guide/

Packaging capacity approx. 4.5 kb

Kim et al. (2014) Genome Res. 24: 1012Lin et al. (2014) eLife, e04766Liang et al. (2015) J. Biotech. 208: 44

Targeting with Cas9 RNPs

Cho et al. (2013) Genetics 195: 1177

This works well in many cell types; no cloning involved.

Gaj et al. (2017) NAR 45: e98

Various types of donor can be used.

RNP applications

C. elegans injectionEmbryo injection – many organisms

Mammalian cell electroporation...

Target P0 Analyzed Mutants %Mutants

dpy-3 A 24 1

B 24 0

C 24 0

D 33 3

E 16 0

Total 121 4 3.3

unc-1 A 32 5

B 24 4

C 18 0

D 22 0

Total 96 9 9.4

Cho et al. (2013) Genetics 195: 1177.

CRISPR-Cas mutagenesis in C. elegans via RNP injection

Cas9 RNP acts and disappears faster

Kim et al. (2014) Genome Res. 24: 1012

Cas9 RNP is more specific

than DNA delivery

Kim et al. (2014) Genome Res. 24: 1012

Liang et al. (2015) J. Biotechnol. 208: 44

Cas9 RNP in various cell types

BFPPEST

CACHis

TACTyr

GFP

ccctcgtgaccaccctgaccCACggcgtgcagtgcttcagccgct

ACggcgtgcagtgcttcagc sgRNA

pCMV

.........GT........... Donor

DeWitt et al. (2017) Methods 121-122: 9

Targeting BFP with RNP

Zuris et al. (2015) NBT 33: 73.

Cationic lipid delivery

Zuris et al. (2015) NBT 33: 73.

Delivery of Cre Cas9 RNP

Bioreducible lipid nanoparticles

Wang et al. (2016) PNAS 113: 2868

Mouse brain injection (Cre)Genome editing (Cas9 RNP)

Mout et al. (2016) ACS Nano 11: 2452

Gold-arginine nanoparticles

Mout et al. (2016) ACS Nano 11: 2452

Cellular localization T7E1 assay

Gold-arginine nanoparticles

Staahl et al. (2017) NBT 35: 431.

Multiple NLSs

NPCs = neural progenitor/stem cells

Staahl et al. (2017) NBT 35: 431.

Mouse brain

injections

Sharma et al. (2015) Blood 126: 1777

Dual AAV in vivo targeting at albumin

Virus 1: ZFNs for Alb intron 1

Virus 2: Donor DNA

Product: Hybrid mRNA

Sharma et al. (2015) Blood 126: 1777

Dual AAV insertion of Factor IX in vivo

Lee et al. (2017) eLife 6: e25312

Linking donor ssODN to crRNA

Lee et al. (2017) eLife 6: e25312

crRNAs tolerate terminal modifications

SpCas9

AsCpf1

Lee et al. (2017) eLife 6: e25312

Fused crRNA-donor DNA (gDonor) is functional

gDonor helps HDR with

nanoparticle delivery

Electroporation

Organism-specific methods

Gonad injection in C. elegans

Gene gun

Agrobacterium transformation in

plants

Embryo injection

Drosophila

Zebrafish

Mouse

Pig

Also electroporation of embryos

Making germline modifications in monkeys

Chen et al. (2015) Human Molecular Genetics 24: 3764

also Kang et al. (2016) J. Assist. Reprod. Genet. 33: 581;Tang et al. (2017) Mol. Genet. Genomics