5’-u gU A A A u agucgg AGCUUAUC GACUG UGUUG cugu g a

caguc UCGGGUAG CUGAC A C AAC ggua c c u

3’ -a UG -- C -- -- u

TALEN Target Mature miR-21 Seed Sequence

TCCATGGCTGTACCACCTTGTCGGGTAGCTTATCAGACTGATGTTGACTGTTGAAAGGTACCGACATGGTGGAACAGCCCATCGAATAGTCTGACTACAACTGACAACTT

HD

NN

NI

NG

HD

NN

NI

HD

HD

NG

HD

NI

NG

HD

NI

NG

HD

NN

NI

NG

HD

NN

NG

HD

NN

NI

NG

NI

HD

NI

NG

HD

NG

HD

NN

NI

HD

NI

FokI

FokI

C-ter

C-ter

Highlights

• Target microRNAs on the genomic level • Pre-designed TALE Nucleases pairs

• Matched HR donor vectors

• Dual selection of miR-KO clonal lines with RFP and Puromycin in HR Vectors

• Discover novel miR knockout phenotypes

miR-KO - Matched TALE Nucleases & HR Donor Vectors for Knockouts

www.systembio.com/mirko

Surgically disrupt microRNAs for permanent knockdown

Completely ablate microRNA genes on the genomic level

miR-KOs are transcription activator-like effector (TALE) nucleases that precisely edit specific miRNAs in mammalian cells. SBI designed miR-TALE-nucleases to cleave within the miRNA seed region. In the absence of HR donor vectors, the cellular machinery repairs such breaks via non-homologous end joining (NHEJ). This is an error-prone system that typically generates small deletions or insertions (indels) at or near the site of cleavage. Since the seed region (defined as bases 2-8 of the microRNA) directs miRNA binding to its target DNA, indels within the seed region completely abolish miRNA function.

Accelerating discoveries through innovations

MicroRNA Research

A TALEN pair targeting to the seed region of human oncogenic miR-21 was designed and built using SBI’s EZ-TAL assembly kit, TALEN-L (left) and TALEN-R (right). Panel a) depicts the miR-21 precursor hairpin with the seed sequence chosen for targeting and the genomic chromosome 17 locus of the human miR-21 region is shown. Also shown is the miR-21 homologous recombination targeting donor vector featuring RFP and Puro markers. The miR-21 TALEN pair was tested for cleavage activity on 293 genomic DNA (Panel b) and about 2% cutting was observed using the surveyor assay with the CleI enzyme. The miR-21 TALENs and the miR-21 homologous recombination donor vector were co-transfected into HEK293 cells to enable RFP fluorescence of positive

MIR-21 Locus

2 kbMIR21 TUBD1TM49

3’ arm 5’ armInsu InsuEF1 RFP-T2A-PuroLoxP LoxP

Donor targeting vector: MIR-21-EF1-RFP-T2A-Puro-PolyAa) b)

TALEN-L

TALEN-R

500 bp

400 bp

300 bp

200 bp

Control TALENsMM -- + -- + Nuclease

(CleI)430 bp

280 bp

1.8%

1281 bp830 bp

Mock TALENs Mock TALENs MM Donor DNA Donor DNA+ +

1000 bp

500 bp

3000 bp

TALE-Nuclease Activity Tests (Surveyor)

Homology-directed DNA repair(HDDR)

Design of TALEN-mediated human miR-21 knockout

c)

1 2 3 4 5 6

cells as well as puromycin selection for the enrichment of KO clones. Screening for the KOs revealed a mixture of clones that harbored biallelic modifications in most cases. An example of HR recombination junction PCR tests on treated HEK293 genomic DNA in Panel c) show accurate left side insertion of 830 bp product (Lanes 2,3) and right side insertion (Lanes 5,6) at the predicted 1281 bp size.

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We O�er Custom Services for TALE Assembly and HR Donor Vector Cloning

Targeted disruption of the human miR-21 locus

miR-KO microRNA knockout system

Further characterization of the TALEN-treated miR-21 genomic DNA shows mono- and double allele homologous recombination events. The wild-type and predicted KO miR-21 genomic loci are depuected in Panel d) with the primer locations used for analyzing the loci in seveeral clones. Single clones were identified using RFP fluorescence and puromycin selection. Examples of the colony images is hsown in Panel e). Thirty different clones were expanded and genotyped by genomic PCR (Panel f ) for single or double HR events. Clones #1 and #7 showed a single HR event at the miR-21 locus and Clone #5 showed double HR insertions at the miR-21 site. Clones #1 and #7 were further analyzed by sequencing the PCR products generated using primers P1f and P1r that correspond to the wild-type miR-21 locus. Sequence analysis of Clones #1 and #7 revealed that the non-HR chromosomic locus infact had been cut by the TALENs and resulted in miR-21 sequence deletions in Clone #1 and severe sequence changes in Clone #7 (Panel g).

3’ arm 5’ armInsu InsuEF1 RFP-T2A-PuroLoxP LoxP

miR-21

3’ arm 5’ armInsu InsuEF1 RFP-T2A-PuroLoxP LoxP

5’

5’

3’

3’

d) e)

miR

-

21

KO Locus

P1f P1r

P2fP2r P3f

P3r

Phase

Phase

RFP

RFPMM WT 1# 5#* 6# 7#

MM 30# 27# 22# 21# 9#

KOWT/NHEJ

KOWT/NHEJ

f)

TCGGGT AGC TTAT CAGACTGATGTTGACTGTTGAATCTC WT

TCGGGT ---C ---AT CAGACTGATGTTGACTGTTGAATCTC 1#

TCGGGC TGAATCTT CAGACTGATGTTGACTGTTGAATCTC 7#TALE

N

g)

PCR genotyping of mono- or double allele KO clones

Sequence genotyping of mono- or double allele KO clones

RFP-positive and puromycin resistantsingle cell-derived clones

Wild-typemiR-21 locus

h) Quantitative PCR analysis of miR-21expression in KO clones

00.10.20.30.40.50.60.70.80.9

1

miR-21

293 cells

Clone #1

Clone #5

Clone #7

Nor

mal

ized

miR

-21

expr

essi

on le

vels

Clon

e #1

Clon

e #5