CRISPR Gene Editing in Stem Cells

containing 9,400 recombinant human proteins (Human Proto-

Array). Using a stringent cut-off (

score > 3), we identified 12

candidates that strongly interacted with the WT transcript (Fig-

ures 4A and S4B; Table S2). Notably, four of these candidates

(CNBP, HNRNPF, SFRS9, and KCNAB2) showed dramatically

decreased binding when the array was probed with the

bvht

dAGIL

transcript (Figures 4B and 4C). These proteins are conserved be-

tween mouse and human and are highly expressed across the

differentiation time course except for KCNAB2 (Figure S4C).

We previously showed that

Bvht

interacts with the Polycomb

Figure 4.

Bvht

Interacts with CNBP, a Zinc-Finger TF

(A) Protein microarray analysis detecting

Bvht

-interacting proteins. Cy5-labeled

Bvht

and

bvht

dAGIL

transcripts were incubated with a human recombinant protein

microarray.

scores of fold-change signal intensity over background are depicted in the scatterplot. The dashed blue line represents the

score cutoff used to

select significant RNA-protein binding events. The significant

Bvht

-binding proteins are colored in red. The horizontal axis is

Bvht

, and the vertical axis for

bvht

dAGIL

. A logarithmic scale was used to display both axes.

(B) Quantification of human protein microarray showing fold changes of signal intensity over background for indicated proteins. Values are the average of

duplicate protein spots.

(C) Image of human protein microarray (left) and enlarged subarray (right) showing that mutation of AGIL motif dramatically reduces the interaction between

Bvht

and CNBP, HNRNPF, and SFRS9. Alexa Fluor 647-labeled rabbit anti-mouse IgG or anti-human IgA2 in corners and middle edge of each subarray are used for

reference.

(D) RNA immunoprecipitation showing the interaction between

Bvht

and CNBP, HNRNPF, and SFRS9 in ESCs. Flag-tagged CNBP, HNRNPF, or SFRS9 was

constitutively expressed in both WT and

bvht

dAGIL

ESCs. Immunoblot analysis using anti-Flag antibody shows equal expression levels of Flag-tagged CNBP,

HNRNPF, and SFRS9 in indicated ESCs. Mouse IgG was used for negative control.

All experiments were performed in triplicate and data are represented as mean values ± SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 (two-tailed Student’s

t test).