Expanding the Pool

Characterizing LAGLIDADG Homing Endonuclease Orthologs

Picking the ORF Regions to Use

Intron

ORF• OK active sites• 50-60+ % homology• Regions homologous to Ani

• Optimization– Yeast expression, restriction– Surface expression?

Expression & Relatedness

Expression problems

• Mutagenesis & directed evolution to regain & improve expression

• Glycosylation changes?– Tas Tin and Vin are predicted to be lacking

glycosylation in regions glycosylated in Pno, Ach, Hje, and Ani

– Tas and Tin are predicted to be glycosylated at DNA binding regions

• Poor surface expressers but otherwise OK

Target Determination• Target for Ani at intron/exon junctions

• By Homology…

• Central 4 are hard to predict

Ach cytb gene2423 bp

Exon 1 Exon 2Sequence Used

Ani apocytB (cobA) Gene2694 bp

Exon 1 Ani Exon2

TGAGGAGGTT T CT CTGTAAA

TGGGGAGG TTT TTCAGTATC

Binding Vs Ani Target &Predicted Targets

0.1 1 10 100 10000

100

200

300

400

500

600

700

800

900

1000Against Ani target

Ach

Hje

Pno

Tas

Tin

Vin

Ani (WT)

Ani (E148D)

concentration (nM)

MFI

(APC

)

0.1 1 10 100 10000

500

1000

1500

2000

2500Against own target

Ach

Hje

Pno

Tas

Tin

Vin

Ani (WT, against SCID)

Ani (E148D)

concentration (nM)

MFI

(APC

)

Conclusion: Tas, Tin, and Vin are not producing a viable surfac-expressed HE

CleavageAc

h

Hje

Pno

Ani

E148

D

Ach

Hje

Pno

Ach

Hje

Pno

Ani

37°C, against Predicted Targ 37°C, against Ani Targ

30°C, againstPredicted Targ

Target Only

What Changes Are Responsible?

• Variance at DNA-interacting domains– Insertions relative to Ani

Differences at DNA-Binding Regions

• 5Å interactions as spheres• Unchanged residues in green• Conserved Residues in Purple

+5, +9 & +10 positions (white) +2 & +5 positions (white)

Insertions• Directly change DNA-interacting regions (left)• May realign directly interacting regions (right)

– Harder to obtain by directed evolution alone

+5, +9 & +10 positions (white) -8 position (white)

Rosetta Predictions

• -8 A -> G• K24N & T29K

• D73N Conserved residue changed

Future Directions• Mutagenesis & directed evolution to improve surface

expression and/or activity• Comparing Ani ability to cleave predicted targets to the

corresponding enzyme’s cleavage to evaluate actual changes• Determine specificity with 1-off panels• DNA shuffling to generate hybrid HE’s• Begin to look at which changes are tolerated and which aren’t

Take-home Message• We can use homology searches to find functional homing

endonucleases with different targets• This can help us determine what AA changes affect what target

specificities