towards predictable editing: influence of target sequence ...€¦ · towards predictable editing:...
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Towards predictable editing: influence of target sequence, cell type, and choice of type II nuclease on
desired repair outcomesLuis Barrera, Barrett Steinberg, Derek Cerchione, Christopher Wilson, Cecilia Cotta-Ramusino, Vic Myer, Hari Jayaram
Editas RNP lead discoveryFigure 1.
RNP for Spy and Sau Cas9
variants is delivered to
HEK293T and T-cells (T cell
data shown) and efficacy
determined by NGS
sequencing in dose
response mode.
Number of active guides
across locus for both Spy
Cas9 and Sa Cas9.
Indel profiles depend on cell type and gRNA locus
Figure 2. The indel spectrum of Cas9 edits across a
series of gRNA in T-cells. gRNA range from those that
result in a dominant indel to those that have a wider
spectrum of indels.
T-cells prefer deletions and HEK-293T cells insertions
Figure 4. Probability density of edit sizes in T-cells vs
HEK293T cells. The skewing of indel patterns under
equivalent conditions indicates a cell type dependence.
Targeted DNA cleavage by type IICRISPR nucleases has enabled avariety of genome editingapplications.
However, the apparently stochasticrepair of DNA breaks can create abroad range of edited sequences,providing a unique challenge shoulda specific edit be desired.
Here, we investigated thedistributions of insertions anddeletions following cleavage by Cas9from S. pyogenes (Sp) and S. aureus(Sa) when paired with different guideRNAs in primary human T-cells andHEK 293T cell line.
We quantified the degree to whichspecific editing conditions createhighly dominant alleles and observedsignificant variability. In addition, weobserved shifts in the types andfrequencies of repair outcomes,particularly when comparing editingin primary cells and cell lines.
The analysis of repair outcomedistributions provides opportunitiesfor improving models of non-homologous end joining andsuggests mechanistic hypotheses forhow factors inherent to the nucleaseactivity influence the repair process. Figure 3. Comparison of guides with dominant indels
between T-cells and HEK-293T cells. Key deletions
indicated by arrows show similarities and differences
between cell types.
Abstract
Dominance of the +1 edit shown by Spy Cas9
Question?
References:1. In vivo genome editing using Staphylococcus aureus Cas9.
Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ,
Zetsche B, Shalem O, Wu X, Makarova KS, Koonin EV, Sharp PA,
Zhang F.
Nature. 2015 Apr 9;520(7546):186-91. doi: 10.1038/nature14299.
Given the wealth of CRISPR
nucleases, how do we pick
between them to engineer a
therapeutic edit?
HEK293 T cells
R1 R2 R2R1
Predominant common 1 bpinsertion
HEK-preferred7 bpdeletion
T-cell-preferred17 bpdeletion HEK293 T cells
R1 R2 R2R1
Inde
l typ
e
Position (relative to cut site):Indellength
Guide 1 Guide 2
More control Less control
Variant, gRNA sequence and cell type influence edit
BCL11Ae−CUNAM−FUGY BCL11Ae−FUVOJ−LEDA BCL11Ae−GEKOD−NYDO BCL11Ae−GUDUN−XORA
BCL11Ae−HUKAT−PAPA BCL11Ae−HUSYB−SALA BCL11Ae−HYFIH−VOMI BCL11Ae−KAQYV−GEZY
BCL11Ae−LUQUG−CISY BCL11Ae−MUXID−TYGE BCL11Ae−NAQOW−VUNA BCL11Ae−PIDAR−GIXU
BCL11Ae−QABOV−KOZA BCL11Ae−QUTYJ−GIRA BCL11Ae−RETEQ−CEFU BCL11Ae−VAJOT−JEGI
BCL11Ae−XOVIS−GUHA BCL11Ae−ZUJUP−LEBA
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−60 −40 −20 0 20 40 −150 −100 −50 0 50 −100 −50 0 50 −25 0 25 50
−200 −150 −100 −50 0 50 −100 −50 0 50 −50 −25 0 25 50 −25 0 25 50
−50 −25 0 25 50 −50 −25 0 25 50 −25 0 25 50 −25 0 25 50
−200 −150 −100 −50 0 50 −30 0 30 60 −25 0 25 50 −200 −150 −100 −50 0 50
−150 −100 −50 0 50 −250 −200 −150 −100 −50 0 50Size
dens
ity
Cell.TypeHEK293T cell
Edit change in size (bp)
InsertionsDeletions
Prob
abilit
y de
nsity
Cell type
Figure 5. Nuclease dependent staggered cutting possibly
leads to predominant +1 edit in Spy Cas9 over Sau Cas9.
*
ConclusiongRNA range from showing a
dominant edit to a large
spectrum of edits dependent on
CRISPR variant and cell type.
Understanding these
relationships can allow
streamlining of gRNA screening
and engineering efforts.
*
Guide 1 Guide 2 Guide 3
Guide 4 Guide 5 Guide 6
guides
T1 T3 T4 T5T2