Enhancing Genome Editing with Engineered CRISPR Enzymes Sunday April 28 th , 2019
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Enhancing Genome Editing with Engineered CRISPR Enzymes
Sunday April 28th, 2019
Introduction: Genome editing and targeting range
Outline: Expanding the genome editing toolbox
Part 2: Challenges & applications
Russell Katie Joey
Kleinstiver Lab
• assay development• protein engineering• molecular medicines
Part 1: Engineering improved CRISPR-Cas12a enzymes
Safe / specificBroadly targetableHigh activity
Ideal genome editing properties
Samson et al. 2013 Nat. Rev. Micro
Protein engineeringto impart desirable
properties
PAM preference can limit editing applications
SpCas9 PAM = NGG =𝟏𝟖 𝒃𝒑
=𝟏𝟑𝟐 𝒃𝒑
=𝟏𝟒𝟑 𝒃𝒑
SaCas9 PAM = NNGRRT
Cas12a PAM = TTTV
Ways to expand targeting via protein engineering
wild-type SpCas9 = NGG
SpCas9-VRQR = NGA
SpCas9-VRER = NGCGKleinstiver et al., Nature, 2016
Kleinstiver et al., Nature, 2015
[ 1 ]alter PAM
SELECTIVE
xCas9 = NGN
SpCas9-NG = NGNHu et al., Nature, 2018
Nishimasu et al., Science, 2018
[ 2 ]relax PAM
EXPANDED
Introduction: Genome editing technologies
Outline: Expanding the genome editing toolbox
Part 2: Challenges & applications
Russell Katie Joey
Kleinstiver Lab
• assay development• protein engineering• molecular medicines
Part 1: Engineering improved CRISPR-Cas12a enzymes
PAM requirement prohibits wide use of Cas12a
CRISPR typeSize (# AA)
PAMDNA breakguide RNA
multiplex
type V-A~1300
TTTV (5’)5’ overhang, PAM distal
~40nt, single RNAsimple
type II-A1368
NGG (3’)blunt, PAM prox.~100nt (sgRNA)
challenging
type II-A1053
NNGRRT (3’)blunt, PAM prox.~100nt (sgRNA)
challenging
Kleinstiver et al., Nature Biotechnol., 2019
Improving CRISPR-Cas12a targeting range
enAsCas12 à expanded PAM preference
Methods to improve properties:a. Orthologsb. Engineering
i. Directed evolutionii. Structure-guided
RussellAlex
Kleinstiver et al., Nature Biotechnol., 2019
Expanded targeting in human cells with enAsCas12a
AsCas12a = TTTVenAsCas12a = TTTV, TTTT, TTCN, VTTV, TRTV, and more
~7-fold expanded targeting range[ 2 ] relax PAM preference
Kleinstiver et al., Nature Biotechnol., 2019
Improved nuclease activity with enAsCas12a
enAsCas12a
~7-fold expanded targeting range~2-3 fold improved on-target activity
Improved base editing with enAsCas12a
Kleinstiver et al., Nature Biotechnol., 2019
enAsCas12a ~5-10 fold improved base editor activity
Summary 1: Improved activities of enAsCas12a
• ~7-fold expanded targeting range• ~2-3 fold improved on-target activity• enhanced activities at lower temperatures• can improve other variants (~2-fold, enRVR & enRR)• improved base editing & gene activation• efficient editing in primary human T cells
enAsCas12a
Kleinstiver et al., Nature Biotechnology, 2019
epigenome editingbase editing
Introduction: Genome editing technologies
Outline: Expanding the genome editing toolbox
Part 2: Challenges & applications
Russell Katie Joey
Kleinstiver Lab
• assay development• protein engineering• molecular medicines
Part 1: Engineering improved CRISPR-Cas12a enzymes
Important properties for allele-specific editing
low medium high0
20
40
60
80
100
% e
dite
d
AB
activitytargeting range edit outcome
Challenge: Single allele editing
Mut-GATCCAATCGGAATCGGATTTCGWT-GATCCAATCGGAATCGTATTTCG
Position the SNP in PAMNGG
Need library of PAM selective variants
SpCas9
AsCas12a
eAsCas12a
0
20
40
60
80
100%
TRAC
kno
ckou
t
SpCas9AsCas12aeAsCas12a
Challenge: Assessing improved tools in primary cells
Building a primary human T cell editing workflow
Kleinstiver et al., Nature Biotechnol., 2019
RNP delivery of enAsCas12a in human cells
Kleinstiver et al., Nature Biotechnol., 2019
Cas12a
SpCas9
AsCas12a
eAsCas12a
0
20
40
60
80
100
% TRAC
kno
ckou
t
SpCas9AsCas12aeAsCas12a
RNP delivery of CRISPR nucleases in primary T cells
CRISPR typeSize (# AA)
PAMDNA breakguide RNA
multiplex
type V-A~1300
TTTV (5’)5’ overhang, PAM distal
~40nt, single RNAsimple
type II-A1368
NGG (3’)blunt, PAM prox.~100nt (sgRNA)
challenging
Introduction: Genome editing technologies
Summary 2: Addressing challenges of genome editing
Part 2: Challenges & applications
Russell Katie Joey
Kleinstiver Lab
• assay development• PAM selective Cas9 variants• Activities in 1o T cells
Part 1: Engineering improved CRISPR-Cas12a enzymes
Molecular medicines + applications
Protein engineering to enhance CRISPR enzymes
Genome editing technologies
Summary + Research in the Kleinstiver lab
Joung LabAlexander Sousa
Russell WaltonMoira WelchEsther TakJoy Horng
Jon Hsu, et al.Martin Aryee – MGH
Jose Lopez, Sara Garcia
Luca Pinello – MGHKendell Clement
Marcela Maus - MGHIrene Scarfo
Kleinstiver Lab
Colleagues & CollaboratorsFriends & Family
Thanks to those who help ask & answer questions
Margaret Q. LandenbergerResearch Foundation
RussellWalton
KatieChristie
JoeyRissman
Recruiting postdocs,students, & technicians
www.kleinstiverlab.org
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