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