1. CRISPR-Cas systems: exploiting genome context to infer novel functions
An exercise in system thinking
Eugene V Koonin
National Center for Biotechnology Information, NIH, Bethesda, MD
Symposium on “The Brave New World of Smart Data and Semantics in the Life Sciences”
Wageningen University
January 24, 2019

2. Evolutionary Genomics Research group at the NCBI

3. The way CRISPR works
Makarova et al. Nature Rev Microbiol 2011

4. CRISPR-Cas adaptive immunity system
CRISPR – Clustered Regularly Interspaced Short Palindromic Repeats
Cas – CRISPR ASsociated proteins
crRNA – CRISPR RNA, the guide RNA
Mohanraju et al. Science 2016

5. CRISPR-Cas as a system Generic system properties Specific features
Defined set of core components
Extensive, elaborate, specific interactions between components
Non-linear causality
Clear purpose: antivirus immunity
Emergent properties: purpose achieved only through interaction between modules
Specific features
Modularity
Enormous architectural flexibility: multiple ancillary components plugged in
Functional flexibility: mechanistic diversification,
re-purposing

6. “Current” classification of CRISPR-Cas systems
Multisubunit
crRNA-effector
complexes
(Cascade)
Single-subunit
crRNA-effector
complexes
(Cas9-like)
Type V introduced
Makarova et al., NRMicro 2015

7. Computational pipeline for comprehensive discovery of Class 2 CRISPR-Cas
Shmakov et al, Mol Cell 2015
Shmakov et al. NRMicro 2017

8. New CRISPR-Cas classification: Class 1
Makarova et al,
CRISPR J 2018

9. New CRISPR-Cas classification: Class 2
Makarova et al,
CRISPR J 2018

10. Discovery of novel CRISPR-Cas systems in genomic and metagenomic databases
At least 10 new subtypes of CRISPR-Cas systems with
substantial architectural and functional diversity
discovered: from 16 to 26 CRISPR subtypes
from 4 to ~14 subtypes of Class 2 – and counting:
new opportunities for genome-editing, RNA-targeting
and regulatory tools
Cas gene list amendment (effector gene renaming):
Cas12: TnpB, no HNH nuclease
Cpf1=Cas12a, C2c1=Cas12b, C2c3=Cas12c…V-U=???
Cas13: 2xHEPN
C2c2=Cas13a, C2c4=Cas13b, C2c5=Cas13c…
Shmakov et al, NRMicro 2017

11. Comparison of Class 2 CRISPR-Cas effector nucleases: substantial functional diversity
Nuclease domains
tracrRNA
PAM/PFS
Substrate
Target cut
Type II/Cas9
TnpB/RuvC+HNH
Yes
3’, GC-rich
dsDNA
Blunt ends
Type V-A/Cas12a (Cpf1)
TnpB/RuvC+
Nuc No
5’, AT-rich
Staggered ends, 5’ overhangs
Type V-B/Cas12b (C2c1)
TnpB/RuvC
Type VI-A/Cas13a (C2c2)
Type VI-B/Cas13b+
Csx27/28
2xHEPN
5’,nonG
5’, nonU, 3’NAN,NNA
ssRNA
U-specific RNA cuts + collateral RNA cleavage
Regulated U-specific RNA cuts + collateral RNA cleavage
Substantial diversity despite the common overall design
Diverse opportunities for genome editing/engineering
Type V-A: simplest known system
Type VI-A,B: specific RNA cleavage
Applications developed for Cas12a,b Cas13a,b
Screening of growing genome collections continues
Shmakov et al, NRMicro 2017
Smargon et al, Mol Cell 2017

12. Source of diversity: Independent origins of type V
effectors from transposon-encoded TnpB nucleases
Shmakov et al,
NRMicro 2017

13. Proposed path to maturity
Koonin, Makarova, Phil Trans Roy Soc, in press

14. Systematic exploration of small Cas12 proteins, likely
evolutionary intermediates between TnpB and “mature” Cas12
Yan WX, Hunnewell P, Alfonse LE, Carte JM, Keston-Smith E, Sothiselvam S, Garrity AJ, Chong S, Makarova KS, Koonin EV, Cheng DR, Scott DA. Functionally diverse type V CRISPR-Cas systems. Science Jan 4;363(6422):88-91

15. Diverse activities of type V subtypes: TnpB-Cas12 intermediates
Robust ssRNA cleavage
requiring RuvC domain
no tracrRNA
Collateral ssRNA/DNA cleavage
Yan WX, Hunnewell P, Alfonse LE, Carte JM, Keston-Smith E, Sothiselvam S, Garrity AJ, Chong S, Makarova KS, Koonin EV, Cheng DR, Scott DA. Functionally diverse type V CRISPR-Cas systems. Science Jan 4;363(6422):88-91

16. Complementary study of small Cas12
Harrington LB, Burstein D, Chen JS, Paez-Espino D, Ma E, Witte IP, Cofsky JC, Kyrpides NC, Banfield JF, Doudna JA. Programmed DNA destruction by miniature
CRISPR-Cas14 enzymes. Science Oct 18
Here we present a set of CRISPR-Cas systems from uncultivated
archaea that contain Cas14, a family of exceptionally compact RNA-guided
nucleases ( amino acids). Despite their small size, Cas14 proteins are
capable of targeted single-stranded DNA (ssDNA) cleavage without restrictive
sequence requirements.

17. Exploitation of genome context to discover new CRISPR-associated functions including non-defense ones

18. Subtype VI-D: new opportunities for RNA editing – and a new insight into CRISPR regulation
Arbor Biotechnologies
David Cheng, David Scott, Winston Yan
Putative accessory proteins:
WYL, a regulatory domain,
found in contexts similar to
or together with CARF
Yan WX, Chong S, Zhang H, Makarova KS, Koonin EV, Cheng DR, Scott DA. Cas13d
Is a Compact RNA-Targeting Type VI CRISPR Effector Positively Modulated by a
WYL-Domain-Containing Accessory Protein. Mol Cell Apr 19;70(2):
Konermann S, Lotfy P, Brideau NJ, Oki J, Shokhirev MN, Hsu PD.
Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR Effectors.
Cell Apr 19;173(3):

19. Cas13d: Sister group of Cas13a
Yan WX, Chong S, Zhang H, Makarova KS, Koonin EV, Cheng DR, Scott DA. Cas13d
Is a Compact RNA-Targeting Type VI CRISPR Effector Positively Modulated by a
WYL-Domain-Containing Accessory Protein. Mol Cell Apr 19;70(2):

20. Subtype VI-D: phylogeny and locus organization
Figure 1
Cas13d: the smallest RNA-targeting
CRISPR effector – by far
Subtype VI-D CRISPR-Cas Systems and Diversity of Type VI Subtypes
(A) Schematic representation of a maximum likelihood tree topology for a selected subset of Cas13d, with the genomic arrangement of the genes encoding predicted protein components of subtype VI-D system components shown to the right. Each locus sequence is identified by a protein accession or gene number with the species name provided where available. Key proteins and CRISPR arrays are color coded as follows: blue, Cas13d; light orange, WYL-domain-containing protein; light blue, Cas1; green, Cas2; dark gray/gray, CRISPR array.
(B) Schematic tree comparing the different type VI subtype locus structures. Gene arrows are shown roughly proportional to size. Labels denote the following: HTH, helix-turn-helix domain; WYL, WYL domain; HEPN, HEPN nuclease domain; TM, transmembrane domains of Csx27–Csx28. Key proteins and CRISPR arrays are color coded as follows: blue, Cas13d; gray, Csx accessory proteins (differentiated by colored domains); light blue, Cas1; green, Cas2; dark gray/gray, CRISPR array. Figure S1 contains the sequence alignment and phylogeny of Cas13d compared to Cas13a, and Figure S2 has the same analysis for the WYL domain proteins.
(C) Size comparison for Cas13 proteins from the four type VI subtypes; error bars specify the mean and standard deviation.
Yan WX, Chong S, Zhang H, Makarova KS, Koonin EV, Cheng DR, Scott DA. Cas13d
Is a Compact RNA-Targeting Type VI CRISPR Effector Positively Modulated by a
WYL-Domain-Containing Accessory Protein. Mol Cell Apr 19;70(2):
Molecular Cell  , e5DOI: ( /j.molcel )
Copyright © 2018 Elsevier Inc. Terms and Conditions

21. Stimulatory effect of the WYL domain protein on Cas13d
Yan WX, Chong S, Zhang H, Makarova KS, Koonin EV, Cheng DR, Scott DA. Cas13d
Is a Compact RNA-Targeting Type VI CRISPR Effector Positively Modulated by a
WYL-Domain-Containing Accessory Protein. Mol Cell Apr 19;70(2):
WYL domains can also regulate type I systems:
Hein S, Scholz I, Voß B, Hess WR. Adaptation and modification of three CRISPR
loci in two closely related cyanobacteria. RNA Biol May;10(5):852-64
All six systems are associated with a gene for a possible transcriptional repressor.
Indeed, we identified one of these genes, sll7009, as encoding a negative regulator
specific for the CRISPR1 subtype I-D system in Synechocystis sp PCC6803

22. Discovering the functions of CRISPR “ancillary” proteins
The Cas10-CARF signaling pathway
non-specific RNA degradation/
toxicity? PCD?
Niewoehner O, Garcia-Doval C, Rostøl JT, Berk C, Schwede F, Bigler L, Hall J,
Marraffini LA, Jinek M. Type III CRISPR-Cas systems produce cyclic oligoadenylate
second messengers. Nature Aug 31;548(7669):
Kazlauskiene M, Kostiuk G, Venclovas Č, Tamulaitis G, Siksnys V. A cyclic
oligonucleotide signaling pathway in type III CRISPR-Cas systems. Science. 2017
Aug 11;357(6351):
Koonin EV, Makarova KS. Discovery of Oligonucleotide Signaling Mediated by
CRISPR-Associated Polymerases Solves Two Puzzles but Leaves an Enigma. ACS Chem
Biol Sep 27

23. Origin of CRISPR effector module from a signal transduction system,
possibly, a suicidal one
Koonin, Makarova, Phil Trans Roy Soc in press

24. Discovering the functions of CRISPR “ancillary” proteins
Csn2 ATPase
Arslan et al. Double-strand DNA end-binding and sliding of the
toroidal CRISPR-associated protein Csn2. Nucleic Acids Res.
2013
Bernheim et al. Inhibition of NHEJ repair by type II-A CRISPR-Cas
systems in bacteria. Nat Commun. 2017
Makarova, Koonin, unpublished

25. With the growth of microbial genome database, new CRISPR ancillary proteins can be expected to pop up Can we systematically identify ancillary genes in CRISPR-cas loci, predict protein functions and distinguish functional associations from spurious ones?
Shmakov. Makarova, Wolf, Severinov, Koonin, PNAS 2018

26. CRISPRicity: a “guilt by association” approach
360 families
Shmakov. Makarova, Wolf, Severinov, Koonin, PNAS 2018

27. Outstanding questions
How do we decide whether a gene is functionally linked to CRISPR-Cas system or just encoded in a “defense island”?
How do we rule out “historical” synteny conservation?

28. CorA (50 protein clusters): membrane connection of subtype III-B
CorA, structure
CorA, metal ion transporter (MIT) superfamily
Tan K, Sather A, Robertson JL, Moy S, Roux B, Joachimiak A. Structure and
electrostatic property of cytoplasmic domain of ZntB transporter. Protein Sci.
2009 Oct;18(10):
DHH – nanoRNA nuclease, metal-dependent
CorA and DHH, model
Uemura Y, Nakagawa N, Wakamatsu T, Kim K, Montelione GT, Hunt JF, Kuramitsu S,
Masui R. Crystal structure of the ligand-binding form of nanoRNase from
Bacteroides fragilis, a member of the DHH/DHHA1 phosphoesterase family of
proteins. FEBS Lett Aug 19;587(16):
LabA – NYN domain RNA nuclease, metal-dependent
Anantharaman V, Aravind L. The NYN domains: novel predicted RNAses with a PIN
domain-like fold. RNA Biol Jan-Mar;3(1):18-27.
Function?
CorA is a phage DNA transporter
CorA is a sensor of phage entry through the membrane
CorA is a metal transporter and regulates activities of metal-dependent nucleases
CorA/DHH is a suicide machine, making membrane leaky and cleaving cellular RNAs if CRISPR immunity fails

29. CorA: horizontal mobility together with III-B effector module
Insertion of unrelated or distantly related type III systems next to cas6 gene
No cas1, cas2 and CRISPR arrays
Cas6 stays in situ and could be aligned on the nucleotide level
Flanking genes mostly stay the same
Puigbò P, Makarova KS, Kristensen DM, Wolf YI, Koonin EV. Reconstruction of the evolution of microbial defense systems. BMC Evol Biol. 2017

30. New membrane-associated CARF (29 clusters)
>WP_ hypothetical protein [Thermocrinis ruber] MWKFWEIELKHFKTLLESGKLDDHIEGLYSQFWELPPSHQYELVKYSKEKEVFPSIQTFRKVFKVSEETAVKFFKEKHITFE
FPVVSSDGKGELIKAVAIKNLKEVITNLKNIKRHFNPIKEFLKTGFAVFFDREFAGASFQLPTVLNLYVENLPQDALFGAIDKK
GNIKSVDGIEEKKKLAKELGLRLVEPYYLSTVDDLKAWFDAESYDVPLYITKTQDRWEGEFKSFLKATGISKEQLTKLEVLS
GLETKPIITGQLAGDVWKNVLEEFWRRFKETEQKLHNKERF
HIAINGPVALAFAIGVLFGSQKP
FVFYHYQNNIYHPITVENVRELKERKESLEKIEQHFQKGGKSLVVMLSFAHHEMESDVKNYISRKVENPSYLLLRAKSS
GNIAVEDMKEVATETASVIQNIRREHSFEDF
HFFLSTPVPIAFMGGLSFGHY
GEGYIYNYAGGTYEPVVSFSFLKALREGKYVLSEV
Some have only membrane-associated CARF domain
CARF – OligoA-binding domain, CRISPR associated Rossmann fold
Membrane CARF, model
Makarova KS, Anantharaman V, Grishin NV, Koonin EV, Aravind L. CARF and WYL domains: ligand-binding regulators of prokaryotic defense systems. Front Genet Apr 30;5:102
Niewoehner O, Garcia-Doval C, Rostøl JT, Berk C, Schwede F, Bigler L, Hall J, Marraffini LA, Jinek M. Type III CRISPR-Cas systems produce cyclic oligoadenylate second messengers. Nature Aug 31;548(7669):
Kazlauskiene M, Kostiuk G, Venclovas Č, Tamulaitis G, Siksnys V. A cyclic oligonucleotide signaling pathway in type III CRISPR-Cas systems. Science Aug 11;357(6351):
Function? ?
Membrane-associated stress signaling?

31. Membrane protein specific for III-B in Actinobacteria (3 clusters)
No HEPN,
CARF alone

32. Complex organization of type III loci: challenge for further study of CRISPR biology beyond adaptive immunity

33. Type IV-B and CysH-like proteins (10 clusters)
Actinomyces
Aureimonas
Cellulosimicrobium
Gordonia
Haloterrigena
Leptospira
Mycobacterium
Rhodococcus
Rubinisphaera
Ruminococcus
Spirochaeta
Tetrasphaera
Thermoanaerobacter
Xylanimonas
CysH belongs to Adenine nucleotide alpha hydrolases superfamily (ATP sulphurylases, tRNA methyl transferase, Universal Stress Response protein)
Presence/absence of cysH in type IV loci
(+/- 20 genes from type IV genes)
VIP2 - ART superfamily (NAD:arginine ADP-ribosyltransferase –like)

34. STAND NTPase linked to interference-deficient I-E system
STAND: Signal Transduction ATPases with Numerous Domains
Leipe DD, Koonin EV, Aravind L. STAND, a class of P-loop NTPases including animal and plant regulators of
programmed cell death: multiple, complex domain architectures, unusual phyletic patterns, and
evolution by horizontal gene transfer. J Mol Biol. 2004

35. Phylogenetic analysis of Cas5 family from I-E systems: Cas1-less loci

36. Branch A gene neighborhood analysis: convergent loss of Cas3/interference
Degradation and
loss of Cas3/
interference

37. Distinct STAND NTPase associated with Cas1-less I-E systems in actinobacteria. STAND-like NTPases are known to be involved in apoptosis in eukaryotes, but the bacterial homologs remain virtually uncharacterized. These systems lack Cas3 and accordingly, the interference capacity.
Leipe DD, Koonin EV, Aravind L. STAND, a class of P-loop NTPases including animal and plant regulators of programmed cell death:
multiple, complex domain architectures, unusual phyletic patterns, and evolution by horizontal gene transfer. J Mol Biol. 2004
The Cas5 phylogeny shows that the branch associated with the STAND NTPase includes primarily Cas1-less loci (Branch A). In addition, in this subtree, there are at least 3 large clades of Cas1-less I-E systems (branches B,C,D). All sequences in these branches are from actinobacteria.
Branch A includes a small subtree where Cas1-less systems are linked to another STAND-like NTPase, which is only distantly related to the first one.
The second STAND-like NTPase is fused to a caspase, a protease known in to be involved in apoptosis in eukaryotes.
Several complete genomes that encode these Cas1-less system lack other CRISPR-Cas systems
Defense without interference, via PCD? Non-defense signal transduction roles?

38. Differentiating functional association from historical synteny conservation or spurious co-occurrence through coevolution analysis
Type III loci
For each pair of loci, derive distances from the trees of: 1) universal marker (16S), 2) CRISPR effector, 3) Candidate ancillary cas gene
𝐿 𝑖 , 𝐿 𝑗 : 𝐸𝑓𝑓 𝑖 𝑥 𝐸𝑓𝑓 𝑗 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 (𝐸𝑓𝑓𝑒𝑐𝑡𝑜𝑟 𝑚𝑜𝑑𝑢𝑙𝑒);
𝑀𝑦𝐺𝑒𝑛𝑒 𝑖 𝑥 𝑀𝑦𝐺𝑒𝑛𝑒 𝑗 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒; 16𝑠 𝑟𝑅𝑁𝐴 𝑖 𝑥 16𝑠 𝑟𝑅𝑁𝐴 𝑗 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒
Where 𝐸𝑓𝑓 𝑖 , 𝐸𝑓𝑓 𝑗 and 𝑀𝑦𝐺𝑒𝑛𝑒 𝑖 , 𝑀𝑦𝐺𝑒𝑛𝑒 𝑗 are comparable (alignable)

39. Functional association vs synteny through pairwise distance comparison
In assumption of gene reshuffling
So we need to show correlation and gene reshufling
Need to show high correlation/coevolution between putative new ancillary gene and cas genes (association) and low correlation between same gene and species tree (16S rRNA)

40. Cas5-Cas10 ClustDist – distance between pairs of Cas5
Cas5 vs Cas10
16s rRNA vs Cas10
16s rRNA vs Cas5
0.84 correlation between the cas5 and cas10
Low correlation with 16S rRNA (0.36, 0.34)
3,218,150 loci pairs found (100k sampled)
ClustDist – distance between pairs of Cas5
Cas10Dist – distance between pairs of Cas10
RRNADist – distance between 16s rRNA

41. New ancillary genes: CorA
CorA vs Cas10
16s rRNA vs Cas10
16s rRNA vs CorA
R=0.86 correlation between the genes
weak correlation with 16s RRNA
2,969 loci pairs found
ClustDist – distance between pairs of CorA
Cas10Dist – distance between pairs of Cas10
RRNADist – distance between 16s rRNA
Clear evidence of coevolution with CRISPR-Cas

42. New ancillary proteins: Membrane CARF
CARF vs Cas10
16s rRNA vs Cas10
16s rRNA vs RT
High correlation for every component
282 pairs of loci
ClustDist – distance between pairs of CARF proteins
Cas10Dist – distance between pairs of Cas10
rRNADist – distance between 16s rRNA
Apparent vertical evolution of new accessory gene

43. Systematic procedure developed for detection of
CRISPR-associated genes - flexible parameters,
approaches for detecting functionally relevant associations,
fully generalizable
Core cas genes already known but many diverged subfamilies
remain undetected
Many new CRISPR accessory genes identified
Type III systems are most interesting/complex:
-great majority of new genes
-membrane association
-various forms of signaling
What is special about Type III?
-ancestral?
-functional versatility?

44. Non-interfering CRISPR-Cas systems: no enzymes for target cleavage
Programmed cell death/signal transduction? – type I-E STAND ATPase-containing
Transposon integration? – “minimal” type I-F (and some type I-B) Tn7-like transposon-encoded
Peters JE, Makarova KS, Shmakov S, Koonin EV. Recruitment of CRISPR-Cas systems by Tn7-like transposons. PNAS 2017
Plasmid maintenance? – type IV
Makarova et al. An updated evolutionary classification of CRISPR-Cas systems. NRMicro unpublished
Gene expression regulation? – type V-U5 – inactivated RuvC domains
Shmakov et al. Diversity and evolution of class 2 CRISPR-Cas systems. NRMicro 2017
Shmakov. Makarova, Wolf, Severinov, Koonin, PNAS 2018 in press + unpublished

45. Take home
The basic CRISPR mechanisms are reasonably well understood, and
the most common CRISPR variants are known although a plethora of
interesting new ones remain to be discovered
However, we are only starting to scratch the surface of the broader
CRISPR biology, particularly, connections with microbial signal
transduction
There are many ways CRISPR are used in the arms race and far beyond –
remains to be investigated
Genome context analysis/guilt by association/icity metrics (to be refined)
offer many clues. Attempts to complement with deep learning underway

46. Feng Zhang and http://www.ncbi.nlm.nih.gov/research/groups/koonin/
Collaborators
NCBI: Evolutionary
Genomics Group
Kira Makarova
Sergey Shmakov
Yuri Wolf
Guilhem Faure
Feng Zhang and
lab (Broad/MIT)
Konstantin Severinov
(Skolkovo/Rutgers)
Joe Peters (Cornell)
Winston Yan, David Scott,
David Cheng, Shaorong Chong,
Huaibin Zhang
Arbor Biotechnologies,
Cambridge, MA
Funding: NIH intramural program