Cas9 Proofreads
Larry H Bernstein, MD, FCAP, Curator
LPBI
2.2.15 Cas9 Proofreads, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 2: CRISPR for Gene Editing and DNA Repair
Cas9 Proofreads Gene Edits
In this week’s Science, a multi-institute team publishes a study revealing new details about how the DNA-cutting protein used in the genome-editing technology CRISPR/Cas9 effectively ignores off-target sites. Although the protein’s specificity was well known, the mechanisms behind its effect were not. The researchers used time-lapsed single-molecule imaging to track Cas9 in vivo, and used the data to calculate the likelihood that Cas9 would remain in one place. They found that the protein spends very little time on non-target gene sequences and that it travels more slowly through dense heterochromatic regions of DNA, although it retains the ability to bind to targets in these regions.
The gene-editing CRISPR/Cas9 system has three checkpoints to ensure it alters the right section of DNA.
| November 13, 2015
http://www.the-scientist.com//?articles.view/articleNo/44467/title/Cas9-Proofreads-Gene-Edits/
CRISPR/Cas9, now widely used as a precise way to edit DNA, involves the Cas9 protein locating pre-programmed sequences of base pairs before slicing and dicing the genome. Two recent reports, one published in Nature (October 28) and the other in Science (November 12), unveil details about how Cas9 ensures it targets the right sequences.
In the Science paper, CRISPR pioneer Jennifer Doudna from the University of California, Berkley, and her colleagues explored how the Cas9 protein searches for a single pattern of base pairs in an entire genome. Cas9 operates in conjunction with an RNA, programmed to guide Cas9 to a specific 20-nucleotide section of DNA. “It’s crazy that the Cas9 complex manages to scan the vast space of eukaryotic genomes,” study coauthor Spencer Knight of Berkeley said in a press release.
The team visualized Cas9’s binding patterns with single-particle tracking, finding that once the protein recognizes a primer sequence in the DNA, it will bind to incorrect areas that look like its actual target—but Cas9 tends to disengage from these mistakes in less than a second. These steps serve as the first and second checks on the gene-editing system.
In the Nature paper, Doudna and others investigated how Cas9 governs its DNA cleavage abilities. Once it binds to a region of DNA, the Cas9 protein will change shape based on how closely the DNA region matches Cas9’s programmed target. “We found that RNA-guided Cas9 can bind some off-target DNA sequences, which differ from the correct target by just a few mutations, very tightly. Surprisingly, though, the region of Cas9 that does the cutting is inhibited because of the imperfect match,” study coauthor Samuel Sternberg of Berkeley said in the press release.
This “proofreading mechanism,” the authors wrote in Nature, “serves as a final specificity checkpoint before DNA double-strand break formation.”
Conformational control of DNA target cleavage by CRISPR–Cas9
Samuel H. Sternberg, Benjamin LaFrance, Matias Kaplan & Jennifer A. Doudna
Nature 527, 110–113 (05 Nov 2015) http://dx.doi.org:/10.1038/nature15544
Cas9 is an RNA-guided DNA endonuclease that targets foreign DNA for destruction as part of a bacterial adaptive immune system mediated by clustered regularly interspaced short palindromic repeats (CRISPR)1, 2. Together with single-guide RNAs3, Cas9 also functions as a powerful genome engineering tool in plants and animals4, 5, 6, and efforts are underway to increase the efficiency and specificity of DNA targeting for potential therapeutic applications7, 8. Studies of off-target effects have shown that DNA binding is far more promiscuous than DNA cleavage9, 10, 11, yet the molecular cues that govern strand scission have not been elucidated. Here we show that the conformational state of the HNH nuclease domain directly controls DNA cleavage activity. Using intramolecular Förster resonance energy transfer experiments to detect relative orientations of the Cas9 catalytic domains when associated with on- and off-target DNA, we find that DNA cleavage efficiencies scale with the extent to which the HNH domain samples an activated conformation. We furthermore uncover a surprising mode of allosteric communication that ensures concerted firing of both Cas9 nuclease domains. Our results highlight a proofreading mechanism beyond initial protospacer adjacent motif (PAM) recognition12 and RNA–DNA base-pairing3 that serves as a final specificity checkpoint before DNA double-strand break formation.
Figure 1: Full-length sgRNA drives inward lobe closure of Cas9
http://www.nature.com/nature/journal/v527/n7576/carousel/nature15544-f1.jpg
a, Domain organization of S. pyogenes Cas9 (top) and X-ray crystal structure of sgRNA/DNA-bound Cas9 (Protein Data Bank (PDB) accession number 4UN3, ref. 16) (bottom), with HNH domain omitted for clarity. BH, bridge helix; REC, recognit…
Figure 2: FRET experiments reveal an activated conformation of the HNH nuclease domain.
http://www.nature.com/nature/journal/v527/n7576/carousel/nature15544-f2.jpg
a, Design of Cas9HNH-1 FRET construct. Measured distances between S355 and S867 in the sgRNA/DNA-bound Cas9 structure16 and a model of the HNH domain docked at the cleavage site are indicated, as are putative conformational changes of t…
……
Dynamics of CRISPR-Cas9 genome interrogation in living cells
Spencer C. Knight1, Liangqi Xie2, Wulan Deng3,4, Benjamin Guglielmi2, Lea B. Witkowsky2, Lana Bosanac2, Elisa T. Zhang2, …., Jennifer A. Doudna1,2,6,7,8,*,Robert Tjian2,3,4,6,9,*
Science 13 Nov 2015; 350(6262):823-826 DOI: http://dx.10..org:/1126/science.aac6572
The RNA-guided CRISPR-associated protein Cas9 is used for genome editing, transcriptional modulation, and live-cell imaging. Cas9-guide RNA complexes recognize and cleave double-stranded DNA sequences on the basis of 20-nucleotide RNA-DNA complementarity, but the mechanism of target searching in mammalian cells is unknown. Here, we use single-particle tracking to visualize diffusion and chromatin binding of Cas9 in living cells. We show that three-dimensional diffusion dominates Cas9 searching in vivo, and off-target binding events are, on average, short-lived (<1 second). Searching is dependent on the local chromatin environment, with less sampling and slower movement within heterochromatin. These results reveal how the bacterial Cas9 protein interrogates mammalian genomes and navigates eukaryotic chromatin structure.
Genome editing with a Cas9 scalpel
The Cas9 nuclease forms the heart of the CRISPR-Cas genome editing system. Cas9 binds small guide RNAs that direct it to its target sites, where the nuclease either cleaves or binds to genomic DNA. Knight et al. used single-molecule imaging to track Cas9 in living cells. Cas9 searches the genome for its target sites using rapid threedimensional diffusion. It spends very little time binding to off-target sites, which explains the high accuracy of the CRISPRCas9 editing machine. Science, this issue p. 823
GEN News Highlights Nov 13, 2015 CRISPR Triple Checks Its Target before Cutting
Several hundred Cas9 enzymes (red dots) searching the nucleus of a live mammalian cell for a particular DNA sequence. They become white when they bind briefly before moving on. The lines show the paths these enzymes have taken, color coded according to time. The Cas9 tracks show that the enzymes search by diffusing through the nucleus, and that off-target binding events are predominately short lived. Motion is slowed to half normal speed. [Spencer Knight video, UC Berkeley]
https://youtu.be/YjuMDtjElfY?t=7
From the co-discoverer of the CRISPR-Cas9 system and her colleagues at UC Berkeley comes new data that should instill a greater level of confidence that the genome editing tool won’t inadvertently excise off-target DNA.
The two new reports from the Berkeley investigators show in great detail how the Cas9 protein searches through billions of base pairs in a cell to find the right DNA sequence, and how Cas9 determines whether to bind or bind and cut, thereby initiating gene editing. From the collected data, the researchers were able to surmise that Cas9 appears to have at least three ways of checking to make sure it finds the right target DNA before it takes the irrevocable step of making a cut.
“CRISPR-Cas9 has evolved for accurate DNA targeting, and we now understand the molecular basis for its seek-and-cleave activity, which helps limit off-target DNA editing,” explained senior author of both articles and co-discover of the CRISPR-Cas9 system Jenifer Doudna, Ph.D., a Howard Hughes Medical Institute investigator and professor of molecular and cell biology and of chemistry at UC Berkeley.
The findings from this study were published recently in two papers: the first in Science through an article entitled “Dynamics of CRISPR-Cas9 genome interrogation in living cells” and the second in Nature in an article titled “Conformational control of DNA target cleavage by CRISPR–Cas9.”
In the Science article, the researchers tracked Cas9-RNA molecules within the nucleus of mammalian cells as they rapidly searched through the entire genome to find and bind to their prescribed target sequence.
“It’s crazy that the Cas9 complex manages to scan the vast space of eukaryotic genomes,” remarked lead author of the Science paper and graduate student Spencer Knight. “There is a lot of off-target binding by Cas9, but we found that these interactions are very brief—from milliseconds to seconds—before Cas9 moves on.”
The Berkeley team estimated that a few thousand CRISPR-Cas9 complexes can scour the entire genome extremely rapidly to find one targeted stretch of DNA. Additionally, Cas9 must also recognize a short three-base-pair DNA sequence immediately following the primer sequence, or PAM, which occurs roughly 300 million times within the human genome.
“If Cas9 bound for tens of seconds or minutes at each off-target site, it would never, ever be able to find a target and cut in a timely manner,” Knight said.
In the Nature article, the investigators observed that once Cas9 binds to a region of DNA, it performs an additional check before two distal sections of the Cas9 protein complex come together, like the blades of a scissors, to precisely align the active sites that cut double-stranded DNA.
“We found that RNA-guided Cas9 can bind some off-target DNA sequences, which differ from the correct target by just a few mutations, very tightly. Surprisingly, though, the region of Cas9 that does the cutting is inhibited because of the imperfect match. But when the correctly matching DNA is located, Cas9 undergoes a large structural change that releases this inhibition and triggers DNA cutting,” noted the lead author of the Nature paper Samuel Sternberg, Ph.D., former graduate student in Dr. Doudna’s laboratory.
“We think that this structural change is the last checkpoint, or proofreading stage, of the DNA targeting reaction,” Dr. Sternberg continued. “First, Cas9 recognizes a short DNA segment next to the target—the PAM—then the target DNA is matched up with the guide RNA via Watson-Crick base-pairing. Finally, when a perfect match is identified, the last part of the protein swings into place to enable cutting and initiate genome editing.”
When asked about the impact of the current researcher, Knight responded that the data “suggests that you have more than one checkpoint to ensure correct Cas9 binding. There’s not just sequence regulation, there is also temporal regulation: it has to engage with the DNA and park long enough that it can actually rearrange and cut.”
The discoveries from the Berkeley teams should allow for greater insight into the molecular mechanisms that lead to off-target events that can occur during genome editing applications—an critical area for researches to grasp an understanding of should they hope to use CRISPR-Cas9 in a wider clinical capacity.
Another Telomere-Regulating Enzyme Found
Researchers identify a novel protein that helps maintain the length of chromosome-capping telomeres.
| November 12, 2015
http://www.the-scientist.com/images/Nutshell/November2015/chromosome.jpg
ATM kinase, an enzyme known to be involved in DNA repair, is required for telomere elongation, according to a study published this week (November 12) in Cell Reports. The results could have implications for diverse diseases, from cancer, which is typically linked to overly long telomeres, to lung and bone marrow disorders that are associated with shortened telomeres.
“We’ve known for a long time that telomerase doesn’t tell the whole story of why chromosomes’ telomeres are a given length, but with the tools we had, it was difficult to figure out which proteins were responsible for getting telomerase to do its work,” Carol Greider, a director at the Johns Hopkins Institute for Basic Biomedical Sciences and a corecipient of the 2009 Nobel Prize in Physiology or Medicine for the discovery of telomerase, said in apress release.
Identifying enzymes involved in maintaining telomere length in mammalian cells has been slow going, requiring blocking individual proteins’ functions then growing the cells in the lab and looking for differences in telomere length between experimental and control cells. In addition to taking at least three months before detectable differences arose, blocking proteins that were required for cell survival would kill the cells before such differences could be seen.
Greider and her colleagues devised a way to expedite the process by cutting telomeres, then looking for elongation by telomerase—a strategy that has been previously used in yeast cells. Using this method, which the researchers dubbed addition of de novo initiated telomeres (ADDIT), the group investigated the role of ATM kinase. Blocking ATM kinase in mouse cells, then cutting down the telomeres, the group found that the enzyme was required to lengthen the chromosome caps—a result verified using the old, three-month-long method. Conversely, activating ATM kinase with a PARP1 inhibitor spurred telomere elongation.
Greider’s team now plans to further explore the role of ATM kinase in telomere elongation and look for other components important in telomere maintenance. “The potential applications are very exciting,” Stella Suyong Lee, a graduate student in the Greider lab who spearheaded the development of ADDIT, said in the release. “Ultimately ADDIT can help us understand how cells strike a balance between aging and the uncontrolled cell growth of cancer, which is very intriguing.”
2012pharmaceutical
Amandeep Kaur
Aashir Awan, Phd
Abhisar Anand
Adina Hazan
Alan F. Kaul, PharmD., MS, MBA, FCCP
alexcrystal6
anamikasarkar
apreconasia
aptubman
Arav Gandhi
aviralvatsa
David Orchard-Webb, PhD
danutdaagmailcom
Demet Sag, Ph.D., CRA, GCP
Dror Nir
dsmolyar
Ethan Coomber
evelinacohn
Gail S Thornton
Irina Robu
jayzmit48
jdpmd
jshertok
kellyperlman
Ed Kislauskis
larryhbern
Madison Davis
marzankhan
megbaker58
ofermar2020
Dr. Pati
pkandala
ritusaxena
Rick Mandahl
sjwilliamspa
Srinivas Sriram
stuartlpbi
Dr. Sudipta Saha
tildabarliya
zraviv06
zs22
