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'''CRISPR/Cpf1''' is a [[DNA]]-editing technology. It works analogously to [[CRISPR]]/[[Cas9]] which has revolutionized research in [[genome editing]]. Like its predecessor, it is derived from a mechanism that bacteria use to acquire [[Adaptive immune system | immunity]] from viruses. CRISPR/Cpf1 is potentially better than CRISPR/Cas9 as Cpf1 is a smaller and simpler [[endonuclease]] (a type of [[enzyme]]) than Cas9. That simplifies delivery to the cells whose genes need modifying.<ref name=econ>{{Cite news|title = Even CRISPR|url = http://www.economist.com/news/science-and-technology/21668031-scientists-have-found-yet-another-way-edit-genomes-suggesting-such-technology-will|newspaper = The Economist|access-date = 2015-10-24|issn = 0013-0613 |date=October 3, 2015}}</ref> |
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'''Clustered Regularly Interspaced Short Palindromic Repeats from Prevotella and Francisella 1''' or '''CRISPR/Cpf1''' is a [[DNA]]-editing technology analogous to the [[CRISPR]]/[[Cas9]] system, characterized in 2015 by [[Feng Zhang]]‘s group from the Broad Institute and MIT. Cpf1 is a RNA-guided [[endonuclease]] of a class II CRISPR/Cas system. This acquired immune mechanism found in [[Prevotella]] and [[Francisella]] bacteria is used to prevent genetic damage from viruses. Cpf1 genes are associated with the CRISPR locus, coding for an [[endonuclease]] that use a guide RNA to find and cleave viral DNA, disabling viruses.<ref name=":2">{{Cite web|url=http://news.meta.com/?p=863|title=CRISPR-Based Genetic Engineering Gets a Kick in the Cas|date=2015-09-29|website=Meta Science News|language=en-US|access-date=2016-05-03}}</ref> Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming some of the CRISPR/Cas9 system limitations. CRISPR/Cpf1 could have multiple applications, including treatment of genetic illnesses and degenerative conditions.<ref name=":3">{{Cite news|url=http://www.economist.com/news/science-and-technology/21668031-scientists-have-found-yet-another-way-edit-genomes-suggesting-such-technology-will|title=Even CRISPR|newspaper=The Economist|issn=0013-0613|access-date=2016-05-03}}</ref> |
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Endonuclease-based systems such as CRISPR/Cpf1 consist of a pair of enzymatic “scissors” (the Cpf1) and a guide sequence made of DNA (the [[CRISPR]]) that positions the scissors at the correct spot on the double [[helix]]. Since cells must protect themselves against a host of viruses (to remove viral DNA), it has been hypothesized that each such virus has a complementary endonuclease, offering the possibility that many more types of scissors remain to be discovered.<ref name=econ/> |
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== Description == |
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| ⚫ | Cas9 requires two [[RNA]] molecules to cut DNA while Cpf1 needs one. The proteins also cut DNA at different places, offering researchers more options when selecting an editing site. Cas9 cuts both strands in a DNA molecule at the same position, leaving behind ‘blunt’ ends. Cpf1 leaves one strand longer than the other, creating 'sticky' ends that are easier to work with. Cpf1 appears to be more able to insert new sequences at the cut site, compared to Cas9.<ref name=nat/> |
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| ⚫ | CRISPR/Cpf1 was found by searching a published database of bacterial genetic sequences for promising bits of DNA. Cpf1 appeared in many species. the ultimate Cpf1 endonuclease that was developed into a tool for genome editing was taken from one of 16 bacteria that harbored it.<ref name=nat>{{Cite journal|title = Alternative CRISPR system could improve genome editing|url = http://www.nature.com/doifinder/10.1038/nature.2015.18432|journal = Nature|pages = 17-17|volume = 526|issue = 7571|doi = 10.1038/nature.2015.18432|first = Heidi|last = Ledford}}</ref> Two candidate enzymes from [[Acidaminococcus|''Acidaminococcus'']] and ''Lachnospiraceae'' display efficient genome-editing activity in human cells.<ref name=":3" /> |
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Cpf1 is a single RNA-guided endonuclease that lacks [[tracrRNA]]. It utilizes a T-rich [[protospacer]]-adjacent motif. Cpf1 cleaves DNA via a staggered DNA double-stranded break. |
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It has been recently reported the discovery a smaller version of Cas9 in the bacterium ''Staphylococcus aureus'', becoming a potential alternative to Cpf1. <ref name="nat" /> |
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=== Classification === |
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| ⚫ | CRISPR/Cpf1 was found by searching a published database of bacterial genetic sequences for promising bits of DNA. Cpf1 appeared in many species |
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The systems CRISPR/Cas are separated into two classes. Class 1 uses several Cas proteins together with the crRNA to build a functional endonuclease. On the other hand, Class 2 CRISPR systems use a single Cas protein with a crRNA. Cpf1 has been recently identified as a Class II, Type V CRISPR/Cas systems containing a 1,300 amino acid protein.<ref name=":0">Makarova, Kira S., et al. "An updated evolutionary classification of CRISPR-Cas systems." Nature Reviews Microbiology (2015).</ref> |
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Two candidate enzymes from ''[[Acidaminococcus]]'' and ''Lachnospiraceae bacterium'' display efficient genome-editing activity in human cells.<ref>{{cite journal|last1=Zetsche|first1=Bernd|last2=Gootenberg|first2=Jonathan S.|last3=Abudayyeh|first3=Omar O.|last4=Slaymaker|first4=Ian M.|last5=Makarova|first5=Kira S.|last6=Essletzbichler|first6=Patrick|last7=Volz|first7=Sara E.|last8=Joung|first8=Julia|last9=van der Oost|first9=John|last10=Regev|first10=Aviv|last11=Koonin|first11=Eugene V.|last12=Zhang|first12=Feng|title=Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System|journal=Cell|date=October 2015|volume=163|issue=3|pages=759–771|doi=10.1016/j.cell.2015.09.038}}</ref> |
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== |
== Structure == |
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The Cpf1 locus contains a mixed alpha/beta domain, a RuvC-I followed by a helical region, a RuvC-II and a zinc finger-like domain.<ref name=":1">{{cite journal|last2=Gootenberg|first2=Jonathan S.|last3=Abudayyeh|first3=Omar O.|last4=Slaymaker|first4=Ian M.|last5=Makarova|first5=Kira S.|last6=Essletzbichler|first6=Patrick|last7=Volz|first7=Sara E.|last8=Joung|first8=Julia|last9=van der Oost|first9=John|date=October 2015|title=Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System|journal=Cell|volume=163|issue=3|pages=759–771|doi=10.1016/j.cell.2015.09.038|last1=Zetsche|first1=Bernd|last10=Regev|first10=Aviv|last11=Koonin|first11=Eugene V.|last12=Zhang|first12=Feng}}</ref> The Cpf1 protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9. Furthermore, Cpf1 does not have a HNH endonuclease domain, and the N-terminal of Cpf1 does not have the alfa-helical recognition lobe of Cas9.<ref name=":0" /> |
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*[[CRISPR]] |
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Cpf1 CRISPR-Cas domain architecture shows that Cpf1 is functionally unique, being classified as Class 2, type V CRISPR system. The Cpf1 loci encode Cas1, Cas2, and Cas4 proteins more similar to types I and III than from type II systems. Database searches suggest the prevalence of Cpf1-family proteins in many bacterial species.<ref name=":0" /> |
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Functional Cpf1 doesn’t need the tracrRNA, therefore, only the crRNA is required. This has advantage for genome editing because Cpf1 is not only smaller than Cas9, but also it has a smaller sgRNA molecule (proximately half of the nucleotides number than Cas9).<ref name=":4">{{Cite web|url=http://epigenie.com/cpf1-takes-crispr-bigger-by-going-smaller/?utm_campaign=coschedule&utm_source=facebook_page&utm_medium=EpiGenie&utm_content=Cpf1+Takes+CRISPR+Bigger+by+Going+Smaller|title=Cpf1 Moves in on Cas9 for Next-Gen CRISPR Genome Editing|website=epigenie.com|access-date=2016-05-03}}</ref> |
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Cpf1-crRNA complex cleave target DNA by identification of a T-rich [[Protospacer adjacent motif|protospacer-adjacent motif (PAM)]], in contrast to the G-rich PAM targeted by Cas9. After identification of PAM, Cpf1 introduces a sticky-end-like DNA double- stranded break of 4 or 5 nucleotides overhang.<ref name=":1" /> |
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| ⚫ | |||
The CRISPR/Cpf1 system consist of a Cpf1 enzyme and a guide RNA that finds and positions the complex at the correct spot on the double helix to cleave target DNA. CRISPR/Cpf1 systems activity has three stages: <ref name="nat" /> |
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# Adaptation: Cas1 and Cas2 proteins facilitate the adaptation of small fragments of DNA into the CRISPR array. . |
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# Formation of crRNAs: processing of pre-cr-RNAs producing of mature crRNAs to guide the Cas protein. |
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# Interference: the Cpf1 is bound to a crRNA to form a binary complex that will identify and cleave a target DNA sequence. |
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=== Ca9 vs. Cpf1=== |
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| ⚫ | Cas9 requires two [[RNA]] molecules to cut DNA while Cpf1 needs one. The proteins also cut DNA at different places, offering researchers more options when selecting an editing site. Cas9 cuts both strands in a DNA molecule at the same position, leaving behind ‘blunt’ ends. Cpf1 leaves one strand longer than the other, creating 'sticky' ends that are easier to work with. Cpf1 appears to be more able to insert new sequences at the cut site, compared to Cas9.<ref name="nat" /> Although the CRISPR/Cas9 system can efficiently disable genes, it is challenging to insert genes or generate a knock-in.<ref name=":2" /> Cpf1 lacks [[tracrRNA]], utilizes a T-rich PAM and cleaves DNA via a staggered DNA DSB.<ref name=":4" /> |
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In summary, some important different between Cpf1 and Cas9 systems are: <ref name=":5">{{Cite web|url=http://www.labcritics.com/crisprcpf1-new-simpler-crispr-protein-may-cut-patent-fights-short-4925/|title=CRISPR/CPF1: New Simpler CRISPR Protein May Cut Patent Fights Short|date=2015-09-27|website=Labcritics|access-date=2016-05-03}}</ref> |
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* Cpf1 recognizes a T-rich PAM. Cas9 recognizes G-rich PAM. Therefore, Cpf1 enables new targeting possibilities. |
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* Cpf1 creates 4-5 nt long sticky ends, instead of blunt ends produced by Cas9; enhancing the efficiency of genetic insertions and specificity during NHEJ or HDR. |
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* Cpf1 cuts target DNA further away from PAM, further away from the Cas9 cutting site. This enables new possibilities for cleaving the DNA. |
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{| class="wikitable" |
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|- |
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! Feature !! Cas9 !! Cpf1 |
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|- |
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| '''Structure''' || Two RNA required || One RNA required |
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|- |
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| '''Cutting Mechanism''' || Blunt end cuts || Staggered end cuts |
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|- |
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| '''Cutting site''' || Proximal to recognition site || Distal from recognition site |
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|- |
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| '''Target sites''' || G-rich PAM || T-rich PAM |
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|- |
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| '''Cell type''' || Fast growing cells, including cancer cells || Non-dividing cells, including nerve cells |
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|}<ref name=":5" /> |
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== Tools == |
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Multiple aspects influence target efficiency and specificity when using the CRISPR system genome editing, including guide RNA design. Many design models for guide RNA has been suggested and an increasing number of tools are facilitating their optimized design. Computational tools for the general use of CRISPR systems includes [[SgRNA designer]], [[CRISPR MultiTargeter]], [[SSFinder]] and other software that facilitates target sequence finding and optimization.<ref>{{Cite journal|last=Graham|first=Daniel B.|last2=Root|first2=David E.|date=2015-01-01|title=Resources for the design of CRISPR gene editing experiments|url=http://dx.doi.org/10.1186/s13059-015-0823-x|journal=Genome Biology|volume=16|pages=260|doi=10.1186/s13059-015-0823-x|issn=1474-760X|pmc=4661947|pmid=26612492}}</ref> |
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== Conflict of interest == |
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Intellectual property concerns are keeping distant agricultural and drug companies from using CRISPR/Cas9. However, CRISPR/Cpf1 is new approached without legal matters, encouraging industries to invest and develop the Cpf1-based system''.''<ref name=":3" /> |
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== References == |
== References == |
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{{reflist|30em}} |
{{reflist|30em}} |
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Revision as of 02:55, 19 May 2016
Clustered Regularly Interspaced Short Palindromic Repeats from Prevotella and Francisella 1 or CRISPR/Cpf1 is a DNA-editing technology analogous to the CRISPR/Cas9 system, characterized in 2015 by Feng Zhang‘s group from the Broad Institute and MIT. Cpf1 is a RNA-guided endonuclease of a class II CRISPR/Cas system. This acquired immune mechanism found in Prevotella and Francisella bacteria is used to prevent genetic damage from viruses. Cpf1 genes are associated with the CRISPR locus, coding for an endonuclease that use a guide RNA to find and cleave viral DNA, disabling viruses.[1] Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming some of the CRISPR/Cas9 system limitations. CRISPR/Cpf1 could have multiple applications, including treatment of genetic illnesses and degenerative conditions.[2]
Description
Discovery
CRISPR/Cpf1 was found by searching a published database of bacterial genetic sequences for promising bits of DNA. Cpf1 appeared in many species. the ultimate Cpf1 endonuclease that was developed into a tool for genome editing was taken from one of 16 bacteria that harbored it.[3] Two candidate enzymes from Acidaminococcus and Lachnospiraceae display efficient genome-editing activity in human cells.[2]
It has been recently reported the discovery a smaller version of Cas9 in the bacterium Staphylococcus aureus, becoming a potential alternative to Cpf1. [3]
Classification
The systems CRISPR/Cas are separated into two classes. Class 1 uses several Cas proteins together with the crRNA to build a functional endonuclease. On the other hand, Class 2 CRISPR systems use a single Cas protein with a crRNA. Cpf1 has been recently identified as a Class II, Type V CRISPR/Cas systems containing a 1,300 amino acid protein.[4]
Structure
The Cpf1 locus contains a mixed alpha/beta domain, a RuvC-I followed by a helical region, a RuvC-II and a zinc finger-like domain.[5] The Cpf1 protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9. Furthermore, Cpf1 does not have a HNH endonuclease domain, and the N-terminal of Cpf1 does not have the alfa-helical recognition lobe of Cas9.[4]
Cpf1 CRISPR-Cas domain architecture shows that Cpf1 is functionally unique, being classified as Class 2, type V CRISPR system. The Cpf1 loci encode Cas1, Cas2, and Cas4 proteins more similar to types I and III than from type II systems. Database searches suggest the prevalence of Cpf1-family proteins in many bacterial species.[4]
Functional Cpf1 doesn’t need the tracrRNA, therefore, only the crRNA is required. This has advantage for genome editing because Cpf1 is not only smaller than Cas9, but also it has a smaller sgRNA molecule (proximately half of the nucleotides number than Cas9).[6]
Cpf1-crRNA complex cleave target DNA by identification of a T-rich protospacer-adjacent motif (PAM), in contrast to the G-rich PAM targeted by Cas9. After identification of PAM, Cpf1 introduces a sticky-end-like DNA double- stranded break of 4 or 5 nucleotides overhang.[5]
Mechanism
The CRISPR/Cpf1 system consist of a Cpf1 enzyme and a guide RNA that finds and positions the complex at the correct spot on the double helix to cleave target DNA. CRISPR/Cpf1 systems activity has three stages: [3]
- Adaptation: Cas1 and Cas2 proteins facilitate the adaptation of small fragments of DNA into the CRISPR array. .
- Formation of crRNAs: processing of pre-cr-RNAs producing of mature crRNAs to guide the Cas protein.
- Interference: the Cpf1 is bound to a crRNA to form a binary complex that will identify and cleave a target DNA sequence.
Ca9 vs. Cpf1
Cas9 requires two RNA molecules to cut DNA while Cpf1 needs one. The proteins also cut DNA at different places, offering researchers more options when selecting an editing site. Cas9 cuts both strands in a DNA molecule at the same position, leaving behind ‘blunt’ ends. Cpf1 leaves one strand longer than the other, creating 'sticky' ends that are easier to work with. Cpf1 appears to be more able to insert new sequences at the cut site, compared to Cas9.[3] Although the CRISPR/Cas9 system can efficiently disable genes, it is challenging to insert genes or generate a knock-in.[1] Cpf1 lacks tracrRNA, utilizes a T-rich PAM and cleaves DNA via a staggered DNA DSB.[6]
In summary, some important different between Cpf1 and Cas9 systems are: [7]
- Cpf1 recognizes a T-rich PAM. Cas9 recognizes G-rich PAM. Therefore, Cpf1 enables new targeting possibilities.
- Cpf1 creates 4-5 nt long sticky ends, instead of blunt ends produced by Cas9; enhancing the efficiency of genetic insertions and specificity during NHEJ or HDR.
- Cpf1 cuts target DNA further away from PAM, further away from the Cas9 cutting site. This enables new possibilities for cleaving the DNA.
| Feature | Cas9 | Cpf1 |
|---|---|---|
| Structure | Two RNA required | One RNA required |
| Cutting Mechanism | Blunt end cuts | Staggered end cuts |
| Cutting site | Proximal to recognition site | Distal from recognition site |
| Target sites | G-rich PAM | T-rich PAM |
| Cell type | Fast growing cells, including cancer cells | Non-dividing cells, including nerve cells |
Tools
Multiple aspects influence target efficiency and specificity when using the CRISPR system genome editing, including guide RNA design. Many design models for guide RNA has been suggested and an increasing number of tools are facilitating their optimized design. Computational tools for the general use of CRISPR systems includes SgRNA designer, CRISPR MultiTargeter, SSFinder and other software that facilitates target sequence finding and optimization.[8]
Conflict of interest
Intellectual property concerns are keeping distant agricultural and drug companies from using CRISPR/Cas9. However, CRISPR/Cpf1 is new approached without legal matters, encouraging industries to invest and develop the Cpf1-based system.[2]
References
- ^ a b "CRISPR-Based Genetic Engineering Gets a Kick in the Cas". Meta Science News. 2015-09-29. Retrieved 2016-05-03.
- ^ a b c "Even CRISPR". The Economist. ISSN 0013-0613. Retrieved 2016-05-03.
- ^ a b c d Ledford, Heidi. "Alternative CRISPR system could improve genome editing". Nature. 526 (7571): 17–17. doi:10.1038/nature.2015.18432.
- ^ a b c Makarova, Kira S., et al. "An updated evolutionary classification of CRISPR-Cas systems." Nature Reviews Microbiology (2015).
- ^ a b Zetsche, Bernd; Gootenberg, Jonathan S.; Abudayyeh, Omar O.; Slaymaker, Ian M.; Makarova, Kira S.; Essletzbichler, Patrick; Volz, Sara E.; Joung, Julia; van der Oost, John; Regev, Aviv; Koonin, Eugene V.; Zhang, Feng (October 2015). "Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System". Cell. 163 (3): 759–771. doi:10.1016/j.cell.2015.09.038.
- ^ a b "Cpf1 Moves in on Cas9 for Next-Gen CRISPR Genome Editing". epigenie.com. Retrieved 2016-05-03.
- ^ a b "CRISPR/CPF1: New Simpler CRISPR Protein May Cut Patent Fights Short". Labcritics. 2015-09-27. Retrieved 2016-05-03.
- ^ Graham, Daniel B.; Root, David E. (2015-01-01). "Resources for the design of CRISPR gene editing experiments". Genome Biology. 16: 260. doi:10.1186/s13059-015-0823-x. ISSN 1474-760X. PMC 4661947. PMID 26612492.
{{cite journal}}: CS1 maint: unflagged free DOI (link)