CRISPR-RT: A web service for designing CRISPR-C2c2 ...although C2c2 may also cleave collateral RNAs (Abudayyeh et al., 2016). Researchers have applied the CRISPR-C2c2 system to sensitively

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CRISPR-RT: A web service for designing CRISPR-C2c2 crRNA with improved target specificity

Houxiang Zhu1, Emily Richmond2, Chun Liang1, 2*

1Department of Biology, Miami University, Oxford, United States; 2Department of Software

Engineering, Miami University, Oxford, United States

*For correspondence: [email protected]

HZ: [email protected]

ER: [email protected]

CL: [email protected]

.CC-BY-NC-ND 4.0 International licenseacertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under

The copyright holder for this preprint (which was notthis version posted January 12, 2017. ; https://doi.org/10.1101/099895doi: bioRxiv preprint

https://doi.org/10.1101/099895

http://creativecommons.org/licenses/by-nc-nd/4.0/

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Abstract CRISPR-Cas systems have been successfully applied in genome editing. Recently,

the CRISPR-C2c2 system has been reported as a tool for RNA editing. Here we describe

CRISPR-RT (CRISPR RNA-Targeting), the first web service to help biologists design the

crRNA with improved target specificity for the CRISPR-C2c2 system. CRISPR-RT allows users

to set up a wide range of parameters, making it highly flexible for current and future research in

CRISPR-based RNA editing. CRISPR-RT covers major model organisms and can be easily

extended to cover other species. CRISPR-RT will empower researchers in RNA editing. It is

available at http://bioinfolab.miamioh.edu/CRISPR-RT.



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Introduction

The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-

associated) system is an adaptive immune system of prokaryotes, which is used to resist foreign

genetic elements in bacteria and archaea (Marraffini and Sontheimer, 2010; Barrangou and

Marraffini, 2014). During the past several years, CRISPR-Cas systems have been successfully

applied in genome editing (Hwang et al., 2013; Jiang et al., 2013; Cho et al., 2013; Cong et al.,

2013; Bikard et al., 2014; Wan et al., 2015), but no systems have been reported for RNA editing.

Therefore, new CRISPR-Cas systems that regulate RNA activities are necessary for studying the

roles of RNA molecules. Recently, the CRISPR-C2c2 system has been demonstrated as a tool for

RNA targeting (Abudayyeh et al., 2016).

CRISPR-C2c2 was discovered in 21 bacterial genomes and belongs to the type VI of

Class 2 CRISPR systems (Shmakov et al., 2015). Researchers have characterized the CRISPR-

C2c2 system from the bacteria Leptotrichia shahii (Abudayyeh et al., 2016). The L. shahii C2c2

locus is simply organized, including C2c2, Cas1, Cas2 and a CRISPR array (Figure 1). Cas1

and Cas2 play an important role in spacer acquisition (Nuñez et al., 2015; Heler et al., 2015;

Nuñez et al., 2014; Díez-Villaseñor et al., 2013; Yosef et al., 2012; Datsenko et al., 2012). C2c2,

which contains two higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domains,

mainly functions as a sole effector protein mediating single-strand RNA cleavage (Abudayyeh et

al., 2016). The CRISPR array consists of many short palindromic repeat elements and spacer

sequences. Similar to other Class 2 systems, the CRISPR array of CRISPR-C2c2 is first

transcribed into pre-crRNA (Shmakov et al., 2015). Differently, the pre-crRNA is processed by

C2c2 into mature crRNAs without attaching to trans-activating crRNAs (East-Seletsky et al.,

2016). The mature crRNA binds to C2c2 and guides it to target a specific single-strand RNA.



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Then, in the target site, the HEPN domains of C2c2 mediate cleavage of the single-strand RNA

(Abudayyeh et al., 2016). The crRNA stem-loop structure is also required for cleavage. Thus, the

palindromic repeat length of crRNA needs to be longer than 24 nt to maintain the stem loop

(Abudayyeh et al., 2016). In addition, C2c2 combined with a 22-28 length of the target

complementarity region of crRNA would effectively mediate cleavage (Abudayyeh et al., 2016).

The seed region is located in the center of the crRNA-target duplex, where is more sensitive to

mismatches than the non-seed region (Abudayyeh et al., 2016). Single mismatch can be fully

tolerated by C2c2, but if double mismatches are located in the seed region, C2c2 is unable to

cleave the single-strand RNA (Abudayyeh et al., 2016). C2c2 can even tolerate 3 consecutive

mismatches in the non-seed region (Abudayyeh et al., 2016). Currently, there is no research

about gaps (insertions or deletions) tolerated by C2c2, which may be explored in the near future.

The CRISPR-C2c2 system prefers H (A, U, or C) for the 3’ protospacer flanking site (PFS)

sequence of one single base length to mediate single-strand RNA cleavage (Abudayyeh et al.,

2016). CRISPR-C2c2 has been reprogrammed to knockdown the specific mRNA in vivo,

although C2c2 may also cleave collateral RNAs (Abudayyeh et al., 2016). Researchers have

applied the CRISPR-C2c2 system to sensitively detect transcripts in complex mixtures (East-

Seletsky et al., 2016). C2c2 can be easily converted into an inactive RNA-binding protein

(dC2c2) by mutating any of the two HEPN domains (Abudayyeh et al., 2016). Compared to the

wild type C2c2, dC2c2 has more potential applications as an RNA-binding protein (Abudayyeh

et al., 2016). The CRISPR-C2c2 system is opening a new chapter for the programmable CRISPR

tools of RNA editing. However, until now there is no available software for designing crRNAs

of the CRISPR-C2c2 system.



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We have developed CRISPR-RT (CRISPR RNA-Targeting), a web service to help

biologists design the crRNA for the CRISPR-C2c2 system. To maximize the flexibility for

current and future research in CRISPR-based RNA editing, CRISPR-RT allows users to set up a

wide range of parameters, such as length of the target complementarity region of crRNA, length

of the seed region, the PFS, and the number of mismatches or gaps tolerated by off targets. After

setting up the required parameters, CRISPR-RT will find target candidates from the input RNA

sequence and employ rigorous alignment algorithms to search on- and off-target sites for each

target candidate within the reference transcriptome (Langmead and Salzberg, 2012). The results

are displayed in highly interactive graphical interfaces. Users can rank target candidates by the

total number of target sites in the reference transcriptome, which help them choose the target

candidate based on the minimum effect of off targets. In addition, users are able to validate the

on- and off-target sites in the background of annotated genome and transcript features by data

visualization through JBrowse (Skinner et al., 2009).

Results

Graphic Input Interface

Figure 2 shows the home page of CRISPR-RT. If users click “C2c2” on the left side

menu, the setting page of the CRISPR-C2c2 system will be displayed (Figure 3). First, users

input an RNA sequence that they want to target in FASTA format into the text area. Users can

also convert a cDNA sequence to the corresponding RNA sequence by clicking the “cDNA?”

link. After converting, they then copy the converted RNA sequence back into the text area as the

input sequence. If users want to use an example sequence, they can click the “Example Sequence”

button. Second, users select a reference transcriptome. If the transcriptome that users want to



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choose does not show up in the drop-down menu and this transcriptome is available in

ENSEMBL genome annotation (www.ensembl.org), they can click the “others?” link to submit a

requirement so that the transcriptome will be added to the web server manually soon. Third, in

terms of our current understanding of the CRISPR-C2c2 system architecture (Figure 1), users

can set up the PFS sequence and crRNA requirements for the CRISPR-C2c2 system properly.

The on- and off- target PFS sequence can be set respectively, which accepts any single letter or

combination of the international union of pure and applied chemistry (IUPAC) nucleotide codes

except “T” and “Z”. Users then set up the length of the target complementarity region of crRNA

and the length of the seed region. The seed region is located in the center of the crRNA-target

duplex, and its length should not be greater than the length of the target complementarity region

of crRNA. Fourth, users can choose an off-target setting (“Basic settings” or “Specific settings”).

For “Basic settings”, the number of mismatches or gaps tolerated by off targets as well as by the

seed region can be set respectively. The number of consecutive mismatches or gaps in the seed

or non-seed region tolerated by off targets can also be configured. For “Specific settings”, users

can set up more detailed parameters in the seed and non-seed region separately, such as the

number of mismatches in the seed or non-seed region tolerated by off targets. Users can also set

up the search sensitivity of Bowtie2 (Langmead and Salzberg, 2012), which is related to the

alignment options setting of Bowtie2, to search target sites in the reference transcriptome. Higher

sensitivity setting causes alignments to be more sensitive, but it usually results in a slower search

time. CRISPR-RT maximizes flexible and adjustable settings to help biologists conduct current

and future experiments with more in-depth understanding of the CRISPR-C2c2 system. After

setting up all the parameters, users click the “Find targets!” button which will run programs in

the background to get the results. If users want to retrieve a recent job, they can click “Retrieve



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Jobs” on the left side menu to enter the “Job ID”, which is generated in the result page, for

retrieving results in later times. The results will be kept in our server for only one week and will

be deleted automatically afterwards.

Graphic Output Interface

Figure 4A shows all the target candidates and relevant information for the user input

RNA sequence. Users can view the input sequence by clicking the “Input Sequence Viewer”

button. In the sequence viewer, users can search and highlight any subsequence such as the target

candidate sequence (Figure 4B). The “Job ID” can be used to retrieve users’ recent jobs from

CRISPR-RT and all relevant data will be stored for a week in our server. Users can also

download the target candidates file by clicking the “Download” link. In the table, the protospacer

and PFS of each target candidate are labeled by different colors. The corresponding crRNA of

each target candidate can be accessed by clicking “crRNA”; a graph will appear to help users

design their own crRNAs (Figure 5). The table also displays the start position, end position, and

GC content of each target candidate sequence. The last column of the table shows the number of

target sites in the transcriptome for each target candidate. By clicking the “#Targets” header

users are able to rank all the target candidates based on the number of target sites. Target

candidates with fewer number of target sites have higher target specificity in the transcriptome.

If the number of target sites is 1, the corresponding table cell will be highlighted with green

background color to indicate that the target candidate is highly specific in the whole

transcriptome. By clicking the number of target sites users can view the detailed information of

target sites for each target candidate (Figure 4C). Because CRISPR-RT has converted the

transcriptome mapping result to a genome mapping result, the information of target sites are

displayed in genomic context with genomic coordinates and gene annotations, including mapped



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gene, chromosome, strand, start/end position, and transcript isoforms. Users can click the

“transcript ID” link or “gene ID” link of each target site to view the detailed description of

transcript or gene where the target site located. The number of mismatches or gaps in each target

site is also shown in the table. To visualize and manually validate the target sites, users can click

the “JBrowse” link to visualize each target site in the background of genome and transcript

features annotated by ENSEMBL (www.ensembl.org) (Figure 4D).

Discussion

The CRISPR-C2c2 system has been reported as a tool for RNA editing (Abudayyeh et al.,

2016). Additionally, CRISPR-C2c2 has already been successfully used for specific RNA

knockdown in E. coli (Abudayyeh et al., 2016), and it has been applied for RNA detection in

human total RNAs (East-Seletsky et al., 2016). The dC2c2, just like dCas9 (Bikard et al., 2013;

Qi et al., 2013; Gilbert et al., 2013; Piatek et al., 2015; Maeder et al., 2013; Perez-Pinera et al.,

2013; Mali et al., 2013; Cheng et al., 2013; Polstein and Gersbach, 2015; Nihongaki et al., 2015;

Zetsche et al., 2015; Gao et al., 2016), also has many potential applications as an RNA-binding

protein, such as bringing effectors to specific RNAs to regulate their translation and tracking

specific RNAs by fluorescent tag (Abudayyeh et al., 2016). Therefore, the CRISPR-C2c2 system

has been viewed as a powerfully programmable tool for current and future RNA editing

(Abudayyeh et al., 2016; East-Seletsky et al., 2016; Nainar et al., 2016; Wang and Qi, 2016;

Puchta, 2017). However, so far no available software was developed to design crRNAs for the

CRISPR-C2c2 system.

In this project, we studied the architecture and features of CRISPR-C2c2 from the

bacteria L. shahii. By emphasizing the flexible parameters input and the target specificity of



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crRNAs, we developed CRISPR-RT, the first web-based bioinformatics tool to help biologists

design the crRNA for the CRISPR-C2c2 system. Specifically, CRISPT-RT accepts a wide range

of parameters such as the number of mismatches or gaps tolerated by off targets, but maintains a

simple interface, making it highly flexible for current and future research in CRISPR-based RNA

editing and targeting. The powerful alignment tool - Bowtie2 (Langmead and Salzberg, 2012) is

used to search all the target sites within the reference transcriptome for each target candidate.

Researchers are able to choose the target candidate with minimum off-target effect for crRNA

designing. In addition, the on and off-target sites can be validated by data visualization through

JBrowse with proper gene and transcript annotations (Skinner et al., 2009).

CRISPR-RT currently covers 10 model organisms including human and can be easily

extended to cover other species. The availability of CRISPR-RT will help researchers improve

the target specificity of crRNA design for the CRISPR-C2c2 system and empower biologists in

CRISPR-based RNA editing and targeting.

Methods

CRISPR-RT is essentially composed of many web interfaces and a backend pipeline.

Web interfaces are implemented by PHP and JavaScript code, which are used to accept user

inputs and display the results interactively. The backend pipeline is implemented by Perl code,

which is used to process user input data and generate multiple result files.

After setting up proper parameters and clicking the “Find targets!” button in the

parameter setting page of CRISPR-C2c2 (Figure 3), all of the parameters stored in PHP code are

passed to the main Perl script, which invokes other specific Perl scripts or commands to execute

specific functions. As shown in Figure 6, first, the Perl script for target candidate search is called



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to find target candidates of specified length with the PFS in the input RNA sequence. Second,

Bowtie2 (Langmead and Salzberg, 2012) is used to map each target candidate sequence to the

reference transcriptome, which is extracted from the genome by RSEM (Li and Dewey, 2011)

using Ensembl gene annotation, to search for on- and off-target sites within the transcriptome.

Third, the Perl scripts for result filtration based on the input parameters are invoked to filter out

the off targets that do not meet the requirements set by users. Next, RSEM is used to convert the

transcriptome mapping result to a genome mapping result, which can be displayed properly in

JBrowse (Skinner et al., 2009). Then, the main Perl script separates the file storing target sites of

all target candidates into many single files that store target sites for each target candidate

respectively. The main Perl script also processes the file of target candidates and the files of

target sites for each target candidate to generate Ajax format files, which are required by

DataTables (a table plug-in for jQuery). After getting all of those files, the PHP and JavaScript

code is used to display detailed information of target candidates and corresponding target sites in

DataTables. The on- and off-target sites can be visualized in JBrowse.



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Figure 1. The configuration and architecture of the CRISPR-C2c2 system. The CRISPR array of CRISPR-C2c2 is

first transcribed into pre-crRNA. Then, the pre-crRNA is processed by C2c2 into mature crRNAs without attaching

to trans-activating crRNAs. The mature crRNA binds to C2c2 and guides C2c2 to target a specific single-strand

RNA at a proper target site.

C2c2

5’

Pre-crRNA

5’

Mature crRNA

C2c2

5’ 3’ PFS

Seed

Target RNA

C2c2 Cas1 Cas2 CRISPR array Leptotrichia shahii CRISPR-C2c2 locus

3’

3’



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Figure 2. The home page of CRISPR-RT web service.



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Figure 3. The parameter setting page of CRISPR-C2c2. First, users input an RNA sequence in FASTA format into

the text area. Second, users select a reference transcriptome. Third, users set up the PFS and crRNA requirements

for the CRISPR-C2c2 system. Fourth, users choose an off-target setting (either “Basic settings” or “Specific

settings”).



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Figure 4. The result web interfaces of CRISPR-RT. (A) The detailed information of all the target candidates in the

user input RNA sequence. (B) User input RNA sequence viewer. (C) The detailed information of target sites in the

transcriptome for each target candidate. (D) Visualization of on- and off-targets with gene and transcript annotations

in JBrowse.

B

C

A

D



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Figure 5. The basic process of crRNA design based on the target candidate sequence (protospacer). The target

complementarity sequence of crRNA is labelled in purple color. The stem-loop sequence of crRNA comes from

previous research (Abudayyeh et al., 2016). The designed crRNA binds to C2c2 to form a crRNA-C2c2 complex for

RNA targeting.



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Figure 6. The flowchart of CRISPR-RT backend pipeline that processes user input RNA sequence and displays

results in the interfaces. All of input parameters, including the RNA sequence, are passed to the main Perl script.

The main Perl script invokes other specific Perl scripts or commands to execute specific functions. Finally, the

results will be displayed in DataTables and JBrowse in the web interfaces.



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Acknowledgements

This project is funded by Office for the Advancement of Research and Scholarship (OARS) and

Biology Department, Miami University, Oxford, Ohio, USA.

Authors’ contributions

CL managed and coordinated the whole project. HZ and CL participated in CRISPR-RT design.

HZ implemented the backend pipeline in Perl. HZ, ER, and CL participated in web interfaces

implementation. HZ, ER, and CL prepared the manuscript. All authors have read and approved

the final manuscript.

Competing interests

The authors declare no competing financial interests.

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Documents

CRISPR-RT: A web service for designing CRISPR-C2c2 ...although C2c2 may also cleave collateral RNAs (Abudayyeh et al., 2016). Researchers have applied the CRISPR-C2c2 system to sensitively