CaSilico: In silico crRNA design for CRISPR-Cas12 (A, B and F1...

In mrb20045/CaSilico: In silico crRNA design for different CRISPR-Cas subtypes

In silico crRNA design for CRISPR-Cas12 (A, B and F1 subtypes) and CRISPR-Cas13 (A, B and D subtypes).

Description

In silico crRNA design for CRISPR-Cas12 (A, B and F1 subtypes) and CRISPR-Cas13 (A, B and D subtypes).

Usage

CaSilico(
  ResultsFolder = "CaSilico_output",
  TargetFasta = NULL,
  TargetAccession = NULL,
  TargetCoordinate = list(chromosome = NA, start = NA, end = NA, strand = NA, species =
    NA),
  CrisprTypes,
  ConservationMethod = 1,
  ConservationThreshold = 0.95,
  OffTarget = F,
  OffAsk = F,
  Organism = NULL,
  LocalOff = F,
  LocalFasta = c("Path1", "Path2"),
  LocalName = c("Organism1", "Organism2"),
  Threads=4
)

Arguments

ResultsFolder	Name of the folder to save the final results; Default: ReaultsFolder="CaSilico_output".
TargetFasta	Full path to the target sequence(s) to design crRNA for; Example: TargetFasta="/usr/mrb/casilico/genome.fasta"
TargetAccession	Genome/gene/transcript accession numbers(s)(RefSeq or ENSEMBL ID)for the target sequence(s) to design crRNA for; Example: TargetAccession=c("U15717", "U15718").
TargetCoordinate	Genomic coordinate(s)for the target sequence(s) to design crRNA for. CaSilico automatically retrieves the sequences based on the coordinates and their species; Example: TargetCoordinate=list(chromosome=c("1","5"), start=c("2000","12000"), end=c("2150","15500"), strand=c("-","-"), species = c("bos_taurus","bos_taurus")).
CrisprTypes	A vector of CRSPR subtypes to design crRNAs based on their characteristics. One or more subtypes can be selected; Example: CrisprTypes=c("casVI_A","casVI_B","casVI_D","casV_A","casV_B","casV_F1").
ConservationMethod	Desired method (1 or 2) to detect the conserved regions when more than one target sequence is submitted. See detailes for more information; Default: 1.
ConservationThreshold	Desired threshold (between 0 and 1) to detect the conserved regions when more than one target sequence is submitted; Default: 0.95.
OffTarget	If TRUE, CaSilico search for off-targets in the sequences specified by the Organism or LocalOff argument. This analysis may take a long time based on the number of subtypes in CrisprTypes argument and number of the selected sequences to be searched (Organism or LocalFasta arguments); Default: FALSE.
OffAsk	If TRUE, in online mode before starting the off target analysis for each subtype, CaSilico ask from the user if he/she wants to continue or not; Default: FALSE.
Organism	A vector of desired organisms for online off target analysis. CaSilico automatically search the identified crRNAs on the sequences(s)(NR or Refseq database, based on the CRISPR-Cas subtype) of the specified organismes; Example: Organism=c("Homo sapiens","Bos taurus",...).
LocalOff	If TRUE, CaSilico search for off targets in local mode and Organism argument dose not works. LocalFile have to be set. Default: FALSE.
LocalFasta	Full path to the fasta file sequence(s) to be used for local off target analysis. One or more fasta file can be addressed; Example: LocalFasta=c("/yourpath1/filename1.fasta","/yourpath2/filename2.fasta").
LocalName	A vector including desired names related to the LocalFasta argument to determine the name of the results. If it is'nt specified by the user, Casilico considers sequence1, sequence2 and etc based on the number of fasta files in LocalFasta argument; Example: LocalName=c("Name1", "Name2").
Threads	Number of threads to use, default value is 4.; Example: Threads=8.

Value

Casilico presents the results in HTML and PDF files that can be found in ReaultsFolder.

References

...

Further details

Casilico accepts three kinds of input formats as target sequence to be scanned for all potential target sites: 1) direct sequence(s) (TargetFasta argument); 2) Accession nember (TargetAccession argument) and 3) genomic coordinate (TargetCoordinate argument). One of these arguments have to be determined by the user as TargetFasta is recommended. Also, there are two different approaches to identify conserved regions (using ConservationMethod argument): 1) Most or all positions in the fragments (with length of w) have to be conserved and ≤2 positions can be polymorphic (based on the defined conservation score), as these positions can be occurred in the specific locations of the window, 2) Most or all positions in the fragments (with length of w) have to be conserved and ≤2 positions can be polymorphic (based on the defined conservation score) in everywhere of the fragment (with length of w).

Examples

library("CaSilico")

############# (1) #############
# Run with TargetFasta and without off-target analysis

# Full path of target sequences
data <- paste0(system.file(package = "CaSilico"),"/data/3D.fasta")

# Run CaSilico for subtype VI_A with ConservationThreshold = 0.98 and ConservationMethod = 1
CaSilico(ResultsFolder="Example",
         TargetFasta=data,
         TargetAccession=NULL,
         CrisprTypes=c("casVI_A"),
         ConservationMethod = 1,
         ConservationThreshold=0.98,
         OffTarget = F,
         OffAsk = F)
##############################
#
#
#
############# (2) #############

# Run with TargetFasta and offline off-target analysis

# Full path of target sequences
data <- paste0(system.file(package = "CaSilico"),"/data/3D.fasta")

# Full path to fasta file sequences for local off target analysis
genome_dir <- paste0(system.file(package = "CaSilico"),"/data/genomes_off_target")
Local_Fasta <- file.path(genome_dir,list.files(genome_dir))

# Names related to the LocalFasta argument to determine the name of the results. (optional)
Local_Name <- c("Genome1","Genome2","Genome3")

# Run CaSilico for subtypes V_A and V_B with ConservationThreshold = 0.98 and ConservationMethod = 1
CaSilico(ResultsFolder="Example1",
         TargetFasta=data,
         TargetAccession=NULL,
         CrisprTypes=c("casV_A","casV_B"),
         ConservationMethod = 1,
         ConservationThreshold=0.98,
         OffTarget = T,
         OffAsk = F,
         Organism=NULL,
         LocalOff=T,
         LocalFasta=Local_Fasta,
         LocalName=Local_Name)
##############################
#
#
#
############# (3) #############
# Run with TargetFasta and online off-target analysis

# Full path of target sequences
data <- paste0(system.file(package = "CaSilico"),"/data/3D.fasta")

# Run CaSilico for subtype VI_A with ConservationThreshold = 0.98 and ConservationMethod = 1
# and online off-target analysis for Homo sapiens
CaSilico(ResultsFolder="Example2",
         TargetFasta=data ,
         TargetAccession=NULL,
         CrisprTypes=c("casVI_A"),
         ConservationMethod = 1,
         ConservationThreshold=0.98,
         OffTarget = T,
         OffAsk = F,
         Organism="Homo sapiens",
         LocalOff=F)
##############################
#
#
#
############# (4) #############
 # Run with accession numbers

# Run CaSilico for subtypes VI_A and VI_B with ConservationThreshold = 0.98 and ConservationMethod = 1
# The accession numbers used are: "U15717" and "U15718"
 CaSilico(ResultsFolder="Example3",
          TargetAccession=c("U15717", "U15718"),
          CrisprTypes=c("casVI_A","casVI_B"),
          ConservationMethod = 1,
          ConservationThreshold=0.98,
          OffTarget = F,
          OffAsk = F)
##############################
#
#
#
############# (5) #############
 # Run with sequence coordinate
 
 # Run CaSilico for subtypes VI_A and VI_B with ConservationThreshold = 0.98 and ConservationMethod = 1
 # The desired coordinates are: 
 # (1) chromosome = 1, start = 2000, end = 2150, strand = "-", species = "bos_taurus"
 # (2) chromosome = 1, start = 2000, end = 2150, strand = "-", species = "bos_taurus"
 CaSilico(ResultsFolder="Example4",
          TargetCoordinate=list(chromosome=c("1","1"),
                             start=c("2000","2000"),
                             end=c("2150","2150"),
                             strand=c("-","-"),
                             species = c("bos_taurus","bos_taurus")),
          CrisprTypes=c("casVI_B","casVI_A"),
          ConservationMethod = 1,
          ConservationThreshold=0.98,
          OffTarget = F,
          OffAsk = F)
##############################

mrb20045/CaSilico documentation built on Nov. 1, 2023, 10:34 a.m.