cas9.gRNA.oligo2: A cas9.gRNA.oligo2 Function

In kevinzhongxu/CasOligo: A R package to search the gRNA-target-site oligo for designing the taxon-specific guide RNA used for CRISPR-Cas Selective Amplicon Sequencing (CCSAS) to assess the eukaryotic microbiome of hosts (e.g. metazoans, plant)

A cas9.gRNA.oligo2 Function

Description

This function allows you to design gRNA-target-site oligos for the guding RNA to direct Cas9 nuclease to specifically cut a gene (e.g. the rRNA genes of 16S, 18S, 23S, etc. Or any other genes) of hosts but not of protists and fungi, as long as a reference sequences database of that gene is provided.

Usage

cas9.gRNA.oligo2(inseq, refseq, target = NULL)

Arguments

inseq	'Path/To/Your/Input_sequence_fasta_file'; For example: inseq="/home/kevin/Desktop/data/pacific_oyster_18S_V4.fasta".
refseq	'Path/To/Your/Reference_sequences_database_fasta_file', For example: database="/home/kevin/Desktop/data/silva_v119_18S_99rep_V4.fasta".
target	The host taxonomic group (from species to kingdom) that you think the obtained sgRNA plan to target; If there is space between two word, the sapce should be replace using "_". For example, target="Homo sapiens" need to change to be target="Homo_sapiens". The target is aiming to obtain target range of your probe (how good it is in helping cleaving the sequence of reference database either within same group or higher taxonomy level).

Details

To conduct this function, you need to generate your own reference sequence database for the genes that you want to target.

Please keep in mind that your input sequence and reference sequences must be within same gene region.

If you aim to cut host 18S rRNA genes of V4 region that are generated using 18S primers "TAReuk454FWD1 and TAReukREV3" (Stoeck et al., 2010), you could use easily cas9.gRNA.oligo1() function as it based on reference database of this region.

Examples

##### If you want to predict the gRNA's target range among a host taxonomic group
cas9.gRNA.oligo2(inseq="Path/To/Your/Input_sequence_fasta_file.fasta", refseq="PATH/To/Your/Reference_database_file.fasta", target="Taxonomic_group_of_a_host")

##### If you do NOT want to predict the gRNA's target range among a host taxonomic group
cas9.gRNA.oligo2(inseq="Path/To/Your/Input_sequence_fasta_file.fasta", refseq="PATH/To/Your/Reference_database_file.fasta")


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##### To design sgRNA for your human 18S (other regions of 18S rRNA gene) and predict the sgRNA's host-target range among other "Homo_sapiens" sequences in SILVA.
cas9.gRNA.oligo2(inseq="/home/kevin/Desktop/data/human.fasta", refseq="PATH/To/Your/Reference_database_file.fasta", target="Homo_sapiens")

##### To design sgRNA for your human 18S (other regions of 18S rRNA gene), but if you do not want to predict the sgRNA's host-target range among other taxonomic groups.
cas9.gRNA.oligo2(inseq="/home/kevin/Desktop/data/human.fasta", refseq="PATH/To/Your/Reference_database_file.fasta")

##### To design sgRNA for your oyster 18S (other regions of 18S rRNA gene) and predict the sgRNA's host-target range among other "Crassostrea_gigas" sequences in SILVA.
cas9.gRNA.oligo2(inseq="/home/kevin/Desktop/data/pacific_oyster_18S_V4.fasta", refseq="PATH/To/Your/Reference_database_file.fasta", target="Crassostrea_gigas")

##### To design sgRNA for your oyster 18S (other regions of 18S rRNA gene) and predict the sgRNA's host-target range among other "Ostreidae" sequences in SILVA.
cas9.gRNA.oligo2(inseq="/home/kevin/Desktop/data/pacific_oyster_18S_V4.fasta", refseq="PATH/To/Your/Reference_database_file.fasta", target="Ostreidae")

##### To design sgRNA for your oyster 18S (other regions of 18S rRNA gene) and predict the sgRNA's host-target range among other "Mollusca" sequences in SILVA.
cas9.gRNA.oligo2(inseq="/home/kevin/Desktop/data/pacific_oyster_18S_V4.fasta", refseq="PATH/To/Your/Reference_database_file.fasta", target="Mollusca")

##### To design sgRNA for your oyster 18S (other regions of 18S rRNA gene), but if you do not want to predict the sgRNA's host-target range among other taxonomic groups.
cas9.gRNA.oligo2(inseq="/home/kevin/Desktop/data/pacific_oyster_18S_V4.fasta", refseq="PATH/To/Your/Reference_database_file.fasta")

kevinzhongxu/CasOligo documentation built on Feb. 25, 2025, 8:43 a.m.