Command Line Usage

If Python CRISPR Analyser is installed using pip, then the command line tools are available as follows:

Gather CRISPRs from genome files

For subsequent analysis we need to gather the CRISPRs from the genome files.

To run the Gather command:

crispr_analyser_gather -i <input_fasta> -o <output_file> -p <pam_sequence>

The parameters are:

-i, –ifile - the Input File needs to be a FASTA file containing the genenome sequence. For example GRCh38 which can be downloaded from Ensembl - Required,
-o, –ofile - the Output File which will be a CSV file (without headers) - Required,
-p, –pam - the PAM sequence which can consist of A, C, G, T and N (for any) - Required,
-h, –help - shows the help

For example:

crispr_analyser_gather -i Homo_sapiens.GRCh38.dna.chromosome.18.fa -o chromosome.18.csv -p "NGG"

Notes

the inpul file(s) can only be uncompressed FASTA files
the PAM sequence can only be three characters long e.g. “NGG”

Index gRNA guides in binary format

From the CRISPRs gathered from genome files we create a binary index of gRNA guides for efficient searching of CRISPRs and off-targets.

To run the Index command:

crispr_analyser_index -i <input_csv> -a <assembly> -o <offset> -o <output_bin> -s <species> -e <species_id> -g <guide_length> -p <pam_length>

The parameters are:

-i, –ifile - The Input CSV file, can be declared one or many times (see example below) - Required,
-o, –ofile - The Output binary file - Required,
-a, –assembly - The name of the genome assembly - Required,
-s, –species - The name of the species - Required,
-f, –offset - the offset after which to start the ID for CRISPRS, defaults to 0,
-e, –species_id - The species ID, defaults to 0,
-g, –guide_length - The length of the gRNA, defaults to 20,
-p, –pam_length - The length of the PAM, defaults to 3,
-h, –help - shows the help

for example:

crispr_analyser_index -i chromosome.1.csv -i chromosome.2.csv -o guides.bin -a GRCh38 -s Human

The CSV input file must have the following fields (but no headers):

Chromosome Name as a string e.g. ‘18’
Position Start - an integer indicating the start position offset from the start of the Chromasome (using 5’ to 3’ orientation),
CRISPR Sequence - the string representing the CRISPR including the PAM,
PAM Right? - a 1 or 0 (true or false) indicating if the PAM is on the right side of the CRISPR,
Species ID - this is currently always set to 1.

Note that Species ID is a legacy field and is not used in the current version of the software.

Find CRISPR IDs given a gRNA sequence

In order to find CRISPR IDs given a gRNA sequence we can use the Search command with the binary gRNA guides file created by the Index command.

Run the Search command as follows:

crispr_analyser_search -i <input_bin> -s <gRNA_sequence>

The parameters are:

-i, –ifile - The Input binary guides file - Required,
-s, –search - The gRNA sequence - Required.

Output is split between STDERR and STDOUT. The IDs of the CRISPRs are printed to STDOUT and the summary of the search is printed to STDERR.

For example, output to STDERR might look like:

Version is 3
Assembly is GRCh38 (Human)
File has 1200559014 sequences
Sequence length is 20
Offset is 0
Species id is 1
Loading took 12.25 seconds
Loaded 1200559014 sequences
Found 1 exact matches
Found the following matches:

And the CRISPR IDs found would be printed to STDOUT, separated by newlines:

1200551676
3249821234

Find off-targets for given CRISPR IDs

We can calculate the summary of off-targets for one or more CRISPRs given their ID and using the Align command as follows:

crispr_analyser_align -i <input_bin> [ids]

The parameters are:

-i, –ifile - The Input binary guides file - Required
–no-cuda - Disable CUDA GPU acceleration
[ids] - one or more IDs of the CRISPRs to search for off-targets - Required

As with the Search command, the output is split between STDERR and STDOUT. The summary of the search is printed to STDERR and the off-targets are printed to STDOUT.

For example, output to STDERR might look like:

Assembly is GRCh38 (Human)
File has 1200559014 sequences
Sequence length is 20
Offset is 0
Species id is 1
Loading took 7.33 seconds

And the off-targets found would be printed to STDOUT, separated by newlines. The format for off-targets for each CRISPR ID is:

<CRISPR ID> <Species ID> <Off-Target CRISPR IDS> <Summary>

For example:

1200551673	1	{5631600,12587091,19109163,22079030,23239000,26501597,30414111,33581723,34229111,34705555,37873467,43668607,45229014,50360433,51303777,53203244,53787722,54810107,55678064,56111581,57172117,60111258,65696513,73938350,78186291,78909324,79032225,79131485,79199752,81814531,83282318,89311432,91700045,94095775,104106532,110628740,117838121,119302948,120328606,121417854,123785758,128025190,129308028,129383427,131704114,141154489,143221468,144953871,153584006,156820656,159690561,162463025,165704925,166653098,169556513,169894014,170813225,172596043,173336999,176798557,176900263,179624805,179879820,180325270,183714994,183725616,187191479,188483145,190105865,194567945,195833213,199102585,199290226,204836295,205765766,213995462,215497592,217290825,218224164,218365574,219065926,222205282,228956564,230872099,231530511,233998802,235057869,236017990,242874656,252500366,252748162,261804881,269244299,270042584,272049195,277367575,280089801,284873674,285627010,287098376,288534000,289237233,289343988,289586896,290746480,298407995,299035379,299567888,305616511,308195124,309038582,313972681,314572210,314878175,316358683,316911638,323421292,325962990,326919721,329949795,331922709,334295731,334846296,338562318,341435450,341728613,342597884,343360904,343429153,352681255,353840365,357616747,357754197,358166591,368932159,379035147,380305864,385343331,388328199,392759812,394013711,395936214,408491849,408755686,410174279,414936251,418511742,425649740,427086025,427886947,429883343,435641334,436616343,437452167,441768519,442845158,444661399,444882063,445672975,447255070,464745129,470350960,471383043,473890728,475060642,475095613,476200278,477156460,479489876,490092071,493128577,500044319,503670391,504239932,506502770,511566658,513551025,515010796,516910537,518522314,518874302,527066517,532871367,540555229,545417977,546590944,548995953,554798163,560390395,561444072,562793216,567065284,570069112,571278043,572715670,575189618,578564090,583318535,590254469,595390813,598450204,599871415,600025117,602055868,602803144,604432573,606497148,607805800,609515434,612971857,613980801,614891455,616693924,617497175,618674279,618693888,619233704,620102197,620871357,627733346,643459607,647558077,650210205,654411215,656081271,656174099,657016785,659425058,662332362,666877185,668392011,670105960,671623434,673828082,676419287,678522049,680269126,681812795,682181056,683858037,687904397,688086499,695096280,699808758,700377621,708176275,714939183,724429779,727287866,730342818,734284179,746863119,756989553,764145480,769124497,772312880,772734053,772994362,776138952,778124819,787329528,794112125,801591361,803539013,807757046,810087625,811896473,824317786,827097789,828717444,833506447,835651412,841880152,844083167,845039322,847014004,849545231,851108109,851287298,855567341,856446172,863783477,864162969,873077012,878677577,882676574,884021802,904384677,906818273,908646372,914503814,919559357,922317697,922699712,923562980,923905133,926409128,929053437,929419673,929656450,935525941,948268750,949547398,949827174,950751939,954122925,957141360,964414988,964488502,965562242,968418491,973758282,974537517,978780419,981858792,998620175,1000284084,1006076964,1012073812,1014825674,1016416691,1020485961,1020497125,1022107423,1023413695,1027567634,1030193706,1030733180,1031536226,1034216041,1037422056,1044851271,1048162796,1052896575,1054970838,1060958364,1061603994,1064490807,1065149388,1065786298,1070371661,1072182346,1072279792,1081522519,1082356418,1083654553,1083959717,1087957969,1094427998,1096775589,1099668608,1107229436,1108523358,1113194026,1118220025,1134507117,1135049225,1135867617,1139813789,1142353946,1144755203,1146013879,1147253218,1153982157,1164707102,1165778714,1167191051,1167628030,1170541198,1173456426,1173689054,1176790664,1178223456,1181252406,1200551673}	{0: 1, 1: 0, 2: 4, 3: 18, 4: 352}
910190339	1	{0: 1, 1: 7, 2: 109, 3: 951, 4: 2452}

Note that any CRISPRs with more than 2000 off-targets will not have the off-target CRISPR IDs printed to STDOUT as shown in the second CRISPR above.

GPU Acceleration

The Align command will run on GPUs if it detects a compatible Nvidia GPU. Note that CUDA libraries are only installed on Linux. To disable GPU acceleration use the –no-cuda flag. This software supports CUDA 12 but depending on the minor version of CUDA you may need to run the Align command with the NUMBA_CUDA_ENABLE_PYNVJITLINK=1 environmental variable. For example:

NUMBA_CUDA_ENABLE_PYNVJITLINK=1 crispr_analyser_align -i grch38_ngg.bin 23322 44343