Sorry, I didn't test the code well enough. I thought the capturing parenthesis would capture the look-behind pattern as well. This code will do what I originally intended:

Updated (thanks Cristoforo)

while ( $line =~ / (?<= .{9} [ATCG]{10} G ) G /gsx ) {
    my $match = substr $line, $+[ 0 ] - 21, 21;
    print $KMERS '>crispr_', ++$count, "\n$match\n"
        unless $unique_data{ $match }++;
    }
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