TABLE 3.

Principles of crRNA designing for type VI-A/B/D CRISPR-Cas system.

Rule No	Parameter
	Positional rule
1	Perfect base pairing with target region a
1–1	Allowing single mismatches into the spacer
	Type VI-A	Central (seed) region or base pairs 13 to 24 LwaCas13a target are intolerant to single mismatches
	Type VI-B	Central (seed) region or base pairs 12 to 26 PspCas13b target are intolerant to single mismatches
	Type VI-D	Central (seed) region or base pairs 2 to 8 RfxCas13d target are intolerant to single mismatches
1–2	Allowing consecutive or nonconsecutive double mismatches into the spacer
	Type VI-A	Central (seed) region or base pairs 8 to 27 LwaCas13a target are intolerant to double mismatches (mismatch occurrence exactly at 5′ or 3′ end is preferred)
	Type VI-B	Central (seed) region or base pairs 12 to 29 PspCas13b target are intolerant to double mismatches (mismatch occurrence exactly at 5′ or 3′ end is preferred)
	Type VI-D	Double mismatches exactly at 5′ or 3′ end are acceptable
2	Protospacer flanking site or sequence (PFS) requirement
	LwaCas13a lacks PFS.
	In PspCas13b, not being D (G, A, or U) at first and second sites of 5′ PFS inhibits single-stranded RNA cleavage and being NAN or NNA at 3′ PFS enhances this activity
	The PFS may not be necessary for RfxCas13d
3	Target nucleotide content
	Cleavage preferentially occurs in uracil-rich regions (poly UU/AU; at uracil bases) for LwaCas13a and RfxCas13d and in adenine-rich regions (at adenine bases) for PspCas13b
4	GC content
Thermodynamics rules
5	Target site accessibility
	Not having any stable secondary structures in target region. This feature is important in type VI-A/B/D that targets RNA
6	Self-complementarity
BLAST rules
7	Searching off-target effects in target organism
8	Searching off-target effects in other organisms

^aG-U wobble base pairing is allowable.