Table 2.

Feature summary for the four selected Cas effector candidates.

Identifier	Source Organism	Features	gRNA
Lwa Cas13a	Leptotrichia wadeii	Size: 1389 aa Structure: not available Nuclease domain: HEPN dCas mutations: D403G, R474A, and R1046A [44] PFS: not required Optimal spacer length: 20–28 nt Specificity: collateral cleavage in vitro but not in mammalian cells Turnover kinetics: not available Others: used for SHERLOCK diagnostics	5′-GATTTAGACTACCCCAAAAACGAAGGGGACTAAAAC-SPACER
Sau Cas9	Staphylococcus aureus	Size: 1053 aa Structure: 5AXW Nuclease domain: RuvC and HNH dCas mutations: D10A (RuvC), N580A (HNH) [26] PAM/PFS: PFS not required for ssRNA targeting; PAM required for dsDNA targeting (5’NNGRRT) Optimal spacer length: 23 nt Specificity: High DNA target specificity due to long PAM; mismatch tolerance characterization available for its RNA-targeting role; no collateral activity Turnover kinetics: DNA (multiple), RNA (single) Others: target secondary structure affects affinity	5′-SPACER- GTTTTAGTACTCTGGAAACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTCGTCAACTTGTTGGCGAGATTT
Lbu Cas13a	Leptotrichia buccalis	Size: 1159 aa Structure: 5XWP Nuclease domain: HEPN dCas mutations: R472A, H477A, R1048A and H1053A [84] PFS: not required Optimal spacer length: 20–24 nt Activity: collateral activity with high turnover (104 turnovers per target RNA recognized) [41] Turnover kinetics: not available for target cleavage but multiple turnover for collateral cleavage	5′- GGCCACCCCAAAAATGAAGGGGACTAAAACA-SPACER
Es Cas13d	Eubacterium siraeum	Size: 954 aa Structure: 6E9F Nuclease domain: HEPN dCas mutations: R295A, H300A, R849A and H854A [71] PFS: not required Optimal spacer length: 20–30 nt Specificity: collateral activity Turnover kinetics: not available Others: robust expression in E. coli; limited activity in mammalian cells [71]	5′ AACTACACCCGTGCAAAAATGCAGGGGTCTAAAAC-SPACER